Journal of Experimental Botany, Vol. 65, No. 18, pp. 5367–5384, 2014doi:10.1093/jxb/eru295 Advance Access publication 19 July, 2014

v

ReseaRch PaPeR

Cucumber metal transport protein MTP8 confers increased tolerance to manganese when expressed in yeast and Arabidopsis thaliana

Magdalena Migocka1,*, Anna Papierniak1, Ewa Maciaszczyk-Dziubińska2, Piotr Poździk1, Ewelina Posyniak1, Arnold Garbiec3 and Sophie Filleur4,5

1 Wrocław University, Institute of Experimental Biology, Department of Molecular Plant Physiology, Kanonia 6/8, 50-328 Wrocław, Poland2 Wrocław University, Institute of Experimental Biology, Department of Genetics and Cell Physiology, Kanonia 6/8, 50-328 Wrocław, Poland3 Wrocław University, Institute of Experimental Biology, Department of Animal Developmental Biology, Sienkiewicza 21, 50-335 Wrocław, Poland4 Institut des Sciences du Végétal, CNRS, 1 Avenue de la Terrasse, Saclay Plant Sciences Labex, 91198 Gif-Sur-Yvette Cedex, France5 Université Paris 7-Denis Diderot, UFR Sciences du Vivant, Saclay Plant Sciences Labex, 91198 Gif-Sur-Yvette Cedex, France

* To whom correspondence should be addressed. E-mail: mmigocka@biol.uni.wroc.pl

Received 17 April 2014; Revised 8 June 2014; Accepted 12 June 2014

Abstract

Cation diffusion facilitator (CDF) proteins are ubiquitous divalent cation transporters that have been proved to be essential for metal homeostasis and tolerance in Archaebacteria, Bacteria, and Eukaryota. In plants, CDFs are des-ignated as metal tolerance proteins (MTPs). Due to the lack of genomic resources, studies on MTPs in other plants, including cultivated crops, are lacking. Here, the identification and organization of genes encoding members of the MTP family in cucumber are described. The first functional characterization of a cucumber gene encoding a member of the Mn-CDF subgroup of CDF proteins, designated as CsMTP8 based on the highest homology to plant MTP8, is also presented. The expression of CsMTP8 in Saccharomyces cerevisiae led to increased Mn accumulation in yeast cells and fully restored the growth of mutants hypersensitive to Mn in Mn excess. Similarly, the overexpression of CsMTP8 in Arabidopsis thaliana enhanced plant tolerance to high Mn in nutrition media as well as the accumulation of Mn in plant tissues. When fused to green fluorescent protein (GFP), CsMTP8 localized to the vacuolar membranes in yeast cells and to Arabidopsis protoplasts. In cucumber, CsMTP8 was expressed almost exclusively in roots, and the level of gene transcript was markedly up-regulated or reduced under elevated Mn or Mn deficiency, respectively. Taken together, the results suggest that CsMTP8 is an Mn transporter localized in the vacuolar membrane, which participates in the maintenance of Mn homeostasis in cucumber root cells.

Key words: CDF (cation diffusion facilitator) family; Cucumis sativus; heavy metals; metal homeostasis; MTP (metal tolerance protein) family; tonoplast.

Introduction

The proteins of the cation diffusion facilitator (CDF; TC 2.A.4) family are ubiquitous membrane transporters identi-fied in all living organisms which participate in the efflux or sequestration of divalent cations within the apoplast or intra-cellular compartments (Nies and Silver, 1995; Anton et al.,

1999; Delhaize et  al., 2003; Hall and Williams, 2003; Nies, 2003; Kambe et al., 2004; Grass et al., 2005). In plants they are designated as metal transport proteins (MTPs), whereas in mammals CDF proteins are known as zinc transporters (ZnTs). Most of the CDF proteins characterized in detail are

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5368 | Migocka et al.

H+(K+)-coupled secondary transporters of different metal cations with ionic radii of 72 pm (Zn2+) to 97 pm (Cd2+) (Guffanti et al., 2002; MacDiarmid et al., 2002; Nies, 2003; Chao and Fu, 2004; Grass et al., 2005). The initial phyloge-netic and functional analyses of representative CDF members from Archaea, Eubacteria, and Eukaryotes allowed for the identification of three major substrate-specific groups within the family: Zn-CDF, Mn-CDF, and Zn/Fe-CDF (Montanini et al., 2007). The further phylogenetic analysis of plant CDF members from Viridiplantae and Rhodophyta (land plants) genomes revealed that all plant MTPs are organized in seven primary groups, G1, G5, G6, G7, G8, G9, and G12, which emerged prior to or simultaneously with land plant evolution (Gustin et al., 2011).

Most of the CDF transporters possess six putative trans-membrane domains (TMDs), with N- and C-termini localized in the cytoplasm and a histidine-rich region either between TMDs IV and V or within the N- or C-termini (Paulsen and Saier, 1997; Anton et al., 1999; Wei and Fu, 2006). However, the subgroup of manganese CDFs in plants (Mn-CDFs) shows a predicted 4–5 TMD homology and lacks the histi-dine-rich domain (Maser et al., 2001; Delhaize et al., 2003) and some human and yeast CDFs are predicted to have 12–15 TMDs (Li and Kaplan, 2001; Cragg et  al., 2002; Kambe et al., 2002). The improved tentative signature for CDF pro-teins covers the second TMD, the following cytosolic loop, and the start of the third TMD (Montanini et al., 2007).

Although bacterial and mammalian CDFs have been widely characterized, only a few members of plant MTPs have been studied in detail. It has been well evidenced that proteins of the MTP family participate in the vacuolar compartmentalization as well as in the efflux of heavy met-als from the cells of plant hyperaccumulators, showing the capacity for uptake and storage of elevated levels of metals in their aerial organs. In the genome of the Ni hyperaccu-mulator Thlaspi goesingense, several allelic variants of the TgMTP1 gene are present, all of which confer resistance to Zn in yeast mutants deficient in ZnTs (zrc1cot1) (Kim et al., 2004). In addition, yeast cells expressing TgMTP1 are more resistant to elevated Cd, Co, and Ni (Persans et  al., 2001). Interestingly, TgMTP1 may contribute to both metal efflux across the plasma membrane and metal sequestration within vacuoles, since the protein localized to the plasma mem-brane of Arabidopsis cells and to the plasma membrane and vacuolar membrane of yeast (Kim et  al., 2004). The zinc hyperaccumulator Arabidopsis halleri harbours five gene copies of MTP1 (Shahzad et al., 2010). Though all the tran-scripts confer more (AhMTP1-A1 and AhMTP1-A2) or less (AhMTP1-B, -C, and -D) increased tolerance to Zn in yeast deficient in zinc transport, only AhMTP1-A and AhMTP1-B appear to be crucial for increased tolerance of A. halleri to ele-vated Zn concentrations (Willems et al., 2007; Shahzad et al., 2010). In both hyperaccumulators, T. goesingense and A. hal-leri, the transcripts of MTP1 genes (TgMTP1, AhMTP1-A, and AhMTP1-B) are much more abundant in comparison with the level of mRNA of their single-copy orthologues in Arabidopsis thaliana (Persans et al., 2001; Shahzad et al., 2010). In contrast to T.  goesingense and A.  halleri, the

member of the MTP family in the legume hyperaccumula-tor Stylosanthes hamata has been shown to localize in the tonoplast of Arabidopsis cells. Overall evidence suggests that ShMTP8 is involved in the enhanced H+-coupled Mn seques-tration within vacuoles of S. hamata cells and thus confers plant resistance to environmental Mn excess (Delhaize et al., 2003). Members of the MTP family are also considered as essential contributors to Brassica juncea increased tolerance to heavy metals. Based on the yeast complementation assay and transcriptional profile of genes encoding the recently identified cation efflux transporters of B.  juncea, it may be suggested that three MTP proteins—BjCET2, BjCET3, and BjCET4—may be involved in the efflux of Zn, Cd, Co, and Ni from plant cells and thus play a substantial role in plant resistance to heavy metal stress (Xu et al., 2009; Lang et al., 2011).

It is assumed that in non-hyperaccumulator MTP trans-porters probably contribute to the basic metal tolerance, allowing the adjustment of heavy metal homeostasis in the cytoplasm within narrow concentration ranges (Clemens, 2001; Clemens et al., 2002). In the genome of the model plant A.  thaliana, 12 genes encoding putative MTP transporters have been identified, despite none of the A.  thaliana acces-sions exhibiting naturally selected metal tolerance (Cho et al., 2003; Becher et al., 2004). Nevertheless, only three Arabidopsis MTPs have been characterized in detail. AtMTP1 and AtMTP3 belonging to the CDF-Zn group have been local-ized to the tonoplast membrane in yeast and plant cells and proved to be involved in Zn and/or Co sequestration within plant cell vacuoles (Kobae et al., 2004; Desbrosses-Fonrouge et al., 2005; Arrivault et al., 2006). In contrast, AtMTP11, a close orthologue of ShMTP8, localizes to trans-Golgi or pre-vacuolar organelles of plant cells and determines the cellular Mn homeostasis (Delhaize et al., 2007; Peiter et al., 2007).

Due to the lack of genomic resources, only single MTP members have been studied in other non-hyperaccumulat-ing plants. MTP1 transporter from hybrid poplar Populus trichocarpa×Populus deltoides localizes to the tonoplast membrane and, similarly to AtMTP1, participates in vacu-olar Zn sequestration, whereas rice MTP1 protein resides at the plasma membrane and displays broader affinity for heavy metals, contributing to the active Zn, Cd, and Ni efflux into the apoplast of plant cells (Blaudez et al., 2003; Yuan et al., 2012). A very recent study revealed that the rice gene OsMTP8.1 encodes a tonoplast-localized Mn trans-porter which confers Mn tolerance through the sequestration of Mn into vacuoles of rice shoot cells (Chen et al., 2013). Nevertheless, studies on members of the plant MTP family in non-model plant organisms are still lacking.

Since CDF proteins in bacteria, yeast, mammals, and humans proved to be important for the homeostasis of vital metals within the cells, and plants are an essential source of microelements for animals and humans, further studies of MTPs in cultivated crops could provide strategies for bioreme-diation and human nutrition and health (Hall and Williams, 2003; Nies, 2003; Kambe et al., 2004). After tomatoes, cab-bage, and onions, cucumbers are the fourth most widely culti-vated vegetable crop in the world (Shetty and Wehner, 2002).

Functional analysis of cucumber metal tolerance protein 8 | 5369

During the last 2 years, the genomes of three cucumber geno-types have been sequenced, including the North American pickling type inbred line Gy14 (http://cucumber.vcru.wisc.edu/), the Chinese long inbred line 9930 (Huang et al., 2009), and the northern European cultivar Borszczagowski line B10 (Woycicki et  al., 2011). The genomic sequences of the two latter cultivars have been deposited in the GenBank database under the accession nos ACHR0100000 (Chinese long) and ACYN01000000 (Borszczagowski). In the present study, the features and phylogeny of cucumber genes encoding putative proteins of the MTP family that have been retrieved through the survey of two cucumber genomic databases are presented. The localization and function of CsMTP8, one of the three cucumber MTPs homologous to members of the Mn-CDF (MTP8–MTP11) subgroup of plant MTPs, are also described. This work presents the first functional characterization of an MTP8-like protein in a non-hyperaccumulator dicot plant and the first survey of the MTP family in cucumber.

Materials and methods

Plant material and growth conditionsCucumber plants (var. Krak) were grown hydroponically as pre-viously described (Migocka et  al., 2011). The nutrient solution was continuously aerated and exchanged twice a week. For organ expression analyses, particular organs of 2-week-old seedlings (roots, hypocotyls, cotyledons, petioles, and leaves) were collected separately and immediately frozen in liquid nitrogen. For short-term manganese treatment, 2-week-old plants were transferred on nutrient solution, pH 5.5, containing 50 μM MnSO4 for 10 h. For long-term Mn stress, plants were grown for 2 weeks on the nutri-ent solution, pH 5.5, with Mn deprivation. For each treatment, four organ samples of 100 mg from four different plants were taken for RNA extraction and immediately frozen in liquid nitrogen before storage at –80 °C.

Arabidopsis thaliana ecotype Columbia were grown in a phy-totron with 16/8 h photoperiod at 250  μmol m−2 s−1 and 23  °C (day)/22 °C (night). Following the stratification of seeds for 3 d at 4  °C, plants were grown on soil (to obtain and propagate plants transformed with CsMTP8 or with empty vector) or on a half-strength Murashige and Skoog (MS) medium (pH 5.8; Sigma, St. Louis, MO, USA), liquid or supplemented with 5 g l−1 Phytagel (Sigma). For the growth tests, the solid MS medium was addition-ally supplemented or not with 4 mM MnSO4.

Expression of CsMTP8 in yeastThe following Saccharomyces cerevisiae strains were used in this study: K667 (cnb1::LEU2 pmc1::TRP1 vcx1Δ), from Cunningham and Fink (1996) and isogenic to W303-1A (MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1) (Wallis et  al., 1989); and zrc1Δ (ZRC1::kanMX4), cot1Δ (COT1::kanMX4), and pmr1Δ (PMR1::kanMX4) all from Euroscarf (Germany), and isogenic to BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) (Brachmann et al., 1998).

For synthesis of cDNAs, total RNA isolated from 2-week-old cucumber roots was transcribed using Superscript III RNase H reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo(dT) primer. For the growth and localization assays, two expression vectors were used: pUG35 and pUG36 (Niedenthal et  al., 1996), which enable the expression of CsMTP8–geen flu-oresent protein (GFP) or GFP–CsMTP8 fusion proteins in yeast, respectively. The full-length cDNA of CsMTP8 was ampli-fied using Phusion polymerase (New England BioLabs) with the

primers introducing EcoI and SalI sites (underlined): forward 5′-AAAGAATTCGATGGAGATTCGGATTTGAGTCCGAA-3′ and reverse 5′-TTTGTCGACTTAGGGCTGAGTGTTTGGTAAC CTG-3′ for ligation to pUG36 and forward 5′-AAAGAATTCA TGGATGGAGATTCGGATTTGAGTCCGAA-3′ and reverse 5′-T TTGTCGACGGGCTGAGTGTTTGGTAACCTG-3′ for ligation to pUG35. The plasmids were introduced into yeast by the LiOAc/PEG method (Gietz and Schiestl, 2007). The expression level of the fusion protein was regulated by adding methionine to the media. The transformants were grown at 30 °C on plates containing a syn-thetic complete medium consisting of yeast nitrogen base (Difco) without uracil, supplemented with amino acids and 2% glucose (SC-U/Glu, pH 7.0), and viewed using a confocal microscope (Olympus FluoView FV1000). For overexpression of CsMTP8 in yeast, the full-length cDNA of CsMTP8 was amplified using Phusion polymerase with the primers introducing CACC at the 5′ end of the amplicon (underlined): forward 5′-CAC CATGG ATGGAGATTCGGATTTGAGTCC-3′ and reverse 5′-GGGCT GAGT GTTTGGTAACCTG-3′. Following amplification, the cDNA was ligated into a Gateway entry vector pENTR/D-TOPO (Invitrogen) which was recombined with the pYES/DEST52 expres-sion vector through the LR reaction (Gateway system, Invitrogen) to obtain the pYES/DEST52-CsMTP8 vector expressing CsMTP8 tagged with the V5 epitope. The assays of CsMTP8 overexpression were performed under the control of the GAL1 promoter in a stand-ard minimal medium (SC-U) supplemented with amino acids and 2% galactose.

Yeast growth assayYeast strains transformed with pUG36-CsMTP8 or pUG36 were grown in liquid SC-U/Glu medium (pH 7.0) supplemented with amino acids up to the stationary phase. For the drop assay, four 10-fold serial dilutions were prepared in the same sterile medium for each culture, and 5 μl of each dilution was spotted onto SC-U/Glu plates supplemented with amino acids (–Met) and different con-centrations of ZnSO4, CdCl2, CoCl2, MnSO4, or NiCl2. The control drop assay was performed on the solid YPD medium. For liquid growth assay, 25 ml of SC-U medium supplemented with amino acids (–Met) and 2% glucose containing 5 mM MnSO4 was inocu-lated with yeast cells to obtain the initial OD600 of 0.2. Cultures were incubated at 30 ºC and the OD600 was measured in samples of 0.5 ml collected after 0, 4, 8, 12, 24, 36, and 48 h.

Metal transport in yeast cellsTo measure Mn uptake, the yeast cells growing exponentially in liquid SC-U medium [with 2% (w/v) glucose] supplemented with amino acids (–Met) were exposed to 5 mM MnSO4 and collected at the time points indicated in the figures. The cells were washed in ice-cold water, pelleted by centrifugation, resuspended in water, and boiled for 10 min. The supernatant collected following the sub-sequent centrifugation of the boiled cells was used to determine the metal content of each sample by a flame atomic absorbtion spec-trometer (AAS 3300, Perkin Elmer). The experiment was performed three times.

Tonoplast isolation from yeast, SDS–PAGE, and immunoblottingYeast cells transformed with plasmids carrying CsMTP8 or empty vectors were grown to the exponential phase in a standard minimal medium supplemented with amino acids (–Met) and 2% glucose (pUG36 vector) or 2% galactose (pYES/DEST52 vector). Crude membrane fractions were prepared from yeast cells essentially as described earlier (Norling, 2000). Tonoplast membrane vesicles were separated from total microsomes using a two-step sucrose gradient (15%/35%) as previously described (Nakanishi et al., 2001). To con-firm the presence of CsMTP8 in yeast tonoplasts, the membranes were subjected to western blot analysis using the antibodies raised

5370 | Migocka et al.

against GFP (pUG36 vector) or V5 (pYES/DEST52 vector). Crude membranes or tonoplast were subjected to SDS–PAGE (Laemmli, 1970) and then transferred to a nitrocellulose membrane (Millipore). After blocking with Blocking-reagent (Roth), the membrane was incubated with the primary antibodies raised against GFP (Roche) or V5 (Invitrogen). After repeated washing, nitrocellulose mem-branes were further incubated for 1 h with secondary antibodies conjugated to horseradish peroxidase (Agrisera) and visualized by staining with diaminobenzidene (DAB; Sigma-Aldrich).

Metal transport in yeast tonoplast membranesThe Mn2+ uptake by tonoplast vesicles was measured as described earlier (Migocka et al., 2011) in two different assays: indirectly, using a spectrophotometer (Beckman DU®640) to measure the changes in acridine orange absorbance at 495 nm reflecting the Mn2+-induced H+ movement across the membranes (assay I), and directly, by atomic absorbtion spectrometry, following the incubation of vesicles with metal and subsequent elution of intravescicular metal content with 3 M HCl (assay II). The uptake assay was performed in buffer containing 0.3 M sorbitol, 5 mM TRIS-MES (pH 7.6), 25 mM KCl, 0.1 mM sodium azide, 0.2 mM sodium orthovanadate, and 100 μg of vesicle protein. In addition, 5 μM acridine orange was included in the reaction medium of assay I. The V-ATPase-mediated generation of the transmembrane H+ gradient was initiated by the addition of 1 mM MgSO4 and 1 mM ATP to the reaction medium. After 3 min, the specific V-ATPase inhibitor bafilomycin (300 nmol) was used to stop ATP-energized H+ translocation. After 3 min, different concen-trations of MnSO4 were added to the reaction to initiate Mn2+/H+ antiport in tonoplast membranes.

Transient expression of CsMTP8 in protoplastsThe full-length cDNA of CsMTP8 was amplified using Phusion polymerase (New England BioLabs) with the for-ward primer introducing a CACC site at the 5′ end (underlined), 5′-CACCATGGATGGAGATTCGGATTTGAGTCC-3′; and reverse primer, without a STOP codon, 5′-GGGCTGAGTGT TTGGTAACCTG-3′. The PCR product was ligated into the pENTR/D-TOPO vector (Invitrogen, Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instruction. Fusion proteins with GFP were produced by the recombination (LR reaction) of entry vectors pENTR/D-TOPO-CsMTP8 with destina-tion vectors pMDC43 (N-terminal GFP) or pMDC83 (C-terminal GFP) (Curtis and Grossniklaus, 2003) using the Gateway system (Invitrogen). Transient expression experiments of GFP fusion pro-teins were performed on protoplasts isolated from A.  thaliana cell suspensions prepared essentially as described earlier (Thomine et al., 2003). Protoplasts containing the plasmids were incubated at 23 ºC for 2–3 d in the dark. Transformed cells were observed using an inverted Leica TCS-SP2 confocal laser scaning microscope (Leica Microsystems) with excitation at 488 nm, and the fluorescence emis-sion signal of GFP was recovered between 500 nm and 525 nm.

Plant transformationThe binary construct pMDC43-CsMTP8 or empty plas-mid pMDC43, harbouring a dual 35S Cauliflower mosaic virus (CaMV) promoter, were introduced into Agrobacterium tumefa-ciens GV3101 through electroporation. The Columbia ecotype of the wild-type line of A.  thaliana (L.) Heynh. was subsequently transformed with use of A.  tumefaciens by the floral dip method (Clough and Bent, 1998). Primary generation transformed seeds were selected in half-strength MS medium, pH 5.8, containing 0.5% (w/v) Phytagel (Sigma) and 100 mg l–1 kanamycin. An average of 15–20 T1 transgenic seedlings were obtained for each transfor-mation and at least 10 were used for selecting T2 transgenic plants resistant to kanamycin with a 3:1 ratio. Using primers specific for CsMTP8 (5′-GATTCA AGAT AAACCTTCTGAAAGTC-3′ and

5′-AGATCTTATCACCAAGAATAGCTG-3′), the two homozy-gous lines expressing CsMTP8 were selected based on PCR and real-time PCR (RT-PCR) analysis of the CsMTP8 transcript level in plants transformed with empty vector (control) or with vector carrying CsMTP8. The gene encoding tubulin AtTUB6 (At5g12250) was used as a reference control with the following prim-ers: forward 5′-GGTGAA GGAA TGGACGAGAT-3′ and reverse 5′-GTCATCTGCAGTTGCGTCTT-3′.

The analysis of Mn content in A. thalianaFor the accumulation assay, 14-day-old A. thaliana plants grown on a half-strength solid MS medium were transferred for 4 d into liq-uid nutrition medium composed as described earlier (Morel et al., 2009), containing or not 50 μM MnSO4. Subsequently, plants were thoroughly rinsed with 10 mM EDTA and distilled water, dried, and mineralized in a microwave in HNO3. The amount of Mn in the samples was determined using a flame atomic absorbtion spectrom-eter (AAS 3300, Perkin Elmer).

RNA isolation, reverse transcription, and qRT-PCRTotal RNA was extracted from cucumber organs using TRI-Reagent according to the manufacturer’s instructions (Sigma-Aldrich). Following digestion of the RNA samples with RNase-free DNase I (Fermentas), RNA was directly used in the one-step semi-quantitative RT-PCR using the Titan One Step RT-PCR System (Roche) according to the protocol provided by the manufacturer (with 25 and 26 cycles and melting temperature 60 ºC for CACS and CsMTP8, respectively). In addition, RNA collected from roots was reverse transcribed into cDNA for qRT-PCR assays. Reverse tran-scription was performed on 2 μg of total RNA using random prim-ers and a High Capacity cDNA synthesis Kit (Applied Biosystems). PCRs were performed using 2× SYBR Green Master Mix B (A&A Biotechnology) in a 96-well plate using Lightcycler 480 (Roche) in a total volume of 20  μl, containing 2  μl of 8-fold diluted cDNA template, 2 μl of each primer (10 μM), 4 μl of nuclease-free water, and 10 μl of 2× Master SYBR® Green I B (A&A Biotechnology). The reaction conditions were as follows: 95 °C for 10 min followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s, 72 °C for 15 s. Samples without template were used as negative controls in the same PCR run for each primer pair. The analysis of dissociation curves and agarose gel electrophoresis of PCR products were performed to con-firm the absence of non-specific by-products. Successive dilutions of the sample with the lowest Cp were used as a standard curve. The amplification efficiency for each primer pair was in a range 1.9–2.0. For each of the two independent RNA extractions, measurements of gene expression were obtained in triplicate. Specific primers for CsMTP8 (5′-GATTCAAGATAAACCTTCTGAAAGTC-3′ and 5′-AGATCTTATCACCAAGAATAGCTG-3′) were carefully designed using LighCycler Probe Design Software 2 (Roche) to amplify only a single PCR product. The gene encoding the clathrin adaptor complex subunit (CACS) was used as internal control with the following primers: 5′-TGGGAAGATTCTTATGAAGTGC-3′ and 5′-CTC GTCA AATTTACACATTGGT-3′. The stability of CACS expression under heavy metal stress was confirmed previ-ously (Migocka and Papierniak, 2011).

Protein determinationProtein was assayed according to Bradford (1976).

Database searching, prediction and analysis of CsMTPsThe nucleic acid sequences of cucumber MTP proteins have been retrieved from GeneBank from whole-genome shotgun reads con-taining the assembly of the cucumber genome using 12 AtMTP cDNA sequences as the initial queries and BlastN with default parameters to obtain high-stringency search results (Tables 1, 2).

Functional analysis of cucumber metal tolerance protein 8 | 5371

Tab

le 1

. Th

e lis

t of c

ontig

s an

d C

sMTP

gen

es r

etrie

ved

from

the

asse

mbl

y of

the

cucu

mbe

r ge

nom

e (C

hine

se lo

ng c

ultiv

ar, l

ine

9930

), sh

owin

g si

gnifi

cant

hom

olog

y w

ith

Ara

bido

psis

thal

iana

MTP

gen

es

MT

P g

ene

A. t

hal

ian

aG

ene

ID in

G

enB

ank

Acc

essi

on

no. o

f co

ntig

s co

ntai

ning

cu

cum

ber

M

TP

s

Str

and

o

rien

tati

on

Po

siti

on

of

pre

dic

ted

C

sMT

P g

enes

w

ithi

n co

ntig

No

. of

bas

e p

airs

in

pre

dic

ted

co

din

g

seq

uenc

es o

f C

sMT

P g

enes

No

. of

amin

o

acid

s in

p

red

icte

d

CsM

TP

s

No

. of

pre

dic

ted

ex

ons

No

. of

pre

dic

ted

in

tro

ns

Co

vera

ge

(%)

E-v

alue

Iden

tity

of

Ara

bid

opsi

s an

d c

ucum

ber

ho

mo

log

ues

(%)

No

men

clat

ure

of

nove

l cu

cum

ber

M

TP

s

AtM

TP1

(AtZ

AT1)

At2

g468

00A

CH

R01

0077

40–

7902

–914

912

4841

51

093

4e-1

1273

CsM

TP1

AC

HR

0100

7875

11 6

52–1

2 81

211

6138

61

036

3e-1

842

CsM

TP4

AC

HR

0103

1515

4572

–581

912

4841

51

093

4e-1

1273

CsM

TP1

AC

HR

0104

9518

4391

–555

111

6138

61

036

3e-1

842

CsM

TP4

AtM

TP2

(AtM

TPa1

)A

t3g6

1940

AC

HR

0100

7740

–79

02–9

149

1248

415

10

902e

-57

69C

sMTP

1A

CH

R01

0078

7511

652

–12

812

1161

386

10

653e

-12

44C

sMTP

4A

CH

R01

0315

1545

72–5

819

1248

415

10

902e

-57

69C

sMTP

1A

CH

R01

0495

1843

91–5

551

1161

386

10

653e

-12

44C

sMTP

4A

tMTP

3 (A

tMTP

a2)

At3

g588

10A

CH

R01

0077

40–

7902

–914

912

4841

51

074

2e-9

667

CsM

TP1

AC

HR

0100

7875

11 6

52–1

2 81

211

6138

61

031

3e-0

743

CsM

TP4

AC

HR

0103

1515

4572

–581

912

4841

51

074

2e-9

667

CsM

TP1

AC

HR

0104

9518

4391

–555

111

6138

61

031

3e-0

743

CsM

TP4

AtM

TP4

(AtM

TPb1

)A

t2g2

9410

AC

HR

0100

7740

–79

02–9

149

1248

415

10

562e

-09

42C

sMTP

1A

CH

R01

0078

7511

652

–12

812

1161

386

10

236e

-53

50C

sMTP

4A

CH

R01

0315

1545

72–5

819

1248

415

10

562e

-09

42C

sMTP

1A

CH

R01

0495

1843

91–5

551

1161

386

10

236e

-53

50C

sMTP

4A

tMTP

5 (A

tMTP

c2)

At3

g121

00A

CH

R01

0008

47–

24 5

92–2

9 89

211

9739

89

831

5e-1

663

CsM

TP5

AtM

TP6

(AtM

TPc1

)A

t2g4

7830

AC

HR

0100

0689

–95

126

–99

394

1587

528

1312

578e

-21

61C

sMTP

6A

tMTP

7 (A

tMTP

c4)

At1

g516

10A

CH

R01

0051

11–

88 4

54–9

3 80

015

0049

914

1359

2e-2

768

CsM

TP7

AtM

TP8

(AtM

TPc3

)A

t3g5

8060

AC

HR

0101

1125

–21

991

–25

437

1209

402

76

462e

-39

68C

sMTP

8A

tMTP

9A

t1g7

9520

AC

HR

0100

3497

+21

556

–23

399

1230

409

65

682e

-47

62C

sMTP

9A

CH

R01

0099

52+

4650

–949

211

8839

56

543

2e-2

653

CsM

TP11

AtM

TP10

At1

g163

10A

CH

R01

0034

97+

21 5

56–2

3 39

912

3040

96

564

5e-4

260

CsM

TP9

AC

HR

0100

9952

+46

50–9

492

1188

395

65

451e

-24

52C

sMTP

11A

tMTP

11A

t2g3

9450

AC

HR

0100

3497

+21

556

–23

399

1230

409

65

362e

-22

55C

sMTP

9A

CH

R01

0099

52+

4650

–949

211

8839

56

582

2e-7

875

CsM

TP11

AtM

TP12

At2

g046

20A

CH

R01

0006

89+

109

178–

111

346

2169

722

10

811e

-167

62C

sMTP

12

5372 | Migocka et al.

Tab

le 2

. Th

e lis

t of c

ontig

s an

d C

sMTP

gen

es r

etrie

ved

from

the

asse

mbl

y of

the

cucu

mbe

r ge

nom

e (B

orsz

czag

owsk

i cul

tivar

, lin

e B

10),

show

ing

sign

ifica

nt h

omol

ogy

with

A

rabi

dops

is th

alia

na M

TP g

enes

MT

P g

ene

A. t

hal

ian

aG

ene

ID in

G

enB

ank

Acc

essi

on

no. o

f co

ntig

s co

ntai

ning

cu

cum

ber

MT

P

gen

es

Str

and

o

rien

tati

on

Po

siti

on

of

pre

dic

ted

C

sMT

P g

enes

w

ithi

n co

ntig

No

. of

bas

e p

airs

in

pre

dic

ted

co

din

g

seq

uenc

es o

f C

sMT

P g

enes

No

. of

amin

o

acid

s in

p

red

icte

d

CsM

TP

p

rote

ins

No

. of

pre

dic

ted

ex

ons

No

. of

pre

dic

ted

in

tro

ns

Co

vera

ge

(%)

E-v

alue

Iden

tity

of

Ara

bid

opsi

s an

d c

ucum

ber

ho

mo

log

ues

(%)

No

men

clat

ure

of

nove

l cu

cum

ber

M

TP

s

AtM

TP1

(AtZ

AT1)

At2

g468

00A

CY

N01

0019

40–

15 6

89–1

6 93

612

4841

51

093

4e-1

1273

CsM

TP1

AC

YN

0100

2902

1744

–290

436

4e-1

742

CsM

TP4

AtM

TP2

(AtM

TPa1

)A

t3g6

1940

AC

YN

0100

1940

–15

689

–16

936

1248

415

10

902e

-57

69C

sMTP

1A

CY

N01

0029

02+

1744

–290

411

6138

61

063

3e-1

144

CsM

TP4

AtM

TP3

(AtM

TPa2

)A

t3g5

8810

AC

YN

0100

1940

–15

689

–16

936

1248

415

10

742e

-96

67C

sMTP

1A

CY

N01

0029

02+

1744

–290

411

6138

61

031

3e-0

743

CsM

TP4

AtM

TP4

(AtM

TPb1

)A

t2g2

9410

AC

YN

0100

2902

+17

44–2

904

1161

386

10

562e

-51

50C

sMTP

4A

CY

N01

0019

4015

689

–16

936

232e

-09

42C

sMTP

1A

tMTP

5 (A

tMTP

c2)

At3

g121

00A

CY

N01

0031

11+

27 5

41–3

2 83

811

9739

89

831

5e-1

663

CsM

TP5

AtM

TP6

(AtM

TPc1

)A

t2g4

7830

AC

YN

0100

5409

+75

50–1

1 81

815

8752

813

1257

8e-2

161

CsM

TP6

AtM

TP7

(AtM

TPc4

)A

t1g5

1610

AC

YN

0100

1432

+61

304

–66

708

1566

521

1413

682e

-27

68C

sMTP

7A

tMTP

8 (A

tMTP

c3)

At3

g580

60A

CY

N01

0068

19+

2734

–617

712

0940

27

646

2e-3

968

CsM

TP8

AtM

TP9

At1

g795

20A

CY

N01

0000

92–

25 6

40–2

7 48

212

3040

96

546

2e-4

762

CsM

TP9

AC

YN

0100

3516

+27

610

–32

482

1197

398

65

791e

-23

53C

sMTP

11A

tMTP

10A

t1g1

6310

AC

YN

0100

0092

_25

640

–27

482

1230

409

65

645e

-42

60C

sMTP

9A

CY

N01

0035

16+

27 6

10–3

2 48

211

9739

86

545

7e-2

152

CsM

TP11

AtM

TP11

At2

g394

50A

CY

N01

0000

92_

25 6

40–2

7 48

212

3040

96

536

2e-2

255

CsM

TP9

AC

YN

0100

3516

+27

610

–32

482

1197

398

65

823e

-76

75C

sMTP

11A

tMTP

12A

t2g0

4620

AC

YN

0100

4363

+12

568

–14

730

2163

720

10

812e

-171

62C

sMTP

12

Functional analysis of cucumber metal tolerance protein 8 | 5373

Deduced untranslated regions (UTRs), exons, introns, and proteins encoded by cucumber MTP genes were obtained from the further analysis of the selected contigs with FGENESH and FGENESH+ tools (Softberry, Inc., Mount Kisco, NY, USA; www.softberry.com). Functional annotations were made by BLASTP searches against GenBank protein data sets with final full-length MTP sequences. The sequences of MTPs from other plants (Supplementary Table S1 available at JXB online) were retrieved from the GenBank data-base (Vitis vinifera, Brachypodium diastychon, and Zea mays), the Rice Annotation Genome Project (RAGP) database (Oryza sativa), Phytozome v.8.0 (Sorghum bicolor), and The Populus Genome Integrative Explorer PopGenIE (Populus trichocarpa). Protein sequence alignments and analysis were performed using ClustalW with Gonnet as protein weight matrix, and the phylogenetic trees were constructed with MEGA5.0 software (Tamura et  al., 2011) using the maximum likelihood method with the bootstrap (1000 replicates).

Statistical analysisUnpaired and paired Student’s t-tests and analysis of variance (ANOVA; Excel) were used for statistical analyses. A  GraphPad Prism program (GraphPad Software, Inc.) was used to fit the data directly to the Michaelis–Menten equation using non-linear regres-sion and to display data with Lineweaver–Burk plots.

Results

Identification and features of cucumber MTPs

The distribution of genes encoding CsMTPs was analysed in two genomes of different cucumber cultivars. Using 12 A.  thaliana cDNAs encoding AtMTP1–AtMTP12 as the query sequences, nine (Borszczagowski cultivar) and 11 (Chinese long cultivar) contigs among two cucumber whole-genome shotgun reads, which significantly matched with AtMTP genes, were selected (Tables 1, 2). The whole contigs were screened to identify the full putative genomic sequences of CsMTP genes including transcription start sites, exons, introns, and polyadenylation sites using the FGENESH tool (www.softberry.com). The tool also provided the full amino acid sequences of cucumber MTPs that were further sub-jected to phylogenetic analysis (Fig. 1) using the previously annotated MTPs from A. thaliana, S. bicolor, P. trichocarpa, and O.  sativa (Montanini et  al., 2007; Gustin et  al., 2011) and the nomenclature of the additional sets of MTPs from Z. mays, V. vinifera, and B. distachyon was assigned based on annotated MTP sequences from A. thaliana (Supplementary Table S1 at JXB online). All CsMTPs were annotated based on the results of BLASTP searches against GenBank protein data sets using full cucumber MTPs and on the output of the phylogenetic analysis including MTPs from monocots and eudicots (Tables 1, 2; Fig. 1).

The genomic organization of cucumber CsMTP genes newly identified in genomes of two cucumber cultivars is pre-sented in Tables 1 and 2. The average lengths of introns, exons, and their numbers in cucumber genes are almost the same for the line B10 and 9930 genomes, except for the genes encod-ing the proteins annotated as CsMTP1 and CsMTP4. In the genome of the 9930 line, two different contigs contain identi-cal full-length cDNAs encoding CsMTP1 (ACHR01007740 and ACHR01031515). Similarly, other contigs of this line

contain two identical genes encoding putative protein hom-ologues to MTP4 (ACHR01007875 and ACHR01049518) (Table  1). In comparison, only single contigs containing

Fig. 1. The phylogenetic relationships between the MTPs from monocot and eudicots plants. The analysis was performed using the maximum likelihood method with 1000 bootstraps in the MEGA5 software. Shaded blocks indicate seven plant-specific groups defined by Gustin et al. (2011). (This figure is available in colour at JXB online.)

5374 | Migocka et al.

genes encoding CsMTP1 (ACYN01001940) and CsMTP4 (ACYN01002902) were identified within the genome of line B10 (Table  2). All cDNAs encoding putative members of the cucumber MTP family are 1161–1587 bp long, except for CsMTP12, which is almost twice as long (2163 bp), like the MTP12 genes in other plants (Tables 1, 2). The genes encod-ing the three proteins CsMTP1, CsMTP4, and CsMTP12 do not contain intron sequences, whereas the number of introns in other CsMTP genes varies from five (CsMTP9 and CsMTP11) to 12–13 (CsMTP6 and CsMTP7) (Tables 1, 2). Phylogenetic analysis of the MTPs from monocots and eud-icots including the novel cucumber MTPs confirms that plant MTPs are organized into seven different subgroups that are more or less closely related (Fig. 1). The tree branches into two main clades: the first containing MTP1–MTP4 (G1), MTP5 (G5), and MTP12 (G12), and the second composed of MTP7 (G7), MTP6 (G6), MTP8 (G8), and MTP9–MTP11 (G9) (Fig.  1). The first clade includes the proteins of the group designated as Zn-CDF (Montanini et al., 2007; Gustin et al., 2011), whereas the second clade encompasses the Zn/Fe-CDF and Mn-CDF groups of plant MTPs. Similar to the previous phylogenetic analysis of plant MTPs (Gustin et al., 2011), the Zn/Fe-CDF group includes MTP6 and MTP7, whereas MTP8 and MTP9–MTP11 form the third Mn-CDF group. Until now, only members belonging to the Zn-CDF or the Mn-CDF group have been functionally characterized in plants. For each of the two groups, the conserved residues in TMDII and TMDV have been identified, according to Montanini et al. (2007): the motif recognized in the proxim-ity of TMDV contains the highly conserved aspartate and histidine or aspartate located four residues upstream from it, which could determine metal specificity of the cucumber pro-teins belonging to Zn-CDFs or to the Mn-CDFs, respectively (Supplementary Fig. S1 at JXB online). These conservative residues as well as the tentative signature sequence were iden-tified within all cucumber MTP proteins with the exception of CsMTP7, for which the conservative motif specific for TMDV was not recognized (Supplementary Fig. S2).

Molecular features of CsMTP8

The CsMTP8 cDNA amplified by PCR on cDNA pre-pared from cucumber Krak was slightly different from the sequences generated by FGENESH from the genomes of cucumbers Borszczagowski and Chinese long. The PCR-amplified CsMTP8 cDNA contained 18 additional nucleo-tides between nucleotides 369 and 388 (Supplementary Fig. S3 at JXB online). As a result, the deduced amino acid sequence of MTP8 from cucumber Krak contained an additional amino acid sequence VLLLLK between amino acids 124 and 130 (Fig. 2A). Both the amplified nucleotide sequence of the cucumber gene CsMTP8 and the putative protein encoded by this gene are available in GenBank (accession no. JQ618099). The putative CsMTP8 is composed of 408 amino acids and possesses all the features common for Mn-CDF transporters including five putative TMDs, with a cytoplasmic N-terminus, a signature sequence between TMDs I and II, and two con-servative motifs characteristic for MTP8-like transporters:

DSLLD within the putative TMDII, and DHYFD within the cytosolic loop preceding TMDV (Montanini et al., 2007) (Fig.  2B). The presence of the motifs characteristic for the Mn-CDF subgroup including the specific Mn transporters ShMTP8, AtMTP11, and OsMTP8.1 strongly suggests that CsMTP8 is a putative Mn transporter in cucumber.

Expression of CsMTP8 in yeast complements the manganese-sensitive phenotype

To investigate the cellular function of CsMTP8, the cucumber protein was expressed in S. cerevisiae yeast mutants lacking the endogenous metal transporters for Zn (zrc1), Co (cot1), and Mn (pmr1), and the strain highly sensitive to Ni, Mn, and Cd (cnb1 vcx1 pmc1) (Fig. 3A). The ZRC1 gene encodes a tonoplast transporter that sequesters Zn into the vacuole (Kamizono et al., 1989; Li and Kaplan, 1998); hence the dele-tion of this gene renders the mutant highly sensitive to Zn (Fig.  3A). Cot1 mediates the efflux of Zn and Co into the vacuole of yeast cells; hence the deletion of the COT1 gene renders the mutants hypersensitive to both metals (Conklin et al., 1992; Li and Kaplan, 1998; Lyons et al., 2000). Pmr1 is an ATP-dependent yeast secretory pathway pump responsi-ble for transporting Ca2+ and Mn2+ ions into the Golgi, which is the major pathway for eliminating toxic Mn2+ involved in the detoxification of yeast cells from Mn excess; loss of PMR1 renders the cells extremely sensitive to Mn2+ toxicity (Lapinskas et al., 1995; Ton et al., 2002; Kellermayer et al., 2003). The cnb1 vcx1 pmc1 strain (K667) lacks the endoge-nous vacuolar Ca2+-ATPase Pmc1, the regulatory calcineu-rin subunit Cnb1p, and the vacuolar calcium and manganese antiporter Vcx1, and thus is defective in vacuolar Ca2+ and Mn2+ transport and highly sensitive to Cd or high Ca2+ and Mn2+ concentrations (Cunningham and Fink, 1996; Pittman et al., 2004). In a preliminary assay, the cnb1 vcx1 pmc1 strain appeared to be hypersensitive to increased Ni concentration, probably as a result of the inactive vacuolar Mn2+/Ca2+/Cd2+ transporter Mnr1 (Vcx1). Only the Mn-sensitive phenotype of the pmr1 and cnb1 vcx1 pmc1 mutants was fully rescued by CsMTP8, suggesting that cucumber protein is a highly specific Mn transporter and functional calcineurin is not required for CsMTP8 activity (Fig. 3A–C). When the wild type and K667 mutant were exposed to 5 mM Mn2+, CsMTP8-expressing cells accumulated significantly more Mn2+ than control cells over the initial 4 h of exposure (Fig.  3C), suggesting that CsMTP8 detoxifies yeast cells by internal sequestration of Mn2+ rather than through an efflux of Mn2+ to the exter-nal medium. Indeed, the subcellular localization of cucum-ber proteins in yeast used for growth tests in metal-enriched media confirmed the presence of CsMTP8 in the intracellu-lar compartments and not in the plasma membrane. When C-terminal (Fig. 4A) or N-terminal (Fig. 4B) GFP CsMTP8 fusion proteins were expressed from a pUG36 or pUG35 vec-tor, respectively, the fluorescence was observed only in the endomembranes of yeast cells. The western blot assay with the antibody raised against GFP clearly confirmed the pres-ence of CsMTP8 in the vacuolar-enriched membrane vesicles isolated from the K667 strain expressing the chimeric protein

Functional analysis of cucumber metal tolerance protein 8 | 5375

GFP–CsMTP8 (Fig. 4C). In yeast expressing GFP–CsMTP8, the cucumber protein of ~45 kDa was present in homodi-meric form (~90 kDa) (Fig. 4C). In the cells overexpressing V5- (GKPIPNPLLGLDST) tagged CsMTP8 fusion protein, two forms of the transporter (monomeric and dimeric) were detected (Fig. 4D).

To confirm that the Mn2+ resistance phenotype observed in Δpmr1 and K667 strains expressing CsMTP8–GFP resulted from the increased accumulation of Mn2+ ions within the vacuole, the Mn2+/H+ transport activity was measured in the vacuolar membrane vesicles isolated from the K667 strain lacking the endogenous vacuolar Mn transporter Vcx1. The uptake of Mn into the membranes was measured using two different methods: indirectly, with acridine orange as the

pH-sensitive probe reflecting Mn-induced H+ fluxes across the tonoplast; and directly, with an atomic absorption spec-trophotometer. The proton motive force was generated using tonoplast V-ATPase, and all experiments were per-formed in the presence of vanadate in order to inhibit any P-ATPase, which could contaminate the tonoplast prepara-tion. Significant Mn2+/H+ exchange activity was observed in vesicles from CsMTP8-expressing K667 (Fig. 5A, C) but was absent in the strain that was expressing empty vector alone (Fig.  5B, C). H+-coupled Mn transport was inhib-ited in the presence of the protonophore gramicidin (data not shown) and was dependent on the concentration of Mn added to the tonoplast vesicles (Fig. 5A, C). The first acrid-ine orange absorbance change induced by Mn was observed

Fig. 2. Sequence analysis of CsMTP8 in cucumber. (A) The predicted amino acid sequences of the CsMTP8 from cucumbers Krak (1227 bp), Chinese long, and Borszczagowski (1209 bp) were aligned using Multalin version 5.4.1 (Corpet, 1988). The additional six amino acid long sequence specific only for the Krak cultivar is shown between amino acids 123 and 129. (B) The predicted topology of CsMTP8 from cucumber Krak (TMHMM server 2.0). The motifs characteristic for the Mn-CDF subgroup of the CDF family (DxxxD) are marked in circles. (This figure is available in colour at JXB online.)

5376 | Migocka et al.

Fig. 3. Effect of CsMTP8 expression on metal tolerance and Mn accumulation of Saccharomyces cerevisiae. (A) Complementation of yeast mutants on solid medium containing heavy metals. The mutant strains of S. cerevisiae sensitive to different metals were transformed with the empty vector pUG36 as controls and with the vector carrying the cucumber gene CsMTP8. Yeast cultures were adjusted to OD600=0.3, and 3 μl of serial dilutions (10-fold, from left to right in each panel) were spotted on SC-U/Glu medium supplemented with NiCl2, ZnSO4, CoCl2, MnSO4, or CdCl2 or on the YPD medium (control) without the supplementation. The plates were incubated for 2–5 d at 30 ºC. The images are representative for three independent experiments. (B) Complementation of the K667 mutant in liquid SC-U/Glu medium containing 10 mM MnSO4. The medium was inoculated with the wild-type (squares) and mutant (circles) cells to an initial OD600=0.2 and was subsampled after 0, 4, 8, 12, 24, 36, and 48 h to monitor growth of yeast in the presence of metal. Open symbols represent the growth of yeast transformed with the empty vector, and filled symbols represent the growth of yeast expressing CsMTP8. (C) Mn accumulation in wild-type (squares) and K667 (circles) strains transformed with the empty vector (open symbols) or vector carrying CsMTP8 (filled symbols). The liquid SC-U/Glu medium containing yeast cultures at an initial OD600 ~1.0 was supplemented with MnSO4 (5 mM) and the subsamples were collected at the time intervals shown. Data represent the means and standard deviation (n=3).

Functional analysis of cucumber metal tolerance protein 8 | 5377

at a concentration of the metal in the reaction medium of 0.75 μM and the maximal rate of absorbance recovery was obtained with 4–5 μM MnSO4 (Fig. 5A). Hence, the phenom-enon of Mn transport into tonoplast vesicles from CsMTP8-expressing K667 cells exhibited saturation kinetics with an apparent Km of ~2.5 ± 0.6 μM (Fig. 5D).

CsMTP8 localizes to the vacuolar membrane of plant cells

The subcellular localization of cucumber protein in plants was assayed using A. thaliana cells as the plant heterologous expression system. To analyse the localization of cucumber protein in Arabidopsis cultured cells, transition transfor-mants of protoplasts were generated using vectors encod-ing CsMTP8 fusion proteins tagged with GFP at the N- or C-terminus under the constitutive 2 × 35S promoter. In pro-toplasts transformed with GFP-CsMTP8 (Fig.  6A) and CsMTP8-GFP (Fig. 6B), the GFP signal was clearly detected in the vacuolar membrane. The data clearly indicate that CsMTP8 is primarily localized to the tonoplast of plant cells.

The expression of CsMTP8 is root specific and regulated by manganese

Two independent assays for expression analysis, semi-quan-titative PCR and RT-PCR, revealed that the expression of CsMTP8 occurs predominantly in cucumber roots (Fig. 7A). In order to determine further the function and regulation of CsMTP8, the effect of Mn stress (elevated Mn or Mn defi-ciency) on the level of CsMTP8 mRNA was investigated.

Compared with plants grown under control conditions, root MTP8 transcript levels were increased in response to Mn excess (>2-fold increase) and reduced upon Mn deficiency (2-fold decrease) (Fig.  7B). The Mn-dependent changes in CsMTP8 expression suggest that the protein encoded by this gene may be engaged in increased or decreased Mn accumu-lation within the vacuoles of cucumber roots cells under Mn excess or deficiency, respectively.

Expression of CsMTP8 in Arabidopsis confers Mn2+ tolerance

To determine whether the enhanced expression of CsMTP8 may be responsible for the enhanced Mn accumulation in the vacuoles leading to the increased Mn tolerance in plants, GFP-CsMTP8 was expressed in A. thaliana. To test the effect of Mn excess on the Mn accumulation and plant growth, two independent homozygous lines were used with confirmed expression of the CsMTP8 gene (Fig.  8A), whereas plants transformed with the empty vector were used as a control. A  4 d exposure of plants to increased MnSO4 concentra-tion resulted in the enhanced accumulation of Mn in two lines expressing CsMTP8 (Fig.  8B). In comparison, plants transformed with empty vector accumulated significantly less Mn in their tissues (Fig.  8B). In addition, the expres-sion of the cucumber gene in Arabidopsis conferred increased Mn2+ tolerance (Fig. 8C). Compared with controls, the lines expressing CsMTP8 was more tolerant to Mn excess since their growth was not disturbed by the presence of toxic Mn in the nutrition medium (Fig. 8C). In contrast, the seedlings transformed with the empty vector grew poorly in toxic

Fig. 4. Vacuolar membrane localization of CsMTP8 in yeast. (A) The localization of CsMTP8–GFP (A) and GFP–CsMTP8 (B) in the cells of the yeast mutant K667. (1) Transmission images of the cells expressing CsMTP8 fused with GFP; (2) the fluorescence of the same cell; (3) overlay: the fluorescence matches tonoplast membranes in the same cells. (C) Immunodetection of CsMTP8 in the total microsomal fractions (M) and in the fractions enriched in vacuolar membranes (T) isolated from the K667 mutant expressing GFP-CsMTP8. The expression was induced or not in the absence (–Met) or in the presence (+Met) of methionine in the growing medium. The tonoplast fraction was obtained from the total microsomes following sucrose density gradient centrifugation at 120 000 g. The proteins in the fractions were electrophoresed and immunodetected with antibodies against GFP. The position of the GFP–CsMTP8 dimers is indicated by an arrow. (D) Immunodetection of CsMTP8 in the tonoplast fraction isolated from the K667 mutant expressing CsMTP8-V5 under the Gal-inducible promoter. Tonoplast protein was isolated from yeast grown in SC-U/Gal medium for 0, 2, 4, 6, or 8 h, electrophoresed, and immunodetected with antibodies against the V5 epitope. The position of the CsMTP8-V5 monomers and dimers is indicated by arrows. (This figure is available in colour at JXB online.)

5378 | Migocka et al.

Mn2+ concentrations (Fig.  8C). Since CsMTP8 localizes in the vacuolar membrane of A. thaliana protoplasts, it may be suspected that the increased Mn accumulation and resistance phenotype of plants expressing CsMTP8 results from the CsMTP8-mediated transport of Mn into the vacuoles of the transformant cells.

Discussion

The MTP family in cucumber is represented by nine proteins

It has been previously shown that the representative mem-bers of the CDF family from all living kingdoms (Archaea, Eubacteria, and Eukaryotes) show partition into three major functional groups: Zn-CDF, Zn/Fe-CDF, and Mn-CDF (Montanini et  al., 2007; Migeon et  al., 2010). Members of each group have been found in cucumber (Tables 1, 2). According to Gustin et  al. (2011), CsMTP1, CsMTP4, CsMTP5, and CsMTP12 represent the Zn-CDF group, CsMTP6 and CsMTP7 form the Zn/Fe-CDF group, and the remaining CsMTP8–CsMTP11 belong to the Mn-CDFs (Fig.  1). The most recent estimation of the phylogeny of CDF proteins including more sequences from Viridiplantae and Rhodophyta confirmed that these metal transporters group into three primary clades, consistent with the previ-ously defined Zn-CDF, Fe/Zn-CDF, and Mn-CDF groups. However, it also revealed that MTPs from land plants clearly form seven different subgroups within the clades (Gustin et al., 2011). Members of each of the seven groups are pre-sent in cucumber; the proteins that form Zn-CDF trans-porters cluster in group G1 (CsMTP1, CsMTP4), group G5 (MTP5), and group G12 (MTP12); hence the Zn-CDF clus-ter has been divided into three smaller clusters. The proteins of the Mn-CDF cluster have also been further divided into two different phylogenetic groups: group G8 (CsMTP8) and group G9 (CsMTP9 and CsMTP11) (Gustin et  al., 2011). The last proteins, CsMTP6 and CsMTP7, have been assigned to the Fe/Zn cluster as the representatives of separate groups G6 (MTP6) and G7 (MTP7) (Montanini et al., 2007; Gustin et al., 2011) (Fig. 1). Following this classification and nomen-clature of plant MTPs, only two genes encode proteins of

Fig. 5. The properties of H+-coupled Mn transport in yeast expressing CsMTP8. The Mn2+/H+ activity was measured in K667 yeast lacking the vacuolar Ca2+/Cd2+/Mn2+ transporter VCX1 (MNR1). (A, B) The acridine orange absorbance increase reflecting Mn2+-induced proton transport activity in tonoplast vesicles prepared from yeast expressing CsMTP8 (A) or yeast transformed with the empty plasmid vector (B). To induce metal/antiport activity, different concentrations (μM) of MnSO4 were introduced into the reaction medium following the establishment of a pH

gradient, as indicated by arrows. The values presented are representative for the results obtained in 3–4 independent experiments, with each experiment done in triplicate. (C) The rate of Mn accumulation in tonoplast vesicles isolated from yeast transformed with the empty plasmid vector (white bars), or yeast expressing CsMTP8 (dark bars) determined by atomic absorbtion spectrometry. Values are the means ±SE (n=5–6 measurements from 4–6 independent tonoplast preparations). Asterisks above indicate a significant difference (P<0.05) between proton-coupled Mn transport activities in tonoplast membranes incubated with different concentrations of manganese. (D) The kinetics of proton-coupled Mn transport across tonoplast membranes isolated from yeast expressing CsMTP8. The apparent Km value (2.5 μM) for the CsMTP8-mediated Mn2+ transport was estimated by Lineweaver–Burk plot. The Km and R2 values were calculated using GraphPrism software. The –1/Km value is indicated by an arrow. Inset: the rate of acridine absorbance increase versus Mn2+ concentration during the initial time (60 s) after the addition of metal to the reaction media containing ΔpH-energized tonoplast vesicles.

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group G1 in cucumber. The first gene generated from the contigs ACHR01007740 and ACHR01031515 (line 9330) and ACYN01001940 (line B10) encode the protein that is most closely related to MTP1s, whereas the second gene identi-fied within the contigs ACHR01007875 and ACHR01049518 (line 9330) and ACYN01002902 (line B10) encodes the pro-tein most closely related to MTP4-like transporters (Tables 1, 2). Similarly, the number of genes encoding group G9 of the Mn-CDF subgroup is reduced in cucumber when compared with A. thaliana: only three putative homologues of MTP8, MTP11, and MTP9/10 genes have been found in each of the two cucumber genomes (Tables 1, 2). The number of MTPs also varies in other plant species, as shown for the monocots O. sativa (10), B. distachyon (10), S. bicolor (9), and Z. mays (7), and the eudicots P. trichocarpa (21) and V. vinifera (13) (Supplementary Table S1 at JXB online). The poplar fam-ily is particularly large since it contains approximately twice the number of genes encoding MTPs found in other species (Supplementary Table S1). It has already been suggested that the genome of P. trichocarpa is evolving at a 6-fold slower rate than the A. thaliana genome and thus has a slower rate of loss of duplicated genes (Tuskan et al., 2006). Consequently, the poplar MTP paralogues of groups G1, G8, and G9 might still be highly redundant (Gustin et al., 2011). It is generally assumed that the recent duplication or gene losses could have led to such a complex distribution pattern of CDF isoforms in plants as well as in other living organisms (Montanini et al., 2007; Gustin et al., 2011). Interestingly, two copies of the MTP1 gene and two copies of the MTP4 gene were found only in the genome of Chinese long line (Table 1). Multiple copies of the MTP1 gene have been reported so far only in plant heavy metal hyperaccumulators. Five MTP1 paral-ogues were found to be present in A. halleri, AhMTP1-A1,

-A2, -B, -C, and -D, and at least three paralogues encode MTP1 in T.  goesingense (Kim et  al., 2004; Shahzad et  al., 2010). The increase and maintenance of a number of cop-ies of genes encoding MTP1 in both hyperaccumulators probably contributed to the increased adaptation of both plants to elevated Zn (A.  halleri) or Zn and Ni concentra-tion (T. goesingense) in the environment. Cucumber is a crop widely cultivated in regions of highly divergent climate and soil conditions, ranging from the northern areas of the tem-perate climate zone to subtropical and tropical habitats. Such diverse environmental conditions required various adaptive mechanisms which are reflected in polymorphisms within the 367 Mbp cucumber genome (Arumuganathan and Earle, 1991). The different copy number of MTP1 and MTP4 genes in the two cucumber lines North-European Borszczagowski and Chinese long most probably results from the exposure of both cultivars to different environmental conditions and pressure. In addition, the molecular analysis of MTP fami-lies in both cucumber genomes revealed very slight changes in the nucleotide composition and the length of CsMTP7, CsMTP11, and CsMTP12 cDNAs (Tables 1, 2). Similar dif-ferences between the same genes in differentially grown varie-ties of some plant species were also reported. A survey of 15 A.  thaliana ecotypes revealed that the gene encoding heavy metal ATPase 3 (AtHMA3) is present as a 1626 bp long pseu-dogene in Columbia, Limeport, and Hau-O ecotypes, and as a functional full-length gene (2283 bp) increasing Cd toler-ance in the remaining ecotypes (Morel et al., 2009).

Similar to G1, group G9 is represented by two proteins in cucumber, whereas the remaining groups G5, G6, G7, G8, and G12 contain single CsMTPs. Beside cucumber and Arabidopsis, other plants used for the phylogenetic analysis possess 2–4 proteins homologous to MTP8-like

Fig. 6. Vacuolar membrane localization of CsMTP8 in planta. Arabidopsis thaliana protoplasts were transformed with pMDC43 and pMDC83 plasmids carrying CsMTP8 to enable the expression of GFP–CsMTP8 (A1–A3) and CsMTP8–GFP (B1–B3) fusion proteins, respectively, or with empty pMDC43 (A4) or pMDC83 (B4). (1) Transmission images of the cells expressing CsMTP8 fused with GFP; (2) the fluorescence of the same cell; (3) overlay: the fluorescence matches tonoplast membranes in the same cells; (4) the fluorescence of GFP alone. t, tonoplast; n, nucleus; c, cytoplasm; v, vacuole. (This figure is available in colour at JXB online.)

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MTPs (Fig.  1; Supplementary Table S1 at JXB online). Nevertheless, not all the MTP8 paralogues may be func-tional genes, for example PtMTP8.4, which is present in a poplar genome in a significantly truncated form lacking the crucial functional domains (Gustin et  al., 2011). Similarly, the number of members of G9 is different in different plants (Fig. 1; Supplementary Table S1). In cucumber, this group is represented by the proteins designated as CsMTP9 and CsMTP11, based on the phylogenetic analysis, whereas other plants, except monocots, possess additional MTP10-like pro-tein (Fig. 1; Supplementary Table S1). Moreover, MTP11 is encoded by two paralogous genes in all the analysed plants except Arabidopsis and cucumber (Fig.  1; Supplementary Table S1). It has already been suggested that the expansion of genes of groups G8 and G9 was related to the primary

radiation of plants onto land and had a significant role for Mn homeostasis in plants (Gustin et al., 2011).

Functional characterization of cucumber protein MTP8

Based on the current phylogenetic and functional analyses of MTPs in plants, the metal specificity of newly identified cucumber MTPs can be deduced from their classification into one of the three groups: Zn-CDF, Zn/Fe-CDF, or Mn-CDF. According to that, the function of proteins of groups G1, G5, and G12 should be related to zinc transport, the pro-teins from groups G6 and G7 can participate in zinc and iron translocation, whereas members of G8 and G9 could be engaged in cellular Mn homeostasis (Montanini et  al., 2007; Gustin et al., 2011). Since only the functional charac-terization of novel plant MTPs can confirm this hypothesis, a detailed functional and biochemical study of cucumber protein CsMTP8 homologous to the confirmed Mn trans-porters from rice (OsMTP8.1) and S. hamata (ShMTP8) was performed in order to elucidate whether the MTP8-like pro-teins in other plants are also highly specific Mn transporters. Both S.  hamata and rice are able to grow in environments with very high Mn availability, since they evolved some molecular adaptations to toxic Mn (de Carvalho et al., 1980; Delhaize et al., 2003; Chen et al., 2013). It has been shown that MTP8s in both plants are engaged in the increased accu-mulation of Mn within vacuoles of the shoots (rice) and roots (S. hamata) (Delhaize et al., 2003; Chen et al., 2013). Mn tolerance that involves the MTP8-mediated sequestration of Mn into the vacuoles could determine constitutive traits for continuous Mn detoxification of Mn hyperaccumula-tors. It is not clear, however, how MTP8-like proteins func-tion in plants with high or moderate sensitivity to Mn. To address this question, the function of CsMTP8 in yeast and Arabidopsis expressing the cucumber gene was studied. The full-length cDNA of CsMTP8 in cucumber Krak encodes a membrane protein with at least five TMDs (TMHMM server 2.0) containing a signature sequence specific for the CDF family (SIAIAASTLDSLLDLMAGGILWF THLYMKQVNIYKYPIGKLRVQ) as well as short, motifs of five amino acids characteristic only for the Mn-CDF subgroup: DSLLD and DHYFD within TMDII and the cytosolic loop preceding TMDV, respectively (Montanini et al., 2007) (Fig. 2). These consensus residues designated as DxxxD (x=any amino acid) are also present in the function-ally characterized Mn transporters OsMTP8.1, AtMTP11, and ShMTP8 (Delhaize et al., 2003, 2007; Chen et al., 2013), strongly suggesting the role of CsMTP8 in Mn transport. Indeed, the heterologous expression of CsMTP8 in S. cerevi-siae resulted in enhanced tolerance to Mn, but not to other heavy metals (Fig. 3A). Subsequent tonoplast localization of cucumber protein in yeast (Fig. 4) suggested that CsMTP8 is involved in vacuolar sequestration of Mn excess within yeast cells. Indeed, the yeast expressing cucumber protein accumu-lated more Mn inside the cells and isolated tonoplast mem-branes (Figs 3C, 5). The H+-coupled Mn transport across the tonoplast was detected only in membranes isolated from yeast expressing CsMTP8 (Fig.  5), confirming that the protein

Fig. 7. The pattern of CsMTP8 expression in cucumber plants. (A) Steady-state level of CsMTP8 expression in the roots, hypocotyls, cotyledons, petioles, and leaves of 2-week old cucumber plants. In quantitative real-time PCR analysis, the average level of CsMTP8 transcript was normalized according to the constitutively expressed control gene CACS and calculated using the ΔΔCt method (Lightcycler 480 software) from three independent biological replicates, each consisting of material pooled from four different seedlings. Inset: the level of CsMTP8 and CsCACS expression in the same samples measured by semi-quantitative RT-PCR. The genes were amplified using 25 cycles for CsCACS and 28 cycles for CsMTP8. Experiments were repeated three times, and a representative gel is shown. (B) Quantitative real-time PCR analysis of the CsMTP8 transcript level in bulk roots of 2-week-old cucumbers grown under temporary Mn excess (50 μM MnCl2 for 10 h) or Mn deficiency. Bars represent average transcripts levels relative to the respective constitutively expressed reference gene encoding CsCACS. Data come from three independent biological replicates, each consisting of material pooled from four different seedlings. Asterisks indicate significant differences between control plants and plants grown under Mn excess or deprivation (P <0.05). The experiment was repeated twice and provided similar results.

Functional analysis of cucumber metal tolerance protein 8 | 5381

encoded by this gene acts as a H+/Mn2+ antiporter with high specificity for Mn. Since CsMTP8 localizes to the tonoplast of Arabidopsis cells (Fig. 6) and confers increased tolerance of A.  thaliana plants to Mn excess (Fig. 8), it may be con-cluded that cucumber protein also contributes to the vacu-olar Mn sequestration in plants. The homologous proteins from the Mn-CDF group in plants which have been charac-terized to date also localize to the tonoplast (ShMTP8 and OsMTP8.1) or the pre-vacuolar compartment and/or trans-Golgi (AtMTP11, PtMTP11.1, and PtMTP11.2), and confer the increased tolerance of yeast and plants to elevated Mn (Delhaize et al., 2003, 2007; Peiter et al., 2007; Chen et al., 2013). Using yeast mutants defective in vacuolar acidification,

Delhaize et al. (2003) provided the first indirect evidence that ShMTP8 transports Mn2+. Using isolated yeast membrane vesicles, the authors recently confirmed that both ShMTP8 and AtMTP11 transport Mn2+ and function as proton anti-porters (Delhaize et al., 2007). Based on the current data, it may be suggested that MTP8-like proteins act as Mn2+/H+ antiporters specifically engaged in the vacuolar sequestration of Mn excess. In fact, most of the CDF proteins characterized to date function as proton antiporters. The yeast S.  cerevi-siae CDF transporter Zrc1 accumulates Zn2+ within vacuoles in a way dependent on the transmembrane proton gradient generated by tonoplast V-ATPase (MacDiarmid et al., 2002). Studies on ZitB from Escherichia coli also revealed that this

Fig. 8. Effect of CsMTP8 expression in Arabidopsis thaliana on Mn2+ accumulation and tolerance. (A) The level of CsMTP8 expression in different homozygous lines of A. thaliana as determined by semi-quantitative RT-PCR. The A. thaliana plants were transformed with plasmid pMDC43 carrying CsMTP8 (L1, L2) or with the empty vector (L3). (B). The accumulation of Mn in homozygous lines 1 and 2 expressing CsMTP8 and in line 3 transformed with empty vector. Plants were grown on nutrient agar for 14 d and then transferred into liquid half-strength MS medium supplemented with 100 μM MnSO4 for 24 h. Data shown are the means and standard deviation (n=30). Significant differences between lines expressing CsMTP8 and lines transformed with empty vector were calculated using Student’s t-test and are indicated by asterisks (P<0.05). (C) The growth of 14-day-old homozygous lines expressing CsMTP8 and a control line transformed with empty vector on solid half-strength MS medium containing basal or 4 mM MnSO4. (This figure is available in colour at JXB online.)

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bacterial CDF protein transports Cd2+ in a proton-dependent manner (Guffanti et al., 2002). The H+-coupled mechanism of Zn2+ transport through ZitB and Fie (another CDF pro-tein from E.  coli) was also supported in experiments using everted membrane vesicles (EMVs) overexpressing bacterial proteins. Zn2+ accumulated in these vesicles only in the pres-ence of NADH, ATP having no effect (Anton et  al., 2004; Grass et al., 2005).

There is also increasing evidence that the members of the CDF family function as dimers or oligomers. FieF from E. coli was detected as a 68 kDa protein, twice the predicted mass of the fieF gene product, suggesting that this trans-porter is present in a homodimeric form in bacterial cells (Wei et al., 2004). The formation of dimeric and oligomeric forms was also shown for plant CDFs. The high level expres-sion of TgMTP1b::HA in yeast gave rise to multiple size variants of TgMTP1b::HA, consistent with the presence of monomer, dimer, and trimer protein complexes (Kim et al., 2004). Similar complexes have also been observed for the Zn2+-transporting plant CDFs PtdMTP from the Populus trichocarpa×P.  deltoids poplar hybrid and AtMTP1 from A. thaliana when expressed in a high copy number in S. cer-evisiae and E.  coli, respectively (Bloss et  al., 2002; Blaudez et  al., 2003), and for Mn2+-transporting OsMTP8.1, when studied in rice (Chen et al., 2013). The lower expression of CsMTP8 from the single-copy vector pUG36 produced only a doublet band of the size predicted for a GFP–CsMTP8 monomer, whereas the high level expression of cucumber protein from the pYES-DEST52 vector gave rise to two size variants consistent with the presence of monomer and dimer protein complexes (Fig. 4C, D). Similarly, only one dimeric form of TgMTP1b::HA, was present in yeast with a lower level expression of TgMTP1b::HA (Kim et  al., 2004), sug-gesting that homodimers might be the main functional forms of MTP proteins.

Though the mechanism of metal transport through CDF proteins seems to be partially elucidated, the kinetics of this process are largely unknown and the data already obtained are confusing (Haney et  al., 2005). Studies on proteoli-posomes indicated the Km for ZitB-mediated Zn2+ transport to be 105 μM (Chao and Fu, 2004), whereas the experiments using everted membranes suggested this Km value to be ~1 μM (Anton et al., 2004). As yet there are no data indicating the Km value for MTP-mediated Mn transport. Using the tono-plast vesicles isolated from yeast expressing CsMTP8, the H+/Mn2+ antiport was measured in two different assays and the obtained data indicate a Km close to ~2.5 ± 0.6 μM for this process (Fig. 5D). Since it is assumed that the concentration of free heavy metal ions within the cells is in the femtomolar range (Outten and O’Halloran, 2001; McCranor et al., 2014), this high Km value suggests that CsMTP8 functions in condi-tions of Mn stress, when the level of cellular Mn is markedly increased. This is consistent with the expression data showing that the level of CsMTP8 transcript in cucumber roots is sig-nificantly increased upon Mn excess (Fig. 7B). However, the techniques used here to study Mn transport do not faithfully reproduce the physiological conditions of the cellular envi-ronment under which the protein may function in a different

mode. Interestingly, taking into account the range of Km values, the present results are similar to the data obtained in experiments with everted membranes isolated from bacte-rial cells, suggesting that the information on CDF-mediated transport obtained using similar experimental procedures could be compared. To the best of the authors’ knowledge, the results of this work are the first indication of the kinetics of Mn transport mediated by MTP8-like protein in plants.

Altogether, the obtained data suggest that cucumber MTP8 is a functional homologue of MTP8s from S. hamata and rice. However, the expression of CsMTP8 was highly spe-cific for cucumber roots and was stimulated under Mn excess (Fig.  7), whereas the OsMTP8.1 transcript was the most abundant in the shoots of rice and was slightly affected by elevated Mn (Chen et al., 2013). In contrast, the homologous protein in S.  hamata localized predominantly in the tono-plast of endodermal and trichomal cells, which are usually involved in heavy metal accumulation (Delhaize et al., 2003). It seems that cucumber protein CsMTP8 and ShMTP8 may be functional homologues engaged in Mn sequestration in roots under Mn stress, whereas OsMTP8 participates in Mn homeostasis in shoots.

Supplementary data

Supplementary data are available at JXB online.Figure S1. The conserved residues in TMDs II and V of

Zn-CDF and Mn-CDF groups of cucumber MTPs..Figure S2. The characteristic motifs in cucumber MTPs.Figure S3. Nucleic acid sequences of the CsMTP8 from

cucumbers Krak (1227 bp) and Chinese long (1209 bp).Table S1. The accession numbers and nomenclature of

MTPs from monocots and dicots used for the construction of the vphylogenetic tree

AcknowledgementsWe gratefully acknowledge Dr Sophie Filleur and Dr Sébastien Thomine [Institut des Sciences du Végétal (ISV), Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, France] for giving us the opportunity to work with Arabidopsis cultured cells and transformation of A.  thaliana plants, and Dr Jon Pittman (Manchester University, UK) for the K667 mutant strain. We thank Chi Tam Nguyen [Institut des Sciences du Végétal (ISV), Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, France] for excellent technical assistance with protoplast preparation. This work was supported by the Polish Ministry of Science and Higher Education (grant no. IP2010 026470).

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