Environmental and Experimental Botany 72 (2011) 131139

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Environmental and Experimental Botany

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Metallo nutoleran ia c

K. SekhaCentre for Plan

a r t i c l

Article history:Received 4 FebReceived in reAccepted 17 F

Keywords:Arabidopsis thaCajanus cajanMetallothioneMetal accumuMetal tolerancSubtracted cD

ds mern blonscripations imphyll cessinworthP) tr

ons. Tby p

rst report of its kind, deals with the isolation, characterization and functional validation of CcMT1 inheterologous systems. As such, CcMT1 may be deployed as a potential candidate for enhancement ofmetal tolerance in diverse crop plants.

2011 Elsevier B.V. All rights reserved.

1. Introdu

Metal iotial for normcrucial roleenzymes anessential anetc., are knoHigher orgadetoxify thcompartmeModicatioport and acpossibilityorganisms.

In eukarmajor role(and were btochelatins(414kDa),et al., 2002

CorresponE-mail add

0098-8472/$ doi:10.1016/j.ction

ns such as iron (Fe), zinc (Zn) and copper (Cu) are essen-al growth and development of plants as they play ain the catalytic and structural properties of variousd proteins. However, higher concentrations of bothd non-essential metals like cadmium (Cd), lead (Pb),wn to cause toxic effects and inhibition of plant growth.nisms have evolved diverse adaptive mechanisms toe excessive metals at the cellular level by secretion,ntalization or chelation by metal ligands (Hall, 2002).n of the characteristics such as metal uptake, trans-cumulation by genetic engineering approach offers thefor increasing the metal tolerance capacity of diverse

yotes, various specialized peptides were found to plays) in heavy metal homeostasis and their detoxication,roadly classied as metallothioneins (MTs) and phy-(PCs). MTs were identied as low molecular weightcysteine-richmetal-bindingproteins (Kagi, 1993;Coyle), and were found to occur in a wide variety of organ-

ding author. Tel.: +91 40 27098087; fax: +91 40 27096170.ress: rao kv1@rediffmail.com (K.V. Rao).

isms (Cobbett and Goldsbrough, 2002). Also, MTs were shown toplay vital role(s) in different organisms through detoxication ofexcess Cu (Ecker et al., 1989); by conferring protection against Cdtoxicity (Palmiter, 1998); by maintaining Zn homeostasis (Suhyet al., 1999; Coyle et al., 2002); and by controlling reactive oxy-gen species (Tamai et al., 1993; Wong et al., 2004). In differentMTs, Cys residues were found to occur in metal-binding motifs,containing Cys-Cys, Cys-Xaa-Cys or Cys-Xaa-Xaa-Cys, which fur-nished sulphydryl ligands for coordination of divalent metal ions(Cobbett and Goldsbrough, 2002). Based on the arrangement ofCys residues, plant MTs were classied into type-I, II, III and IV(Cobbett and Goldsbrough, 2002). The organization/distribution ofcysteine residues in diverse MTs was found to confer the abilityto bind and sequester various metal ions for detoxication andhomeostasis. Unlike MTs, PCs represent a family of enzymaticallysynthesized, cysteine-rich peptides with a general structure of (-Glu-Cys)211-Gly (Guo et al., 2008), and were deemed to constitutean essential component of Cd-detoxication in various organisms(Cobbett, 2000).

Effects of various metals on MT expression disclosed widevariation from species to species. Molecular mechanisms under-lying enhancement, depression or stasis of the transcript levelsin response to metal stresses, remain largely unknown in plants(Cobbett and Goldsbrough, 2002). In different plants, MT geneexpression was strongly induced by Cu treatment followed by Cd

see front matter 2011 Elsevier B.V. All rights reserved.envexpbot.2011.02.017thionein 1 (CcMT1) of pigeonpea (Cajace to copper and cadmium in Escherich

r, B. Priyanka, V.D. Reddy, K.V. Rao

t Molecular Biology, Osmania University, Hyderabad 500007, A.P., India

e i n f o

ruary 2010vised form 16 February 2011ebruary 2011

liana

in 1 genelationeNA library

a b s t r a c t

A cDNA clone, encoding 75-amino acithe cDNA library of pigeonpea. Northerevealed increased levels ofCcMT1 trametal tolerance and higher accumulCcMT1 in transgenic Arabidopsis plantby higher plant biomass and chloropcontrol plants. Transgenic plants exprroots as compared to the shoots. Nocopper (AtZIP2) and cadmium (AtNRAMunder stressed andunstressed conditithe detoxication of metal ions, therelocate /envexpbot

s cajan, L.) confers enhancedoli and Arabidopsis thaliana

tallothionein type 1 protein (CcMT1), has been isolated fromt analysis of pigeonpea plants subjected to heavymetal stressts. Escherichia coli cells expressingCcMT1exhibited enhancedof metal ions than that of control cells. Over-expression ofarted increased copper and cadmium tolerance as evidencedontent besides profuse root growth when compared to theg CcMT1 disclosed higher accumulations of Cu and Cd ions inwhile differences were observed in the expression levels ofansporter genes of CcMT1-transgenics and control plants bothheoverall results amply indicate that CcMT1 is contributing toroviding marked tolerance against metal stresses. This study,

132 K. Sekhar et al. / Environmental and Experimental Botany 72 (2011) 131139

and Zn (Hsieh et al., 1995; Roosens et al., 2005; Usha et al., 2009;Xue et al., 2009). Abiotic stresses caused by absciscic acid (ABA),drought, salt, heat, cold, excessive light, wounding and senescencewere shown to alter the expression of MT transcript levels in var-ious plantset al., 2004;et al., 2010)by their oveet al., 1992)2005), andet al., 2005)

Pigeonpthe tropicallent deep-rdrought, saand Sheila,from well-aoffer scopetolerance ametallothiosubtractiveerologous Ethe pigeonpas increased

2. Materia

2.1. Plants,

A highlobtained froresponsivemercuric chsterile watetaining stertransferredunder contrstress wasing water fowater stress1964).

2.2. ConstruCcMT1 from

Total RNand waterdinium thioThe mRNAoligo (dT) pcDNA libraemployingand ve par(driver) placDNAwereUnbound surst strand2010). TheZAP vector,E. coli cells acDNA librarcDNA fragmand nucleoting BLASTperformed

tor the stress-inducible nature of metallothionein gene, pigeonpeaplants were treated with 200M of CuSO4/CdSO4/ZnSO4 for 24h.

2.3. Southern and northern blot analyses

therolateopsisas blorn bd frodCTP

oning

codyingTTCnded Ecenc

2 (veindep

etal t

rnigm (1C. Aftd byconcLaterdilutdiumm cotesw

tima

measf E. cilutecilindwiO4/Cy cend twfollowt of cAS93

nstruidop

l-lengNACTGTCTAGCcMT100presnediew,I121e Agr. Tra(Buchanan-Wollaston, 1994; Choi et al., 1996; AkashiJin et al., 2006; Quan et al., 2008; Xue et al., 2009; Dong. Functional properties of plantMTswere demonstratedr-expression in Escherichia coli as fusionproteins (Evans, through complementation studies in yeast (Ma et al.,by RNAi knock down approach in Arabidopsis (Zimeri.ea (Cajanus cajan L.) is a major grain legume crop ofand subtropical regions of the world. It has an excel-

oot system with profuse laterals and is well known as alinity and alkalinity tolerant crop among legumes (Nene1990). Isolation and cloning of genes for stress tolerancedapted plants to different environmental conditionsfor genetic engineering of crop plants for enhanced

gainst different abiotic stresses. In this investigation, anein type 1 gene (CcMT1) has been isolated from thecDNA library of pigeonpea and over-expressed in het-. coli and Arabidopsis systems. In both the organisms,ea CcMT1 bestowed enhanced metal tolerance as wellaccumulation of Cu and Cd ions.

ls and methods

materials and treatments

y drought tolerant pigeonpea accession ICP 8744,m ICRISAT (India), was used for the isolation of stress-genes. Seeds were surface-sterilized with 0.1% (w/v)loride for 5min and were washed thoroughly withr. Sterilized seeds were germinated in petri plates con-ilewet blotting paper, and one-week old seedlingswereto pots. Plants were maintained in the green houseolled conditions at 282 C and70% humidity.Watergiven to four-week old pigeonpea plants by withhold-r 4 d. Relative water content (RWC) of the control andedplantswasmeasured as described (Scholander et al.,

ction of subtractive cDNA library and isolation ofpigeonpea

Awas isolated from four-week old control (80% RWC)stressed (5060% RWC) pigeonpea plants by guani-cynate (GTC) method (Sambrook and Russell, 2001).was isolated from the total RNA through biotin labeledrobeusingmRNA isolation kit (Promega,Madison,USA).ry was constructed through subtractive hybridizationone part of poly (A)+ RNA from stressed (tester) plantsts of 5-biotinylated rst strand cDNA from unstressednts. The poly (A)+ RNAcDNA hybrids and excessiveimmobilized onto streptavidin-coatedmagnetic beads.btracted poly (A)+ RNAs were used to synthesize thefollowed by the second strand cDNA (Priyanka et al.,resultant cDNA fragments were ligated to a lambda-in vitro packaged and allowed to infect XL1 blue MRFsper themanufacturers instructions, usingUni-ZAPXRy construction kit (Stratagene, Lajolla, CA, USA). Clonedentswere sequenced using automatedDNA sequencer,ide and amino acid sequences were analyzed employ-and ExPASy tools. Multiple sequence alignment wasemploying CLUSTALW using Bioedit software. Tomoni-

SouDNA isArabidand wNortheisolate-32P-

2.4. Cl

TheemploGCGAAsites (uNdeI anproteinpTYB1BL21)

2.5. M

Ovemediuat 37

inducea nal37 C.serialLB memediuThe pla

2.6. Es

Totures owere damphiinduceof CuSered bwashesuredconten(GBCA

2.7. Coof Arab

FulPfu DTCTAG5-GCTlined).the pRThe exand clotain VThe pBinto thmatingn blot analysis was carried out using 10g of genomicd from the control (vector alone) and CcMT1-transgenicplants. GenomicDNAwas digestedwith BamHI enzymetted as per the method (Sambrook and Russell, 2001).

lot was carried out with 20g and 10g of total RNAm pigeonpea and Arabidopsis plants, respectively. The-labeled CcMT1 cDNA was used as a probe.

of CcMT1 for expression in E. coli

ing region of the CcMT1 was amplied using PCRprimers, 5-GCCATATGTCTAGCTGTGGGTGT-3 and 5-TCACTTGCAGTTGCATGG-3 containing NdeI and EcoRIrlined), respectively. The PCR fragment digested withoRI was cloned into pTYB12 vector containing the inteinoding sequence. The pTYB12-CcMT1 fusion product andctor) plasmids were introduced into E. coli cells (strainendently.

olerance assays in E. coli

ht cultures (100l) were inoculated into fresh liquid LB0.0ml) containing amphicilin (50mg/l) andwere growner reaching an optical density (OD)600 of 0.6, cells werethe addition of isopropyl -d-thiogalactoside (IPTG) toentration of 1mM and were allowed to grow for 3h at, cultures were diluted to an OD600 of 0.2 from whichions (1, 101, 102 and 103) were made with fresh, and 5l from each dilution was spotted on LB solidntaining 1.5mM CuSO4/0.1mM CdSO4/1.0mM ZnSO4.ere incubated at 37 C for 12h andwere photographed.

tion of metal ions accumulations in E. coli

ure the metal content in E. coli cells, overnight cul-oli (BL 21) containing pTYB12/pTYB12-CcMT1 plasmidsd to 1:10 in 50ml fresh LBmedium supplemented with(50mg/l). After reaching an OD600 of 0.6, cells wereth 1mMIPTGandwere cultured in thepresence of 1mMdSO4/ZnSO4 for 3h at 37 C. Later, cells were recov-trifugation at 5000 g for 10min. The cell pellets wereice in LB medium and dry weight of cells was mea-ing their dehydration for 48h at 80 C. The metal ionells was determined by atomic emission spectrometry2).

ction of plant expression vector and transformationsis plants

th CcMT1 coding sequence was amplied withpolymerase using primers, 5-GCCTCGAGATG-

GGGTGT-3 (forward, XhoI site underlined) andATCACTTGCAGTTGCATGG-3 (reverse, XbaI site under-

T1 coding region was cloned into XhoI and XbaI sites ofplasmid (Topfer et al., 1987) in the sense orientation.

sion unit, 35S: CcMT1: PolyA, was excised with HindIIIinto HindIII site of the pBI121 vector (Clontech, Moun-CA, USA) containing gusA and nptII expression units.and CcMT1-pBI121 constructs were then mobilized

obacterium tumefaciens strain (EHA105) by tri-parentalnsformation of Arabidopsis thaliana (ecotype Columbia)

K. Sekhar et al. / Environmental and Experimental Botany 72 (2011) 131139 133

was performed via the vacuum inltration method (Bechtold andPelletier, 1998). Seeds were harvested plant wise and were platedon kanamycin (50mg/l) selection medium to identify the putativetransgenic plants. Kanamycin resistant T1 transgenic plants werescreened for the presence of T-DNA by GUS staining of seedlings,and were also conrmed by molecular analyses. Two transgeniclines of Mth3 and Mth6 (T3 generation) along with the vectorcontaining line (control) were selected for further stress tolerancestudies.

2.8. Functional analysis of transgenics for metal-stress tolerance

Seeds obtained from T2 transgenic lines and control plants weresurface-sterilized and were grown onMSmedium (Murashige andSkoog, 1962) or in soil (mixture of 1 vermiculite:1 perlite:1 soil-rite), and were kept at 4 C in dark for 3 d for stratication. Later,theywere transferred into Conviron growth chamber (TC16) underlong day conditions (16h light/8h dark cycles)with orescent light(7000 lux at 20 cm). To test the metal tolerance, seeds were germi-nated on 0.5 MS medium and medium supplemented with 50and 100Mof CuSO4 and CdSO4, or 200 and 300Mof ZnSO4, andwere kept at 201 C. After culturing for 23 d, seedling phenotypeswere observed visually, and seedling fresh and dry weights wererecorded. For determination of root growth, seeds of control andtransgenicsthen transfewith 50 anbated at 20was measur

Leaf diswere oate(50/100Mtemperaturing chloropdimethylsuthree times

2.9. Measur

For meaand CcMT1or supplemplants wererinsedwithharvested safter thorou

HNO3, and Cu and Cd contents were measured by atomic emissionspectrometry. These experimentswere repeated three times undersimilar conditions.

2.10. Quantitative real time PCR (qRT-PCR)

qRT-PCR was performed for ZIP2/NRAMP1 genes of A.thaliana associatedwith copper/cadmium transport using oligonu-cleotide primers of 5-TTTCTCCTTGATCCTAGCTCACGGC-3 and5-GGAGAAACGACTCGTTCCATCGGTA-3 for AtZIP2; 5-CAGTA-AGCGGGGCTGTTTGTAATG-3 and 5-CCATGGCTCGAGTCTGAGATCAAGA-3 for AtNRAMP. First strand cDNA was synthesized fromRNA samples of control and transgenic Arabidopsis seedlings sub-jected to CuSO4 and CdSO4 (50M) stresses for 10 d as well asfrom unstressed plants. The resultant cDNAs were used as tem-plates for qRT-PCR analysis. Real time PCR analysis was carried outusing Eurogenetec SYBR Green qPCR Master mix with Realplex 4(Eppendorf, USA) at 94 C (15 s), 58 C (30 s) and 68 C (45 s) for40 cycles. Later, the products were analyzed through a melt curveanalysis to check the specicity of PCR amplication. Each reactionwas performed twice and the relative expression ratio was calcu-lated using 2Ct method employing actin gene as a reference.Oligonucleotide primers of 5-GGCGATGAAGCTCAATCCAAACG-3

and 5-GGTCACGACCAGCAAGATCAAGACG-3 were used for ampli-of a

tatis

an vap of ptions

ults

arac

NAolypn inibrarit ha(Cc

d thdiffC-te

tic am

Fig. 1. Compa T1) wCajanus cajan (PisumNo. NP 172239 are shwere germinated on normal MS medium for 4 d, andrredonto0.5MSmediumandmediumsupplementedd 100M of CuSO4 and CdSO4. The plates were incu-1 C in vertical position for 2weeks. The root lengthed from 20 seedlings in each treatment.cs from 4-week old transgenic and control plantsd on 20ml solutions of CuSO4 (50/100M), CdSO4), or water (experimental control), for 96h at roome (282 C). Leaf discs were then used for measur-hyll content spectrophotometrically after extraction inlphoxide (DMSO) for 2h. All experimentswere repeatedunder identical conditions.

ement of metal content in Arabidopsis plants

surement of Cu and Cd ion content, 4-week old control-transgenic plants were grown on MS basal medium,ented with 50M of CuSO4 or CdSO4 for 10 d. Later,thoroughly washedwith 5mMCaCl2 at 4 C for 1h andsterilewater. Shoots and roots from treatedplantswereeparately, and dry weights of samples were recordedgh drying at 80 C for 2d. Samples were digested with

cation

2.11. S

Methehelcalcula

3. Res

3.1. Ch

A cDfor a pdomaicDNA lclone,1 generevealesentingN- andaroma

rison of the deduced amino acid sequences of Cajanus cajan metallothionein (CcMmetallothionein (CcMT1) with GmMT1 (Glycine max Acc. No. BAD18376); PsMT1); OsMT1 (Oryza sativa Acc. No. ABA99658). Identical and conserved amino acidsctin gene.

tical analysis

lues, standard errors and t-test were computed withre-loaded software in excel, programmed for statistical(www. Physics.csbsju.edu/stats/t-test.html).

terization of Cajanus cajan metallothionein 1 gene

clone of 228bp sequences (Acc No. GU444043), codingeptide of 75 amino acids containing a cysteine-richN- and C-terminal regions, has been isolated from they of pigeonpea. Based on sequence analysis of the cDNAs been designated as Cajanus cajan metallothioneinMT1). The deduced amino acid sequence of CcMT1e presence of six Cys-Xaa-Cys motifs (with Xaa repre-erent amino acids) which are equally distributed in therminal domains. Also, a spacer region containing 40ino acids was observed between N- and C-terminal

ith type 1 metallothioneins of different plant species. Alignment ofsativum Acc. No. BAD18382) AtMT1A (Arabidopsis thaliana Acc.

own in dark and grey colours, respectively.

134 K. Sekhar et al. / Environmental and Experimental Botany 72 (2011) 131139

Fig. 2. Transcript proles of CcMT1 in pigeonpea plants subjected to metal stress.RNA-gel blot analysis of total RNA isolated from four-week old pigeonpea plantssubjected to 200M of CuSO4/CdSO4/ZnSO4 for 24h. About 20g of total RNA wasused for northern blot analysis. C represents unstressed pigeonpea plants. The blotwas hybridized with the cDNA fragment of CcMT1. Ethidium bromide stained rRNAis shown for equal amount of RNA loading.

domains. The BLAST search of CcMT1 revealed 100% similaritywith that of GmMT1 of Glycine max, >70% similarity with PsMT1of Pisum sativum, >50% similarity with AtMT1A of A. thaliana and>41% similaritywithOsMT1 ofOryza sativa type 1metallothioneins(Fig. 1). Increased levels of CcMT1 transcripts were observed inmetal-stressed pigeonpea plants compared to the unstressedplants (Fig. 2).

3.2. Metal tolerance and ion accumulation in E. coli

To test the ability of CcMT1 in metal tolerance, E. coli cells har-bouring pTYB12-CcMT1/pTYB12 alone (control) were subjected to1.5mM CuSO4/0.1mM CdSO4/1.0mM ZnSO4 stress. Under normalconditions, the growth pattern of cells containing pTYB12-CcMT1was similar to that of control (Fig. 3a). However, under metal

stress, E. coli cells expressing CcMT1 showedhigher levels of copperand cadmium tolerance as compared to the control cells (Fig. 3a).The copper ion accumulation in E. coli cells expressing CcMT1was 3-fold higher (44034.0g/g DW) than that of controlcells (15017.0g/gDW). Cells harbouringpTYB12-CcMT1 accu-mulated 25031.8g/g DW of cadmium, which was >2.5-foldhigher than thatof control (9517.6g/gDW) (Fig. 3b).Whereas,under zinc stress, nonoteworthydifferenceswereobserved for zinctolerance and Zn ion accumulation between E. coli cells expressingCcMT1 and the control cells (Fig. 3b).

3.3. Development of stable CcMT1 transgenics in Arabidopsis

TransformationofA. thalianawas carried outwithpBI121 vectoralone (control) and pBI121 carrying CcMT1 gene (Fig. 4a). Trans-formed seedlings were selected on MS medium supplementedwith kanamycin (50mg/l). PCR analysis using the genomic DNAof T1 and T2 transgenic plants, employing CcMT1 primers, revealed>200bp amplied fragment, while no such band was observed inthe control plants (Fig. S1a). Transformed lines of CcMT1 and con-trol plants disclosed GUS expression as evidenced by intense bluecolour (Fig. S1b). Southern blot analysis of six independent trans-genic lines (T1), probedwith the CcMT1 coding sequence, exhibiteddifferent hybridizable bands ranging from >4 to 9.0Kb (Fig. 4b).Northern blot analysis of transgenic lines (T3), containing singlecopy of the CcMT1, revealed varied levels of transgene expression(Fig. 4c). Transgenic lines (Mth3 and Mth6) with higher levels ofCcMT1 transcripts were chosen for stress tolerance studies.

Fig. 3. Metal ton medium co101, 102 andZnSO4. The plathe presence odifferences inolerance and accumulation of E. coli expressing CcMT1 subjected to metal stress. (a) Gntaining CuSO4/CdSO4/ZnSO4. Cultures (pTYB12 and pTYB12-CcMT1 expressing E. coli103) were made with fresh LB medium, and 5l from each dilution was spotted on LB stes were incubated at 37 C for 12h and were photographed. (b) Accumulation of Cu, Cdf 1mM CuSO4/CdSO4/ZnSO4 for 3h at 37 C. Data represents mean S.E. of three indepecomparison with the control at P

K. Sekhar et al. / Environmental and Experimental Botany 72 (2011) 131139 135

Fig. 4. Structure of the T-DNA region of pBI121 containing CcMT1 expression unitand molecular analysis of CcMT1-transgenic Arabidopsis plants. (a) Restriction mapof the CcMT1 expression cassette used for Arabidopsis transformation. Nos pro: Nospromoter; Nos: Nos terminator; RB (right) and LB (left) borders of T-DNA. (b) South-ern blot and (c) Northern blot analysis of CcMT-transgenics of Arabidopsis plants.Each lane was loaded with 10g of genomic DNA/RNA. C represents vector trans-formed Arabidopsis and Mth1 to Mth6 represent six independent CcMT1-transgeniclines. The blots were hybridized with the cDNA fragment of CcMT1. Ethidium bro-mide stained rRNA is shown for equal amount of RNA loading.

3.4. Functional validation of CcMT1 transgenic lines for stresstolerance

To evaluate the stress tolerance of CcMT1 transgenics, seedsobtained from transgenics and control plants were germinated on0.5 MS medium with 0, 50 and 100M CuSO4 and CdSO4; and0, 200 and 300M ZnSO4, for 23 d. Under unstressed conditions,both shoot and root growth of control and transgenic plants werefound similar. However, transgenic seedlings cultured undermetalstress (CuSO4/CdSO4) disclosed increased tolerance towards heavymetals when compared to the control seedlings (Fig. 5ad). Boththe transgenic lines, when subjected to copper stress, showed sig-nicant increases in the total biomass of 180% (50M CuSO4)and 300% (100M CuSO4) compared with the control seedlingsunder identical stress conditions (Fig. 6a). Similarly, transgeniclines exhibited increased total biomass of 230% (50M CdSO4)and 220% (100M CdSO4) treatments as compared to the con-trol seedlings (Fig. 6a). Whereas, no noticeable differences wereobserved between controls and transgenic plants subjected to Znstress (data not shown).

Transgenic lines disclosed substantial increases in the rootgrowth by 120% (50M CuSO4) and by 75% (100M CuSO4) ascompared to the control seedlings (Fig. 6b). Transgenics also dis-closed signicant increases in the root growth by 80% (50MCdSO4) and by 60% (100M CdSO4) compared to the controlseedlings (Fig. 6b).

Leaf disks of four-week old Mth3 and Mth6 transgenic linesrevealed higher chlorophyll content of 70% (50M CuSO4) and72% (100M CuSO4) in comparison with the control plants(Fig. 6c). Similarly, transgenicsdisclosedhigher chlorophyll content

Fig. 5. Evaluation of CcMT1-transgenics of Arabidopsis for Cu and Cd tolerance. (a and b). Control and t50M and 100M CuSO4, respectively, for 23 d. (c and d) Control and transgenics seedlings grown orespectively, for 23 d. C represents control; Mth3 and Mth6 represent CcMT1-Arabidopsis transgenic lineransgenic seedlings grown on 0.5 MS medium supplemented withn 0.5 MS medium supplemented with 50M and 100M CdSO4,s.

136 K. Sekhar et al. / Environmental and Experimental Botany 72 (2011) 131139

Fig. 6. Effectsof control andCcMT1-transgeData represen(b) For root lenwithout or wit20 seedlings frdeterminedonin 0, 50, 100Data representrol; Mth3 andDW representdifferences in

of 72% (50the control

3.5. Estimacontrol plan

Accumuhigher in roever, shootaccumulatitransgenicaccumulatiion accumucompared t(46% andlines compof CuSO4 and CdSO4 on biomass, root length and chlorophyll contentCcMT-transgenics of Arabidopsis. (a) For total biomass, control andnics were grown on MS with 0/50/100M of CuSO4/CdSO4 for 23 d.ts mean S.E. of 20 seedlings from three independent experiments.gth, 4 d old control and transgenic seedlings were transferred to MSh 50/100Mof CuSO4/CdSO4 for 14 d. Data represents mean S.E. ofom three independent experiments. (c) Total chlorophyll contentwas4weekold control and transgenic leaf discs (20each) after incubationM of CuSO4/CdSO4 solutions at room (282 C) temperature for 96h.ts mean S.E. of three independent experiments. C represents con-Mth6 represent CcMT1-transgenics, WS represents without stress,

s dry weight and FW represents fresh weight. *Indicates signicantcomparison with the control at P

K. Sekhar et al. / Environmental and Experimental Botany 72 (2011) 131139 137

Table 1Distribution of Cu and Cd ions in transgenic and control plants under metal stress conditions.

Control and transgenicplants

Shoot Root Shoot-to-Root ratioof metal ions

g/g D

Control 6.5Mth3 30.3Mth6 21.2

Values represe

plants showconditions.

CcMT1-tshowed red(0.660.68)in control p

3.6. Express

CcMT1-tplants, subunstressedsion levelstransporterdifferencesand roots ofunstressed

4. Discussi

Metallotmetal-bindof diverse lisequenceshave distindiffer fromand Goldsbfound to plaet al., 1989reports areysis of planterms of the

In our eaferent abiotlibrary of pet al., 2010mum similametallothio(Fig. 1), sugevolved froysis of pigehybridizablCu, Cd andin the unstionein is inmetallothioabiotic streandheavy (gene of wilseveredroubidopsis plaand AtMT2 gand Taiz, 19expressionby Zn, Cu, a

thebserivergrse

condr-exandons iesultred tsingppeThreuponandCxprein (Hu andthate deectoincreparered tot syigherac).ce at obsrole

y. Eargainsgingss, AontesionsulteTheoMT) iet alHMAot to010)3) ins (NiopsisCd a-typCu ion (g/g DW) Cd ion (g/g DW) Cu ion (

84.0 6.2 699 39.1 120.6 123.6 10.6 871 52.9 207.4 111.4 17.0 910 68.4 211.6

nt mean S.E.

ed lower levels of Cd ion accumulations under normal

ransgenics subjected to copper and cadmium stressesuced shoot to root ratios for Cu (0.520.59) and Cdions compared to 0.69 for Cu and 0.98 for Cd observedlants under stressed conditions (Table 1).

ion proles of Cu and Cd transporters of Arabidopsis

ransgenic lines and transformed control (vector alone)jected to 50M CuSO4 and CdSO4 stresses andconditions, were analyzed by qRT-PCR for the expres-of AtZIP2 (NM 125344) and NRAMP1 (AY128347)

s for copper and cadmium, respectively. Noworthwhilewere observed in the transcript levels between shootstransgenics and control plants both under stressed andconditions (data not shown).

on

hioneins (MTs) are low-molecular-weight, Cys-riching proteins which are involved in the metal toleranceving organisms. However, unlike the highly conservedof mammalian MTs (Klaassen et al., 1999), plant MTsctive arrangement(s) of Cys residues, and as such theyeach other in their base sequence and function (Cobbettrough, 2002). In yeast and animal systems, MTs werey key role in the metal homeostatic mechanism (Ecker; Borrelly et al., 2002; Coyle et al., 2002). Till date, noavailable on the characterization and functional anal-t type-1 metallothioneins in heterologous systems inir mechanism of action for metal detoxication.rlier study, an array of cDNA clones associatedwith dif-ic stress tolerances have been isolated from the cDNAigeonpea plants subjected to drought stress (Priyanka). The amino acid sequence of CcMT1 disclosed maxi-rity with that of previously characterized type-1 plantneins of G. max, P. sativum, A. thaliana and O. sativagesting that these genes have plausibly originated andm a common ancestral gene source. Northern blot anal-onpea plants, using CcMT1 as a probe, revealed intensee signals owing to enhanced transcript levels underZn stresses compared to the weak signals observed

ressed plants, implying that the pigeonpea metalloth-duced by different heavy metals (Fig. 2). In rice, anein-like rgMT gene was highly induced by differentsses caused by salts (NaCl and NaHCO3), drought (PEG)Cu, Cd andZn)metals (Jin et al., 2006). The type-2CLMT2

ent forthese ohave dthe coustress

OvecoppermulatiE. coli rcompaexpresboth co2006).and 3,Cd, ZnOver-e2 proteboth Cstratefrom th

Theveyedas comcompafuse roand h(Fig. 6tolerancontentectivetoxicitcells ascavenCu strephyll cExpresfaba re2004).gene ((Wangand Atthe roet al., 2(FeMTCd ionArabidtive toof wildd watermelon was induced by intense light as well asght conditions (Akashi et al., 2004). Similarly,whenAra-nts were subjected to Cu and Cd stresses, both AtMT1enes disclosed increased levels of expression (Murphy95). In mangrove species (Bruguiera gymnorrhiza), theprole of type 2metallothionein (BgMT2)was regulatednd Pb, yet the regulation pattern proved to be differ-

invalutus, wcopper) yeametal ionsof PiMT1 incopper streB. juncea Bjmium tolerW) Cd ion (g/g DW) Cu Cd

708 53.1 0.69 0.981275.8 33.3 0.59 0.681363.4 52.1 0.52 0.66

three metals (Huang and Wang, 2009). An overview ofvations indicates that different plant metallothioneinsed into various forms from a common source duringof evolution to cope with the adverse effects of diverseitions encountered by the plants.pression of CcMT1 gene not only imparted enhancedcadmium tolerance but also increased metal ion accu-n E. coli cells (Fig. 3a and b). Expression of the PsMtA ined in8-fold increase in the copper accumulationwheno the control cells (Evans et al., 1992). Similarly, E. coliBjMT2 of Brassica juncea exhibited higher tolerances tor and cadmium than that of control cells (Zhigang et al.,e different types of Prosopis juliora MTs, viz., PjMT 1, 2expression in E. coli, revealed differential binding withuand sequestrationof themetal ions (Usha et al., 2009).ssion of recombinantHevea brasiliensismetallothioneinbMT2) in E. coli cells resulted in increased tolerance toZn ions (Zhu et al., 2010). These results amply demon-

the pigeonpea metallothionein protects bacterial cellsleterious effects of heavy metals.pic expression of CcMT1 gene in Arabidopsis plants con-ased tolerance to both copper and cadmium stressesd to the control plants (Fig. 5). CcMT1-transgenic lines,o control, could produce healthy seedlings with pro-stem, enhanced plant biomass, increased root growthchlorophyll content under metal stress conditionsConversely, these transgenics could not provide anygainst the stress exerted by Zn. The higher chlorophyllerved in the transgenic plants is attributable to the pro-of CcMT1 at cellular level from heavy metal-inducedlier, itwas reported that plantmetallothioneins protectt the oxidative stress through their hydroxyl radical-activity (Akashi et al., 2004; Wong et al., 2004). Underrabidopsis expressing BjMT2 exhibited higher chloro-nt than that of wild-type plants (Zhigang et al., 2006).of AtMT2a and AtMT3 genes in the guard cells of Viciad in enhanced tolerance against cadmium (Lee et al.,ver-expressionof Puccinellia tenuiorametallothioneinn tobacco plants conferred enhanced resistance to Cd., 2009). In tobacco, the combined expression of AtMT2b4 signicantly increased Cd tolerance and enhancedshoot translocation of both Cd and Zn ions (Grispen. The expression of type3metallothionein of buckwheatE. coli and yeast conveyed increased tolerance to Cu andkolic et al., 2010). Three MT1 knockdown mutants of, viz., MT1a, MT1b and MT1c, were found hypersensi-nd accumulated lower levels of metal ions than thate plants (Zimeri et al., 2005). PiMT1 gene of Paxillus

hen expressed in the hypersensitive (for cadmium andst mutant, bestowed increased tolerance to both the(Bellion et al., 2007). Also, the constitutive expressionHebeloma cyclindrosporum showed higher tolerance toss (Bellion et al., 2007). Although over-expression ofMT2 in Arabidopsis caused increased copper and cad-ance, but it was associated with reduced root growth

138 K. Sekhar et al. / Environmental and Experimental Botany 72 (2011) 131139

under unstressed conditions (Zhigang et al., 2006).Whereas, trans-genic Arabidopsis lines expressing CcMT1 exhibited normal rootgrowth both under stressed and unstressed conditions, owing tothe absence of negative effects of Cajanus metallothionein 1 on theroot growth

Arabidop(CuSO4 andlation of m(Figs. 7 andions from sions in thereported eaWhen wildgrown in tof Cu accumpared to wof metals bviz., increashoot-to-ronal detoximetal tolertration and1999, 2004;tration/homconclusivellevels of Attransgenicsconditions,has no roleexpressionshoot-to-rostress, resuoverall resumaintenanchomeostasi

5. Conclus

In this igene (CcMTbeen validaand was inconditions.explicit meCd ions, impingly, the Cbestowing m

Acknowled

We exteBiotechnoloassistance.Osmania Unions of theof Departmgestions an

Appendix A

Supplemthe online v

References

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, V., Pt Arab266.m, F.Vke, R.idopsiM., Coctionebelom, G.P.M2002.reticu430n-Wong leaKim, Hmetalnding, C., 20n. Cu, C., Gy met, Philcein. Ce.J., Waioneinegr. P.J., Bulation.M., GaessionidopsisMTA, V.M.d expal tranhoot t10.101., BunallothscencJ., Mesof indrance.G.Y., Wangrore 77, 2002Bot. 5.M., Ligene fheng,ein oftic str.R., 1hionealloth, C.D.rotectH., Zhes of

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ion

nvestigation, the functional role of a metallothionein1), isolated from the cDNA library of pigeonpea, hasted. The CcMT1 belongs to the type 1 metallothioneinsduced in pigeonpea plants grown under metal-stressExpression of CcMT1 in E. coli and Arabidopsis affordedtal tolerance besides increased accumulations of Cu andlicating its crucial role in metal detoxication. Accord-cMT1 seems promising as a prime candidate gene foretal tolerance in crop plants.

gements

nd our thanks to the Andhra PradeshNetherlandsgy Programme, Hyderabad, for their generous nancialWe thank Mr. G. Raghu, Department of Biochemistry,iversity, for the help rendered in measuring the metal

samples. The authors are grateful to Prof. T. Papi Reddyent of Genetics, Osmania University, for his helpful sug-d for critical evaluation of the manuscript.

. Supplementary data

entary data associatedwith this article can be found, inersion, at doi:10.1016/j.envexpbot.2011.02.017.

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Metallothionein 1 (CcMT1) of pigeonpea (Cajanus cajan, L.) confers enhanced tolerance to copper and cadmium in Escherichia...1 Introduction2 Materials and methods2.1 Plants, materials and treatments2.2 Construction of subtractive cDNA library and isolation of CcMT1 from pigeonpea2.3 Southern and northern blot analyses2.4 Cloning of CcMT1 for expression in E. coli2.5 Metal tolerance assays in E. coli2.6 Estimation of metal ions accumulations in E. coli2.7 Construction of plant expression vector and transformation of Arabidopsis plants2.8 Functional analysis of transgenics for metal-stress tolerance2.9 Measurement of metal content in Arabidopsis plants2.10 Quantitative real time PCR (qRT-PCR)2.11 Statistical analysis

3 Results3.1 Characterization of Cajanus cajan metallothionein 1 gene3.2 Metal tolerance and ion accumulation in E. coli3.3 Development of stable CcMT1 transgenics in Arabidopsis3.4 Functional validation of CcMT1 transgenic lines for stress tolerance3.5 Estimation of Cu and Cd ion accumulations in transgenic and control plants3.6 Expression profiles of Cu and Cd transporters of Arabidopsis

4 Discussion5 ConclusionAcknowledgementsAppendix A Supplementary dataAppendix A Supplementary data