, Volume 235, Issue 3, pp 523–537 | Cite as

Overexpression of Nelumbo nucifera metallothioneins 2a and 3 enhances seed germination vigor in Arabidopsis

  • Yuliang Zhou
  • Pu Chu
  • Huhui Chen
  • Yin Li
  • Jun Liu
  • Yu Ding
  • Edward W. T. Tsang
  • Liwen Jiang
  • Keqiang Wu
  • Shangzhi HuangEmail author
Original Article


Metallothioneins (MTs) are small, cysteine-rich and metal-binding proteins which are involved in metal homeostasis and scavenging of reactive oxygen species. Although plant MTs have been intensively studied, their roles in seeds remain to be clearly established. Here, we report the isolation and characterization of NnMT2a, NnMT2b and NnMT3 from sacred lotus (Nelumbo nucifera Gaertn.) and their roles in seed germination vigor. The transcripts of NnMT2a, NnMT2b and NnMT3 were highly expressed in developing and germinating sacred lotus seeds, and were dramatically up-regulated in response to high salinity, oxidative stresses and heavy metals. Analysis of transformed Arabidopsis protoplasts showed that NnMT2a-YFP and NnMT3-YFP were localized in cytoplasm and nucleoplasm. Transgenic Arabidopsis seeds overexpressing NnMT2a and NnMT3 displayed improved resistance to accelerated aging (AA) treatment, indicating their significant roles in seed germination vigor. These transgenic seeds also exhibited higher superoxide dismutase activity compared to wild-type seeds after AA treatment. In addition, we showed that NnMT2a and NnMT3 conferred improved germination ability to NaCl and methyl viologen on transgenic Arabidopsis seeds. Taken together, these data demonstrate that overexpression of NnMT2a and NnMT3 in Arabidopsis significantly enhances seed germination vigor after AA treatment and under abiotic stresses.


Arabidopsis Metallothionein Oxidative stresses Sacred lotus Salt stress Seed germination vigor 



Accelerated aging


Bio-array resource for plant biology




Days after pollination


Expressed sequence tags




Methyl viologen


Nitro blue tetrazolium


Superoxide dismutase


Yellow fluorescent protein



This study was supported by Natural Science Foundation of China (30370912), Natural Science Foundation of Guangdong Province (2006B20101010 and 9151027501000075) and Guangdong Agriculture Science and Technology Team Project to S. Huang and Guangdong Provincial Science and Technology Program (2010D020301003) to J Liu.

Supplementary material

425_2011_1527_MOESM1_ESM.pdf (645 kb)
Online Resources 1–7 (PDF 644 kb)


  1. Akashi K, Nishimura N, Ishida Y, Yokota A (2004) Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon. Biochem Biophys Res Commun 323:72–78PubMedGoogle Scholar
  2. Bailly C, Benamar A, Corbineau F, Come D (1996) Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated aging. Physiol Plantarum 97:104–110Google Scholar
  3. Bailly C, El-Maarouf-Bouteau H, Corbineau F (2008) From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology. C R Biol 331:806–814PubMedGoogle Scholar
  4. Bhalerao R, Keskitalo J, Sterky F et al (2003) Gene expression in autumn leaves. Plant Physiol 131:430–442PubMedGoogle Scholar
  5. Brkljacic JM, Samardzic JT, Timotijevic GS, Maksimovic VR (2004) Expression analysis of buckwheat (Fagopyrum esculentum Moench) metallothionein-like gene (MT3) under different stress and physiological conditions. J Plant Physiol 161:741–746PubMedGoogle Scholar
  6. Byrd HW, Delouche JC (1971) Deterioration of soybean seed in storage. Proc Assoc Offic Seed Analysts 61:41–57Google Scholar
  7. Clendennen SK, May GD (1997) Differential gene expression in ripening banana fruit. Plant Physiol 115:463–469PubMedGoogle Scholar
  8. Clerkx EJ, Vries HB, Ruys GJ, Groot SP, Koornneef M (2003) Characterization of green seed, an enhancer of abi3–1 in Arabidopsis that affects seed longevity. Plant Physiol 132:1077–1084PubMedGoogle Scholar
  9. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743PubMedGoogle Scholar
  10. Cobbett C, Goldsbrough P (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu Rev Plant Biol 53:159–182PubMedGoogle Scholar
  11. Coyle P, Philcox JC, Carey LC, Rofe AM (2002) Metallothionein: the multipurpose protein. Cell Mol Life Sci 59:627–647PubMedGoogle Scholar
  12. Debeaujon I, Leon-Kloosterziel KM, Koornneef M (2000) Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis. Plant Physiol 122:403–414PubMedGoogle Scholar
  13. Dickson MH (1980) Genetic aspects of seed quality. Hortic Sci 15:771–774Google Scholar
  14. Ding Y, Cheng H, Song S (2008) Changes in extreme high-temperature tolerance and activities of antioxidant enzymes of sacred lotus seeds. Sci China Ser C Life Sci 51:842–853Google Scholar
  15. Finkel T, Holbrook NJ (2000) Oxidants, oxidative stress and the biology of ageing. Nature 408:239–247PubMedGoogle Scholar
  16. Grennan AK (2011) Metallothioneins, a diverse protein family. Plant Physiol 155:1750–1751PubMedGoogle Scholar
  17. Guo WJ, Bundithya W, Goldsbrough PB (2003) Characterization of the Arabidopsis metallothionein gene family: tissue-specific expression and induction during senescence and in response to copper. New Phytol 159:369–381Google Scholar
  18. Guo WJ, Meetam M, Goldsbrough PB (2008) Examining the specific contributions of individual Arabidopsis metallothioneins to copper distribution and metal tolerance. Plant Physiol 146:1697–1706PubMedGoogle Scholar
  19. Holdsworth MJ, Bentsink L, Soppe W (2008) Molecular networks regulating Arabidopsis seed maturation, after-ripening, dormancy and germination. New Phytol 179:33–54PubMedGoogle Scholar
  20. Huang S, Tang X, Zhang L, Fu J (2003) Thermotolerance and activity of antioxidative enzymes in lotus seeds. J Plant Physiol Mol Biol 29:421–424 (in Chinese)Google Scholar
  21. Klaassen CD, Liu J, Choudhuri S (1999) Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu Rev Pharmacol Toxicol 39:267–294PubMedGoogle Scholar
  22. Koh M, Kim HJ (2001) The effects of metallothionein on the activity of enzymes involved in removal of reactive oxygen species. Bull Korean Chem Soc 22:362–366Google Scholar
  23. Kumar G, Knowles NR (1993) Changes in lipid peroxidation and lipolytic and free-radical scavenging enzyme activities during aging and sprouting of potato (Solanum tuberosum) seed-tubers. Plant Physiol 102:115–124PubMedGoogle Scholar
  24. Lee YP, Baek KH, Lee HS, Kwak SS, Bang JW, Kwon SY (2010) Tobacco seeds simultaneously over-expressing Cu/Zn-superoxide dismutase and ascorbate peroxidase display enhanced seed longevity and germination rates under stress conditions. J Exp Bot 61:2499–2506PubMedGoogle Scholar
  25. Les DH, Garvin DK, Wimpee CF (1991) Molecular evolutionary history of ancient aquatic angiosperms. Proc Natl Acad Sci USA 88:10119–10123PubMedGoogle Scholar
  26. McDonald MB (1999) Seed deterioration: physiology repair and assessment. Seed Sci Technol 27:177–238Google Scholar
  27. Miao Y, Jiang L (2007) Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells. Nat Protoc 2:2348–2353PubMedGoogle Scholar
  28. Mir G, Domenech 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 55:2483–2493PubMedGoogle Scholar
  29. Møller IM, Jensen PE, Hansson A (2007) Oxidative modifications to cellular components in plants. Annu Rev Plant Biol 58:459–481PubMedGoogle Scholar
  30. Oge L, Bourdais G, Bove J et al (2008) Protein repair l-isoaspartyl methyltransferase1 is involved in both seed longevity and germination vigor in Arabidopsis. Plant Cell 20:3022–3037PubMedGoogle Scholar
  31. Orr WC, Sohal RS (1994) Extension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogaster. Science 263:1128–1130PubMedGoogle Scholar
  32. Palmiter RD (1998) The elusive function of metallothioneins. Proc Natl Acad Sci USA 95:8428–8430PubMedGoogle Scholar
  33. Rajjou L, Debeaujon I (2008) Seed longevity: survival and maintenance of high germination ability of dry seeds. C R Biol 331:796–805PubMedGoogle Scholar
  34. Raymond AD, Gekonge B, Giri MS et al (2010) Increased metallothionein gene expression, zinc, and zinc-dependent resistance to apoptosis in circulating monocytes during HIV viremia. J Leukocyte Biol 88:589–596PubMedGoogle Scholar
  35. Robinson NJ, Tommey AM, Kuske C, Jackson PJ (1993) Plant metallothioneins. Biochem J 295:1–10PubMedGoogle Scholar
  36. Rosnoblet C, Aubry C, Leprince O, Vu BL, Rogniaux H, Buitink J (2007) The regulatory gamma subunit SNF4b of the sucrose non-fermenting-related kinase complex is involved in longevity and stachyose accumulation during maturation of Medicago truncatula seeds. Plant J 51:47–59PubMedGoogle Scholar
  37. Samardzic JT, Nikolic DB, Timotijevic GS, Jovanovic ZS, Milisavljevic MD, Maksimovic VR (2010) Tissue expression analysis of FeMT3, a drought and oxidative stress related metallothionein gene from buckwheat (Fagopyrum esculentum). J Plant Physiol 167:1407–1411PubMedGoogle Scholar
  38. Sattler SE, Gilliland LU, Magallanes-Lundback M, Pollard M, DellaPenna D (2004) Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination. Plant Cell 16:1419–1432PubMedGoogle Scholar
  39. Shen-Miller J, Mudgett MB, Schopf JW, Clarke S, Berger R (1995) Exceptional seed longevity and robust growth: ancient sacred lotus from China. Am J Bot 82:1367–1380Google Scholar
  40. Shen-Miller J, Schopf JW, Harbottle G, Cao R, Ouyang S, Zhou K, Southon JR, Liu G (2002) Long-living lotus: germination and soil γ-irradiation of centuries-old fruits, and cultivation, growth, and phenotypic abnormalities of offspring. Am J Bot 89:236–247PubMedGoogle Scholar
  41. Shin JH, Kim SR, An G (2009) Rice aldehyde dehydrogenase7 is needed for seed maturation and viability. Plant Physiol 149:905–915PubMedGoogle Scholar
  42. Suhy DA, Simon KD, Linzer D, Halloran TV O (1999) Metallothionein is part of a zinc-scavenging mechanism for cell survival under conditions of extreme zinc deprivation. J Biol Chem 274:9183–9192PubMedGoogle Scholar
  43. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedGoogle Scholar
  44. Tellmann G (2006) The E-method: a highly accurate technique for gene-expression analysis. Nat Methods 3:i–iiGoogle Scholar
  45. Thornalley PJ, Vasak M (1985) Possible role for metallothionein in protection against radiation-induced oxidative stress Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim Biophys Acta 827:36–44PubMedGoogle Scholar
  46. Tucker SL, Thornton CR, Tasker K, Jacob C, Giles G, Egan M, Talbot NJ (2004) A fungal metallothionein is required for pathogenicity of Magnaporthe grisea. Plant Cell 16:1575–1588PubMedGoogle Scholar
  47. Waterworth WM, Masnavi G, Bhardwaj RM, Jiang Q, Bray CM, West CE (2010) A higher plant DNA ligase is an important determinant of seed longevity. Plant J 63:848–860PubMedGoogle Scholar
  48. Wharton MJ (1955) The use of tetrazolium test for determining the viability of seeds of the genus Brassica. Proc Int Seed Test Assoc 20:81–88Google Scholar
  49. 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 135:1447–1456PubMedGoogle Scholar
  50. Wu Y, Wang Q, Ma Y, Chu C (2005) Isolation and expression analysis of salt up-regulated ESTs in upland rice using PCR-based subtractive suppression hybridization method. Plant Sci 168:847–853Google Scholar
  51. Xue T, Li X, Zhu W, Wu C, Yang G, Zheng C (2009) Cotton metallothionein GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast. J Exp Bot 60:339–349PubMedGoogle Scholar
  52. Yang X, Doser TA, Fang CX, Nunn JM, Janardhanan R, Zhu M, Sreejayan N, Quinn MT, Ren J (2006) Metallothionein prolongs survival and antagonizes senescence-associated cardiomyocyte diastolic dysfunction: role of oxidative stress. FASEB J 20:1024–1026PubMedGoogle Scholar
  53. Yang Z, Wu Y, Li Y, Ling HQ, Chu C (2009) OsMT1a, a type 1 metallothionein, plays the pivotal role in zinc homeostasis and drought tolerance in rice. Plant Mol Biol 70:219–229PubMedGoogle Scholar
  54. Yuan J, Chen D, Ren Y, Zhang X, Zhao J (2008) Characteristic and expression analysis of a metallothionein gene, OsMT2b, down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice. Plant Physiol 146:1637–1650PubMedGoogle Scholar
  55. Zhigang A, Cuijie L, Yuangang Z, Yejie D, 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 57:3575–3582PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Yuliang Zhou
    • 1
  • Pu Chu
    • 1
  • Huhui Chen
    • 1
  • Yin Li
    • 1
  • Jun Liu
    • 2
  • Yu Ding
    • 3
  • Edward W. T. Tsang
    • 4
  • Liwen Jiang
    • 3
  • Keqiang Wu
    • 5
  • Shangzhi Huang
    • 1
    Email author
  1. 1.Guangdong Provincial Key Laboratory of Plant Resource, School of Life SciencesSun Yat-Sen UniversityGuangzhouChina
  2. 2.Guangdong Academy of Agricultural SciencesGuangzhouChina
  3. 3.Centre for Cell and Developmental Biology, School of Life SciencesThe Chinese University of Hong KongHong KongChina
  4. 4.Plant Biotechnology InstituteNational Research Council of CanadaSaskatoonCanada
  5. 5.Institute of Plant Biology, College of Life ScienceNational Taiwan UniversityTaipei 106Taiwan

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