Engineering Copper Hyperaccumulation in Plants by Expressing a Prokaryotic copC Gene

Engineering Copper Hyperaccumulation in Plants by Expressing aProkaryotic copC GeneIgnacio D. Rodríguez-Llorente,† Alejandro Lafuente,† Bouchra Doukkali, Miguel A. Caviedes,and Eloisa Pajuelo*

Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla, 41012-Sevilla, Spain

*S Supporting Information

ABSTRACT: In this work, engineering Cu-hyperaccumulation in plants was approached. First,the copC gene from Pseudomonas sp. Az13, encoding a periplasmic Cu-binding protein, wasexpressed in Arabidopsis thaliana driven by the CaMV35S promoter (transgenic lines 35S-copC).35S-copC lines showed up to 5-fold increased Cu accumulation in roots (up to 2000 μg Cu. g−1)and shoots (up to 400 μg Cu. g−1), compared to untransformed plants, over the limitsestablished for Cu-hyperaccumulators. 35S lines showed enhanced Cu sensitivity. Second, copCwas engineered under the control of the cab1 (chlorophyll a/b binding protein 1) promoter, inorder to drive copC expression to the shoots (transgenic lines cab1-copC). cab1-copC linesshowed increased Cu translocation factors (twice that of wild-type plants) and also displayedenhanced Cu sensitivity. Finally, subcellular targeting the CopC protein to plant vacuoles wasaddressed by expressing a modified copC gene containing specific vacuole sorting determinants(transgenic lines 35S-copC-V). Unexpectedly, increased Cu-accumulation was not achievedneither in roots nor in shootswhen compared to 35S-copC lines. Conversely, 35S-copC-Vlines did display greatly enhanced Cu-hypersensitivity. Our results demonstrate the feasibility ofobtaining Cu-hyperaccumulators by engineering a prokaryotic Cu-binding protein, but they highlight the difficulty of altering theexquisite Cu homeostasis in plants.

■ INTRODUCTION

Copper has a dual effect on plant cells: it is an essentialelement, cofactor of many enzymatic reactions; and it isinvolved in physiological processes such as photosynthesis,mitochondrial respiration, superoxide scavenging, cell wallmetabolism, ethylene perception, and development ofreproductive organs.1,2 However, Cu is also one of the mosttoxic heavy metalsmedian toxic concentration around 2.0 μMin solution.3 Cu is at the top of the Irving-Williams series andtightly binds to polypeptides. Moreover, the redox coupleCu(I)/Cu(II) causes severe oxidative damage by producingharmful reactive oxygen species (ROS).To conciliate both aspects, plants have developed finely

regulated homeostatic networks controlling Cu import,distribution to target proteins, and detoxification.4,5 At lowCu concentrations, the family of copper transporters COPT1-COPT6 (highly specific for Cu(I)) are involved in the high-affinity Cu uptake by roots. FRO metalloreductases seem to beinvolved in the reduction of Cu(II) to Cu(I) at the plantplasma membrane.6 Cu is then bound to metallochaperones,including ATX1, CCH, and CCS1, which mediate Cu deliveryto specific proteins.4 In the case of Cu deficiency, plants canmobilize vacuolar Cu reserves through the Cu transporter at thetonoplast, COPT5.7,8 Under severe Cu deficiency, higher plantsprioritize Cu delivery to plastocyanin and dispensable copperproteins are substituted by other metalloproteins assuming anidentical role (for instance, Cu/ZnSOD by FeSOD, laccases,and plantacyanin).9 In fact, the expression of both COPT and

Cu/ZnSOD is regulated by a transcription factor of theSQUAMOSA family (SPL7), which is a master regulator inresponse to low Cu that recognizes a GTAC core motif in thepromoters.10,11 By contrast, if a Cu excess in the cytoplasmoccurs, Cu(I) effluxeither to the apoplast or to storagecompartmentsis mediated by P-type ATPases, such asHMA5.12 Other P-type ATPases are RAN1, mediating Cudelivery to ethylene receptors,13 and PAA1 and PAA2,mediating Cu delivery to chloroplasts and tylacoids, respec-tively.14 At the same time, Cu binding peptides such asphytochelatins, and preferably metallothioneins, bind excesscopper.15 Furthermore, besides its role as electron transport inphotosynthesis, plastocyanin PC2 acts as a sink in bufferingexcess Cu.16 This fine regulation leads to an oscillation of Cuconcentration in the cytoplasm, coupled to the circadian clock.1

Concerning Gram-negative bacteria, Cu resistance inPseudomonas is determined by the copRZABCD operon. Withinthis operon, copC encodes a periplasmic Cu-binding protein.The protein of Pseudomonas putida was postulated to display a1:1 (polypeptide:Cu atoms) stoichiometry.17 More recentstudies with crystallized protein from Pseudomonas syringaepathovar. tomato demonstrated a structure of β-barrel with twohighly specific Cu binding sites at the ends, one for Cu(I) and

Received: March 2, 2012Revised: August 29, 2012Accepted: September 28, 2012Published: September 28, 2012

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the other one for Cu(II).18,19 Within the protein, the ligandsthat coordinate Cu ions have been identified. The Cu(I) siteshows a tetrahedral geometry and it is formed by two−three ofthe four methionines (Met-40, Met-43, Met-46, and Met-51)plus His48. The Cu(II) binding site has a tetragonal geometry,involving His1 and His91, together with Asp89 and Glu27. Awater molecule in the apical position confers stability to thissite.18,19 In the periplasm oxidizing environment, only when theprotein is fully loaded with Cu(I)/Cu(II) are the Cu speciesstable. If only the Cu(I) site is occupied, Cu(I) is rapidlyoxidized to Cu(II), just in the presence of oxygen. Theavailability of an unoccupied site of higher affinity induces anintermolecular transfer of either Cu(I) or Cu (II), whilebuffering the concentrations of cupric ion to subpicomolarlevels.19 This peculiar biochemistry, and the fact that CopC caninteract with the rest of the proteins codified by the operon,confers to the CopC protein the structural liability to transferCu (with or without redox switch) in the periplasm of Gram-negative bacteria. The presence of Cu-binding periplasmproteins (CopC and CopA, codified by the same operon)allows the accumulation of copper in the periplasm of Cu-resistant Gram-negative bacteria. In some Pseudomonas strains,Cu accumulation occurs in such large amounts (up to 12%)that colonies turn blue.20

In the past two decades, phytoremediation has emerged as asuitable green biotechnology to clean up contaminatedenvironments.21 Concerning metals, the two main phytor-emediation techniques are phytoextraction (using (hyper)-accumulator plants)22 and phytostabilization (using metalexcluders).23 Few plant species able to hyperaccumulate Cuhave been described, such as the moss Scopelophila cataractae,24

and the higher plants Ipomoea alpine,25 Commelia communis,26

Elsholtzia splendens,27 and, more recently, Crassula helmsii, ableto accumulate up to 9000 μg Cu.g−1.28 In contrast to otherhyperaccumulators, which preferably accumulate metals inshoots, most of these plants are characterized by a preferentialaccumulation of Cu in roots.Transgenic plants expressing bacterial genes have been

successfully developed for phytoremediation.29,30 Increasedarsenic accumulation was achieved by expressing a prokaryoticarsenate reductase and a plant γ-glutamylcysteine synthetase.31

Also, mercury volatilization was achieved by expressingmodified bacterial merA and merB genes (although thesegenes were optimized for plants).32

The aim of this work was expressing the copC gene fromPseudomonas sp. Az13 in A. thaliana under the control of aconstitutive promoter in order to increase Cu accumulation inthe plant. Furthermore, tissue-specific expression of copC andsubcellular targeting of the CopC protein were approached.

■ EXPERIMENTAL SECTIONGeneration of copC Constructs. Pseudomonas sp. strain

Az13 was first isolated from the rhizosphere of legumes grownin metal polluted soils.33 It is able to tolerate up to 4.5 mMCu34 and harbors a copABCD operon (acc. no EF587902).Comparison of the CopC protein sequence with the database ispresented as Supporting Information (Figure S1).The copC gene was amplified using Pfu polymerase and

primers FEcop (forward, 5′-GGGAATTCATGTTGCTCCT-CAGCAGTC-3′) and RBcop (reverse, 5′-CCAGATCTT-CACGTCACTTTGAACGT-3′). Restriction sites for EcoRIand BglII (underlined in the sequences) were included forcloning. The fragment was cloned into the EcoRI and BglII sites

of pCambia1390 (pCambia-copC). copC was placed under thecontrol of the CaMV35S promoter by mobilizing a 35Spromoter fragment from pPZPY112 35 to pCambia-copCplasmid as a HindIII-BamHI fragment (pCambia-35S- copC,SI Figure S2).To direct the expression of copC to green tissues, the gene

was placed under the control of the cab1 (chlorophyll a/bbinding protein) gene promoter, which is a light-inducible andorgan-specific promoter.36,37 The cab1 promoter sequence wasamplified from Arabidopsis thaliana genomic DNA usingprimers CABF1 (forward, 5′-GGAAGCTTCTCCTCAATCA-CACTCCTATAG-3′) and CABR2 (reverse, 5′-GGGGATC-CAGGTTGAGTAGTGCAGCAC-3′). The PCR product(1120 bp), flanked by HindIII and BamHI restriction sites(underlined in the primers), was cloned into the correspondingsites of the pCambia-copC (pCambia- cab -copC, SI Figure S2).To target the CopC protein to the vacuole, specific vacuole

sorting determinants were added both at the N- and C-terminiof the protein. The sequence NPIRL was added at the N-terminus of the protein38 and the sequence EIPDIATVV at theC-terminus.39 Amplification of copC was performed usingprimers FEcopV (forward, 5′-GGGAATTCATGAATCCAAT-TAGACTTTTGCTCCTCAGCAGTC-3′) and RBcopV (re-verse, 5′-CCAGATCTTCAAACAACAGTAGCAATATCTG-GAATTTCCGTCACTTTGAACGT-3′), where the italicsequences represent the vacuole sorting determinants. Thefragment was cloned into the EcoRI and BglII sites ofpCambia1390 (pCambia-copC-V). copC-V was placed underthe control of the CaMV35S promoter by mobilizing the 35Spromoter fragment from pPZPY112 as described above(pCambia-35S-copC-V, SI Figure S2.C-D).

Transformation of A. thaliana Plants and Selection ofTransgenic Lines. Constructs were electroporated intoAgrobacterium tumefaciens EHA10540 for A. thaliana trans-formation by the floral dip method.41 Seeds were sterilized for 8min with 15% (v/v) household bleach containing a few dropsof Tween 20, followed by three washes with sterile water. T1generation seedlings were germinated on plates containing 1/2MS (MS medium with one-half-strength macrosalts42)supplemented with hygromycin (30 mg/L) at 21 °C under a12 h light/12 h dark regime. Plants surviving selection weregrown on substrate in the greenhouse and T1 seeds werecollected. The seeds were sown on 1/2 MS-hygromycinmedium, and four hygromycin-resistant transgenic plant linesper construct (whose progeny segregated 3:1 for hygromycinresistance) were selected and allowed to set T2 generationseeds. Homozygous T3 lines were obtained as describedpreviously.43 The presence of copC was confirmed by PCRusing FEcop/RBcop primers for lines containing copC andFEcopV/RBcopV primers for those containing the modifiedgene copC-V.

RNA Expression Analysis. For RNA studies, plants weregrown in square plates containing 1/2 MS medium,supplemented with 50 μM Cu. Plates were incubated in aplant growth chamber at day/night temperatures of 22/18 °Cand 16 h/8 h light/dark photoperiod. The root part of theplants was protected from light with black paper. Thirty-day-oldseedlings were harvested at approximately 4 h after the lighttransition, for a high functionality of the cab1 promoter in cab1-copC lines.

RT-PCR. RNA was isolated from roots and shoots using theRNeasy Plant Mini Kit (Qiagen) following the manufacturer'sinstructions. Three independent RNA samples were obtained.

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For cDNA synthesis, 100 ng of DNase I-treated total RNA wasused, and reverse transcription was carried out with the gene-specific reverse primers RBcop and RBcopV, using theQuantiTect Reverse Transcription Kit (Qiagen) following themanufacturer’s instructions. Double-stranded cDNA wassynthesized using FEcop/RBcop and FEcopV/RBcopV primerspairs, and concentration was adjusted to 75 ng/μL using aThermo Scientific NanoDrop Spectrophotometer. RT-PCRwas carried out using the QuantiFast Multiplex PCR Kit(Qiagen), primers pairs FEcop/RBcop or FEcopV/RBcopV,and a TGradient termocycler (Biometra), following themanufacturer’s instructions. PCR conditions were as follows:an initial denaturation at 95 °C for 2 min followed by 25 cycles

at 95 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min, and a finalstep at 72 °C for 10 min.

qRT-PCR. Levels of mRNA were quantified by qRT-PCR aspreviously described44 using internal primers copC-q-F (5′-ACCCTGGTCACGCAGTTTT-3′) and copC-q-R (5′-CGCTGACTTTGGCTTTCAT-3′). RT-PCR was carried outusing the Eco Real-Time PCR System (Illumina), following themanufacturer’s instructions. PCR conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95 °C for 15 s, 55 °Cfor 30 s, 72 °C for 10 s (reading), and a final step at 72 °C for 5min, followed by a reading program consisting of 95 °C for 15s, 50 °C for 15 s, and 30 min at increasing temperature (1.5 °Cper min) and reading, and a final step at 95 °C for 15 s.Amplification of three independent housekeeping genes (actin2

Figure 1. Characteristic features of A. thaliana transgenic lines expressing copC under the control of 35S promoter. (A) RT-PCR amplification ofcopC in shoots and roots of 35S lines. No copC transcript was detected in wild-type (wt) plants (M: molecular weight marker). (B) Quantitative RT-PCR of copC gene in shoots and roots of 35S lines. Amplification of the F-box transcript was used as a control to normalize expression levels. Resultsare the means ± SE of three different experiments. (Similar results were obtained using ACT2 and AT4G26410 as housekeeping genes.) (C) Aspectof wt and transgenic 35S-3 plants grown in medium containing 75 μM CuSO4. A representative set of plants is shown. Note the reduction of rootlength in transgenic plants. (D) Root length of 7-day-old wild-type and transgenic seedlings grown in the absence or presence of Cu (upper panel).Fresh weight of 14-day-old wt and transgenic seedlings grown in the absence or presence of Cu (lower panel). Data are means ± SE of 60 seedlings(20 representative seedlings × 3 plates). (E) Cu accumulation in shoots and root of wt and 35S transgenic plants. Plants were grown in a hydroponicsystem for one month and then transferred to 150 μM Cu for one week. Data are means ± SE of three independent determinations. Significantdifferences from wt plants as determined by Student’s t test are indicated by one asterisk (P < 0.05). The two asterisks indicate differences betweentransgenic lines.



ACT2, F-Box and the unknown protein AT4G26410) was usedto normalize results from different samples. Primers andconditions for ACT2 (actin-2 gene), F-Box, and the unknownprotein AT4G26410 amplification have been described.45,46

Each assay was repeated twice (independent biologicalsamples), and each measurement was performed in triplicate.Cu Tolerance Assays. Cu tolerance was evaluated as

previously described,47 where the Cu concentrations wereoptimized. Seeds were germinated vertically on 1/2 MS plates.After three days, seedlings were transferred to 1/2 MS platescontaining different concentrations of CuSO4 from 25 to 200μM (at 25 μM intervals). Plates were incubated in a plantgrowth chamber at 16 h light/8 h dark photoperiod attemperature 22 °C/18 °C. Roots were protected from light bycovering the lower part of the plate with black paper. Growth ofthe primary root was measured after 4 days (7-day-oldseedlings), and biomass was determined in 14-day-old seed-lings. Only relevant data are presented in the Results section atconcentrations 50 and 75 μM: below 50 μM, minor differencesbetween wild-type and transgenic lines were observed, and over100 μM, phytotoxicity symptoms started to appear also in wild-type plants (SI Figure S3).Cu Accumulation Assays. To estimate Cu accumulation, a

higher Cu concentration (150 μM) was used. Since this Cuconcentration did not allow long-term Arabidopsis survival,plants were grown in a hydroponic system for one month in 1/2 MS medium (without additional Cu) and then transferred to150 μM Cu for one week (1 week exposure). The systemconsisted in a container for the liquid 1/2 MS medium and afine silk mesh floating onto the liquid surface. The system wasplaced inside transparent magenta boxes provided with a gas-permeable membrane that allowed gas exchange, andautoclaved. The lower part of the boxes was protected fromlight. Sterilized seeds from wild-type plants or from transgeniclines were spread on top of the mesh and incubated in a plantgrowth chamber at day/night temperatures of 22/18 °C and 16h light/8 h dark photoperiod. After 1 month, the solution wassubstituted by fresh 1/2 MS medium containing 150 μM Cu,and plants were further cultivated for 1 week. After this period,plants were harvested at approximately 4 h after the lighttransition; roots and leaves were separated, exhaustively washedand dried; and metal content in both tissues was determined byICP-OES as described.48

Targeting the Green Fluorescent Protein to theVacuoles of Onion Cells. To test the correct targeting ofproteins containing the vacuole sorting determinants, anotherconstruct was generated by replacing the copC gen in pCambia-35S-CopC-V-plasmid by the green fluorescent protein gene(gfp). gfp was amplified from plasmid pMON3004949 usingprimers FEgfpV (forward, 5′-GGGAATTCATGAATCCAAT-TAGACTTGGCAAGGGCGAGGACTG-3′) and RBgfpV (re-verse, 5′ CCAGATCTTCAAACAACAGTAGCAATATCTG-GAATTTCCTTGTAGAGTTCATCCATGCC-3′), containingthe same restriction sites and vacuole sorting determinants ascopC-V. The PCR fragment was cloned into the EcoRI-BglIIsites of pCambia-CopC-V, thus replacing copC by gfp (pCambia-35S-gfp-V, Figure S1).pCambia-35S-gfp-V or pCambia-35S-gfp were delivered onto

onion cells by gold particle bombardment as described.50,51 1.0μm gold particles (Bio-Rad) were coated with 2 μL of a 1 μg/μL solution of plasmid DNA. Particles were bombarded directlyonto onion pieces using a Biolistic PDS-1000/He system (Bio-Rad) with 1100 psi rupture discs under a vacuum of 28 in Hg.13

24 h after bombardment, the upper cell layer of onion pieceswas peeled off and observed under an epifluorescencemicroscope Olympus BX61 provided with a camera DP70.

■ RESULTSTransgenic A. thaliana Plants Expressing Bacterial

copC Gene Showed Enhanced Cu Accumulation. Fourindependent randomly selected T1 transgenic lines (designated35S-2, 35S-3, 35S-4, and 35S-5) were analyzed for copCinsertion. All selected lines contained the transgene (SI FigureS4), whereas untransformed plants did not show the PCRproduct. As a first approach, Cu content was determined inshoots of T1 lines (SI Table S1). All lines showed 2- to 4-foldincreased Cu accumulation with regard to the untransformedcontrol. Lines 35S-3 and 35S-5, presenting the highest Cuaccumulation in shoots, were selected. Homozygous T3 lines of35S-3 and 35S-5 were obtained and used for furthercharacterization.RT-PCR analysis showed that A. thaliana was able to express

the copC gene under the control of the 35S promoter, both inshoots and in roots (Figure 1A). No expression was found inwild-type plants. These data were further confirmed byquantitative qRT-PCR (Figure 1B). Similar expression levelsin shoots and roots were observed in both lines.To examine whether the expression of copC influenced

seedling tolerance to Cu, root growth inhibition and freshweight were determined. No significant differences in rootlength were observed at 50 μM Cu (Figure 1C), whiletransgenic lines presented a reduction of around 20% in freshweight (Figure 1D). At 75 μM Cu, 25% reduction in rootlength and 30% reduction in fresh weight were observed intransgenic lines with regard to the control (Figure 1D). Theseresults suggested that expression of copC in A. thalianadecreases seedling tolerance toward Cu. Nevertheless, theseplants still retained a relatively great biomass and were able tocomplete their life cycle in the presence of Cu (see Figure 3Din next section).As expected, wild-type plants showed a preferential

accumulation of Cu in the roots. The concentration of Cuaccumulated in the roots was up to 5-fold higher than that ofthe shoot (TF: translocation factor of 0.2 as root-to-shoot Cuconcentration ratio) (Figure 1E). Transgenic lines 35S-copCshowed enhanced Cu accumulation. Line 35S-5 was able toaccumulate 2-fold more Cu, both in shoots and in roots. Inparticular, line 35S-3 showed the highest level of Cuaccumulation, up to 400 μg Cu.g−1 in shoots and 1800 μgCu.g−1 in roots (up to 5-fold increase in metal accumulation inshoots and roots with regard to control untransformed plants)(Figure 1E). Furthermore, translocation factors in bothtransgenic lines did not significantly change (0.22 and 0.23for 35S-3 and 35S-5, respectively, compared to 0.20 for controluntransformed plants, SI Table S2).It can be concluded that heterologous expression of the copC

gene in A. thaliana plants led to a slight, although significant,decrease in copper tolerance, and at the same time, there was amarked increase of Cu accumulation in plant tissues.

Transgenic A. thaliana Plants Expressing copC underthe Control of the Light-Inducible and Tissue-Specificcab1 Promoter Displayed Higher Translocation Factors.Transgenic lines expressing copC under the control of the cab1promoter could be an interesting model for Cu phytoex-traction. Four randomly selected T1 lines (confirmed by PCR,not shown) were grown in the presence of Cu, and those



showing higher Cu accumulation in shoots, designated cab1−4and cab1−6, were selected (SI Table S1). Homozygous T3lines were obtained and used for further characterization.The level of expression of the copC gene was analyzed by RT-

PCR (Figure 2A). Both lines seemed to have higher expressionof copC in shoots than in roots. qRT-PCR was also performed(Figure 2B). It can be seen that the level of copC expression wasnot higher in shoots than in roots. By contrast, a higherexpression in roots was observed in cab1−6. The experimentwas repeated several times, and every time, the same result wasobtained. Thus, qRT-PCR did not allow us to confirm a higherexpression of the copC gene under the control of the tissue-specific promoter cab1 in green tissues.The sensitivity to copper was analyzed in T3 seedlings. There

was a slight reduction (about 10−20%) in root length and adecrease in plant biomass (between 20% and 30%) (Figure2C). These results again suggest a higher sensitivity oftransgenic lines cab1−4 and cab1−6 to Cu, when compared

to control untransformed plants, quantitatively similar to thatfound in 35S-copC lines.Cu accumulation in plant tissues was determined (Figure

2C). Thirty-day-old transgenic seedlings were able toaccumulate 2−3 times more Cu in the green tissues whencompared to control plants, reaching values of Cu accumulationaround 185−240 μg Cu.g−1. By contrast, root Cu accumulationwas only up to 1.3−1.6-fold as compared to controluntransformed plants (between 500 and 600 μg Cu.g−1).These data suggested that the expression of copC under thecontrol of the light-inducible and tissue-specific promoter cab1allowed higher Cu accumulation in shoots, with a maximumtranslocation factor of 0.41 for the transgenic line cab1−6 (SITable S2). Moreover, total Cu accumulation in transgenic linescab1−4 and cab1−6 were lower when compared to 35S lines,probably as a consequence of cab1 being a weaker promoterthan 35S.

Figure 2. Characteristic features of A. thaliana transgenic lines expressing copC under the control of cab1 promoter. (A) RT-PCR amplification ofcopC in shoots and roots of cab1 lines. No copC transcript was detected in wild-type (wt) plants (M: molecular weight marker). (B) QuantitativeRT-PCR of copC gene in shoots and roots of cab1-copC lines. Amplification of the F-box transcript was used as a control to normalize expressionlevels. Results are the means ± SE of three different experiments. (Similar results were obtained using ACT2 and AT4G26410 as housekeepinggenes). (C) Root length of 7-day-old wild-type and transgenic seedlings grown in the absence or presence of Cu (upper panel). Fresh weight of 14-day-old wt and transgenic seedlings grown in the absence or presence of Cu (lower panel). Data are means ± SE of 60 seedlings (20 representativeseedlings × 3 plates). (D) Cu accumulation in shoots and roots of wt and cab1-copC transgenic plants. Plants were grown in a hydroponic systemfor one month and then transferred to 150 μM Cu during one week. Values of TF (Cu shoot accumulation/Cu root accumulation) are given. Dataare means ± SE of three independent determinations. Significant differences from wt plants as determined by Student’s t test are indicated by oneasterisk (P < 0.05).



Subcellular Targeting of CopC to the Vacuole Led toCu Hypersensitivity. CopC protein was targeted to thevacuole by adding specific vacuole sorting determinants. Fourrandomly selected transgenic lines were confirmed by PCR (SIFigure S4) and those with the highest level of Cu accumulation,designated 35S−V-4 and 35S−V-6, were chosen (SI Table S1).T3 homozygous plants were obtained and used for furthercharacterization.The expression of the chimerical copC-V gene was confirmed

by qRT-PCR. Similar levels of expression were found in shootsand roots of both transgenic lines 35S−V-4 and 35S−V-6 (datanot shown). In order to confirm that the vacuole sortingdeterminants were able to target soluble proteins to thevacuole, the construct pCambia-gf p-V was delivered bybombardment onto onion epidermal cells, and fluorescenceof the GFP protein due to transient expression was investigated.In the absence of vacuole sorting determinants (control),fluorescence was localized in the nucleus and in the cytoplasm(displaced to a peripheral position by the large central vacuole)(Figure 3A). Fluorescence was also observed in transvacuolarstrands, typical of untargeted GFP, since these structures

contain cytoplasm.50,51 When the vacuole sorting determinantswere added, fluorescence showed a diffuse aspect and occupiedall the central cell volume, coincident with the position of thelarge central vacuole (Figure 3B). As previously reported,52,53

no fluorescence was observed in the nucleus.Cu accumulation was measured in tissues of transgenic lines

35S−V-4 and 35S−V-6. In both lines, Cu accumulation was upto 2-fold as compared to control untransformed plants, both inshoots and in roots (Figure 3C). Translocation factors werealso similar to those of the control untransformed plants and35S transgenic lines (SI Table S2).The sensitivity toward Cu of transgenic lines 35S−V was

analyzed in plantlets and mature plants. There was a reductionof 35−40% in the length of the root and around 30−40% inbiomass (SI Figure S5). These results suggested a highersensitivity to Cu, as compared to control untransformed plantsand also to 35S-copC plants.Transgenic 35S-copC and 35S-copC-V lines (containing the

cytoplasmic or the vacuolar CopC) were compared for theirsensitivity toward Cu in two developmental stages: 7-day-oldplantlets and 1-month-old mature plants. Stronger Cu

Figure 3. Characteristic features of A. thaliana transgenic lines expressing copC-V (with vacuole sorting determinants) under the control of 35Spromoter. (A) Transient expression of control cytoplasmic GFP in onion epidermal cells. Fluorescence appears inside the nucleus (N), in thecytoplasm, and in transvacuolar strands (TVS) containing cytoplasm. The cytoplasm is located in the periphery of the cell (cyt). Lower right corner:close-up view of a cell expressing the nontargeted GFP. (B) Transient expression of gfp-V fusion in onion epidermal cells. Fluorescence appearsdiffusely in the cells, coincident with the position of the central vacuole. Fluorescence was not observed in the nucleus or in TVS. Lower right cornershows some cells in which the tonoplast is separated from the plasma membrane (indicated by an asterisk). (C) Cu accumulation in shoots and rootsof wt and 35S−V transgenic plants. Plants were grown in a hydroponic system for one month and then transferred to 150 μM Cu during one week.Data are means ± SE of three independent determinations. Significant differences from wt plants as determined by Student’s t test are indicated byone asterisk (P < 0.05). (D−G) Comparison of transgenic lines 35S and 35S−V grown in the presence of 150 μM Cu. Note the symptoms of Cuhypersensitivity in 35S−V lines (D,E): lower biomass and size, small and brown-purple leaves and early flowering. (F,G) Comparison of rosetteleaves of transgenic lines 35S (F) and 35S−V (G): leaves are brown-purple and smaller in 35S−V lines. (H,I) Comparison of 7-day-old transgenic35S (H) and 35S−V (I) seedlings grown in the presence of 75 μM Cu. Cu hypersensitivity in transgenic 35S−V lines was observed as shorter andbranched root systems.



sensitivity was observed in mature 35S−V lines. There was aconsiderable reduction in plant biomass (Figure 3D,E), leaveswere smaller and brown-purple (Figure 3F,G), and plantspresented a shorter life cycle (early flowering), smallerseedpods, and a lower production of seeds when comparedto 35S transgenic lines (Figure 3D,E). This enhanced Cusensitivity was also remarkable in 7-day-old plantlets: 35S−Vlines showed shorter and branched roots (Figure 3H,I).

4. DISCUSSIONCu hyperaccumulation is not a frequent trait in plants: up tothe present date, 35 plant taxa belonging to 15 families weredescribed as Cu (hyper)accumulators,54 although this numbermight be overestimated.55 Attempts to increase Cu accumu-lation/tolerance by expressing methallotioneins genes weremade.29,30,47 In most cases, increased Cu tolerance wasachieved, although Cu accumulation was not significantlyimproved.In this work, the first transgenic plants expressing a

prokaryotic Cu binding protein have been generated.Expression of copC under the control of 35S promoter led toa maximum 5-fold increase in Cu accumulation in shoots androots, without alteration of the TF values. These plants can beconsidered Cu hyperaccumulators, since the threshold for Cuhyperaccumulation has been recently established as 300 μgCu.g−1.54 However, they showed lower Cu tolerance than thecontrol untransformed plants at the seedling stage, as deducedfrom the reduction in root length and total biomass, whichcould be a handicap for phytoremediation. Nevertheless, theseplants were able to complete their life cycles.Bacterial genes have been expressed in plants with variable

phenotypes. For example, the expression of arsenate reductasearsC gene led to arsenic hypersensitivity.31 Analogously, theexpression of merC from Acidithobacillus ferrooxidans (encodinga mercury uptake pump) led to enhanced Hg accumulation andhypersensitivity.56 Conversely, the expression of a bacterialheavy metal transporter in A. thaliana led to enhancedresistance and decreased uptake.57 Finally, the expression ofthe metal binding protein MerP from Bacillus megateriumallowed both metal (Hg, Cd, and Pb) tolerance andaccumulation, due to metal biosorption to MerP on the plantcell surface.58

The biochemistry of the CopC protein might be related tothe phenotype of 35S-copC plants, since CopC can exchangecopper between the two Cu binding sites activated by a redoxswitch in the presence of O2.

18,19 Due to its inherent toxicity,Cu(I), the most abundant Cu species in the cytoplasm, isalways bound to ligands. The concentration of free Cu(I) in thecytosol of yeast cells is below 10−18 M.59 The partitioning ofCu(I) in the cytoplasm would depend on the relative bindingaffinities of Cu complexes with the array of cuproproteins,which have been recently investigated,60 and with theintroduced protein CopC. In the cytoplasm, with a low redoxpotential, Cu/ZnSOD1 and metallothioneins are the proteinswith higher affinity for Cu(I) (dissociations constants, Kd ∼0.23and 0.41 and × 1015 M, respectively).60 After these proteins,some others also bind Cu(I) very tightly, such as the chaperoneCCS.60 Under Cu excess, Cu(I) could still be available forbinding to CopC [binding constant k ∼10−7 to 10−13 M forCu(I)].19 However, when this reaction occurs, the CopC-Cu(I)loaded protein must not be stable in air, and it must quicklyoxidize to CopC-Cu(II) in a intermolecular transfer reactionwith redox change.18,19 The generation of ROS could increase

the oxidative stress of the plant, which must cope with thisoxidative damage. In particular, the cuproenzyme Cu/Znsuperoxide dismutase (Cu/ZnSOD) is involved in thedetoxification of ROS and receive Cu through protein−proteininteraction with the copper chaperone for superoxide dismutase(CCS).61 Cu trafficking between different cuproproteins hasbeen shown to depend, not only on affinity gradients, but alsoon protein−protein specific recognition.60 Maybe the prokary-otic CopC protein cannot deliver Cu to plant cuproproteins,such as Cu/ZnSOD, due to a lack of specific protein−proteininteractions. Furthermore, the binding constant of the CopC-Cu(II) complex (k ∼10−13 M)19 is much lower than that of theCopC-Cu(I) complex, indicating much more tightly boundCu(II) to CopC. Thus, in this context, and in spite of the highexternal Cu concentration, an intracellular “Cu-depletion”situation may have been created (due to both thermodynamicand kinetic reasons), which may be another reason for Cu-sensitivity.Alternatively, enhanced Cu accumulation in transgenic lines

could damage the photosynthetic apparatus. For example, Cuexcess has been reported to decrease quantum efficiency ofphotosystem II, net photosynthetic rate, stomatal conductance,and pigment concentrations in Spartina densiflora.62 This effectis attributed to a substitution of chlorophyll Mg2+ by Cu2+ andto oxidative stress.63,64 Further investigation is required toelucidate these possibilities. For example, the expression ofgenes depending on SPL7 (FeSOD, COPTs, CCS, Cu-microRNAs) could be analyzed in order to know whether a“Cu-depletion situation” is sensed by the plants. On thecontrary, analyzing the levels of metallothioneins, HMA5 andPC2, which are known to be involved in Cu detoxification,could be done as an indication of copper excess.12,15,16 Inaddition, enzymes involved in redox stress can be studied.In an attempt to drive the expression of copC to shoots, it

was cloned downstream of the cab1 promoter.36 Apparently, ahigher expression of copC in green tissues could be detected byRT-PCR. At the same time, further increases in thetranslocations factors were achieved. These results suggestedthat these plants limit Cu accumulation in roots and uploadmore Cu to the xylem, maybe in response to a hypothetical“Cu-depletion” situation due to Cu tightly bound to CopC ingreen tissues. Expression levels of Cu transporters that uploadCu into the xylem, such as HMA5,12 might be investigated.Again, the expression of copC driven by cab1 led to Cu-sensitivity. It would be interesting to know whether there isadditional damage to the photosynthetic apparatus in cab1-linesdue to increased TFs. Under Cu excess, plastocyanin2 (PC2)has been shown to participate, not only in electron transferduring photosynthesis, but also in buffering Cu excess.16 Levelsof PC2, which accumulates after Cu addition, can beinvestigated to test this possibility. However, Cu-depletioncould also explain a Cu-sensitive phenotype, having a greatinfluence on photosynthesis by modulating plastocyaninlevels.14

Although speculative, the use of a stronger tissue-specificpromoter could further increase Cu accumulation in shoots,making plants more useful for Cu phytoextraction. The light-induced tissue-specific promoter SRS1p driving the expressionof arsC to the shoots resulted in As hyperaccumulation andhypersensitivity.31

Finally, subcellular targeting was addressed as a strategy toaccumulate the metal in the vacuole, one of the maindetoxification mechanisms in plants.9 Since the vacuole-sorting



determinants used in this work38,39 were able to target GFP tothe vacuole of onion cells, it could be assumed that CopC wastargeted to the vacuole as well, although further confirmationusing a fusion protein or antibodies could be approached.Subcellular targeting of proteins to the endoplasmic reticulumor chloroplast were shown to improve Hg phytoremedia-tion.65,66 In our study, transgenic plants with vacuole-targetedCopC showed 2-fold increased Cu accumulation, with nosignificant alteration of TF values. Surprisingly, an enhanced Cuaccumulation was not achieved with regard to 35S lines. Itseems that a great part of the copper is transported to the cellwall and bound to pectins.67,68 Alternatively, targeting CopC asa secreted protein to the cell wall may be attempted.65,69

The most remarkable characteristic of 35S−V lines was thehigh Cu hypersensitivity, especially in mature plants (lowbiomass, inhibited root growth, early flowering, and poor seedproduction). Since a higher Cu accumulation compared to 35S-copC lines was not seen, it could be due to other reasons, suchas a higher oxidative stress or damage to the photosyntheticapparatus.4,5 Besides, a situation of Cu-depletion might havebeen provoked by irreversible copper sequestration by CopCinside the vacuole. Whereas in the cytoplasm Cu is bound to−S ligands (such as metallothioneins), in the vacuole it isthought to be bound to N− and O− ligands like organic acidsand nicotinamide. Cu is also bound to −N and −O ligands ofCopC. The vacuole Cu transporter COPT5 is involved inremobilizing Cu from the vacuoles.7,8 Again, the vacuolarprokaryotic CopC might not be able to deliver Cu to COPT5,and Cu cannot be reallocated to the cytoplasm. For instance,Cu deprivation is known to affect photosynthesis, pigmentssuch as chlorophyll and carotenoids, and reproductive organsdue to pollen development defects70 (in our case, 35S-copC-Vtransgenic plants have lower biomass, purple-brown leaves, anddecreased seed production). However, the addition of moreexternal Cu supply did not alleviate the hypersensitivity towardCu (SI Figure S3). Furthermore, analyzing the expression levelsof SPL7 (or other genes previously mentioned, such as FeSOD,CCH, and Cu-microRNAs) could shed some light on thisphenotype, since the transcription factor SPL7 is a masterregulator of Cu starvation.In this work, evidence is presented that Cu hyper-

accumulation in plants can be achieved by expressing theprokaryotic copC gene. Expression of copC in deep-rootedplants with higher biomass production and adapted toparticular environments could be interesting in Cu phytostabi-lization, since they accumulated huge Cu concentrations inroots. Furthermore, the light-inducible and tissue-specificpromoter cab1 can drive the expression of the gene to theshoots, leading to increased TF for Cu accumulation, adesirable trait in Cu phytoextraction. The main disadvantageof transgenic plants expressing copC is the elevated Cusensitivity, which could be related with one or several aspectsof Cu homeostasis (redox unbalance, Cu hyperaccumulation, oreven cytoplasmic “Cu-depletion”). Some possibilities toalleviate Cu hypersensitivity may be crossing these plants tometallotionein-expressing plants30,47 or buffering redox stressby coexpressing antioxidative enzymes, such as Fe superoxidedismutase (FeSOD), ascorbate peroxidase (APX), catalase(CAT), and glutathione reductase (GR), involved in ROSscavenging in hyperaccumulators.71

■ ASSOCIATED CONTENT*S Supporting InformationAdditional information and figures. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Tel.: +34954556924. Fax: +34954628162. E-mail: [email protected] Contributions†These authors contributed equally to this work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was financed by the MICIIN (BIO2009/7766) andJunta de Andalucia (P06−CVI-01850). FEDER founding isacknowledged. Authors are grateful to the Microanalysis andBiology Services of the CITIUS (University of Sevilla) for Cudeterminations and particle bombardment equipment. Com-ments and suggestions made by anonymous reviewers areacknowledged.

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Engineering Copper Hyperaccumulation in Plants by Expressing a Prokaryotic copC Gene