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Induction of reactive oxygen species and the potential role of NADPH oxidase in hyperhydricity of garlic plantlets in vitro

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Abstract

Hyperhydricity is a physiological disorder associated with oxidative stress. Reactive oxygen species (ROS) generation in plants is initiated by various enzymatic sources, including plasma membrane-localized nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, cell wall-bound peroxidase (POD), and apoplastic polyamine oxidase (PAO). The origin of the oxidative burst associated with hyperhydricity remains unknown. To investigate the role of NADPH oxidases, POD, and PAO in ROS production and hyperhydricity, exogenous hydrogen peroxide (H2O2) and inhibitors of each ROS-producing enzyme were applied to explore the mechanism of oxidative stress induction in garlic plantlets in vitro. A concentration of 1.5 mM H2O2 increased endogenous ROS production and hyperhydricity occurrence and enhanced the activities of NADPH oxidases, POD, and PAO. During the entire treatment period, NADPH oxidase activity increased continuously, whereas POD and PAO activities exhibited a transient increase and subsequently declined. Histochemical and cytochemical visualization demonstrated that specific inhibitors of each enzyme effectively suppressed ROS accumulation. Moreover, superoxide anion generation, H2O2 content, and hyperhydric shoot frequency in H2O2-stressed plantlets decreased significantly. The NADPH oxidase inhibitor was the most effective at suppressing superoxide anion production. The results suggested that NADPH oxidases, POD, and PAO were responsible for endogenous ROS induction. NADPH oxidase activation might play a pivotal role in the oxidative burst in garlic plantlets in vitro during hyperhydricity.

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Abbreviations

BSA:

Bovine serum albumin

CaCl2 :

Calcium chloride

CBA:

Sodium cacodylate

CeCl3 :

Cerous chloride

DAB:

3,3-Diaminobenzidine

DPI:

Diphenylene iodonium

DTT:

dl-Dithiothreitol

EDTA:

Ethylene diamine tetraacetic acid

EGTA:

Ethylene glycol tetraacetic acid

GUA:

Guazatine

H2O2 :

Hydrogen peroxide

IWF:

Infiltrated washing fluid

KCl:

Potassium chloride

KH2PO4 :

Monopotassium phosphate

KI:

Potassium iodide

MOPS:

3-[N-morpholino] propanesulfonic acid

NADPH:

Nicotinamide adenine dinucleotide phoshate

NaN3 :

Sodium azide

NBT:

Nitro blue tetrazolium

O2 :

Superoxide anion

O2 :

Singlet oxygen

PAO:

Polyamine oxidase

PEG:

Polyethylene glycol

pH:

Potential of hydrogen

PMSF:

Phenylmethylsulfonyl fluoride

POD:

Peroxidase

PVP:

Polyvinylpyrrolidone

ROS:

Reactive oxygen species

TCA:

Trichloroacetic acid

XTT:

3′-[1-[Phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (31372056) and Doctoral Fund of Ministry of Education of China (200803071012).

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Authors and Affiliations

  1. College of Horticulture, Ministry of Agriculture, Nanjing Agricultural University/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Nanjing, 210095, People’s Republic of China

    Jie Tian, Yaqi Cheng, Xiangyu Kong, Min Liu, Fangling Jiang & Zhen Wu

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  1. Jie Tian
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  2. Yaqi Cheng
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  3. Xiangyu Kong
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  4. Min Liu
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  5. Fangling Jiang
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Correspondence to Zhen Wu.

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Tian, J., Cheng, Y., Kong, X. et al. Induction of reactive oxygen species and the potential role of NADPH oxidase in hyperhydricity of garlic plantlets in vitro. Protoplasma 254, 379–388 (2017). https://doi.org/10.1007/s00709-016-0957-z

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  • DOI: https://doi.org/10.1007/s00709-016-0957-z

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