Journal of Plant Research

, Volume 129, Issue 5, pp 963–978 | Cite as

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

Regular Paper

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.

Keywords

Copper Nitric oxide Reactive oxygen species Programed cell death 

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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Copyright information

© The Botanical Society of Japan and Springer Japan 2016

Authors and Affiliations

  1. 1.Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and AgroecologyChinese Academy of SciencesHarbinChina

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