Environmental Science and Pollution Research

, Volume 26, Issue 1, pp 299–311 | Cite as

Characterization of differentially expressed genes to Cu stress in Brassica nigra by Arabidopsis genome arrays

  • Birsen Cevher-KeskinEmail author
  • Yasemin Yıldızhan
  • Bayram Yüksel
  • Eda Dalyan
  • Abdul Razaque Memon
Research Article


Phytoremediation is an efficient and promising cleanup technology to extract or inactivate heavy metals and several organic and inorganic pollutants from soil and water. In this study, different Brassica nigra L. ecotypes, including Diyarbakır, collected from mining areas were exposed to different concentrations of copper and harvested after 72 h of Cu stress for the assessment of phytoremediation capacity. The Diyarbakır ecotype was called as “metallophyte” because of surviving at 500 μM Cu. To better understand Cu stress mechanism, ArabidopsisATH1 genome array was used to compare the gene expression in root and shoot tissues of B. nigra under 25 μM Cu. The response to Cu was much stronger in roots (88 genes showing increased or decreased mRNA levels) than in leaf tissues (24 responding genes). These genes were classified into the metal transport and accumulation-related genes, signal transduction and metabolism-related genes, and transport facilitation genes. Glutathione pathway-related genes (γ-ECS, PC, etc.) mRNAs were identified as differentially expressed in root and shoot tissues. QRT-PCR validation experiments showed that γ-ECS and PC expression was upregulated in the shoot and leaf tissues of the 100 μM Cu-subjected B. nigra-tolerant ecotype. This is the first study showing global expression profiles in response to Cu stress in B. nigra by Arabidopsis genome array. This work presented herein provides a well-illustrated insight into the global gene expression to Cu stress response in plants, and identified genes from microarray data will serve as molecular tools for the phytoremediation applications in the future.


B. nigra Phytoremediation Heavy metals Microarray Affymetrix GeneChip Copper Metal tolerance Real-time PCR γ-ECS PC 



This project was supported by The Scientific and Technological Council of Turkey (TUBITAK-1040211) and the COST Action 859 “Phytotechnologies to promote sustainable land use and improve food safety”.

Supplementary material

11356_2018_3577_Fig8_ESM.png (463 kb)
Suppl. Data 1

The effect of 500 μM Cu concentration in leaf tissue of non-tolerant Brassica nigra cv. CGN06619. Leaf necrosis was observed althought Cu content remained stable. (PNG 462 kb)

11356_2018_3577_MOESM1_ESM.tif (669 kb)
High resolution image (TIF 668 kb)
11356_2018_3577_Fig9_ESM.png (392 kb)
Suppl. Data 2

Pearson’s Correlation analysis was performed after the normalization of the signals from root and leaf tissues of 25 μM Cu treated B. nigra 6619. (PNG 392 kb)

11356_2018_3577_MOESM2_ESM.tif (34 kb)
High resolution image (TIF 34 kb)
11356_2018_3577_MOESM3_ESM.docx (24 kb)
Suppl. Data 3 Expression levels of significantly up (-) and down (+) regulated Cu –stress responsive transcripts in B.nigra 6619 ecotype roots exposed to 72 h 25 μM Cu stress compared to control conditions (Hoagland Solution-no copper) obtained with Affymetrix GeneChip Arabidopsis Genome Array (ATH1-121501 GeneChip) and three biological replicates. (DOCX 23 kb)


  1. Alaoui-Sosse B, Genet P, Vinit-Dunand F, Toussaint ML, Epron D, Badot PM (2004) Effect of copper on growth in cucumber plants (Cucumis sativus) and its relationships with carbohydrate accumulation and changes in ion contents. Plant Sci 166:1213–1218Google Scholar
  2. Andrés-Colás N, Sancenón V, Rodríguez-Navarro S, Mayo S, Thiele DJ, Ecker JR, Puig S, Peñarrubia L (2006) The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots. Plant J 45(2):225–236CrossRefGoogle Scholar
  3. Baker AJM, McGrath SP, Reeves RD, Smith JAC (2000) Metal hyperaccumulator plants: a review of the ecology and physiology of a biological resource for phytoremediation of metal-polluted soils. In: Terry N, Banuelos G (eds) Phytoremediation of contaminated soil and water. Lewis Publishers Inc, Boca RatonGoogle Scholar
  4. Balandin T, Castresana C (2002) AtCOX17, an Arabidopsis homolog of the yeast copper chaperone COX17. Plant Physiol 129:1852–1857Google Scholar
  5. Becher M, Talke IN, Krall L, Kramer U (2004) Cross-species microarray transcript profiling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri. Plant J 37:251–268CrossRefGoogle Scholar
  6. Cevher-Keskin B (2013) ARF1 and SAR1 GTPases in endomembrane trafficking in plants. Int J Mol Science 14:18181–18199CrossRefGoogle Scholar
  7. Cevher-Keskin B, Yuca E, Ertekin O, Yuksel B, Memon AR (2012) Expression characteristics of ARF1 and SAR1 during development and the de-etiolation process. Plant Biol 14:24–32Google Scholar
  8. Cevher-Keskin B, Yıldızhan Y, Külen O, Onarıcı S (2015) Quantitative expression analysis of TaMPK4 and TaTIP1 genes in drought tolerant and non-tolerant wheat (Triticum aestivum L.) cultivars. Plant Omics 8:270–277Google Scholar
  9. Chen J, Shafi M, Li S, Wang Y, Wu J, Ye Z, Peng D, Yan W, Liua D (2015) Copper induced oxidative stresses, antioxidant responses and phytoremediation potential of Moso bamboo (Phyllostachys pubescens). Sci Rep 5:13554–13563CrossRefGoogle Scholar
  10. Cherian S, Oliveira MM (2005) Transgenic plants in phytoremediation: recent advances and new possibilities. Environ Sci Technol 39:9377–9390CrossRefGoogle Scholar
  11. Clemens SE, Kim J, Neumann D, Schroeder JI (1999) Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J 18:3325–3333CrossRefGoogle Scholar
  12. Clemens S, Palmgren MG, Kramer U (2002) A long way ahead: understanding and engineering plant metal accumulation. Trends Plant Sci 7:309–315CrossRefGoogle Scholar
  13. Cobbett CS (2000) Phytochelatin biosynthesis and function in heavy-metal detoxification. Curr Opin Plant Biol 3:211–216CrossRefGoogle Scholar
  14. DalCorso G, Manara A, Furini A (2013) An overview of heavy metal challenge in plants: from roots to shoots. Metallomics 5:1117–1132Google Scholar
  15. Dalyan E, Yüzbaşıoğlu E, Cevher Keskin B, Yıldızhan Y, Memon A, Ünal M, Yüksel B (2017) The identification of genes associated with Pb and Cd response mechanism in Brassica juncea L. by using Arabidopsis expression array. Environ Exp Bot 139:105–115CrossRefGoogle Scholar
  16. Deng F, Yamaji N, Xia J, Ma JF (2013) A member of the heavy metal P-type ATPase OsHMA5 is involved in xylem loading of copper in rice. Plant Physiol 163:1353–1362CrossRefGoogle Scholar
  17. Dietz K-J, Kra¨mer U, Baier M (1999) Free radicals and reactive oxygen species as mediators of heavy metal toxicity. In: Prasad MNV, Hagemeyer J (eds) Heavy metal stress in plants: from molecules to ecosystems. Springer-Verlag, Berlin, pp 73–97CrossRefGoogle Scholar
  18. Fidalgo F, Azenha M, Silva AF, de Sousa A, Santiago A, Ferraz P (2013) Copper-induced stress in Solanum nigrum L. and antioxidant defense system responses. Food Energy Secur 2(1):70–80CrossRefGoogle Scholar
  19. Flocco CG, Lindblom SD, Smits E (2004) Overexpression of enzymes involved in glutathione synthesis enhances tolerance to organic pollutants in Brassica juncea. Int J Phytorem 6:289–304CrossRefGoogle Scholar
  20. Foyer CH, Noctor G (2003) Redox sensing and signaling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria. Physiol Plantarum 119:355–364CrossRefGoogle Scholar
  21. Foyer CH, Noctor G (2005) Oxidant and antioxidant signaling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ 28:1056–1071CrossRefGoogle Scholar
  22. Gasic K, Korban SS (2007a) Expression of Arabidopsis phytochelatin synthase in Indian mustard (Brassica juncea) plants enhances tolerance for Cd and Zn. Planta 225:1277–1285CrossRefGoogle Scholar
  23. Gasic K, Korban SS (2007b) Expression of Arabidopsis phytochelatin synthase (AtPCSI) in Indian mustard (Brassica juncea) enhances As and Cd tolerance. Plant Mol Biol 64:361–369CrossRefGoogle Scholar
  24. Godfrey D, Able AJ, Dry IB (2007) Induction of a grapevine germin-like protein (VvGLP3) gene is closely linked to the site of Erysiphe necator infection: a possible role in defense? Mol Plant-Microbe Inter 20:1112–1125CrossRefGoogle Scholar
  25. Guerra F, Duplessis S, Kohler A, MartinF, Tapia J, Lebed P, Francisco Z, Gonzáleza E (2009) Gene expression analysis of Populus deltoides roots subjected to copper stress. Environmental and Experimental Botany 67(2):335-344Google Scholar
  26. Haag-Kerwer A, Schäfer HJ, Heiss S, Walter C, Rausch T (1999) Cadmium exposure in Brassica juncea causes a decline in transpiration rate and leaf expansion without effect on photosynthesis. J Exp Bot 50:1827–1835CrossRefGoogle Scholar
  27. Hall JL (2002) Cellular mechanisms for heavy metal detoxification and tolerance. J Exp Bot 53:1–11CrossRefGoogle Scholar
  28. Heiss S, Schäfer HJ, Haag-Kerwer A, Rausch T (1999) Cloning sulfur assimilation genes of Brassica juncea L.: cadmium differentially affects the expression of a putative low affinity sulfate transporter and isoforms of ATP sulfurylase and APS reductase. Plant Mol Biol 39:847–857CrossRefGoogle Scholar
  29. Heiss S, Wachter A, Bogs J, Cobbett C, Rausch T (2003) Phytochelatin synthase (PCS) protein is induced in Brassica juncea leaves after prolonged Cd exposure. J Exp Botany 54:1833–1839CrossRefGoogle Scholar
  30. Hoagland DR, Arnon D (1938) The water culture method for growing plants without soil. UC College of Agriculture, Ag. Exp. Station, Berkeley, CA. Circular. 347:1–39Google Scholar
  31. Hurkman WJ, Tanaka CK (1996) Germin gene expression is induced in wheat leaves by powdery mildew infection. Plant Physiol 111:735–739CrossRefGoogle Scholar
  32. Ichimura K. et al. (2002) Mitogen-activated protein kinase cascades in plants: A new nomenclature. Trends Plant Sci 7: 301–308Google Scholar
  33. Jonak C, Nakagami H, Hirt H (2004) Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium. Plant Physiol 136:3276–3283CrossRefGoogle Scholar
  34. Katsuhara M, Koshio K, Shibasaka M, Hayashi Y, Hayakawa T, Kasamo K (2003) Over-expression of a barley aquaporin increased the shoot/root ratio and raised salt sensitivity in trans-genic rice plants. Plant Cell Physiol 44:1378–1383CrossRefGoogle Scholar
  35. Keskin BC, Sarikaya AT, Yuksel B, Memon AR (2010) Abscisic acid regulated gene expression in bread wheat (Triticum aestivum L.). Aust J Crop Sci 4:617–625Google Scholar
  36. Kobayashi Y, Kuroda K, Kimura K, Southron-Francis J L, Furuzawa A, Kimura K, Iuchi S, Kobayashi M, Taylor G J, Koyama H (2008) Amino Acid Polymorphisms in Strictly Conserved Domains of a P-Type ATPase HMA5 Are Involved in the Mechanism of Copper Tolerance Variation in Arabidopsis. Plant Physiol 148:969–980Google Scholar
  37. Kramer U (2005) Phytoremediation: novel approaches to cleaning up polluted soils. Curr Opin Biotech 16:133–141CrossRefGoogle Scholar
  38. Krämer U, Talke IN, Hanikenne M (2007) Transition metal transport. FEBS Lett 581:2263–2272CrossRefGoogle Scholar
  39. Lagercrantz U (1998) Comparative mapping between Arabidopsis thaliana and Brassica nigra indicates that Brassica genomes have evolved through extensive genome replication accompanied by chromosome fusions and frequent rearrangements. Genetics 150:1217–1228Google Scholar
  40. Lagercrantz U, Lydiate D (1996) Comparative genome mapping in Brassica. Genetics 144:1903–1910Google Scholar
  41. Lee SM, Kang BS (2005) Phytochelatin is not a primary factor in determining copper tolerance. J Plant Biol 48:32–38CrossRefGoogle Scholar
  42. Lee SM, Moon JS, Domier LL, Korban SS (2002) Molecular characterization of phytochelatin synthase expression in transgenic Arabidopsis. Plant Physiol Biochem 40:727–733CrossRefGoogle Scholar
  43. Luu DT, Maurel C (2005) Aquaporins in a challenging environment: molecular gears for adjusting plant water status. Plant Cell Environ 28:85–96CrossRefGoogle Scholar
  44. Malecka A, Piechalak A, Mensinger A, Hanc A, Baralkiewicz TB (2012) Antioxidative defense system in Pisum sativum roots exposed to heavy metals (Pb, Cu, Cd, Zn). Pol. J. Environ. Stud. 21(6):1721–1730Google Scholar
  45. Martins LL, Mourato MP, Baptista S, Reis R, Carvalheiro F, Almeida AM, Fevereiro P, Cuypers A (2014) Response to oxidative stress induced by cadmium and copper in tobacco plants (Nicotiana tabacum) engineered with the trehalose-6-phosphate synthase gene (AtTPS1). Acta Physiol Plant 36:755–765CrossRefGoogle Scholar
  46. Noctor G, Hamdi A, Chaouch S, Han Y, Neukermans J, Marquez-Garcia B, Queval G, Foyer CH (2012) Glutathione in plants: an integrated overview. Plant Cell Environ 35:454–484CrossRefGoogle Scholar
  47. Pilon-Smits EAH, Zhu YL, Sears T, Terry N (2000) Overexpression of glutathione reductase in Brassica juncea: effects on cadmium accumulation and tolerance. Physiol Plantarum 110:455–460CrossRefGoogle Scholar
  48. Polle A, Schützendübel A (2003) Heavy metal signalling in plants: linking cellular and oganismic responses. In: Hirt H, Shinozaki K (eds) Plant responses to abiotic stress, vol 4. Springer-Verlag, Berlin, pp 187–121CrossRefGoogle Scholar
  49. Qadir S, Qureshi MI, Javed S, Abdin MZ (2004) Genotypic variation in phytoremediation potential of Brassica juncea cultivars exposed to Cd stress. Plant Sci 167:1171–1181CrossRefGoogle Scholar
  50. Raskin I, Kramer U, Smith RD, Salt DE, Schulman R (1997) Phytoremediation and mechanisms of metal accumulation in plants. Plant Physiol 114:1253–1253Google Scholar
  51. Reisinger S, Schiavon M, Terry N, Pilon-Smits EAH (2008) Heavy metal tolerance and accumulation in Indian mustard (Brassica juncea L.) expressing bacterial γ-glutamylcysteine synthetase or glutathione synthetase. Int J Phytorem 10:440–454CrossRefGoogle Scholar
  52. Romero-Puertas MC, Corpas FJ, Rodriguez-Serrano M, Gomez M, del Río LA, Sandalio LM (2007) Differential expression and regulation of antioxidative enzymes by cadmium in pea. J Plant Physiol 164:1346–1357CrossRefGoogle Scholar
  53. Sancenón V, Puig S, Mira H, Thiele DJ, Peñarrubi L (2003) Identification of a copper transporter family in Arabidopsis thaliana. Plant Mol Biol 51:577–587Google Scholar
  54. Schäfer HJ, Greiner S, Rausch T, Haag-Kerwer A (1997) In seedlings of the heavy metal accumulator Brassica juncea Cu2+ differentially affects transcript amounts for γ-glutamylcysteine synthase (γ-ECS) and metallothionein (MT2). FEBS Lett 404:216–220CrossRefGoogle Scholar
  55. Schäfer HJ, Haag-Kerwer A, Rausch T (1998) cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy-metal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial γ-glutamylcysteine synthetase isoform. Plant Mol Biol 37:87–97CrossRefGoogle Scholar
  56. Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW (2000) Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal Biochem 285(2):194–204Google Scholar
  57. Shahid M, Pourrut B, Dumat C, Nadeem M, Aslam M, Pinelli E (2014) Heavy-metal-induced reactive oxygen species: phytotoxicity and physicochemical changes in plants. Rev Environ Contam Toxicol 232:1–44Google Scholar
  58. Tabuchi T, Kumon T, Azuma T, Nanmori T, Yasuda T (2003) The expression of a germin-like protein with superoxide dismutase activity in the halophyte Atriplex lentiformis is differentially regulated by wounding and abscisic acid. Physiol Plant 118:523–531CrossRefGoogle Scholar
  59. Vatamaniuk OK, Mari S, Lu YP, Rea PA (1999) AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proc Natl Acad Sci U S A 96:7110–7115CrossRefGoogle Scholar
  60. Vatamaniuk OK, Mari S, Lu Y-P, Rea PA (2000) Mechanism of heavy metal ion activation of phytochelatin (PC) synthase-blocked thiols are sufficient for PC synthase-catalyzed transpeptidation of glutathione and related thiol peptides. J Biol Chem 275:31451–31459CrossRefGoogle Scholar
  61. Warwick SI, Black LD (1991) Molecular systematics of Brassica and allied genera (subtribeBrassicinae, Brassiceae)—chloroplastgenome and cytodeme congruence. Theor Appl Genet 82:81–92CrossRefGoogle Scholar
  62. Weber M, Harada E, Vess C, Roepenack-Lahaye EV, Clemens S (2004) Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors. Plant J 37:269–281CrossRefGoogle Scholar
  63. Weber M, Trampczynska A, Clemens S (2006) Comparative transcriptome analysis of toxic metal responses in Arabidopsis thaliana and the Cd2+-hypertolerant facultative metallophyte Arabidopsis halleri. Plant Cell Environ 29:950–963CrossRefGoogle Scholar
  64. Williams LE, Mills RF (2005) P(1B)-ATPases: an ancient family of transition metal pumps with diverse functions in plants. Trends Plant Sci 10:491–502CrossRefGoogle Scholar
  65. Wójcik M, Tukiendorf A (2003) Response of wild type of Arabidopsis thaliana to copper stress. Biol Plantarum 46:79–84CrossRefGoogle Scholar
  66. Wójcik M, Tukiendorf A (2011) Glutathione in adaptation of Arabidopsis thaliana to cadmium stress. Biol Plantarum 55:125–132CrossRefGoogle Scholar
  67. Yeh C-M, Hsiao L-J, Huang H-J (2004) Cadmium activates a mitogen-activated protein kinase gene and MBP kinases in rice. cell Plant Cell Physiol 45:1306–1312CrossRefGoogle Scholar
  68. Zhu YL, Pilon-Smits EAH, Tarun AS, Weber SU, Jouanin L, Terry N (1999a) Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing γ-glutamylcysteine synthetase. Plant Physiol 121:1169–1177CrossRefGoogle Scholar
  69. Zhu YL, Pilon-Smits EAH, Jouanin L, Terry N (1999b) Overexpression of glutathione synthetase in Indian mustard enhances cadmium accumulation and tolerance. Plant Physiol 119:73–79CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Birsen Cevher-Keskin
    • 1
    Email author
  • Yasemin Yıldızhan
    • 1
  • Bayram Yüksel
    • 1
  • Eda Dalyan
    • 2
  • Abdul Razaque Memon
    • 3
  1. 1.The Scientific and Technological Research Council of Turkey (TUBITAK); Marmara Research Center; Genetic Engineering and Biotechnology Institute; Plant Molecular Biology and Genetics LaboratoryGebzeTurkey
  2. 2.Faculty of Science, Department of BotanyIstanbul UniversityIstanbulTurkey
  3. 3.Faculty of Science and Arts, Department of Molecular Biology and GeneticsUşak UniversityUşakTurkey

Personalised recommendations