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Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of Brassica napus leaf protoplasts

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Abstract

Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H2O2 and enzymes involved in H2O2 generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H2O2 and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H2O2 was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H2O2 in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H2O2 generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.

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Abbreviations

APX:

Ascorbate peroxidase

AsA:

Ascorbic acid

DAB-POD:

3,3-Diaminobenzidine peroxidase

DHA:

Dehydroascorbic acid

DiOC6 :

3,3′-Dihexyloxacarbocyanine iodide

DPI:

Diphenyleneiodonium chloride

G-POD:

Guaiacol peroxidase

H2DCF-DA:

2′,7′-Dichlorodihydrofluorescein diacetate

ME:

Macerating enzyme

NOX:

NADPH oxidase

PCD:

Programmed cell death

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

XTT:

2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt

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Acknowledgments

This work was financially supported by Japan Society for the Promotion of Science (JSPS) as a postdoctoral fellowship (P08413) to RKT and grant-in-aid for scientific research. Authors are thankful to Prof. Esso Nishino, Chiba University, for his help in preparation of microtome sections of leaf.

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  1. Daisuke Watanabe

    Present address: Department of Medical Pharmacy, Faculty of Pharmaceutical Sciences, Josai International University, 1 Gumyo, Togane, Chiba, 283-8555, Japan

Authors and Affiliations

  1. Laboratory of Plant Nutrition, Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba, 271-8510, Japan

    Rajesh Kumar Tewari, Daisuke Watanabe & Masami Watanabe

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  1. Rajesh Kumar Tewari
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Correspondence to Masami Watanabe.

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Tewari, R.K., Watanabe, D. & Watanabe, M. Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of Brassica napus leaf protoplasts. Planta 235, 99–110 (2012). https://doi.org/10.1007/s00425-011-1495-8

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