Photosynthesis Research

, Volume 125, Issue 1–2, pp 141–150 | Cite as

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

  • I. E. Zlobin
  • V. P. Kholodova
  • Z. F. Rakhmankulova
  • Vl. V. Kuznetsov
Regular Paper


In the present study, the influence of 50 and 100 µM CuSO4 was investigated starting from 3 h till 72 h treatment of 4-weeks Brassica napus plants. High CuSO4 concentrations in nutrient medium resulted in the rapid copper accumulation in plants, especially in roots, much slower and to lower degree in leaves. Copper excess induced early decrease in the leaf water content and temporary leaf wilting. The decrease in content of photosynthetic pigments became significant to 24 h of excessive copper treatments and reached 35 % decrease to 72 h, but there were no significant changes in maximum quantum efficiency of photosystem II photochemistry. The copper excess affected the expression of ten genes involved in heavy metal homeostasis and copper detoxification. The results showed the differential and organ-specific expression of most genes. The potential roles of copper-activated genes encoding heavy metal transporters (ZIP5, NRAMP4, YSL2, and MRP1), metallothioneins (MT1a and MT2b), low-molecular chelator synthesis enzymes (PCS1 and NAS2), and metallochaperones (CCS and HIPP06) in heavy metal homeostasis and copper ion detoxification were discussed. The highest increase in gene expression was shown for NRAMP4 in leaves in spite of relatively moderate Cu accumulation there. The opinion was advanced that the NRAMP4 activation can be considered among the early reactions in the defense of the photosystem II against copper excess.


Brassica napus Copper detoxification Copper excess Photosynthetic pigments Photosystem II photochemistry Gene expression 



Antioxidant 1-like


Copper chaperone for Cu/Zn superoxide dismutase






Copper transporter


Dry weight


Minimal fluorescence yield of dark-adapted state


Maximal fluorescence yield of dark-adapted state


Variable fluorescence = F m − F 0


Maximal quantum yield of PSII photochemistry


Fresh weight


Heavy metal-associated isoprenylated plant protein


Heavy metal


Multidrug resistance-associated protein homolog




Nicotianamine synthase


Natural resistance-associated macrophage protein




Phytochelatin synthase


Reactive oxygen species






Yellow stripe-like


Zrt-, Irt-like protein



This study was supported through funding from the Russian Foundation for Basic Research, project no. 13-04-01001, and the Presidium of the Russian Academy of Sciences (Molecular and Cellular Biology Program).

Supplementary material

11120_2014_54_MOESM1_ESM.doc (1.1 mb)
Supplementary material 1 (DOC 1094 kb)


  1. Abdel-Ghany SE (2009) Contribution of plastocyanin isoforms to photosynthesis and copper homeostasis in Arabidopsis thaliana grown at different copper regimes. Planta 229:767–779PubMedCrossRefGoogle Scholar
  2. Baker N (2008) Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu Rev Plant Biol 59:89–113PubMedCrossRefGoogle Scholar
  3. Bernal M, Roncel M, Ortega J, Picorel R, Yruela I (2004) Copper effect on cytochrome b559 of photosystem II under photoinhibitory conditions. Physiol Plant 120:686–694PubMedCrossRefGoogle Scholar
  4. Burkhead JL, Gogolin Reynolds KA, Abdel-Ghany SE, Cohu CM, Pilon M (2009) Copper homeostasis. New Phytol 182:799–816PubMedCrossRefGoogle Scholar
  5. Burzynski M, Klobus G (2004) Changes of photosynthetic parameters in cucumber leaves under Cu, Cd, and Pb stress. Photosynthetica 42:505–510CrossRefGoogle Scholar
  6. Caspi V, Droppa M, Horvath G, Malkin S, Marder J, Raskin V (1999) The effect of copper on chlorophyll organization during greening of barley leaves. Photosynth Res 62:165–174CrossRefGoogle Scholar
  7. Chettri MK, Cook CM, Vardaka E, Sawidis T, Lanaras T (2014) The effect of Cu, Zn and Pb on the chlorophyll content of the lichens Cladonia convoluta and Cladonia rangiformis. Environ Exp Bot 39:1–10CrossRefGoogle Scholar
  8. Das S, Sen M, Saha C, Chakraborty D, Das A, Banerjee M, Seal A (2011) Isolation and expression analysis of partial sequences of heavy metal transporters from Brassica juncea by coupling high throughput cloning with a molecular fingerprinting technique. Planta 234:139–156PubMedCrossRefGoogle Scholar
  9. de Abreu-Neto JB, Turchetto-Zolet AC, de Oliveira LFV, Zanettini MHB, Margis-Pinheiro M (2013) Heavy metal-associated isoprenylated plant protein (HIPP): characterization of a family of proteins exclusive to plants. FEBS J 280:1604–1616PubMedCrossRefGoogle Scholar
  10. Di Donato RJ Jr, Roberts LA, Sanderson T, Eisley RB, Walker EL (2004) Arabidopsis Yellow Stripe-Like 2 (YSL2): a metal-regulated gene encoding a plasma membrane transporter of nicotianamine–metal complexes. Plant J 39:403–414CrossRefGoogle Scholar
  11. Ducic T, Polle A (2005) Transport and detoxification of manganese and copper in plants. Braz J Plant Physiol 17:103–112CrossRefGoogle Scholar
  12. Gonzalez-Mendoza D, Quiroz Moreno A, Zapata-Perez O (2007) Coordinated responses of phytochelatin synthase and metallothionein genes in black mangrove, Avicennia germinans, exposed to cadmium and copper. Aquat Toxicol 83:306–314PubMedCrossRefGoogle Scholar
  13. Grill E, Mishra S, Srivastava S, Tripathi RD (2007) Role of phytochelatins in phytoremediation of heavy metals. In: Singh SN, Tripathi RD (eds) Environmental bioremediation technologies. Springer, New york, pp 101–146CrossRefGoogle Scholar
  14. Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, Hediger MA (1997) Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388:482–488PubMedCrossRefGoogle Scholar
  15. Guo WJ, Meetam M, Goldsbrough PB (2008) Examining the specific contributions of individual Arabidopsis metallothioneins to copper distribution and metal tolerance. Plant Physiol 146:1697–1706PubMedCentralPubMedCrossRefGoogle Scholar
  16. Ivanova E, Kholodova V, Kuznetsov Vl (2010) Biological effects of high copper and zinc concentrations and their interaction in rapeseed plants. Russ J Plant Physiol 57:864–873CrossRefGoogle Scholar
  17. Kholodova V, Volkov K, Abdeyeva A, Kuznetsov V (2011) Water status in Mesembryanthemum crystallinum under heavy metal stress. Environ Exp Bot 71:382–389Google Scholar
  18. Kulikova A, Kuznetsova N, Kholodova V (2011) Effect of copper excess in environment on soybean root viability and morphology. Russ J Plant Physiol 58:719–727CrossRefGoogle Scholar
  19. Lanquar V, Ramos MS, Lelièvre F, Barbier-Brygoo H, Krieger-Liszkay A, Krämer U, Thomine S (2010) Export of vacuolar manganese by AtNRAMP3 and AtNRAMP4 is required for optimal photosynthesis and growth under manganese deficiency. Plant Physiol 152:1986–1999PubMedCentralPubMedCrossRefGoogle Scholar
  20. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148:350–382Google Scholar
  21. Mendoza-Cózatl DG, Zhai Z, Jobe TO, Akmakjian GZ, Song WY, Limbo O, Russell MR, Kozlovskyy VI, Martinoia E, Vatamaniuk OK, Russell P, Schroeder JI (2010) Tonoplast-localized abc2 transporter mediates phytochelatin accumulation in vacuoles and confers cadmium tolerance. J Biol Chem 285:40416–40426PubMedCentralPubMedCrossRefGoogle Scholar
  22. Molas J (2002) Changes of chloroplast ultrastructure and total chlorophyll concentration in cabbage leaves caused by excess of organic Ni(II) complexes. Environ Exp Bot 47:115–126CrossRefGoogle Scholar
  23. Oláh V, Lakatos G, Bertók C, Kanalas P, Szőllősi E, Kis J, Mészáros I (2010) Short-term chromium(VI) stress induces different photosynthetic responses in two duckweed species, Lemna gibba L. and Lemna minor L. Photosynthetica 48:513–520CrossRefGoogle Scholar
  24. Oomen RJ, Wu J, Lelièvre F, Blanchet S, Richaud P, Barbier-Brygoo H, Aarts MGM, Thomine S (2009) Functional characterization of NRAMP3 and NRAMP4 from the metal hyperaccumulator Thlaspi caerulescens. New Phytol 181:637–650PubMedCrossRefGoogle Scholar
  25. Park J, Song WY, Ko D, Eom Y, Hansen TH, Schiller M, Lee TG, Martinoia E, Lee Y (2012) The phytochelatin transporters AtABCC1 and AtABCC2 mediate tolerance to cadmium and mercury. Plant J 69:278–288PubMedCrossRefGoogle Scholar
  26. Peng H, Kroneck PMH, Küpper H (2013) Toxicity and deficiency of copper in Elsholtzia splendens affect photosynthesis biophysics, pigments and metal accumulation. Environ Sci Technol 47:6120–6128PubMedGoogle Scholar
  27. Perales-Vela HV, González-Moreno S, Montes-Horcasitas C, Cañizares-Villanueva RO (2007) Growth, photosynthetic and respiratory responses to sub-lethal copper concentrations in Scenedesmus incrassatulus (Chlorophyceae). Chemosphere 67:2274–2281PubMedCrossRefGoogle Scholar
  28. Sagardoy R, Morales F, Lopez-Millan AF, Abadìa A, Abadìa J (2009) Effects of zinc toxicity on sugar beet (Beta vulgaris L.) plants grown in hydroponics. Plant Biol 11:339–350PubMedCrossRefGoogle Scholar
  29. Schreiber U (1997) Chlorophyll fluorescence and photosynthetic energy conversion: simple introductory experiments with the TEACHING-PAM Chlorophyll Fluorometer. Heinz Walz GmbH, Effeltrich, p 73Google Scholar
  30. Sochia AL, Guerinot ML (2014) Mn-euvering manganese: the role of transporter gene family members in manganese uptake and mobilization in plants. Front Plant Sci 5:1–16Google Scholar
  31. Stephens BW, Cook DR, Grusak MA (2011) Characterization of zinc transport by divalent metal transporters of the ZIP family from the model legume Medicago truncatula. Biometals 24:51–58PubMedCrossRefGoogle Scholar
  32. Stiborova M, Ditrichova M, Brezinova A (1987) Effect of heavy metal ions on growth and biochemical characteristics of photosynthesis of barley and maize seedlings. Biol Plant 29:5453–5467Google Scholar
  33. Suzuki N, Yamaguchi Y, Koizumi N, Sano H (2002) Functional characterization of a heavy metal binding protein CdI19 from Arabidopsis. Plant J 32:165–173PubMedCrossRefGoogle Scholar
  34. Thomas G, Stark HJ, Wellenreuther G, Dickinson B, Küpper H (2013) Effects of nanomolar copper on water plants—comparison of biochemical and biophysical mechanisms of deficiency and sublethal toxicity under environmentally relevant conditions. Aquat Toxicol 140–141:27–36PubMedCrossRefGoogle Scholar
  35. Vangrosveld J, Clijsters H (1994) Toxic effect of metals. In: Farago MG (ed) Plants and the chemical elements. VCH, Weinheim, pp 149–177CrossRefGoogle Scholar
  36. Wojcik M, Tukiendorf A (2003) Response of wild type of Arabidopsis thaliana to copper stress. Biol Plant 46:79–84CrossRefGoogle Scholar
  37. Wu J, Zhao FJ, Ghandilyan A, Logoteta B, Guzman MO, Schat H, Wang X, Aarts MGM (2009) Identification and functional analysis of two ZIP metal transporters of the hyperaccumulator Thlaspi caerulescens. Plant Soil 325:79–95CrossRefGoogle Scholar
  38. Yruela I (2005) Copper in plants. Braz J Plant Physiol 17:145–156CrossRefGoogle Scholar
  39. Yruela I (2009) Copper in plants: acquisition, transport and interactions. Funct Plant Biol 36:409–430CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • I. E. Zlobin
    • 1
  • V. P. Kholodova
    • 1
  • Z. F. Rakhmankulova
    • 1
  • Vl. V. Kuznetsov
    • 1
    • 2
  1. 1.Timiryazev Institute of Plant PhysiologyRussian Academy of SciencesMoscowRussia
  2. 2.Department of Plant PhysiologyMoscow State UniversityMoscowRussia

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