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Development of Assay Method of Singlet Excited Oxygen in Chloroplasts and Molecular Mechanisms of Its Generation, Scavenging and Action

叶绿体中单线态激发氧测定方法及其产生、清除和作用分子机制的建立

基本信息

批准号：
15370026
负责人：
ASADA Kozi
金额：
$ 6.72万
依托单位：
Fukuyama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Sun light intensity (P) is very variable under natural conditions, not only by diurnal change but also by meteorological factors. When P is higher than those required for CO_2-assimilation (A), excess photons produce reactive oxygen species (ROS) and the ROS inactivates chloroplasts(photoinhibition). To adapt such photon excess stress chloroplasts would equip the relaxation system to suppress the ROS production and the scavenging system of ROS. In this research project, establishment of the detection and assay of singlet oxygen (^1O_2) is the primary object. Because its life time is very short (10 μs ), we used thylakoid membrane-associable fluorescence probes (DPAX and DanePy) and the pulse-amplitude modulation system for its low photodecomposition of the probes and the assay of the fluorescence change of the probes under actinic light illumination.The established method allows to determine at real time the generation of ^1O_2 in thylakoids. Thylakoids generates ^1O_2 immediately after the actinic light illumination indicating that this species of ROS is photoproduced without specific photoinhibition of thylakoids. Further, ^1O_2 generation is increased by 5-6 folds under anaerobic conditions where no electron acceptor for thylakoids is available. In other words, this finding indicates that the electron transfer to oxygen (water-water cycle) suppresses the generation of ^1O_2 in PSII and the ^1O_2-induced photoinhibition. In addition, the relaxation systems to escape from P>A stress have been revealed for the participating enzymes in the cyclic electron flow through PSI and its maintenance by ascorbate, the function of and participating enzymes in the water-water cycle.

在自然条件下，太阳光强度（P）变化很大，不仅受日变化的影响，而且受气象因素的影响。当P高于CO_2同化所需的P（A）时，过量的光子产生活性氧（ROS），使叶绿体失活（光抑制）。为了适应这种光子过度胁迫，叶绿体将配备松弛系统来抑制ROS的产生和ROS的清除系统。本研究的主要目的是建立单线态氧（^1O_2）的检测方法。由于类囊体膜结合荧光探针的寿命很短（10 μs），我们采用了DPAX和DanePy两种荧光探针，并利用其低的光分解性和光化光照射下探针荧光变化的脉冲幅度调制系统，建立了真实的实时测定类囊体中^1O_2产生的方法。类囊体在光化光照射后立即产生^1O_2，表明这种ROS是光产生的，而类囊体没有特异性的光抑制。此外，在无电子受体的厌氧条件下，^1O_2的产生增加了5-6倍。也就是说，电子向氧的转移（水-水循环）抑制了PSII中^1O_2的产生和^1O_2诱导的光抑制。此外，还揭示了参与PSI循环电子流的酶和抗坏血酸维持PSI循环电子流的酶以及参与水-水循环的酶的功能。