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

在自然条件下，太阳光强(P)变化很大，不仅受日变化的影响，还受气象因素的影响。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)，ROS使叶绿体失活(光抑制)。为了适应这种光子过量胁迫，叶绿体将装备松弛系统来抑制ROS的产生和清除ROS的系统。本研究以建立单线态氧(^1O_2)检测分析方法为主要研究对象。由于类囊体膜结合荧光探针(DPAX和DanePy)的寿命很短(10μS)，利用其光解低的特点和脉冲幅度调制系统来测定其在光化光照下的荧光变化，建立了实时测定类囊体中^1O2的方法。类囊体在光化光照后立即产生^1O_2，表明该种ROS是在没有特定光抑制的情况下光产生的。此外，在没有类囊体电子受体的厌氧条件下，^1O_2的产生增加了5-6倍。换句话说，这一发现表明，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循环电子流的酶的逃逸P&GT；A胁迫的松弛系统，以及抗坏血酸维持PSI的松弛系统，水-水循环中的功能和参与酶。

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.