管藻黄素型LHCII参与的假根羽藻非光化学淬灭机制研究

In LHCII of Bryopsis corticulans, a unique siphonaceous marine green alga living in intertidal zones in a large area around the east coast of our country, siphonaxanthin and siphonein replace lutein and xanthophyll cycle pigments to adapt complicated environment where light illumination oscillates between strong and insufficient intensities depending on tidal changes. In previous studies, we demonstrated the light harvesting characteristics and sequences of B. corticulans LHCII, but the mechanism of NPQ (non-photochemical quench) in this unique alga is unknown. In this proposal, we will investigate the NPQ mechanism of B. corticulans, comparing with that in higher plants, by the experiments in alga, intact chloroplast and membrane protein levels. Chlorophyll fluorescence will be measured to analyze the photosynthetic efficiency (Y (II)) and NPQ of B. corticulans, and isolated intact chloroplast will be used for high light and inhibitor treatment to find the NPQ characteristics in this intertidal green alga. We will isolate natural LHCII from B. corticulans and also prepare reconstituted LHCII by expressing the LHCII apo-proteins in E. coli followed by reconstitution with different carotenoids. We will then analyze the properties of the native and reconstituted LHCII in quenched and unquenched states by absorption spectrum, fluorescence spectrum and florescence yield measurement. We will also investigate possible conformational changes of the proteins and carotenoids between quench and unquenched states by means of Circular Dichroism. And Nanosecond time-resolved spectra will help us analyze the triplet states features of siphonaxanthin and siphonein in B. corticulans LHCII involved with NPQ . These studies will enable us to reveal the mechanism of NPQ and the roles of siphonaxanthin and siphonein in LHCII of the unique green alga B. corticulans.

假根羽藻是生长在潮间带的低等绿藻，其LHCII结合管藻黄素和管藻素，替代黄体素和叶黄素循环色素以适应剧烈变化的强光和弱光环境。此前我们研究了假根羽藻LHCII序列和捕获绿光机制，但其非光化学淬灭机制还不清楚。本项目拟在藻体、完整叶绿体和膜蛋白三个层次与高等植物对比研究管藻黄素型LHCII参与的假根羽藻非光化学淬灭机制。通过叶绿素荧光技术研究假根羽藻的光合效率和非光化学淬灭特点，对完整叶绿体进行胁迫处理，进一步了解假根羽藻非光化学淬灭机制。制备天然LHCII和重组不同类胡萝卜素的LHCII，通过吸收光谱、荧光光谱和叶绿素荧光产量测定，研究假根羽藻LHCII在能量淬灭和非淬灭状态下的特征；通过圆二色光谱分析LHCII蛋白构象和类胡萝卜素构象变化；通过纳秒时间分辨光谱研究淬灭和非淬灭状态下LHCII中管藻黄素和管藻素的三重态性质，最终阐明管藻黄素型LHCII参与的假根羽藻非光化学淬灭机制。

假根羽藻是生长在潮间带的低等绿藻，其LHCII结合管藻黄素和管藻素，替代黄体素和叶黄素循环色素以适应剧烈变化的强光和弱光环境。本项目在藻体、完整叶绿体和膜蛋白三个层次与高等植物对比研究了管藻黄素型LHCII参与的假根羽藻光保护机制。在项目执行期间，我们按照计划任务在野外和室内检测假根羽藻的光合生理特征、在实验室内对假根羽藻的藻体、叶绿体和LHCII蛋白进行了生化和光谱学研究；按照计划测定了假根羽藻和拟南芥的NPQ及电子传递速率等，研究了完整叶绿体和LHCII对强光和温度胁迫的响应机制，研究了假根羽藻LHCII 捕光和光保护的超快光谱特征，阐明了假根羽藻适应潮间带光环境的可能机理。. 研究结果揭示了假根羽藻对潮间带环境的适应机制与假根羽藻持续的NPQ、以及PSII核心亚基的快速修复和重整有关(Giovagnetti et al.2018, Planta)；阐明了假根羽藻LHCII 中类胡萝卜素三重态能量传递的特征在于管藻黄素和管藻素同时在两个黄体素位点对叶绿素三重态激发能进行淬灭(Li et al. 2020, BBA - Bioenergetics)；阐释了管藻黄素和管藻素参与的假根羽藻非光化学淬灭与其他捕光天线的不同机制 (Qin et al. 2019, Nature Plants; Wang et al. 2020, FEBS journal)。另外也对假根羽藻LHCII中的叶绿素a/b位点以及超快能量传递进行了研究，证实了假根羽藻LHCII具有较低的传能速度，但保证了PSII的高效运转和光保护 (Akhtar et al. 2020, BBA – Bioenergetics)。