Laboratory of Plant Nutrition, Faculty of Horticulture, Chiba University, 648, Matsudo, Chiba, 271-8510, Japan.
Plant Cell Rep. 2013 Jan;32(1):31-44. doi: 10.1007/s00299-012-1338-5. Epub 2012 Sep 13.
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. 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.
质体中未检测到任何形式的一氧化氮（NO）生成。NO、过氧亚硝酸根（ONOO(-)）和活性氧（O2(-)和 H2O2）在叶绿体中生成。一氧化氮合酶样蛋白似乎参与了 NO 的生成。NO 刺激叶绿素生物合成和叶绿体分化。本研究旨在更好地理解叶片质体和原生质体中 NO 生成的过程。使用特定染料、共聚焦激光扫描和明场显微镜，在原生质体和分离的叶绿体中检测到 NO、过氧亚硝酸根和超氧阴离子。原生质体分离后，NO 水平最高，随后在培养过程中逐渐降低。在 PTIO 存在下抑制 NO 信号表明，二氨基荧光素-2 二乙酸酯（DAF-2DA）检测到的是 NO。过氧亚硝酸根（NO 和超氧阴离子的反应产物）的检测进一步表明 NO 的生成。此外，野生型拟南芥叶绿体中生成了 NO 和过氧亚硝酸根，而 Atnoa1 突变体叶衍生原生质体中不存在或信号较弱，证实了 DAF-2DA 和氨苯基荧光素分别对 NO 和过氧亚硝酸根的反应性。分离的叶绿体也显示出 NO 的信号。在存在 100μM 一氧化氮合酶抑制剂 [L-NNA、Nω-硝基-L-精氨酸和 PBIT、S，S'-1，3-亚苯基-双（1，2-乙二基）-双异硫脲] 的情况下抑制 NO 信号表明，一氧化氮合酶样系统参与了 NO 的合成。在存在环己亚胺的情况下抑制原生质体中 NO 信号表明，在原生质体分离过程中，NO 生成蛋白的从头合成。此外，钨酸钠（250μM）对 NO 生成的抑制作用以及 L-NNA 和 PBIT 的抑制作用表明，在叶片质体和原生质体中，NO 的生成涉及 NOS 样蛋白，而不是硝酸还原酶。
