Lingvay Mónika, Akhtar Parveen, Sebők-Nagy Krisztina, Páli Tibor, Lambrev Petar H
Institute of Plant Biology, Biological Research Centre, Szeged, Hungary.
Doctoral School of Physics, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary.
Front Plant Sci. 2020 Jun 24;11:849. doi: 10.3389/fpls.2020.00849. eCollection 2020.
Excess light causes damage to the photosynthetic apparatus of plants and algae primarily via reactive oxygen species. Singlet oxygen can be formed by interaction of chlorophyll (Chl) triplet states, especially in the Photosystem II reaction center, with oxygen. Whether Chls in the light-harvesting antenna complexes play direct role in oxidative photodamage is less clear. In this work, light-induced photobleaching of Chls in the major trimeric light-harvesting complex II (LHCII) is investigated in different molecular environments - protein aggregates, embedded in detergent micelles or in reconstituted membranes (proteoliposomes). The effects of intense light treatment were analyzed by absorption and circular dichroism spectroscopy, steady-state and time-resolved fluorescence and EPR spectroscopy. The rate and quantum yield of photobleaching was estimated from the light-induced Chl absorption changes. Photobleaching occurred mainly in Chl and was accompanied by strong fluorescence quenching of the remaining unbleached Chls. The rate of photobleaching increased by 140% when LHCII was embedded in lipid membranes, compared to detergent-solubilized LHCII. Removing oxygen from the medium or adding antioxidants largely suppressed the bleaching, confirming its oxidative mechanism. Singlet oxygen formation was monitored by EPR spectroscopy using spin traps and spin labels to detect singlet oxygen directly and indirectly, respectively. The quantum yield of Chl photobleaching in membranes and detergent was found to be 3.4 × 10 and 1.4 × 10, respectively. These values compare well with the yields of ROS production estimated from spin-trap EPR spectroscopy (around 4 × 10 and 2 × 10). A kinetic model is proposed, quantifying the generation of Chl and carotenoid triplet states and singlet oxygen. The high quantum yield of photobleaching, especially in the lipid membrane, suggest that direct photodamage of the antenna occurs with rates relevant to photoinhibition . The results represent further evidence that the molecular environment of LHCII has profound impact on its functional characteristics, including, among others, the susceptibility to photodamage.
过量的光主要通过活性氧对植物和藻类的光合机构造成损害。单线态氧可由叶绿素（Chl）三重态，特别是在光系统II反应中心，与氧相互作用形成。捕光天线复合物中的叶绿素是否在氧化光损伤中起直接作用尚不清楚。在这项工作中，研究了在不同分子环境——蛋白质聚集体、嵌入去污剂胶束或重构膜（蛋白脂质体）中的主要三聚体捕光复合物II（LHCII）中叶绿素的光诱导光漂白。通过吸收光谱和圆二色光谱、稳态和时间分辨荧光以及电子顺磁共振光谱分析强光处理的影响。根据光诱导的叶绿素吸收变化估算光漂白的速率和量子产率。光漂白主要发生在叶绿素上，并伴随着剩余未漂白叶绿素的强烈荧光猝灭。与去污剂溶解的LHCII相比，当LHCII嵌入脂质膜中时，光漂白速率增加了140%。从介质中去除氧气或添加抗氧化剂在很大程度上抑制了漂白，证实了其氧化机制。使用自旋捕获剂和自旋标记分别直接和间接检测单线态氧，通过电子顺磁共振光谱监测单线态氧的形成。发现膜和去污剂中叶绿素光漂白的量子产率分别为3.4×10和1.4×10。这些值与通过自旋捕获电子顺磁共振光谱估计的活性氧产生率（约4×10和2×10）相当。提出了一个动力学模型，对叶绿素和类胡萝卜素三重态以及单线态氧的产生进行量化。光漂白的高量子产率，特别是在脂质膜中，表明天线的直接光损伤以与光抑制相关的速率发生。结果进一步证明，LHCII的分子环境对其功能特性有深远影响，包括对光损伤的敏感性等。
