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Excess copper induces accumulation of hydrogen peroxide and increases lipid peroxidation and total activity of copper–zinc superoxide dismutase in roots of Elsholtzia haichowensis

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Abstract

The effects of excess copper (Cu) on the accumulation of hydrogen peroxide (H2O2) and antioxidant enzyme activities in roots of the Cu accumulator Elsholtzia haichowensis Sun were investigated. Copper at 100 and 300 μM significantly increased the concentrations of malondialdehyde and H2O2, and the activities of catalase (E.C. 1.11.1.6), ascorbate peroxidase (E.C. 1.11.1.11), guaiacol peroxidase (GPOD, E.C. 1.11.1.7) and superoxide dismutase (SOD, E.C. 1.15.1.1). Isoenzyme pattern and inhibitor studies showed that, among SOD isoforms, only copper–zinc superoxide dismutase (CuZn–SOD) increased. Excess Cu greatly increased the accumulation of superoxide anion (O2 ·−) and H2O2 in E. haichowensis roots. This study also provides the first cytochemical evidence of an accumulation of H2O2 in the root cell walls as a consequence of Cu treatments. Experiments with diphenyleneiodonium as an inhibitor of NADPH oxidase, 1,2-dihydroxybenzene-3,5-disulphonic acid as an O2 ·− scavenger, and N-N-diethyldithiocarbamate as an inhibitor of SOD showed that the source of H2O2 in the cell walls could partially be NADPH oxidase. The enzyme can use cytosolic NADPH to produce O2 ·−, which rapidly dismutates to H2O2 by SOD. Apoplastic GPOD and CuZn–SOD activities were induced in roots of E. haichowensis with 100 μM Cu suggesting that these two antioxidant enzymes may be responsible for H2O2 accumulation in the root apoplast.

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Abbreviations

APX :

Ascorbate peroxidase

CAT:

Catalase

CuZn–SOD:

Copper–zinc superoxide dismutase

DAB:

3,3-Diaminobenzidine

DDC:

N-N-diethyldithiocarbamate

DPI:

Diphenyleneiodonium

GPOD:

Guaiacol peroxidase

H2DCFDA :

2′,7′-Dichlorodihydrofluorescein diacetate

MDA :

Malondialdehyde

NBT:

Nitroblue tetrazolium

Tiron:

1,2-Dihydroxybenzene-3,5-disulphonic acid

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Acknowledgments

We would like to thank the Electron Microscopy Unit of the Centre of Biological Instrumentation of Nanjing Agricultural University for providing laboratory facilities and Dr Jianhua Hao for her technical assistance in the microscopy analysis. This research project was supported by the National Natural Science Foundation of China (No. 30471036), the 111 project (No B07030), and the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institutions of MOE.

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  1. College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, China

    Hongxiao Zhang, Yan Xia, Guiping Wang & Zhenguo Shen

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  1. Hongxiao Zhang
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Correspondence to Zhenguo Shen.

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Zhang, H., Xia, Y., Wang, G. et al. Excess copper induces accumulation of hydrogen peroxide and increases lipid peroxidation and total activity of copper–zinc superoxide dismutase in roots of Elsholtzia haichowensis . Planta 227, 465–475 (2008). https://doi.org/10.1007/s00425-007-0632-x

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