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

太阳光照强度(P)在自然条件下变化很大，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(A)所需的P时，多余的光子产生活性氧（ROS）， ROS使叶绿体失活（光抑制）。为了适应这种光子过度胁迫，叶绿体会配备松弛系统来抑制ROS的产生和清除系统。在本研究项目中，建立单线态氧（^ 10_2）的检测和测定方法是主要目标。由于其寿命极短（10 μs），我们采用类囊体膜相关荧光探针（DPAX和DanePy）和脉冲幅度调制系统对探针进行了低光解和光化光照下探针荧光变化的测定。所建立的方法可以实时测定类囊体中^ 10_2的生成。类囊体在光化光照后立即产生^ 10_2，这表明这种ROS是在没有特定的类囊体光抑制的情况下产生的。此外，在无类囊体电子受体的厌氧条件下，^ 10_2的生成增加了5-6倍。换句话说，这一发现表明电子向氧的转移（水循环）抑制了PSII中^ o_2的产生和^ o_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对其维持的松弛系统，以及参与水循环的酶的功能。

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.