Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of Brassica napus leaf protoplasts
Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of Brassica napus leaf protoplasts
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DOI：
10.1007/s00425-011-1495-8
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发表时间：
2011
期刊：
影响因子：
4.3
通讯作者：
R. K. Tewari;Daisuke Watanabe;M. Watanabe
中科院分区：
文献类型：
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作者：
R. K. Tewari;Daisuke Watanabe;M. Watanabe
尽管在过去的几年里进行了广泛的研究，但只有有限数量的植物物种观察到原生质体再生。原生质体在分离过程中经历复杂的代谢修饰。原生质体的分离诱导甘蓝型油菜叶片原生质体产生活性氧。本研究旨在为深入了解B细胞活性氧的产生机制提供新的思路。油菜叶原生质体研究了叶片和离体原生质体中H2 O2和参与H2 O2产生和解毒的酶的体内定位、分子抗氧化剂抗坏血酸及其氧化还原状态和脂质过氧化作用。在浸渍酶（ME）中孵育叶条不同的持续时间（3，6，和12小时）诱导的H2 O2和丙二醛（脂质过氧化，膜损伤的指数）在原生质体中的积累。H_2O_2的水平在原生质体分离后达到最高，随后在培养过程中下降。超氧化物生成NADPH氧化酶（NOX）样活性增强，而超氧化物歧化酶（SOD）和抗坏血酸过氧化物酶（APX）在原生质体相比，叶下降。二氨基联苯胺过氧化物酶（DAB-POD）的活性也较低的原生质体相比，叶片。总抗坏血酸含量，抗坏血酸脱氢抗坏血酸比（氧化还原状态），在原生质体相比，叶增强。原生质体中类NOX酶活性的升高和抗氧化酶（SOD、APX和DAB-POD）活性的减弱导致了H_2O_2在叶绿体中的过量积累。叶绿体NADPH氧化酶样活性介导的永久H2 O2的产生可能诱导了B.油菜叶原生质体的DNA梯状带和线粒体膜电位降低。
Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation inBrassica napusleaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation inB. napusleaf protoplasts. In vivo localization of H2O2and enzymes involved in H2O2generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H2O2and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H2O2was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H2O2in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H2O2generation probably induced apoptotic-like cell death ofB. napusleaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.