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Overproduction of stromal ferredoxin:NADPH oxidoreductase in H2O2-accumulating Brassica napus leaf protoplasts

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Abstract

The isolation of Brassica napus leaf protoplasts induces reactive oxygen species generation and accumulation in the chloroplasts. An activated isoform of NADPH oxidase-like protein was detected in the protoplasts and the protoplast chloroplasts. The purpose of this study is to define the NADH oxidase-like activities in the H2O2-accumulating protoplast chloroplasts. Proteomic analysis of this protein revealed an isoform of ferredoxin:NADPH oxidoreductase (FNR1). While leaves highly expressed the LFNR1 transcript, protoplasts decreased the expression significantly. The protoplast chloroplasts predominantly expressed soluble FNR1 proteins. While the albino leaves of white kale (Brassica oleracea var. acephala f. tricolor cv. white pigeon) expressed FNR1 protein at the same level as B. napus leaves, the protoplasts of albino leaves displayed reduced FNR1 expression. The albino leaf protoplasts of white kale generated and accumulated H2O2 in the cytoplasm and on the plasma membrane. Intracellular pH showed that the chloroplasts were acidic, which suggest that excess H+ was generated in chloroplast stroma. NADPH content of the protoplast chloroplasts increased by over sixfold during the isolation of protoplasts. This study reports a possibility of mediating electrons to oxygen by an overproduced soluble FNR, and suggests that the FNR has a function in utilizing any excess reducing power of NADPH.

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Acknowledgments

The authors are grateful to Prof. Poul Erik Jensen, University of Copenhagen and Prof. Toshiraru Hase, Institute for Protein Research, Osaka University for kindly gifting FNR1 antibody. This study was supported by the Japan Society for the Promotion of Science (JSPS) [Grant-in-Aid for a post-doctoral fellowship (P08413) to R.K.T.] and The Sumitomo Foundation to M.W.

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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. Terrestrial Ecosystem Research, Vienna Ecology Center (VEC), Faculty of Life Sciences, University of Vienna, Althanstrasse 14, 1090, Vienna, Austria

    Rajesh Kumar Tewari

  3. Clinical Proteomics Research Center, Chiba University Hospital, Chiba, Japan

    Mamoru Satoh

  4. Chemical Analysis Center, Chiba University, Chiba, Japan

    Mamoru Satoh & Sayaka Kado

  5. Laboratory of Genetics and Plant Breeding, Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba, 271-8510, Japan

    Kohei Mishina

  6. National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan

    Kohei Mishina

  7. Division of Applied Biological Chemistry, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba, 271-8510, Japan

    Misato Anma, Kazuhiko Enami & Mitsumasa Hanaoka

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  1. Rajesh Kumar Tewari
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Correspondence to Masami Watanabe.

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Tewari, R.K., Satoh, M., Kado, S. et al. Overproduction of stromal ferredoxin:NADPH oxidoreductase in H2O2-accumulating Brassica napus leaf protoplasts. Plant Mol Biol 86, 627–639 (2014). https://doi.org/10.1007/s11103-014-0252-3

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