Laboratory of Plant Nutrition, Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba, 271-8510, Japan.
Plant Mol Biol. 2014 Dec;86(6):627-39. doi: 10.1007/s11103-014-0252-3. Epub 2014 Sep 26.
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.
油菜叶片原生质体的分离诱导叶绿体中活性氧的产生和积累。在原生质体和原生质体叶绿体中检测到一种激活的 NADPH 氧化酶样蛋白同工型。本研究旨在确定积累 H2O2 的原生质体叶绿体中的 NADH 氧化酶样活性。该蛋白的蛋白质组分析揭示了一种铁氧还蛋白：NADPH 氧化还原酶（FNR1）同工型。虽然叶片中 LFNR1 转录本高度表达，但原生质体中的表达显著降低。原生质体叶绿体主要表达可溶性 FNR1 蛋白。虽然白芥（Brassica oleracea var. acephala f. tricolor cv. white pigeon）的白化叶片与油菜叶片表达相同水平的 FNR1 蛋白，但白化叶片的原生质体显示出降低的 FNR1 表达。白芥白化叶片的原生质体在细胞质和质膜上产生和积累 H2O2。细胞内 pH 表明叶绿体呈酸性，这表明叶绿体基质中产生了过量的 H(+)。在原生质体分离过程中，原生质体叶绿体中的 NADPH 含量增加了六倍以上。本研究报告了一种通过过表达可溶性 FNR 将电子传递给氧的可能性，并表明 FNR 在利用 NADPH 的任何过量还原能力方面具有功能。
