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Copper-induced oxidative stress, initiation of antioxidants and phytoremediation potential of flax (Linum usitatissimum L.) seedlings grown under the mixing of two different soils of China

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Flax (Linum usitatissimum L.), one of the oldest cultivated crops, continues to be widely grown for oil, fiber and food. Furthermore, the plants show a metal tolerance dependent on species so is ideal for research. Present study was conducted to find out the influence of copper (Cu) toxicity on plant biomass, growth, chlorophyll content, malondialdehyde (MDA) contents, proline production, antioxidative enzymes and metal up taken by L. usitatissimum from the soil grown under mixing of Cu-contaminated soil with natural soil by 0:1 (control), 1:0, 1:1, 1:2 and 1:4. Results revealed that, high concentration of Cu in the soil affected plant growth and development by reducing plant height, plant diameter and plant fresh and dry biomass and chlorophyll contents in the leaves compared with the control. Furthermore, Cu in excess causes generation of reactive oxygen species (ROS) such as superoxide radical (O) and hydroxyl radicals (OH), which is manifested by high malondialdehyde (MDA) and proline contents also. The increasing activities of superoxidase dismutase (SOD) and peroxidase (POD) in the roots and leaves of L. usitatissimum are involved in the scavenging of ROS. Results also showed that L. usitatissimum also has capability to revoke large amount of Cu from the contaminated soil. As Cu concentration in the soil increases, the final uptake of Cu concentration by L. usitatissimum increases. Furthermore, the soil chemical parameters (pH, electrical conductivity and cation exchange capacity) were increasing to highest levels as the ratio of Cu concentration to the natural soil increases. Thus, Cu-contaminated soil is amended with the addition of natural soil significantly reduced plant growth and biomass, while L. usitatissimum is able to revoke large amount of Cu from the soil and could be grown as flaxseed and a potential candidate for phytoremediation of Cu.

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  1. Aggarwal A, Sharma I, Tripathi B, Munjal A, Baunthiyal M, Sharma V (2012) Metal toxicity and photosynthesis. Photosynthesis: overviews on recent progress and future perspectives, 229–236

  2. Amna MS, Syed JH, Munis MFH, Chaudhary HJ (2015) Phyto-extraction of nickel by Linum usitatissimum in association with Glomus intraradices. Internat J Phytor 17:981–987

  3. Andrade SA, Gratão PL, Azevedo RA, Silveira AP, Schiavinato MA, Mazzafera P (2010) Biochemical and physiological changes in jack bean under mycorrhizal symbiosis growing in soil with increasing Cu concentrations. Environ Exp Bot 68:198–207

  4. Ashraf S, Ali Q, Zahir ZA, Ashraf S, Asghar HN (2019) Phytoremediation: environmentally sustainable way for reclamation of heavy metal polluted soils. Ecotox Environ Safe 174:714–727

  5. Bates LS, Waldren RP, Teare I (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207

  6. Belkhadi A, Hediji H, Abbes Z, Nouairi I, Barhoumi Z, Zarrouk M, Chaïbi W, Djebali W (2010) Effects of exogenous salicylic acid pre-treatment on cadmium toxicity and leaf lipid content in Linum usitatissimum L. Ecotox Environ Safe 73:1004–1011

  7. Bouazizi H, Jouili H, Geitmann A, El Ferjani E (2010) Copper toxicity in expanding leaves of Phaseolus vulgaris L.: antioxidant enzyme response and nutrient element uptake. Ecotox Environ Safe 73:1304–1308

  8. Chandrasekhar C, Ray JG (2017) Copper accumulation, localization and antioxidant response in Eclipta alba L. in relation to quantitative variation of the metal in soil. Acta Physiol Plant 39:205

  9. Chen C-N, Pan S-M (1996) Assay of superoxide dismutase activity by combining electrophoresis and densitometry. Botan Bullet Acad Sinica 37

  10. Chen J, Shafi M, Li S, Wang Y, Wu J, Ye Z, Peng D, Yan W, Liu D (2015) Copper induced oxidative stresses, antioxidant responses and phytoremediation potential of Moso bamboo (Phyllostachys pubescens). Sci Report 5:13554

  11. Goswami S, Das S (2016) Copper phytoremediation potential of Calandula officinalis L. and the role of antioxidant enzymes in metal tolerance. Ecotox Environ Safe 126:211–218

  12. Griga M, Bjelkova M, Tejklova E (2003a) Phytoextraction of heavy metals by fibre crops: Linum usitatissimum L. case study. Proceedings of the 2nd European Bioremediation Conference, Chania, Crete, TU Crete pp 353-356

  13. Griga M, Bjelkova M, Tejklová E (2003b) Potential of flax (Linum usitatissimum L.) for heavy metal phytoextraction and industrial processing of contaminated biomass-a review. Risk assessment and sustainable land management using plants in trace element-contaminated soils. Centre INRA Bordeaux-Aquitaine, Villenave d’Ornon, France 174-180

  14. Habiba U, Ali S, Farid M, Shakoor MB, Rizwan M, Ibrahim M, Abbasi GH, Hayat T, Ali B (2015) EDTA enhanced plant growth, antioxidant defense system, and phytoextraction of copper by Brassica napus L. Environ Sci Pollut R 22:1534–1544

  15. Halliwell B, Gutteridge JM (2015) Free radicals in biology and medicine. Oxford University Press, NewYork, pp 888

  16. Hancock LM, Ernst CL, Charneskie R, Ruane LG (2012) Effects of cadmium and mycorrhizal fungi on growth, fitness, and cadmium accumulation in flax (Linum usitatissimum; Linaceae). Amer J Bot 99:1445–1452

  17. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198

  18. Hosman ME, El-Feky SS, Mohamed M, Shaker EM (2017) Mechanism of phytoremediation potential of flax (Linum usitatissimum L.) to Pb, Cd and Zn. Asian J Plant Sci Re 7:30–40

  19. Houben D, Evrard L, Sonnet P (2013) Mobility, bioavailability and pH-dependent leaching of cadmium, zinc and lead in a contaminated soil amended with biochar. Chemosphere 92:1450–1457

  20. Hradilová J, Řehulka P, Řehulková H, Vrbová M, Griga M, Brzobohatý B (2010) Comparative analysis of proteomic changes in contrasting flax cultivars upon cadmium exposure. Electrophoresis 31:421–431

  21. Husak V (2015) Copper and copper-containing pesticides: metabolism, toxicity and oxidative stress. J Vasyl Stef Precarp Nat Uni 2:38–50

  22. Kaplan ME, Simmons ER, Hawkins JC, Ruane LG, Carney JM (2015) Influence of cadmium and mycorrhizal fungi on the fatty acid profile of flax (Linum usitatissimum) seeds. J Sci Food Agric 95:2528–2532

  23. Khan SU, A-u K, Shah A-u-HA, Shah SM, Hussain S, Ayaz M, Ayaz S (2016) Heavy metals content, phytochemical composition, antimicrobial and insecticidal evaluation of Elaeagnus angustifolia. Toxicol Ind Health 32:154–161

  24. Kolbert Z, Pető A, Lehotai N, Feigl G, Erdei L (2012) Long-term copper (Cu 2+) exposure impacts on auxin, nitric oxide (NO) metabolism and morphology of Arabidopsis thaliana L. Plant Growth Regul 68:151–159

  25. Ku H-M, Tan C-W, Su Y-S, Chiu C-Y, Chen C-T, Jan F-J (2012) The effect of water deficit and excess copper on proline metabolism in Nicotiana benthamiana. Biol Plant 56:337–343

  26. Lajayer BA, Moghadam NK, Maghsoodi MR, Ghorbanpour M, Kariman K (2019) Phytoextraction of heavy metals from contaminated soil, water and atmosphere using ornamental plants: mechanisms and efficiency improvement strategies. Environ Sci Pollut R 26:8468–8484

  27. Li L, Zhang K, Gill RA, Islam F, Farooq MA, Wang J, Zhou W (2018, 2018) Ecotoxicological and interactive effects of copper and chromium on physiochemical, ultrastructural, and molecular profiling in Brassica napus L. BioMed Res Internat

  28. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes, methods in enzymology. Elsevier:350–382

  29. Liu Q, Zheng L, He F, Zhao F-J, Shen Z, Zheng L (2015) Transcriptional and physiological analyses identify a regulatory role for hydrogen peroxide in the lignin biosynthesis of copper-stressed rice roots. Plant Soil 387:323–336

  30. Liu J, Wang J, Lee S, Wen R (2018) Copper-caused oxidative stress triggers the activation of antioxidant enzymes via ZmMPK3 in maize leaves. PLoS One 13:e0203612

  31. Lu R (2000): Analytical methods of soil agrochemistry (in Chinese), China Agriculture Technology Press, pp 305–336

  32. Mahmud S, Hassan MM, Moniruzzaman M, Biswas N, Rahman MM, Haque ME (2013) Study on the accumulation of copper from soil by shoots and roots of some selective plant species. Internat J Biosc 3:68–75

  33. Manousaki E, Kadukova J, Papadantonakis N, Kalogerakis N (2008) Phytoextraction and phytoexcretion of Cd by the leaves of Tamarix smyrnensis growing on contaminated non-saline and saline soils. Environ Res 106:326–332

  34. Martins LL, Mourato MP (2006) Effect of excess copper on tomato plants: growth parameters, enzyme activities, chlorophyll, and mineral content. J Plant Nutr 29:2179–2198

  35. Meng Q, Zou J, Zou J, Jiang W, Liu D (2007) Effect of Cu2+ concentration on growth, antioxidant enzyme activity and malondialdehyde content in garlic (Allium sativum L.). Acta Biol Cracov Ser Bot 49:95–101

  36. Murakami M, Ae N (2009) Potential for phytoextraction of copper, lead, and zinc by rice (Oryza sativa L.), soybean (Glycine max [L.] Merr.), and maize (Zea mays L.). J Hazar Material 162:1185–1192

  37. Nagajyoti PC, Lee KD, Sreekanth T (2010) Heavy metals, occurrence and toxicity for plants: a review. Environ Chem Lett 8:199–216

  38. Nair PMG, Chung IM (2015) Study on the correlation between copper oxide nanoparticles induced growth suppression and enhanced lignification in Indian mustard (Brassica juncea L.). Ecotox Environ Safe 113:302–313

  39. Najmanova J, Neumannova E, Leonhardt T, Zitka O, Kizek R, Macek T, Mackova M, Kotrba P (2012) Cadmium-induced production of phytochelatins and speciation of intracellular cadmium in organs of Linum usitatissimum seedlings. Ind Crop Prod 36:536–542

  40. Pajević S, Borišev M, Nikolić N, Arsenov DD, Orlović S, Župunski M (2016) Phytoextraction of heavy metals by fast-growing trees: a review, phytoremediation. Springer, pp 29–64

  41. Pietrzak U, Uren N (2011) Remedial options for copper-contaminated vineyard soils. Soil Research 49:44–55

  42. Praczyk M, Heller K, Silska G, Baraniecki P (2015) Analysis of accumulation of cadmium in seeds of selected breeding linseed (Linum usitatissimum L.) genotypes cultivated for medicinal purposes. Herba Polonica 61:19–30

  43. Race M, Marotta R, Fabbricino M, Pirozzi F, Andreozzi R, Cortese L, Giudicianni P (2016) Copper and zinc removal from contaminated soils through soil washing process using ethylenediaminedisuccinic acid as a chelating agent: a modeling investigation. J Environ Chem Engin 4:2878–2891

  44. Rehman M, Liu L, Bashir S, Saleem MH, Chen C, Peng D, Siddique KH (2019a) Influence of rice straw biochar on growth, antioxidant capacity and copper uptake in ramie (Boehmeria nivea L.) grown as forage in aged copper-contaminated soil. Plant Physiol Biochem 138:121–129

  45. Rehman M, Liu L, Wang Q, Saleem MH, Bashir S, Ullah S, Peng D (2019b) Copper environmental toxicology, recent advances, and future outlook: a review. Environ Sci Pollut R:1–14

  46. Rehman M, Maqbool Z, Peng D, Liu L (2019c) Morpho-physiological traits, antioxidant capacity and phytoextraction of copper by ramie (Boehmeria nivea L.) grown as fodder in copper-contaminated soil. Environ Sci Pollut R 26:5851–5861

  47. Rizwan M, Ali S, Abbas T, Zia-ur-Rehman M, Hannan F, Keller C, Al-Wabel MI, Ok YS (2016) Cadmium minimization in wheat: a critical review. Ecotox Environ Safe 130:43–53

  48. Rout JR, Sahoo SL (2013) Antioxidant enzyme gene expression in response to copper stress in Withania somnifera L. Plant Growth Regul 71:95–99

  49. Sakharov IY, Ardila GB (1999) Variations of peroxidase activity in cocoa (Theobroma cacao L.) beans during their ripening, fermentation and drying. Food Chem 65:51–54

  50. Saleem MH, Fahad S, Khan SU, Ahmar S, Khan MHU, Rehman M, Maqbool Z, Liu L (2019a) Morpho-physiological traits, gaseous exchange attributes, and phytoremediation potential of jute (Corchorus capsularis L.) grown in different concentrations of copper-contaminated soil. Ecotox Environ Safe.

  51. Saleem MH, Ali S, Seleiman MF, Rizwan M, Rehman M, Akram NA, Liu L, Alotaibi M, Al-Ashkar I, Mubushar M (2019b) Assessing the correlations between different traits in copper-sensitive and copper-resistant varieties of jute (Corchorus capsularis L.). Plants 8:545

  52. Sánchez-Pardo B, Fernández-Pascual M, Zornoza P (2014) Copper microlocalisation and changes in leaf morphology, chloroplast ultrastructure and antioxidative response in white lupin and soybean grown in copper excess. J Plant Res 127:119–129

  53. Sgherri C, Quartacci MF, Navari-Izzo F (2007) Early production of activated oxygen species in root apoplast of wheat following copper excess. J Plant Physiol 164:1152–1160

  54. Shin L-J, Lo J-C, Yeh K-C (2012) Copper chaperone antioxidant protein1 is essential for copper homeostasis. Plant Physiol 159:1099–1110

  55. Sidhu GPS, Bali AS, Singh HP, Batish DR, Kohli RK (2018) Ethylenediamine disuccinic acid enhanced phytoextraction of nickel from contaminated soils using Coronopus didymus (L.) Sm. Chemosphere 205:234–243

  56. Singh PK, Wang W, Shrivastava AK (2018) Cadmium-mediated morphological, biochemical and physiological tuning in three different Anabaena species. Aquat Toxicol 202:36–45

  57. Smolinska B (2015) Green waste compost as an amendment during induced phytoextraction of mercury-contaminated soil. Environ Sci Pollut R 22:3528–3537

  58. Smykalova I, Vrbova M, Tejklova E, Vetrovcova M, Griga M (2010) Large scale screening of heavy metal tolerance in flax/linseed (Linum usitatissimum L.) tested in vitro. Ind Crop Prod 32:527–533

  59. Song B, Zeng G, Gong J, Liang J, Xu P, Liu Z, Zhang Y, Zhang C, Cheng M, Liu Y (2017) Evaluation methods for assessing effectiveness of in situ remediation of soil and sediment contaminated with organic pollutants and heavy metals. Environ Int 105:43–55

  60. Stojek M (2013) The concentration of molybdenum and copper in rocks, soils and plants in the area of Jabłonki (eastern Beskids Mts.). Environ Prot Nat 24:13–17

  61. Sun B-Y, Kan S-H, Zhang Y-Z, Deng S-H, Wu J, Yuan H, Qi H, Yang G, Li L, Zhang X-H (2010) Certain antioxidant enzymes and lipid peroxidation of radish (Raphanus sativus L.) as early warning biomarkers of soil copper exposure. J Hazard Mater 183:833–838

  62. Sun X-H, Yu G, Li J-T, Jia P, Zhang J-C, Jia C-G, Zhang Y-H, Pan H-Y (2014) A heavy metal-associated protein (AcHMA1) from the halophyte, Atriplex canescens (Pursh) Nutt., confers tolerance to iron and other abiotic stresses when expressed in Saccharomyces cerevisiae. Int J Mol Sci 15:14891–14906

  63. Szabados L, Savouré A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15:89–97

  64. Szynkowska M, Rybicki E, Leśniewska E, Pawlaczyk A, Paryjczak T, Matyjas-Zgondek E (2009) Influence of production progress on the heavy metal content in flax fibers. Chem Pap 63:537–542

  65. Tahmasbian I, Sinegani AAS (2016) Improving the efficiency of phytoremediation using electrically charged plant and chelating agents. Environ Sci Pollut R 23:2479–2486

  66. Tanhan P, Kruatrachue M, Pokethitiyook P, Chaiyarat R (2007) Uptake and accumulation of cadmium, lead and zinc by Siam weed [Chromolaena odorata (L.) King & Robinson]. Chemosphere 68:323–329

  67. Thounaojam TC, Panda P, Mazumdar P, Kumar D, Sharma G, Sahoo L, Sanjib P (2012) Excess copper induced oxidative stress and response of antioxidants in rice. Plant Physiol Biochem 53:33–39

  68. Uddin Nizam M, Mokhlesur Rahman M, Kim J-E (2016) Phytoremediation potential of Kenaf (Hibiscus cannabinus L.), Mesta (Hibiscus sabdariffa L.), and jute (Corchorus capsularis L.) in arsenic-contaminated soil. Korean J Environ Agri 35:111–120

  69. ul Hassan Z, Ali S, Rizwan M, Hussain A, Akbar Z, Rasool N, Abbas F (2017) Role of zinc in alleviating heavy metal stress, essential plant nutrients. Springer, pp 351–366

  70. Upadhyay R, Panda SK (2010) Zinc reduces copper toxicity induced oxidative stress by promoting antioxidant defense in freshly grown aquatic duckweed Spirodela polyrhiza L. J Hazard Mater 175:1081–1084

  71. Vangronsveld J, Herzig R, Weyens N, Boulet J, Adriaensen K, Ruttens A, Thewys T, Vassilev A, Meers E, Nehnevajova E (2009) Phytoremediation of contaminated soils and groundwater: lessons from the field. Environ Sci Pollut R 16:765–794

  72. Verma A, Bhatia S (2014) Analysis of some physicochemical parameters and trace metal concentration present in the soil around the area of Pariccha thernal power station in Jhansi. India. Int J Inno Res Sci Eng Technol 3:10482–10488

  73. Volland S, Bayer E, Baumgartner V, Andosch A, Lütz C, Sima E, Lütz-Meindl U (2014) Rescue of heavy metal effects on cell physiology of the algal model system Micrasterias by divalent ions. J Plant Physiol 171:154–163

  74. Vrbová M, Kotrba P, Horáček J, Smýkal P, Švábová L, Větrovcová M, Smýkalová I, Griga M (2013) Enhanced accumulation of cadmium in Linum usitatissimum L. plants due to overproduction of metallothionein α-domain as a fusion to β-glucuronidase protein. Plant Cell Tissue Organ Cult 112:321–330

  75. Waters BM, Armbrust LC (2013) Optimal copper supply is required for normal plant iron deficiency responses. Plant Signal Behav 8:e26611

  76. Wróbel-Kwiatkowska M, Czemplik M, Kulma A, Żuk M, Kaczmar J, Dymińska L, Hanuza J, Ptak M, Szopa J (2012) New biocomposites based on bioplastic flax fibers and biodegradable polymers. Biotech Progr 28:1336–1346

  77. Wuana RA, Okieimen FE (2011) Heavy metals in contaminated soils: a review of sources, chemistry, risks and best available strategies for remediation. Isrn Ecology 2011

  78. Xu J, Yang L, Wang Z, Dong G, Huang J, Wang Y (2006) Toxicity of copper on rice growth and accumulation of copper in rice grain in copper contaminated soil. Chemosphere 62:602–607

  79. Yahaghi Z, Shirvani M, Nourbakhsh F, De La Pena TC, Pueyo JJ, Talebi M (2018) Isolation and characterization of Pb-solubilizing bacteria and their effects on Pb uptake by Brassica juncea: implications for microbe-assisted phytoremediation. J Microbiol Biotechnol 28:1156–1167

  80. Yang W-d, Wang Y-y, Zhao F-l, Ding Z-l, Zhang X-c, Zhu Z-q, Yang X-e (2014) Variation in copper and zinc tolerance and accumulation in 12 willow clones: implications for phytoextraction. J Zhejiang Univ Sci B 15:788–800

  81. Yang Z, Shi W, Yang W, Liang L, Yao W, Chai L, Gao S, Liao Q (2018) Combination of bioleaching by gross bacterial biosurfactants and flocculation: A potential remediation for the heavy metal contaminated soils. Chemosphere 206:83–91

  82. Yurkevich OY, Kirov IV, Bolsheva NL, Rachinskaya OA, Grushetskaya ZE, Zoschuk SA, Samatadze TE, Bogdanova MV, Lemesh VA, Amosova AV (2017) Integration of physical, genetic, and cytogenetic mapping data for cellulose synthase (CesA) genes in flax (Linum usitatissimum L.). front. Plant Sci 8:1467

  83. Zaheer IE, Ali S, Rizwan M, Farid M, Shakoor MB, Gill RA, Najeeb U, Iqbal N, Ahmad R (2015) Citric acid assisted phytoremediation of copper by Brassica napus L. Ecotox Environ Safe 120:310–317

  84. Zhao S, Liu Q, Qi Y, Duo L (2010) Responses of root growth and protective enzymes to copper stress in turfgrass. Acta Biol Cracov Ser Bot 52:7–11

  85. Zhu H, Chen C, Xu C, Zhu Q, Huang D (2016) Effects of soil acidification and liming on the phytoavailability of cadmium in paddy soils of central subtropical China. Environ Pollut 219:99–106

  86. Zlobin I, Kholodova V, Rakhmankulova Z, Kuznetsov VV (2015) Brassica napus responses to short-term excessive copper treatment with decrease of photosynthetic pigments, differential expression of heavy metal homeostasis genes including activation of gene NRAMP4 involved in photosystem II stabilization. Photosynth Res 125:141–150

  87. Zvezdanović J, Marković D, Nikolić G (2007) Different possibilities for the formation of complexes of copper and zinc with chlorophyll inside photosynthetic organelles: chloroplasts and thylakoids. J Serbian Chem Soci 72

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This research was supported by China Agriculture Research System project (CARS-16-E10) and the National Natural Science Foundation of China (31571717).

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Saleem, M.H., Fahad, S., Khan, S.U. et al. Copper-induced oxidative stress, initiation of antioxidants and phytoremediation potential of flax (Linum usitatissimum L.) seedlings grown under the mixing of two different soils of China. Environ Sci Pollut Res (2019).

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  • Flax (Linum usitatissimum)
  • Phytoextraction
  • Flaxseed crop
  • Cu-contaminated soil
  • Natural soil
  • Cu uptake
  • Reactive oxygen species
  • Growth
  • Proline