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Metallothioneins BcMT1 and BcMT2 from Brassica campestris enhance tolerance to cadmium and copper and decrease production of reactive oxygen species in Arabidopsis thaliana

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Abstract

Aims

Metallothioneins are cysteine-rich, metal-binding proteins, but their exact functions are not fully understood. In this study, we isolated two metallothionein genes, BcMT1 and BcMT2 from Brassica campestris to increase our understanding of metal tolerance mechanisms in Brassica plants.

Methods

Semi-quantitative RT-PCR was used to analyze expression of the two BcMTs genes. BcMT1 and BcMT2 were ectopically expressed in Arabidopsis thaliana. Quantitative real-time RT-PCR and GUS-staining method were used to select transgenic Arabidopsis plants. Cd and Cu concentrations were analyzed by flame atomic absorption spectrometry. Histochemical detection of H2O2 and O2 •− were conducted by 3,3-diaminobenzidine and nitroblue tetrazoliu-staining methods.

Results

BcMT1 is expressed predominantly in roots, whereas BcMT2 is expressed mainly in leaves of B. campestris. Expression of BcMT1 was induced by both Cd and Cu, but expression of BcMT2 was enhanced only by Cd. Ectopic expression of BcMT1 and BcMT2 in Arabidopsis thaliana enhanced the tolerance to Cd and Cu and increased the Cu concentration in the shoots of the transgenic plants. Transgenic Arabidopsis accumulated less reactive oxygen species (ROS) than wild-type plants.

Conclusions

BcMT1 and BcMT2 increased Cd and Cu tolerance in transgenic Arabidopsis, and decreased production of Cd- and Cu-induced ROS, thereby protecting plants from oxidative damage.

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References

  • Ahn YO, Kim SH, Lee J, Kim HR, Lee HS, Kwak SS (2012) Three Brassica rapa metallothionein genes are differentially regulated under various stress conditions. Mol Biol Rep 39:2059–2067

    Article  PubMed  CAS  Google Scholar 

  • An ZG, Li CJ, Zu YG, Du YJ, Wachter A, Gromes R, Rausch T (2006) Expression of BjMT2, a metallothionein 2 from Brassica juncea, increases copper and cadmium tolerance in Escherichia coli and Arabidopsis thaliana, but inhibits root elongation in Arabidopsis thaliana seedlings. J Exp Bot 57:3575–3582

    Article  CAS  Google Scholar 

  • Bellion M, Courbot M, Jacob C, Guinet F, Blaudez D, Chalot M (2007) Metal induction of a Paxillus involutus metallothionein and its heterologous expression in Hebeloma cylindrosporum. New Phytol 174:151–158

    Article  PubMed  CAS  Google Scholar 

  • Chae HZ, Uhm TB, Rhee SG (1994) Dimerization of thiol-specific antioxidant and the essential role of cysteine 47. Proc Natl Acad Sci USA 91:7022–7026

    Article  PubMed  CAS  Google Scholar 

  • Choi D, Kim HM, Yun HK, Park JA, Kim WT, Bok SH (1996) Molecular cloning of a metallothionein-like gene from Nicotiana glutinosa L. and its induction by wounding and tobacco mosaic virus infection. Plant Physiol 112:353–359

    Article  PubMed  CAS  Google Scholar 

  • Chyan CL, Lee TTT, Liu CP, Yang YC, Tzen JTC, Chou WM (2005) Cloning and expression of a seed-specific metallothionein-like protein from sesame. Biosci Biotechnol Biochem 69:2319–2325

    Article  PubMed  CAS  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  PubMed  CAS  Google Scholar 

  • Cobbett C, Goldsbrough P (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu Rev Plant Biol 53:159–182

    Article  PubMed  CAS  Google Scholar 

  • Dixit V, Pandey V, Shyam R (2001) Differential antioxidative responses to cadmium in roots and leaves of pea (Pisum sativum L. cv. Azad). J Exp Bot 52:1101–1109

    Article  PubMed  CAS  Google Scholar 

  • Domènech J, Mir G, Huguet G, Capdevila M, Molinas M, Atrian S (2006) Plant metallothionein domains: functional insight into physiological metal binding and protein folding. Biochimie 88:583–593

    Article  PubMed  Google Scholar 

  • Domènech J, Tinti A, Capdevila M, Atrian S, Torreggiani A (2007) Structural study of the zinc and cadmium complexes of a type 2 plant (Quercus suber) metallothionein: insights by vibrational spectroscopy. Biopolymers 86:240–248

    Article  PubMed  Google Scholar 

  • Ekmekçi Y, Tanyolaç D, Ayhan B (2008) Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars. J Plant Physiol 165:600–611

    Article  PubMed  Google Scholar 

  • Foley RC, Liang ZM, Singh KB (1997) Analysis of type 1 metallothionein cDNAs in Vicia faba. Plant Mol Biol 33:583–591

    Article  PubMed  CAS  Google Scholar 

  • Gisbert C, Clemente R, Navarro-Aviñó J, Baixauli C, Ginér A, Serrano R, Walker DJ, Bernal MP (2006) Tolerance and accumulation of heavy metals by Brassicaceae species grown in contaminated soils from Mediterranean regions of Spain. Environ Exp Bot 56:19–27

    Article  CAS  Google Scholar 

  • Guo WJ, Bundithya W, Goldsbrough PB (2003) Characterization of the Arabidopsis metallothioneins gene family: tissue-specific contributions and induction during senescence and in response to copper. New Phytol 159:369–381

    Article  CAS  Google Scholar 

  • Guo WJ, Meetam M, Goldsbrough PB (2008) Examining the specific contributions of individual Arabidopsis metallothioneins to copper distribution and metal tolerance. Plant Physiol 146:1697–1706

    Article  PubMed  CAS  Google Scholar 

  • Hall JL (2002) Cellular mechanisms for heavy metal detoxification and tolerance. J Exp Bot 53:1–11

    Article  PubMed  CAS  Google Scholar 

  • Hassinen VH, Tuomainen M, Peräniemi S, Schat H, Kärenlampi SO, Tervahauta AI (2009) Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens. J Exp Bot 60:187–196

    Article  PubMed  CAS  Google Scholar 

  • Hassinen VH, Tervahauta AI, Schat H, Kärenlampi SO (2011) Plant metallothioneins - metal chelators with ROS scavenging activity? Plant Biol 13:225–232

    Article  PubMed  CAS  Google Scholar 

  • Hsieh HM, Liu WK, Huang PC (1995) A novel stress-inducible metallothionein-like gene from rice. Plant Mol Biol 28:381–389

    Article  PubMed  CAS  Google Scholar 

  • Hsieh HM, Liu WK, Chang A, Huang PC (1996) RNA expression patterns of a type 2 metallothionein-like from rice. Plant Mol Biol 32:525–529

    Article  PubMed  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Kawashima I, Kennedy TD, Chino M, Lane BG (1992) Wheat Ec metallothionein genes: like mammalian Zn2+ metallothionein genes, wheat Zn2+ metallothionein genes are conspicuously expressed during embryogenesis. Eur J Biochem 209:971–976

    Article  PubMed  CAS  Google Scholar 

  • Kim SH, Lee HS, Song WY, Choi KS, Hur YK (2007) Chloroplast-targeted BrMT1 (Brassica rapa Type-1 Metallothionein) enhances resistance to cadmium and ROS in transgenic Arabidopsis plants. J Plant Biol 50:1–7

    Article  Google Scholar 

  • Kim YO, Patel DH, Lee DS, Song Y, Bae HJ (2011) High cadmium-binding ability of a novel Colocasia esculenta metallothionein increases cadmium tolerance in Escherichia coli and tobacco. Biosci Biotech Bioch 75:1912–1920

    Article  CAS  Google Scholar 

  • Kumar PBAN, Dushenkov V, Motto H, Raskin I (1995) Phytoextraction: the use of plants to remove heavy metals from soils. Environ Sci Technol 29:1232–1238

    Article  PubMed  CAS  Google Scholar 

  • Ledger SE, Gardner RC (1994) Cloning and characterization of five cDNAs for genes differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var. deliciosa). Plant Mol Biol 25:877–886

    Article  PubMed  CAS  Google Scholar 

  • Lee J, Shim D, Song WY, Hwang I, Lee Y (2004) Arabidopsis metallothioneins 2a and 3 enhance resistance to cadmium when expressed in Vicia faba guard cells. Plant Mol Biol 54:805–815

    Article  PubMed  CAS  Google Scholar 

  • Lombardi L, Sebastiani L (2005) Copper toxicity in Prunus cerasifera: growth and antioxidant enzymes responses of in vitro grown plants. Plant Sci 168:797–802

    Article  CAS  Google Scholar 

  • Maret W (1994) Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange. Proc Natl Acad Sci USA 91:237–241

    Article  PubMed  CAS  Google Scholar 

  • Marschner H (1995) Mineral nutrition of higher plants, 2nd edn. Academic Press, London

    Google Scholar 

  • McLaughlin MJ, Maier NA, Correll RL, Smart MK, Sparrow LA, McKay A (1999) Prediction of cadmium concentrations in potato tubers (Solanum tuberosum L.) by pre-plant soil and irrigation water analyses. Aust J Soil Res 37:191–207

    Article  CAS  Google Scholar 

  • Metwally A, Safronova VI, Belimov AA, Dietz KJ (2005) Genotypic variation of the response to cadmium toxicity in Pisum sativum L. J Exp Bot 56:167–178

    PubMed  CAS  Google Scholar 

  • Mir G, Domènech J, Huguet G, Guo WJ, Goldsbrough P, Atrian S, Molinas M (2004) A plant type 2 metallothionein (MT) from cork tissue responds to oxidative stress. J Exp Bot 55:2483–2493

    Article  PubMed  CAS  Google Scholar 

  • Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410

    Article  PubMed  CAS  Google Scholar 

  • Morelli E, Scarano G (2004) Copper-induced changes of non-protein thiols and antioxidant enzymes in the marine microalga Phaeodactylum tricornutum. Plant Sci 167:289–296

    Article  CAS  Google Scholar 

  • Murphy A, Taiz L (1995) Comparison of metallothionein gene expression and nonprotein thiols in ten Arabidopsis ecotypes. Plant Physiol 109:945–954

    Article  PubMed  CAS  Google Scholar 

  • Murphy A, Zhou JM, Goldsbrough PB, Taiz L (1997) Purification and immunological identification of metallothionein 1 and 2 from Arobidopsis thaliana. Plant Physiol 113:1293–1301

    Article  PubMed  CAS  Google Scholar 

  • Nishiuchi S, Liu S, Takano T (2007) Isolation and characterization of a metallothionein-1 protein in Chloris virgata Swartz that enhances stress tolerances to oxidative, salinity and carbonate stress in Saccharomyces cerevisiae. Biotechnol Lett 29:1301–1305

    Article  PubMed  CAS  Google Scholar 

  • Obertello M, Wall L, Laplaze L, Nicole M, Auguy F, Gherbi H, Bogusz D, Franche C (2007) Functional analysis of the metallothionein gene cgMT1 isolated from the actinorhizal tree Casuarina glauca. Mol Plant Microbe In 20:1231–1240

    Article  CAS  Google Scholar 

  • Pan AH, Yang MZ, Tie F, Li LY, Chen ZL, Ru BG (1994) Expression of mouse metallothionein-I gene confers cadmium resistance in transgenic tobacco plants. Plant Mol Biol 24:341–351

    Article  PubMed  CAS  Google Scholar 

  • Ren YJ, Zhao J (2009) Functional analysis of the rice metallothionein gene OsMT2b promoter in transgenic Arabidopsis plants and rice germinated embryos. Plant Sci 176:528–538

    Article  CAS  Google Scholar 

  • Robinson NJ, Tommey AM, Kuske C, Jackson PJ (1993) Plant metallothioneins. Biochem J 295:1–10

    PubMed  CAS  Google Scholar 

  • Romero-Puertas MC, Rodríguez-Serrano M, Corpas FJ, Gómez M, Delrío LA, Sandalio LM (2004) Cadmium-induced subcellular accumulation of O2 .- and H2O2 in pea leaves. Plant Cell Environ 27:1122–1134

    Article  CAS  Google Scholar 

  • Roosens NH, Leplae R, Bernard C, Verbruggen N (2005) Variations in plant metallothioneins: the heavy metal hyperaccumulator Thlaspi caerulescens as a study case. Planta 222:716–729

    Article  PubMed  CAS  Google Scholar 

  • Salt DE, Prince RC, Pickering IJ, Raskin I (1995) Mechanisms of cadmium mobility and accumulation in Indian mustard. Plant Physiol 109:1427–1433

    PubMed  CAS  Google Scholar 

  • Samardžić JT, Nikolić DB, Timotijević GS, Jovanović ŽS, Milisavljević MĐ, Maksimović VR (2010) Tissure expression analysis of FeMT3, a drought and oxidative stress related metallothionein gene from buckwheat (Fagopyrum esculentum). J Plant Physiol 167:1407–1411

    Article  PubMed  Google Scholar 

  • Schützendübel A, Polle A (2002) Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. J Exp Bot 53:1351–1365

    Article  PubMed  Google Scholar 

  • Sekhar K, Priyanka B, Reddy VD, Rao KV (2011) Metallothionein 1 (CcMT1) of pigeonpea (Cajanus cajan, L.) confers enhanced tolerance to copper and cadmium in Escherichia coli and Arabidopsis thaliana. Environ Exp Bot 72:131–139

    Article  CAS  Google Scholar 

  • Semane B, Cuypers A, Smeets K, Belleghem FV, Horemans N, Schat H, Vangronsveld J (2007) Cadmium responses in Arabidopsis thaliana: glutathione metabolism and antioxidative defence system. Physiol Plant 129:519–528

    Article  CAS  Google Scholar 

  • Sereno ML, Almeida RS, Nishimura DS, Figueira A (2007) Response of sugarcane to increasing concentrations of copper and cadmium and expression of metallothionein genes. J Plant Physiol 164:1499–1515

    Article  PubMed  CAS  Google Scholar 

  • Sharma RK, Agrawal M, Marshall FM (2008) Heavy metal (Cu, Zn, Cd and Pb) concentration of vegetables in urban India: a case study in Varanasi. Environ Pollut 154:254–263

    Article  PubMed  CAS  Google Scholar 

  • Smeets K, Ruytinx J, Semane B, Belleghem FV, Remans T, Sanden SV, Vangronsveld J, Cuypers A (2008) Cadmium-induced transcriptional and enzymatic alterations related to oxidative stress. Environ Exp Bot 63:1–8

    Article  CAS  Google Scholar 

  • Tewari RK, Kumar P, Sharma PN (2006) Antioxidant responses to enhanced generation of superoxide anion radical and hydrogen peroxide in the copper-stressed mulberry plants. Planta 223:1145–1153

    Article  PubMed  CAS  Google Scholar 

  • Turchi A, Tamantini I, Camussi AM, Racchi ML (2012) Expression of a metallothionein A1 gene Pisum sativum in white poplar enhances tolerance and accumulation of zinc and copper. Plant Sci 183:50–56

    Article  PubMed  CAS  Google Scholar 

  • Uraguchi S, Watanabe I, Yoshitomi A, Kiyono M, Kuno K (2006) Characteristics of cadmium accumulation and tolerance in novel Cd-accumulating crops, Avena strigosa and Crotalaria juncea. J Exp Bot 57:2955–2965

    Article  PubMed  CAS  Google Scholar 

  • van Hoof NALM, Hassinen VH, Hakvoort HWJ, Ballintijn KF, Schat H, Verkleij JAC, Ernst WHO, Karenlampi SO, Tervahauta AI (2001) Enhanced copper tolerance in Silene vulgaris (Moench) Garcke populations from copper mines is associated with increased transcript levels of a 2b-type metallothionein gene. Plant Physiol 126:1519–1526

    Article  PubMed  Google Scholar 

  • Wong HL, Sakamoto T, Kawasaki T, Umemura K, Shimamoto K (2004) Down-regulation of metallothionein, a reactive oxygen scavenger, by the small GTPase OsRac1 in rice. Plant Physiol 135:1447–1456

    Article  PubMed  CAS  Google Scholar 

  • Wu FB, Zhang GP, Dominy P (2003) Four barley genotypes respond differently to cadmium: lipid peroxidation and activities of antioxidant capacity. Environ Exp Bot 50:67–78

    Article  CAS  Google Scholar 

  • **a Y, Lv YY, Yuan YX, Wang GP, Chen YH, Zhang HS, Shen ZG (2012a) Cloning and characterization of a type 1 metallothionein gene from the copper-tolerant plant Elsholtzia haichowensis. Acta Physiol Plant 34:1819–1826

    Google Scholar 

  • **a Y, Qi Y, Yuan YX, Wang GP, Cui J, Chen YH, Zhang HS, Shen ZG (2012b) Overexpression of Elsholtzia haichowensis metallothionein 1 (EhMT1) in tobacco plants enhances copper tolerance and accumulation in root cytoplasm and decreases hydrogen peroxide production. J Hazard Mater 233:65–71

    Google Scholar 

  • Xue TT, Li XZ, Zhu W, Wu C, Yang G, Zheng CC (2009) Cotton metallothionein GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast. J Exp Bot 60:339–349

    Article  PubMed  CAS  Google Scholar 

  • Yang Z, Wu YR, Li Y, Ling HQ, Chu CC (2009) OsMT1a, a type 1 metallothionein, plays the pivotal role in zinc homeostasis and drought tolerance in rice. Plant Mol Biol 70:219–229

    Article  PubMed  CAS  Google Scholar 

  • Zhang HX, **a Y, Wang GP, Shen ZG (2008) Excess copper induces accumulation of hydrogen peroxide and increases lipid peroxidation and total activity of copper-zinc superoxide dismutase in roots of Elsholtzia haichowensis. Planta 227:465–475

    Article  PubMed  CAS  Google Scholar 

  • Zhang HX, Zhang FQ, **a Y, Wang GP, Shen ZG (2010) Excess copper induces production of hydrogen peroxide in the leaf of Elsholtzia haichowensis through apoplastic and symplastic CuZn-superoxide dismutase. J Hazard Mater 178:834–843

    Article  PubMed  CAS  Google Scholar 

  • Zhou JM, Goldsbrough PB (1994) Functional homologs of fungal metallothionein genes from Arabidopsis. Plant Cell 6:875–884

    PubMed  CAS  Google Scholar 

  • Zhou JM, Goldsbrough PB (1995) Structure, organization and expression of the metallothionein gene family in Arabidopsis. Mol Gen Genet 248:318–328

    Article  CAS  Google Scholar 

  • Zhou GK, Xu YF, Liu JY (2005) Characterization of a rice class II metallothionein gene: tissue expression patterns and induction in response to abiotic factors. J Plant Physiol 162:686–696

    Article  PubMed  CAS  Google Scholar 

  • Zhu W, Zhao DX, Miao Q, Xue TT, Li XZ, Zheng CC (2009) Arabidopsis thaliana metallothionein, AtMT2a, mediates ROS balance during oxidative stress. J Plant Biol 52:585–592

    Article  CAS  Google Scholar 

  • Zimeri AM, Dhankher OP, McCaig B, Meagher RB (2005) The plant MT1 metallothioneins are stabilized by binding cadmium and are required for cadmium tolerance and accumulation. Plant Mol Biol 58:839–855

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was financially supported by the Fundamental Research Funds for the Central Universities (KYZ200905), Specialized Research Fund for the Doctoral Program of Higher Education (20070307010, 20090097120035) and the National Natural Science Foundation of China (30571107).

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Correspondence to Yan **a or Zhenguo Shen.

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Responsible Editor: Henk Schat.

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Lv, Y., Deng, X., Quan, L. et al. Metallothioneins BcMT1 and BcMT2 from Brassica campestris enhance tolerance to cadmium and copper and decrease production of reactive oxygen species in Arabidopsis thaliana . Plant Soil 367, 507–519 (2013). https://doi.org/10.1007/s11104-012-1486-y

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