Journal of Plant Research

, Volume 125, Issue 1, pp 165–172 | Cite as

Protein dynamics during seed germination under copper stress in Arabidopsis over-expressing Potentilla superoxide dismutase

  • Tejpal Gill
  • Vivek Dogra
  • Sanjay Kumar
  • Paramvir Singh Ahuja
  • Yelam Sreenivasulu
Regular Paper


Copper (Cu), though an essential micronutrient for plants, poses toxicity at higher concentrations possibly by inducing oxidative stress. With the background that enzyme superoxide dismutase (SOD) ameliorates oxidative stress, the present work focused on understanding physiological and proteomic response of Arabidopsis seeds constitutively over-expressing copperzinc SOD of Potentilla atrosanguinea (PaSOD) during germination in response to varied concentrations of copper sulphate (Cu stress). Transgenics showed higher germination percentage and required less “mean time to germination” under Cu-stress. In response to Cu stress, 39 differentially expressed protein spots were detected by 2-D electrophoresis in proteins of germinating wild type (WT) and transgenic seeds, of which 14 spots appeared exclusively in transgenics. Among the rest 25 protein spots, 14 showed down-regulation, one showed up-regulation, and 10 spots disappeared. MALDI-TOF and subsequent peptide mass fingerprinting analysis revealed that the down-regulated proteins in transgenics were related to oxidative stress, detoxification, germination, intermediary metabolism and regulatory proteins. Up-regulated proteins in WT and down-regulated proteins in transgenic during Cu stress were the same. Changes in key proteins, vis-à-vis alleviation of oxidative stress in transgenic Arabidopsis over-expressing PaSOD possibly alleviated toxicity of Cu-induced stress during seed germination, resulting in higher germination rate and germination percentage.


Arabidopsis thaliana Heavy metal toxicity Oxidative stress Seed germination proteins Transgenics SOD 



Authors thank the Department of Biotechnology, New Delhi, India for financial assistance through the project entitled “Bioprospecting Himalayan Bioresources Through Transgenic and Nutraceutical Technology; BT/PR8876/NDB/52/68/2007”. TG acknowledges receipt of Senior Research Fellowship by the Council of Scientific and Industrial Research (CSIR), New Delhi. The authors are grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi for providing infrastructural support.

Supplementary material

10265_2011_421_MOESM1_ESM.doc (68 kb)
Supplementary table S1 (DOC 67.5 kb)


  1. Ahsan N, Lee DG, Lee SH, Kang KY (2007) Excess copper induced physiological and proteomic changes in germinating rice seeds. Chemosphere 67:1182–1193PubMedCrossRefGoogle Scholar
  2. An YJ (2006) Assessment of comparative toxicities of lead and copper using plant assay. Chemosphere 62:1359–1365PubMedCrossRefGoogle Scholar
  3. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress and signal transduction. Annu Rev Plant Biol 55:373–399PubMedCrossRefGoogle Scholar
  4. Badawi CH, Yamauchi Y, Shimada E, Sasaki R, Kawano N, Tanaka K, Tanaka K (2004) Enhanced tolerance to salt stress and water deficit by overexpressing superoxide dismutase in tobacco (Nicotiana tabacum) chloroplasts. Plant Sci 166:919–928CrossRefGoogle Scholar
  5. Bollag DM, Edeilstein SJ (1991) Protein methods. Wiley, New YorkGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye-binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  7. Brenchley JL, Probert RJ (1998) Seed germination responses to some environmental factors in the seagrass Zostera capricorni from Western Australia. Aquat Bot 62:177–188CrossRefGoogle Scholar
  8. Chen LM, Kao CH (1999) Effect of excess copper on rice leaves: evidence for involvement of lipid peroxidation. Bot Bull Acad Sin 40:283–287Google Scholar
  9. Colombo L, Franken J, Koetje E, Van Went J, Dons HJM, Angenent GC, van Tunenai AJ (1995) The petunia MADS Box gene FBPI7 determines ovule identity. Plant Cell 7:1859–1868PubMedCrossRefGoogle Scholar
  10. De Vos CHR, Vonk MJ, Schat H (1991a) Glutathione depletion due to copper induced phytochelatin synthesis causes oxidative stress in Silene cucubalus. Plant Physiol 98:853–858CrossRefGoogle Scholar
  11. De Vos CHR, Vonk MJ, Vooijs R, Schat H, De KLJ (1991b) Effect of copper on fatty acid composition and peroxidation of lipids in roots of copper tolerant and sensitive Silene cucubalus. Plant Physiol Biochem 31:151–158Google Scholar
  12. Devi S, Prasad M (1998) Copper toxicity in Ceratophyllum demersum L. (Coontail), a free floating macrophyte: response of antioxidant enzymes and antioxidants. Plant Sci 138:157–165CrossRefGoogle Scholar
  13. Devi S, Prasad M (2005) Antioxidant capacity of Brassica juncea plants exposed to elevated levels of copper. Russ J Plant Physiol 52:205–208CrossRefGoogle Scholar
  14. Dominguez-Solis JR, Gutierrez-Alcala G, Vega JM, Romero LC, Gotor C (2001) The cytosolic O-acetylserine(thiol)lyase gene is regulated by heavy metals and can function in cadmium tolerance. J Biol Chem 276:9297–9302PubMedCrossRefGoogle Scholar
  15. Fernendez JC, Henriques FS (1991) Biochemical, physiological and structural effects of copper in plants. Bot Rev 57:246–273CrossRefGoogle Scholar
  16. Fishman WH (1970) Metabolic conjugation and metabolic hydrolysis, vols 1, 2 and 3. Academic Press, LondonGoogle Scholar
  17. Gill T, Sreenivasulu Y, Kumar S, Ahuja PS (2010a) Over-expression of superoxide dismutase exhibits lignifications of vascular structures in Arabidopsis thaliana. J Plant Physiol 167:757–760PubMedCrossRefGoogle Scholar
  18. Gill T, Kumar S, Ahuja PS, Sreenivasulu Y (2010b) Over-expression of Potentilla superoxide dismutase improves salt stress tolerance during germination and growth in Arabidopsis thaliana. J Plant Genet Transgenics 1:1–10Google Scholar
  19. Gillette JR, Conney AH, Cosmides GJ, Estabrook RW, Fouts JR, Mannering GJ (1969) Microsomes and drug oxidations. Academic Press, LondonGoogle Scholar
  20. Gubler F, Kalla R, Roberts JK, Jacobsen JV (1995) Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high-pl [alpha]-amylase gene promoter. Plant Cell 7:1879–1891PubMedCrossRefGoogle Scholar
  21. Hu D, Klann E, Theils E (2007) Superoxide dismutase and hippocampal function: age and isozymes matter. Antioxidant Redox Signal 9:201–210CrossRefGoogle Scholar
  22. Hwang YS, Karrer EE, Thomas BR, Chen L, Rodrigues RL (1998) Three cis-elements required for rice α-amylase Amy3D expression during sugar starvation. Plant Mol Biol 36:331–341PubMedCrossRefGoogle Scholar
  23. Job C, Rajjou L, Lovigny Y, Belghazi M, Job D (2005) Patterns of protein oxidation in Arabidopsis seeds and during germination. Plant Physiol 138:790–802PubMedCrossRefGoogle Scholar
  24. Kawashima CG, Noji M, Nakamura M, Ogra Y, Suzuki KT, Saito K (2004) Heavy metal tolerance of transgenic tobacco plants over-expressing cysteine synthase. Biotechnol Lett 26:153–157PubMedCrossRefGoogle Scholar
  25. Labra M, Gianazza E, Waitt R, Eberini I, Sozzi A, Regondi S, Grassi F, Agradi E (2006) Zea mays L. protein changes in response to potassium dichromate treatments. Chemosphere 60:1234–1244CrossRefGoogle Scholar
  26. Li W, Khan MA, Yamaguchi S, Kamiya Y (2005) Effects of heavy metals on seed germination and early seedling growth of Arabidopsis thaliana. Plant Growth Regul 46:45–50CrossRefGoogle Scholar
  27. Lou LQ, Shen ZG, Li XD (2004) The copper tolerance mechanisms of Elsholtzia haichowensis, a plant from copper-enriched soils. Environ Exp Bot 51:111–120CrossRefGoogle Scholar
  28. Marcshner H (1995) Mineral nutrition in higher plants. Acad. Inc., LondonGoogle Scholar
  29. Martin C, Pazares J (1997) MYB transcription factors in plants. Trends Genetics 13:67–73CrossRefGoogle Scholar
  30. Mitsuhashi W, Sasaki S, Kanazawa A, Yang YY, Kamiya Y, Toyomasu T (2004) Differential expression of acid invertase genes during seed germination in Arabidopsis thaliana. Biosci Biotechnol Biochem 68:602–608PubMedCrossRefGoogle Scholar
  31. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assay with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  32. Ohnuma SI, Hirooka K, Tsuruoka N, Yano M, Ohto C, Nakane H, Nishinok T (1998) A pathway where polyprenyl diphosphate elongates in prenyltransferase. Insight into a common mechanism of chain length determination of prenyltransferases. J Biol Chem 273:26705–26713PubMedCrossRefGoogle Scholar
  33. Sandmann G, Boger P (1980) Copper mediated lipid peroxidation processes in photosynthetic membranes. Plant Physiol 66:797–800PubMedCrossRefGoogle Scholar
  34. Sato N, Albrieux C, Joyard J, Douce R, Kuroiwa T (1993) Detection and characterization of a plastid envelope DNA binding protein which may anchor plastids nucleoids. EMBO J 12:555–561PubMedGoogle Scholar
  35. Sato N, Ohshima K, Watanabe A, Ohta N (1998) Molecular characterization of the PEND protein, a novel bZIP protein present in the envelope membrane that is the site of nucleoid replication in developing plastids. Plant Cell 10:859–872PubMedCrossRefGoogle Scholar
  36. Schubert D, Lechtenberg B, Forsbach A, Gils M, Bahadur S, Schmidt R (2004) Silencing in Arabidopsis T-DNA transformants: the predominant role of a gene-specific RNA sensing mechanism versus position effects. Plant Cell 16:2561–2572PubMedCrossRefGoogle Scholar
  37. Shen ZG, Zhang FQ, Zhang FS (1998) Toxicity of copper and zinc in seedlings of mung bean and inducing accumulation of polyamine. J Plant Nutr 21:1153–1162CrossRefGoogle Scholar
  38. Shi H, Lee BH, Wu SJ, Zhu JK (2003) Overexpression of a plasma membrane Na+/H+ antiporter gene improves salt tolerance in Arabidopsis thaliana. Nat Biotechnol 21:81–85PubMedCrossRefGoogle Scholar
  39. Tang G, Reinhart BJ, Bartel DP, Zamore PD (2003) Biochemical framework for RNA silencing in plants. Genes Dev 17:49–63PubMedCrossRefGoogle Scholar
  40. Teisseire H, Guy V (2000) Copper-induced changes in antioxidant enzymes activities in fronds of duckweed (Lemna minor). Plant Sci 153:65–72CrossRefGoogle Scholar
  41. 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 223:1145–1153PubMedCrossRefGoogle Scholar
  42. Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K (1993) An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell 5:1529–1539PubMedCrossRefGoogle Scholar
  43. Wang W (1994) Rice seed toxicity tests for organic and inorganic substances. J Environ Monit Assess 29:101–107CrossRefGoogle Scholar
  44. Wang Y, Ying Y, Chen J, Wang X (2004) Transgenic Arabidopsis overexpressing Mn-SOD enhanced salt-tolerance. Plant Sci 167:671–677CrossRefGoogle Scholar
  45. Yang P, Fu H, Walker J, Papa CM, Smalle J, Ju YM, Vierstra RD (2004) Purification of the Arabidopsis 26S proteasome. J Biol Chem 279:6401–6413PubMedCrossRefGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer 2011

Authors and Affiliations

  • Tejpal Gill
    • 1
  • Vivek Dogra
    • 1
  • Sanjay Kumar
    • 1
  • Paramvir Singh Ahuja
    • 1
  • Yelam Sreenivasulu
    • 1
  1. 1.Biotechnology DivisionInstitute of Himalayan Bioresource Technology (Council of Scientific and Industrial Research)PalampurIndia

Personalised recommendations