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Early generation of nitric oxide contributes to copper tolerance through reducing oxidative stress and cell death in hulless barley roots

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Abstract

The objective of this study was to investigate the specific role of nitric oxide (NO) in the early response of hulless barley roots to copper (Cu) stress. We used the fluorescent probe diaminofluorescein-FM diacetate to establish NO localization, and hydrogen peroxide (H2O2)-special labeling and histochemical procedures for the detection of reactive oxygen species (ROS) in the root apex. An early production of NO was observed in Cu-treated root tips of hulless barley, but the detection of NO levels was decreased by supplementation with a NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO). Application of sodium nitroprusside (a NO donor) relieved Cu-induced root inhibition, ROS accumulation and oxidative damage, while c-PTIO treatment had a synergistic effect with Cu and further enhanced ROS levels and oxidative stress. In addition, the Cu-dependent increase in activities of superoxide dismutase, peroxidase and ascorbate peroxidase were further enhanced by exogenous NO, but application of c-PTIO decreased the activities of catalase and ascorbate peroxidase in Cu-treated roots. Subsequently, cell death was observed in root tips and was identified as a type of programed cell death (PCD) by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The addition of NO prevented the increase of cell death in root tips, whereas inhibiting NO accumulation further increased the number of cells undergoing PCD. These results revealed that NO production is an early response of hulless barley roots to Cu stress and that NO contributes to Cu tolerance in hulless barley possibly by modulating antioxidant defense, subsequently reducing oxidative stress and PCD in root tips.

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Abbreviations

ASC:

Ascorbate

CAT:

Catalase

Cu:

Copper

c-PTIO:

2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

DAB:

Diaminobenzine

DAPI:

4′,6-Diamidino-2-phenylindole

DAF-FM:

DA 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate

DTT:

Dithiothreitol

DW:

Dry weight

GPX:

Guaiacol peroxidase

GR:

Glutathione reductase

GSH:

Glutathione

H2O2 :

Hydrogen peroxide

MDA:

Malondialdehyde

NBT:

Nitro blue tetrazolium

NO:

Nitric oxide

NOS:

Nitric oxide synthase

NR:

Nitrate reductase

PBS:

Phosphate buffered saline

PCD:

Programmed cell death

PI:

Propidium iodide

POD:

Peroxidase

SNP:

Sodium nitroprusside

TBA:

Thiobarbituric acid

TCA:

Trichloroacetic acid

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Acknowledgments

This work was supported by Open Funds for Key Laboratory of Mollisols Agroecology of Chinese Academy of Sciences and Heilongjiang Province Science Foundation for Youths (QC2015036).

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  1. Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin, 150000, China

    Yanfeng Hu

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Hu, Y. Early generation of nitric oxide contributes to copper tolerance through reducing oxidative stress and cell death in hulless barley roots. J Plant Res 129, 963–978 (2016). https://doi.org/10.1007/s10265-016-0841-0

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