浒苔LHCSR和PSBS蛋白在叶绿素荧光非光化学猝灭中的作用

Photosynthetic organisms respond to strong illumination by activating several photoprotection mechanisms. The fastest and most important among these mechanisms is non-photochemical quenching (NPQ), which dissipates the excess absorbed thermal energy and is activated within seconds after any change in light intensity. In vascular plants NPQ is essentially controlled by low pH in the thylakoid lumen, and further depends on the action of PSBS protein and xanthophylls cycle. PSBS protein acts in chloroplast lumenal pH sensing and in activation of NPQ via conformational changes in the antenna of PSII. However, in unicellular green algae, NPQ is more dependent on lumen pH and a different LHC protein, namely LHCSR. In the green algae Chlamydomonas reinhardtii LHCSR protein combines the functions of pH sensor and the chlorophyll excited state quenching needed for NPQ, which in plants is performed by two distinct protein components: PsbS and Lhcb subunits. Ulva species are intertidal pluricellular green algae in the early evolutionary stage of land colonization, has to adapt to the wide variation in light intensity, making the species particularly rewarding for studying the evolution of photoprotective mechanisms. We have found the accumulation of PSBS and LHCSR transcripts for the first time in the green tide-forming alga Ulva prolifera, but their biochemical properties are still unknown. And in this study, we will analysis biochemical functions of PSBS and LHCSR proteins in U. prolifera and confirm whether these two proteins could sense proton gradient in the thylakoid lumen, form complexes with pigments and active chlorophyll quenching and energy dissipation directly. Also, we will compare the biochemical functions of PSBS and LHCSR proteins in U. prolifera with those in unicellular and land plants, which will contribute to the evolution analysis of photoprotective mechanisms.

叶绿素荧光非光化学猝灭（NPQ）是光合生物中最重要的光保护机制之一。高等植物的NPQ依赖于PSBS蛋白，该蛋白通过感受质子梯度引起捕光复合物系统II构象变化从而激活NPQ。与高等植物不同，单细胞绿藻的NPQ依赖于LHCSR蛋白，与PSBS蛋白不同，LHCSR蛋白不仅能感受质子梯度，而且能结合色素进行直接的能量猝灭。石莼属绿藻是一种潮间带海洋大型藻类和绿潮藻类，生存的光照条件介于单细胞绿藻与陆生植物之间。本课题组首次在我国最主要的绿潮藻种浒苔中发现存在共同表达的LHCSR和PSBS的基因及蛋白，本申请书将在此研究基础上，以LHCSR和PSBS蛋白能否感受质膜质子梯度、能否结合色素、能否直接进行能量猝灭为主要研究内容，旨在揭示这两种蛋白在浒苔非光化学猝灭中的功能，同时与单细胞绿藻和高等植物的NPQ机制进行比较，为完善光合生物的光保护分子机制及其进化历程奠定基础。

叶绿素荧光非光化学猝灭（NPQ）是光合生物中最重要的光保护机制之一。高等植物的NPQ 依赖于PSBS 蛋白，该蛋白通过感受质子梯度引起捕光复合物系统II 构象变化从而激活NPQ。与高等植物不同，单细胞绿藻的NPQ 依赖于LhcSR 蛋白，与PSBS 蛋白不同，LhcSR 蛋白不仅能感受质子梯度，而且能结合色素进行直接的能量猝灭。浒苔是一种潮间带海洋大型藻类和绿潮藻类，生存的光照条件介于单细胞绿藻与陆生植物之间。本研究发现缘管浒苔中LhcSR和PSBS蛋白结构与高等植物类似，分别具有三个和四个跨膜螺旋结构，并具有与高等植物蛋白类似的保守氨基酸序列。系统进化分析发现藻类中的PSBS蛋白与高等植物形成两大类，说明在进化的早期已经分化，推测由于生存环境的不同，PSBS蛋白在藻类和高等植物中有功能分化。对两种蛋白进行定位研究发现其均定位在光合系统的PSII上，通过体外色素结合实验发现LhcSR蛋白具有很强的色素结合能力。通过荧光定量和Western杂交检测LhcSR和PSBS基因及蛋白的表达模式，发现LhcSR和PSBS基因和蛋白均在强光诱导条件下表达量上调，说明在缘管浒苔中两种蛋白可能都发挥光保护的功能，且LhcSR对强光的响应更加显著。缘管浒苔的整个NPQ过程都受ΔpH的激活和调控，其中第一个过程完全由ΔpH激活和调控，而第二个过程主要是受由ΔpH激活的玉米黄素循环调控。相对于莱茵衣藻，缘管浒苔的NPQ响应模式与拟南芥更相近，我们推测缘管浒苔的对强光的响应机制可能更倾向于高等植物。