title: Overproduction of stromal ferredoxin:NADPH oxidoreductase in H2O 2-accumulating Brassica napus leaf protoplasts.
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.
authors.affiliation: Laboratory of Plant Nutrition, Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba, 271-8510, Japan.
authors.forename: Rajesh Kumar
authors.initials: RK
authors.lastname: Tewari
authors.forename: Mamoru
authors.initials: M
authors.lastname: Satoh
authors.forename: Sayaka
authors.initials: S
authors.lastname: Kado
authors.forename: Kohei
authors.initials: K
authors.lastname: Mishina
authors.forename: Misato
authors.initials: M
authors.lastname: Anma
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Enami
authors.forename: Mitsumasa
authors.initials: M
authors.lastname: Hanaoka
authors.forename: Masami
authors.initials: M
authors.lastname: Watanabe
mesh_terms: D029688:Brassica napus / Q000201:enzymology* / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D054046:Plant Stomata / Q000201:enzymology / Q000235:genetics; D040901:Proteomics; D011523:Protoplasts; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D005288:Ferredoxins; D007527:Isoenzymes; D010940:Plant Proteins; D017382:Reactive Oxygen Species; D009249:NADP; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases