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Endogenous nitric oxide generation in protoplast chloroplasts

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Abstract

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NO generation is studied in the protoplast chloroplasts. NO, ONOO − and ROS (O − 2 and H 2 O 2 ) are generated in chloroplasts. Nitric oxide synthase-like protein appears to be involved in NO generation.

Abstract

Nitric oxide stimulates chlorophyll biosynthesis and chloroplast differentiation. The present study was conducted to better understand the process of NO generation in the leaf chloroplasts and protoplasts. NO, peroxynitrite and superoxide anion were investigated in the protoplasts and isolated chloroplasts using specific dyes, confocal laser scanning and light microscopy. The level of NO was highest after protoplast isolation and subsequently decreased during culture. Suppression of NO signal in the presence of PTIO, suggests that diaminofluorescein-2 diacetate (DAF-2DA) detected NO. Detection of peroxynitrite, a reaction product of NO and superoxide anion, further suggests NO generation. Moreover, generation of NO and peroxynitrite in the chloroplasts of wild-type Arabidopsis and their absence or weak signals in the leaf-derived protoplasts of Atnoa1 mutants confirmed the reactivity of DAF-2DA and aminophenyl fluorescein to NO and peroxynitrite, respectively. Isolated chloroplasts also showed signal of NO. Suppression of NO signal in the presence of 100 μM nitric oxide synthase inhibitors [l-NNA, Nω-nitro-l-arginine and PBIT, S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea] revealed that nitric oxide synthase-like system is involved in NO synthesis. Suppression of NO signal in the protoplasts isolated in the presence of cycloheximide suggests de novo synthesis of NO generating protein during the process of protoplast isolation. Furthermore, the lack of inhibition of NO production by sodium tungstate (250 μM) and inhibition by l-NNA, and PBIT suggest involvement NOS-like protein, but not nitrate reductase, in NO generation in the leaf chloroplasts and protoplasts.

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Abbreviations

APF:

Aminophenyl fluorescein

DAF-2DA:

Diaminofluorescein-2 diacetate

H2DCF-DA:

2,7-Dichlorodihydrofluorescein diacetate

l-NNA:

Nω-nitro-l-arginine

NO:

Nitric oxide

NOS:

NO synthase

PBIT:

S,S′-1,3-Phenylene-bis(1,2-ethanediyl)-bis-isothiourea

PCD:

Programmed cell death

PTIO:

2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide

ROS:

Reactive oxygen species

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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. Eisho Nishino and Dr. Kohei Mishina, Chiba University, for their help in preparation of microtome sections of leaf and confocal microscopy of samples, respectively.

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Authors and Affiliations

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

    Rajesh Kumar Tewari & Masami Watanabe

  2. Department of Terrestrial Ecosystem Research, Faculty of Life Sciences, University of Vienna, Althanstrasse 14, 1090, Vienna, Austria

    Rajesh Kumar Tewari & Judith Prommer

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  1. Rajesh Kumar Tewari
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  2. Judith Prommer
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  3. Masami Watanabe
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Correspondence to Rajesh Kumar Tewari or Masami Watanabe.

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Communicated by P. Kumar.

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Tewari, R.K., Prommer, J. & Watanabe, M. Endogenous nitric oxide generation in protoplast chloroplasts. Plant Cell Rep 32, 31–44 (2013). https://doi.org/10.1007/s00299-012-1338-5

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  • DOI: https://doi.org/10.1007/s00299-012-1338-5

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