ORIGINAL ARTICLE

Expression of Ceratophyllum demersum phytochelatin synthase,CdPCS1, in Escherichia coli and Arabidopsis enhances heavymetal(loid)s accumulation

Devesh Shukla & Ravi Kesari & Manish Tiwari &Sanjay Dwivedi & Rudra Deo Tripathi & Pravendra Nath &

Prabodh Kumar Trivedi

Received: 2 January 2013 /Accepted: 2 May 2013# Springer-Verlag Wien 2013

Abstract Phytochelatin synthase (PCS) gene encoding keyenzyme for heavy metal detoxification and accumulationhas been characterised from different sources and used todevelop a technology for bioremediation. Past efforts pro-vided limited success and contradictory results. Therefore,functional characterisation of PCS gene from new sourcesinto different target systems is considered as an importanttask in the area of bioremediation. Earlier, we isolated andfunctionally characterised PCS gene from an aquatic mac-rophyte Ceratophyllum demersum L., a metal accumulatoraquatic plant. Expression of this gene, CdPCS1, in tobaccoenhanced PC synthesis and metal accumulation of transgen-ic tobacco plants. In the present study, we have expressedCdPCS1 in more diverse systems, Escherichia coli andArabidopsis, and studied growth and metal accumulationof transgenic organisms. The expression of CdPCS1 in E.coli offered tolerance against cadmium as well as higher

accumulation accompanied with PCS1 activity. The expressionof CdPCS1 in Arabidopsis showed a significant enhancedaccumulation of heavy metal(loid)s in aerial parts withoutsignificant difference in growth parameters in comparison towild-type Arabidopsis plants. Our study suggests that CdPCS1can be utilised for enhancing bioremediation potential of dif-ferent organisms using biotechnological approaches.

Keywords Arabidopsis . Bioremediation . CdPCS1 .

Phytochelatin synthase . Heavymetal(loid)s

AbbreviationsAs(III) ArseniteAs(V) ArsenateCd CadmiumFW Fresh weightIPTG Isopropyl β-D-1-thiogalactopyranosidePC PhytochelatinPCS Phytochelatin synthaseWT Wild type

Introduction

Bioremediation is considered as a relatively less expensive,eco-friendly, efficient way of cleaning up wastes, sedimentsor soil contaminated with toxic heavy metal(loid)s usingplants and microbes (Bae et al. 2001; Mejare and Bulow2001; Tripathi et al. 2007; Singh et al. 2010; Abhilash et al.2009). However, this ability of bioremediation is limited tofew organisms. A promising way of improving bioremedi-ation potential is to genetically engineer organisms withincreased abilities to tolerate and accumulate toxic heavymetal(loid)s. In past, efforts have been made to engineermicrobes and plants for enhanced metal accumulation

Handling Editor: Bhumi Nath Tripathi

Electronic supplementary material The online version of this article(doi:10.1007/s00709-013-0508-9) contains supplementary material,which is available to authorized users.

D. Shukla : R. Kesari :M. Tiwari : S. Dwivedi : R. D. Tripathi :P. Nath : P. K. Trivedi (*)National Botanical Research Institute (NBRI), Councilof Scientific and Industrial Research (CSIR), Rana Pratap Marg,Lucknow 226001, Indiae-mail: prabodht@hotmail.com

Present Address:D. ShuklaInternational Centre for Genetic Engineering and Biotechnology,Aruna Asaf Ali Marg,New Delhi 110067, India

Present Address:R. KesariBihar Agriculture University,Bhagalpur 813210, India

ProtoplasmaDOI 10.1007/s00709-013-0508-9

potential, but limited success was achieved (Chaurasia et al.2008; Gisbert et al. 2003; Gasic and Korban 2007a, b; Kanget al. 2007; Lee et al. 2003; Li et al. 2004; Sauge-Merle et al.2003; Say et al. 2003; Singh et al. 2008, 2010;Wawrnzynska et al. 2005; Wojas et al. 2008, 2010a, b;Brunetti et al. 2011; Liu et al. 2011, 2012; Wang et al.2012; Kumar et al. 2013a). It was argued that limited suc-cess may be due to the use of genes from organisms whichare not a potential accumulator of heavy metals. Therefore,it was proposed to identify molecular networks and genesfrom various biological sources with the ability to uptake,accumulate and detoxify heavy metals (Meagher 2000;Cobbett and Goldsbrough 2002; Tripathi et al. 2007).

The heavy metal cadmium (Cd) and the metalloid arsenic(As) are among the most toxic carcinogenic elements whichshow high affinity towards thiols (Gill et al. 2012; Tripathiet al. 2013). The role of non-protein thiols, such as γ-glutamylcysteine, glutathione (GSH) and phytochelatins(PCs), in the detoxification of Cd and As has been welldocumented in plants (Cobbett 2000; Tripathi et al. 2007;Mishra et al. 2009; Tuli et al. 2010; Rea 2012; Kumar et al.2013b). Of these, PCs with a general structure of (γ-Glu-Cys)n-X where n=2–11 and X is usually glycine are sulphurcontaining a small metal-binding peptide. Role of PCs inmetal detoxification was confirmed through sensitivity forheavy metals in PC- and GSH-deficient mutants ofSchizosaccharomyces pombe (Glaeser et al. 1991) andArabidopsis (Cobbett 2000). It has been observed that PCschelate heavy metal with higher affinity and binding capacitythan metallothioneins (Mehra and Winge 1991; Zenk 1996;Vatamaniuk et al. 1999; Bae et al. 2001). The synthesis ofphytochelatin is catalysed by phytochelatin synthase (PCS) inthe presence of metal ions using GSH as a substrate.

The genes coding for PCS have been cloned from a varietyof organisms and expressed either alone or with other genes inEscherichia coli and plants for enhanced metal accumulationand tolerance; however, developed transgenic organism pro-vided varying degrees of success and contradictory results(Brunetti et al. 2011; Chaurasia et al. 2008; Kang et al. 2007;Lee et al. 2003; Li et al. 2006; Sauge-Merle et al. 2003; Singhet al. 2008, 2010; Tsai et al. 2012; Wawrnzynska et al. 2005;Wojas et al. 2008, 2010a, b; Wang et al. 2012). It washypothesised that these disparities in metal response in trans-genic plants may be due to different PCS genes withmodulatedactivity and plant species for transformation. Through usingAtPCS1 and CePCS1 and developing tobacco transgenic lines,it was concluded that not all PCS genes would be suitable forthe transformation of all plant species for the enhanced metalaccumulation potential (Wojas et al. 2008).

We hypothesised that isolation and use of PCS genesfrom potential accumulators might help in enhancing metalaccumulation in transgenic plants. Recently, we havecharacterised PCS gene, CdPCS1, from submerged rootless

aquatic macrophyte Ceratophyllum demersum (Shukla et al.2012) which has heavy metal detoxification mechanismsdue to induction of PCs and antioxidant systems (Mishraet al. 2006; 2008a; b; 2009). Expression of CdPCS1 intobacco showed a several-fold increased content of PCsand related molecules with enhanced accumulation of Cdand As without a significant decrease in plant growth(Shukla et al. 2012). In the present study, to validate func-tion of this gene in other biological systems as well as toassess the phytoremediation potential, we expressedCdPCS1 in E. coli and Arabidopsis and studied heavymetal(loid)s tolerance and accumulation. This study demon-strates an application of CdPCS1 gene in both prokaryoticand eukaryotic systems and provides the suitability of thisgene for the phytoremediation purpose. This in contrast toother reports which displayed varying results of PCS genedepending on the host biological systems. Our study sug-gests that expression of CdPCS1 in both the systems pro-vides tolerance with enhanced metal accumulation potential.

Materials and methods

Bacterial strains and cell culture

For expression of recombinant gene in E. coli, pGEX-4T-2vector (GE Healthcare, USA) with tac promoter was used.This tac promoter is a functional hybrid derived from the trpand lac promoters. It is composed of the −35 region of the trppromoter and the −10 region of the lacUV5 promoter/operatorand strongly induced in presence of isopropyl β-D-1-thio-galactopyranoside (IPTG). The promoter is tightly regulatedby the lacI protein repressor. For protein expression andinduction, E. coli BL21 (DE3) strain was used. E. coli strainDH5α was used for plasmid cloning and propagation.

Development of recombinant E. coli expressing CdPCS1and protein accumulation

Plasmid vector pGEX-4T-2 was used to express the proteinencoded by CdPCS1 in E. coli. For the development of aconstruct, oligonucleotides with EcoRI and SalI restrictionenzymes sites, PCS fusion 1 and PCS fusion 2, weredesigned and used for PCR (Supplementary Table S1).The amplified product was purified, digested and ligatedin vector pGEX-4T-2 digested with the same set of en-zymes. Ligation product was transformed into E. coliBL21 cells (DE3) and grown in lysogeny broth (LB) medi-um. Positive colonies were screened through colony PCRwith gene-specific primers, PCS fusion 1 and PCS fusion 2.Positive recombinant cells were inoculated in LB media andgrown at 37 °C, 220 rpm for overnight. Primary culture(100 μl) was inoculated in a secondary culture of 10 ml

D. Shukla et al.

LB and further grown at 37 °C until optical density (OD)600reached to 0.4. Finally, culture was divided into two separatetubes, each containing 5 ml culture (tubes A and B). In tubeB, IPTG was added to a final concentration of 0.4 mM forinduction of expression of fusion protein. Both the tubes(un-induced and induced) were incubated at 28 °C for 10 hwith continuous shaking at 220 rpm. During each hour ofincubation starting from 0 h, 500 μl of culture was takenfrom each tube and harvested by centrifugation inEppendorf tubes. Supernatant was discarded, and pelletwas suspended in the 200 μl of 1× protein loading bufferby vortexing. Samples were heated at 100 °C in a dry bathfor 3 min and centrifuged, and supernatant was chilled onice and analysed on a 12 % sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) afterstaining with Coomassie blue.

Growth kinetics of E. coli under Cd stress

In order to study the response of recombinant bacteriaexpressing CdPCS1 to 50 μM of Cd stress, growth oftransformed E. coli BL21 (DE3) cells with pGEX-4T-2(empty vector), recombinant plasmid pGEX-4T-2-CdPCS1and mock E. coli BL21 (DE3) cells was examined in LBmedium with and without Cd treatment. Primary cultureof these was allowed to grow at 37 °C for overnight.Overnight grown culture (100 μl) was diluted into2.4 ml of LB broth of which 750 μl was inoculated into20 ml of secondary culture and allowed to grow at 37 °Cfor approximately 3 h until OD600 reached up to 0.4. Insecondary culture, a final concentration of 0.1 mM ofIPTG was added for induction at 28 °C. After a gap of1 h, Cd was added to a final concentration of 50 μM.Thereafter, absorbance was measured at 600 nm wavelengthat each interval of 1 h.

Estimation of Cd accumulation in E. coli

For quantitative determination of Cd, E. coli cells grown for5 h in the presence of IPTG and Cd (50 μM) were harvested,rinsed three times using LB medium and dried at 60 °C for2 days. Dried cells were digested in HNO3 (70 %)/HCLO4

(30 %), and mineralisation was carried out in a microwaveoven (Dwivedi et al. 2010). The level of metal was quanti-fied using inductively coupled plasma mass spectrometer(Agilent 7500 cx).

Measurement of CdPCS1 enzyme activity

In vitro enzyme activity of CdPCS1 in recombinantbacteria was determined by measuring the amount ofPC2 in the reaction mixture in presence of Cd(100 μM). The E. coli cells grown in 200 ml of LB

at 37 °C to OD600 of 0.6 were supplemented withIPTG (0.6 mM) and further grown at 28 °C for 6 hto express the protein. Enzyme extraction wasperformed following the protocol of Ogawa et al.(2010). Bacterial culture was centrifuged at 8,000×gfor 10 min, and the harvested pellet was suspended inan extraction buffer solution consisting of 25 mMTris–HCl (pH 8.0), 400 mM NaCl, 10 % glycerol,1 mM 2-mercaptoethanol, 0.1 % Tween-20, 1 mMphenylmethylsulfonyl fluoride. The suspended bacteri-al cells were sonicated and centrifuged at 13,000×gfor 10 min at 4 °C. The supernatant obtained was usedfor determination of PCS activity assay as describedby Chen et al. (1997) with slight modifications. Themodification was of incubation time of 15 min insteadof 30 min . The reac t ions were der ived wi thmonobromobimane, and quantification of the enzymeactivity was measured by the production of PC2 asdetermined by HPLC according to method describedby Minocha et al. (2008). The total cell protein of thelysate was estimated by using the standard protocol ofPeterson (1977).

Plant materials, growth conditions, treatmentsand metal(loid)s estimation

The transgenic seeds of CdPCS1 lines and wild type(WT) were germinated on an agar medium containinghalf-strength Murashige and Skoog (MS) and 1.5 %(w/v) sucrose (pH 5.8) in 135-mm plates. Plates weresupplemented with the different concentrations ofCdCl2 (20 μM), As(V) (30 μM) and As(I I I )(2.5 μM). Plates were kept in the dark for 48 hfollowed by in a growth chamber at 23 °C under a16-h photoperiod provided by cool-white fluorescenttubes at a light intensity of 120 μmol m−2 s−1. Plateswere positioned vertically along the shelf to facilitatemeasurement of root length in developing seedlings.

For estimating heavy metal(loid)s accumulation,sterilised homozygous seeds of CdPCS1-expressing lineswith WT were germinated into plastic glasses containingsoilrite and grown for 20 days. These plants wereallowed to grow for the next 10 days in the presenceof 100 μM CdCl2, 300 μM As(V) and 25 μM As(III)contained in nutrient medium irrigated at each intervalof 3 days. Aerial tissues were harvested and dried andused for metal accumulation experiments. Dried tissues(100 mg) were digested in HNO3 (70 %) and 30 %HClO4 for Cd or 30 % H2O2 for As, and mineralisationwas carried out in a microwave oven. Cd concentrationwas determined on a flame atomic absorption spectro-photometer (GBC Avanta R, Australia) (Mishra et al.2009). For the determination of As concentration, the

CdPCS1 expression enhances heavy metal(loid)s accumulation

atomic absorption spectrophotometer (GBC Avanta,Australia) coupled to the GBC hydride generation sys-tem (HG900) was used (Mishra et al. 2008b).

Plant transformation

Plants were grown for 6 weeks in 10-cm2 potscontaining soilrite and maintained in a growth cham-ber under the same growth conditions as describedabove. These plants were used for transformation bythe floral dip method (Clough and Bent 1998) usingAgrobacterium tumefaciens strain GV3101 harbouringthe plant gene expression construct. Transformed seedswere selected on MS agar medium containing50 mg l−1 kanamycin. Homozygous seeds were col-lected and used in the further experiment.

Expression analysis

Total RNA was extracted from around 100 mg offrozen tissue using the RNeasy Plant Mini kit withon-column DNase digestion (Qiagen, USA). RNA(3 μg) from each sample was used for 20 μl reversetranscription reaction as recommended by the manu-facturer (Fermentas, USA). The reverse transcription(RT)-PCR product for the actin gene was used as acontrol to confirm that equal amounts of RNA wereused in each reaction. PCR was carried out with 3 μlof the RT reaction product using the specific primersCdPCS1RTF and CdPCS1RTR for CdPCS1 gene asdescribed in Supplementary Table S1.

Plant growth during Cd, As(V) and As(III) stress

Seeds of WT and transgenic lines expressing CdPCS1were germinated and grown vertically on Petri platescontaining half-strength MS medium with 1.5 % su-crose and supplemented with different concentrationsof Cd (20 μM), As(V) (30 μM) and As(III) (2.5 μM)as well as without metal supplementation (0 μM).Plates were kept at 4 °C for 2 days in the dark forstratification and then placed vertically right from thebeginning and grown in a culture room at 23 °C on a16-h day/8-h night cycle for 10 days; thereafter, rootlength and fresh weight of seedlings were recorded.The experiment was repeated at least three times, andthere was ten seedlings taken to measure the rootlength from each replicate.

Statistical analysis

Each experiment was carried out under a completelyrandomised design with independent experiments with

at least three replications. The data were analysed byStudent's paired t test, and mean values under eachtreatment were compared at P≤0.05–0.001.

Results

Heterologous expression of CdPCS1 in E. coli

Positive recombinant colony was identified through colonyPCR using gene-specific primers and subsequently used forprotein expression experiments (Supplementary Fig. S1).Full-length CdPCS1 cDNA was expressed as a fusion pro-tein glutathione S-transferase (GST)::PCS1 (Fig. 1a) in E.coli, and the level of CdPCS1 protein accumulation, PCSenzyme activity, growth and metal accumulation in controland recombinant bacterial cells under Cd (50 μM) exposurewas analysed. No induced protein band was observed incontrol E. coli cells, whereas in the E. coli cells which weretransformed with empty vector containing only GST, aprotein band of GST was observed in IPTG-induced cells(Fig. 1b). No GST:PCS1 protein was observed in cellswhich were not induced by IPTG. However, in the case ofrecombinant cells expressing CdPCS1:GST, when inducedwith IPTG, a protein of approximately 81 kDa was observedas a fusion protein which contains GST and CdPCS1 protein(predicted molecular weight by ExPasy tools is 56 kDa)(Fig. 1b). The accumulation of CdPCS1 protein increasedwith time after treatment of IPTG as observed on SDS-PAGE (Fig. 1c).

Expression of CdPCS1 confers Cd tolerance in E. coli

Growth inhibition was observed in Cd-treated control aswell as E. coli cells transformed with pGEX-4T-2 (emptyvector). However, in the absence of Cd, E. coli cellstransformed with pGEX-4T-2 grew normally (Fig. 2a,Supplementary Fig. S2). In contrast, the growth of the Cd-treated E. coli cells expressing GST–PCS1 fusion proteinincreased with time in comparison to control cells (Fig. 2a,Supplementary Fig. S2). This observation suggests growthtolerance against Cd exposure for the cells expressing GST–PCS1 fusion protein (Fig. 2a). To study whether tolerance toCd stress is due to CdPCS1 activity and PC synthesis, invitro enzyme activity was measured in E. coli cellstransformed with pGEX-4T-2 and pGEX4T2:CdPCS1. Thein vitro enzyme activity of PCS was observed in terms ofsynthesis of PC2 using GSH as a substrate in the presence ofCd. HPLC chromatograms demonstrated in vitro synthesisof PC2 as a result of PCS activity (Supplementary Fig. S3)in pGEX:CdPCS1-transformed cells. In control, no PC2 wasdetected; however, the reaction mixture containing mBBr-labelled lysate of recombinant E. coli showed peaks of PC2.

D. Shukla et al.

Activity of PCS was calculated as 1.39±0.5 nmol PC2

synthesised min−1 mg−1 protein (Fig. 2b). These resultssuggest that expression of CdPCS1 confers PC synthesis,providing metal tolerance in bacterial cells in the presenceof Cd. Cd accumulation was also measured in recombinantE. coli cells grown for 5 h under Cd exposure. The accu-mulation of Cd in control E. coli cells was recorded as 13.89±1.53 ppm; however, cells expressing pGEX4T2:CdPCS1accumulated 17.09±2.56 ppm, indicating enhanced intracel-lular Cd level relative to control E. coli cells (Table 1). Wedid not carry out As toxicity experiments on recombinant E.coli as bacterial cells generally accumulate very low arseniclevels because of a highly efficient export pump “ArsAB”(Sauge-Merle et al. 2003).

Expression of CdPCS1 in Arabidopsis

Plant transformation construct developed earlier (Shuklaet al. 2012) was used to develop transgenic Arabidopsisexpressing CdPCS1 gene. Three independent homozy-gous lines were selected for further analysis. Thesehomozygous lines contained CdPCS1 as confirmed bygenomic DNA PCR (Fig. 3(A)) as well as expressedCdPCS1 as observed through semi-quantitative RT-PCRanalysis (Fig. 3(B)). However, no genomic DNA PCRand RT-PCR amplification was observed in WT plantsused for analysis (Fig. 3(A–C)).

Growth of WT and transgenic Arabidopsis plants ex-pressing CdPCS1 was studied after treatments to different

*

**

kDa M -I +I -I +I -I +I

BL

21

pG

EX

pG

EX

::C

dP

CS

10 1 2 3 4 5 6 10 4

+ IPTG - IPTG

**

(a)

(b) (c)

Fig. 1 Expression of CdPCS1 in E. coli. a Construct developed andused for transformation of E. coli. Ptac tac promoter, GST tag gluta-thione transferase tag, CdPCS1 C. demersum PC synthase codingsequence from nucleotides 1 to 1506; Ampr beta-lactamase codingsequence, pBR322 ori origin of replication, lac Iq lac I encoding arepressor protein inhibits the gene expression in the absence of IPTGtreatment. b SDS-PAGE (12 %) gel analysis of PCS protein expressionin E. coli BL21. Lane M, protein marker. BL21, pGEX andpGEX::CdPCS1 represents lanes containing cell lysates from E. colicells (mock), cells containing empty vector pGEX-4T-2 and recombi-nant plasmid pGEX-4-T-2 with CdPCS1, respectively. E. coli cells

were grown until OD600 reached to 0.4 followed by addition of IPTG(+I; −I represents samples without IPTG) for induction of expression offusion protein. Single and double asterisks represent GST and fusionprotein of GST and CdPCS1, respectively. c Time-dependent CdPCS1accumulation in E. coli. SDS-PAGE (12 %) analysis of PCS proteinexpression in E. coli BL21 containing pGEX::CdPCS1 plasmid. E. colicells were grown until OD600 reached to 0.4 followed by addition ofIPTG (+I; −I represents samples without IPTG) for induction of ex-pression of fusion protein. The accumulation of CdPCS1 (shown bydouble asterisks) increased with time post-treatment by IPTG withgrowth of recombinant cells

CdPCS1 expression enhances heavy metal(loid)s accumulation

heavy metals (Fig. 4a–c). No significant phenotypic differ-ence between the CdPCS1-expressing transgenic plants andWT plants was observed when grown in the absence ofheavy metal(loid)s stress (Fig. 4a). Seedling weight and root

length of the transgenic plants were not significantly differ-ent in comparison to WT plants (Fig. 4b, c). To study theeffect of Cd, As(V) and As(III) on CdPCS1-expressingArabidopsis lines, seeds of transgenic plants were ger-minated on Cd (20 μM), As(V) (30 μM) and As(III)(2.5 μM), respectively, and grown for 10 days. Rootlength and fresh weight of CdPCS1-expressing trans-genic lines were not significantly different in compar-ison to WT under different heavy metal(loid)s stresses(Fig. 4b, c). Our results suggest that the expression ofCdPCS1 does not affect growth of Arabidopsis trans-genic plants under heavy metal(loid)s stress.

Heavy metal(loid) accumulation in Arabidopsis

To investigate the heavy metal(loid)s accumulation anddetoxification capabili ty of CdPCS1-expressingArabidopsis plants, the metal(loid) content was estimat-ed in aerial parts of WT and transgenic Arabidopsisplants. Transgenic plants accumulated more than two-fold Cd and As(III) and more than four-fold higherAs(V) as compared to WT (Fig. 5). The accumulationcapacity of CdPCS1-expressing plants appeared to bemuch higher than that of WT plants under heavymetal(loid) stress (Fig. 5) without a significant differ-ence in growth. These results clearly suggest that ex-pression of CdPCS1 can be used to enhance heavymetal(loid) accumulation in aerial tissues of the adultArabidopsis plants for phytoremediation of heavymetal(loid)s.

(b)

***

OD

(60

0 n

m)

Time (hrs)

BL21 -Cd pGEX BL21 -Cd pGEX-PCS1 BL21 –Cd BL21 +Cd pGEX BL21 +Cd pGEX-PCS1 BL21 +Cd

PC

S a

ctiv

ity

(nm

ol P

C2 m

g-1

pro

tein

-1)

1 2 3 4 5

(a)

Fig. 2 Growth and metal accumulation of E. coli under Cd stress. aEffect of Cd on growth of E. coli cells (mock; circle), empty vectorpGEX-4T-2 (triangle) and recombinant plasmid pGEX-4-T-2containing CdPCS1 (rectangle), respectively. Empty and filled markersrepresent with and without Cd treatment. The mean of three indepen-dent experiments are plotted with error bars indicating ±SD. b In vitroactivity assay of PCS in terms of phytochelatin production (PC2) inpresence of Cd (100 μM) from E. coli cells containing empty vector(pGEX-4T-2) and (pGEX-4T-2:CdPCS1) after IPTG induction. Lysatefrom IPTG-induced cells was used for measuring the activity as de-scribed in “Materials and methods”

Table 1 Accumulation of Cd by control and recombinant E. coli

Type of E.coli cells

Cd (μM) treatment Cd accumulation(in ppm)

Control(pGEX4T2)

0 ND

50 13.89±1.5

Recombinant(pGEX4T2:CdPCS1)

0 ND

50 17.09±2.5

ND no data

CdPCS1

CdPCS1

M WT 1 2 3

Actin

(a)

(b)

Fig. 3 Expression of the CdPCS1 in Arabidopsis transgenic lines(Col-0 background). A Confirmation of transgenic Arabidopsis linesthrough genomic DNA PCR using CaMV 35S forward and gene-specific reverse primers. B Expression of the CdPCS1 gene in trans-genic Arabidopsis (Col-0) lines. RT-PCR of total RNA extracted froma leaf of mature Arabidopsis plants grown in the absence of heavymetal. PCR products were obtained after amplification of CdPCS1 andactin cDNAs. Numbers 1, 2 and 3 represent three independent trans-genic lines. M, 100 bp ladder; WT, wild type; L10, L4 and L5 representthree independent transgenic lines

D. Shukla et al.

Discussion

Bioremediation or phytoremediation is an alternative tech-nology that can be used as a cost-effective and promisingway of cleaning up heavy metal(loid)s contaminations.Various efforts have been made in the past to increase themetal accumulation potential in bacterial cells by expressingdifferent genes including PCS and metallothioneins(Chaurasia et al. 2008; Kang et al. 2007; Singh et al. 2008,2010; Sauge-Merle et al. 2003; Say et al. 2003;Wawrnzynska et al. 2005; Gautam et al. 2012). It has beenobserved that expression of metallothioneins in bacteria hasfaced difficulties due to the instability of the cysteine-richprotein (Bae et al. 2001; Mejare and Bulow 2001).

The genes coding for PCS have been cloned from plants,blue green algae and fungi and functionally expressed in E.coli (Chaurasia et al. 2008; Kang et al. 2007; Sauge-Merle etal. 2003; Singh et al. 2008, 2010; Wawrnzynska et al. 2005).It is imperative from earlier studies that the use of diversePCS genes has been worked differentially. These differencesmay be due to limitation or utilisation of GSH which are

required for biosynthesis of PCs (Lee et al. 2003; Pomponiet al. 2006; Shukla et al. 2012). To figure out the functionaldifferences exhibited in different PCS genes, different PCSgenes have been functionally characterised in different het-erologous plant systems. In the present study, the expressionof CdPCS1 in E. coli led to the significant tolerance whenbacterial cells were grown in the presence of cadmium(Figs. 2 and 3) possibly by the synthesis of PCs by PCSactivity (Fig. 2). The expression of CdPCS1 enhanced cel-lular Cd content by 23 % in recombinant E. coli cells.Similar observations were made in the case of Anabaenasp. PCC 7120 PCS gene expressing in E. coli that providedtolerance against Cd, heat and salt stress (Chaurasia et al.2008) as well as in yeast expressing AtPCS1 (Singh et al.2008). As far as tolerance to other stresses and heavy metalis concerned, it was suggested that due to strong nucleophil-ic sulfhydryl groups of the cysteine present in PCs react witha broad spectrum of agents ranging from free radicals,reactive oxygen species to heavy metals (Rabenstein 1989)and detoxifying them, resulting into tolerance (Chaurasia etal. 2008; Gill and Tuteja 2010).

0 µM

0

30

60

90

Roo

t le

ng

th (

mm

) /s

eed

ling

s

0

30

60

90

120

FW

(mg

/10

seed

ling

s)

As(V) 30 µM As(III) 2.5 µMCd 20 µM

L1 L2 L3 WT L1 L2 L3 WT L1 L2 L3 WTL1 L2 L3 WT

Control Cd As(V) As(III) Control Cd As(V) As(III)

(a)

(b) (c)

WT L1L2 L3

WT L1L2 L3

Fig. 4 Growth response of CdPCS1-expressing seedlings to heavymetal stress. a Homozygous seeds were sterilised and plated ontohalf-strength MS containing 0 μM, 30 μM As(V), 2.5 μM As(III)and 20 μM Cd(II). Approximately 12 seeds were germinated andgrown in a vertical orientation for 10 days. CdPCS1 L1, L2 and L3Arabidopsis transgenic lines showed no phenotypic difference relative

to WT. b, Fresh weight of ten seedlings was measured for WT andCdPCS1-expressing transgenic plants under each type of heavy met-al(loid)s stress. c Root length was measured for each seedlings for WTand CdPCS1-expressing transgenic plants under different heavy met-al(loid)s stresses. The data shown are the average SEM of threeindependent experiments. Bars represent SD of mean values

CdPCS1 expression enhances heavy metal(loid)s accumulation

Our previous study with expression of CdPCS1 in tobac-co led to significantly enhanced Cd or As accumulation inroots accompanied by increased PCs content (Shukla et al.2012). Recently, we also demonstrated that CdPCS1 com-plements cad1-3 (PC-deficient) mutant of Arabidopsis tothe similar level of synthetic PC [M(EC)14G] (Shukla et al.2013). In the present study, the heterologous expression ofCdPCS1 in matured plants of Arabidopsis led to a signifi-cant increase in heavy metal(loid)s accumulation in aerialtissues relative to WT (Fig. 5) without significant growthpenalty. In the present study, we also used one additionalform of arsenic, i.e. arsenite As(III), and shown that trans-genic Arabidopsis plants tolerate this metal in addition toAs(V) used in our previous study. There are enough evi-dences which suggest the role of PCs in the long-distancetransport of heavy metal(loid)s via either xylem or phloem(Lappartient and Touraine 1996; Li et al. 2006; Gong et al.2003; Chen et al. 2006; Mendoza et al. 2008). A similarkind of mechanisms which might be involved inCdPCS1-expressing Arabidopsis plants led to higherheavy metal(loid)s accumulation in aerial tissues.

In summary, the present work suggests that the PCS fromC. demersum could be a good candidate for heavy metal(loid) bioremediation or phytoremediation. The heterolo-gous expression of CdPCS1 in E. coli not only improvesthe tolerance but also increases Cd accumulation. In addi-tion, expression in Arabidopsis led to a significant higherlevel of metal(loid)s accumulation in above-ground tissuesrelative to WT without any growth penalty.

Acknowledgments Authors acknowledge the Council of Scientificand Industrial Research (CSIR), Government of India, for providing afinancial support to carry out a study under Network Project(BSC0107). DS and MT acknowledge the Council of Scientific andIndustrial Research and the Indian Council of Medical Research,Government of India, for Senior Research Fellowships, respectively.

Conflict of Interest All the authors declare that we have no conflictof interest.

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WT L1 L2 L3

Cd

As(V)As(III)

**

******

**

*

**

*

***

**

Fig. 5 Estimation of heavy metal(loid)s accumulation in aerial tissues ofadult Arabidopsis expressing CdPCS1 exposed to heavy metal(loid)s for10 days. Sterilised homozygous seeds were germinated into plasticglasses containing soilrite irrigated with Arabidopsis nutrient media andgrown for 20 days. These plants were allowed to grow further for 10 daysin the presence of 100 μM Cd, 300 μM As(V) and 25 μM As(III)contained in nutrient medium which was irrigated at each interval of3 days. The plant samples were dried and digested and subjected to AASwith graphite furnace to determine metal(loid)s concentrations. The datashown are the average ± SD of three independent experiments,normalised to microgram of metal or metal(loid)s per gram of dry planttissue. *P<0.05 and ***P<0.001, differ significantly from their respec-tive controls according to Student's paired t test

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