DataSheet_1_High light-induced changes in whole-cell proteomic profile and its correlation with the organization of thylakoid super-complex in cyclic electron transport mutants of Chlamydomonas reinhardtii.docx

Light and nutrients are essential components of photosynthesis. Activating the signaling cascades is critical in starting adaptive processes in response to high light. In this study, we have used wild-type (WT), cyclic electron transport (CET) mutants like Proton Gradient Regulation (PGR) (PGRL1), and PGR5 to elucidate the actual role in regulation and assembly of photosynthetic pigment–protein complexes under high light. Here, we have correlated the biophysical, biochemical, and proteomic approaches to understand the targeted proteins and the organization of thylakoid pigment–protein complexes in the photoacclimation. The proteomic analysis showed that 320 proteins were significantly affected under high light compared to the control and are mainly involved in the photosynthetic electron transport chain, protein synthesis, metabolic process, glycolysis, and proteins involved in cytoskeleton assembly. Additionally, we observed that the cytochrome (Cyt) b6 expression is increased in the pgr5 mutant to regulate proton motive force and ATPase across the thylakoid membrane. The increased Cyt b6 function in pgr5 could be due to the compromised function of chloroplast (cp) ATP synthase subunits for energy generation and photoprotection under high light. Moreover, our proteome data show that the photosystem subunit II (PSBS) protein isoforms (PSBS1 and PSBS2) expressed more than the Light-Harvesting Complex Stress-Related (LHCSR) protein in pgr5 compared to WT and pgrl1 under high light. The immunoblot data shows the photosystem II proteins D1 and D2 accumulated more in pgrl1 and pgr5 than WT under high light. In high light, CP43 and CP47 showed a reduced amount in pgr5 under high light due to changes in chlorophyll and carotenoid content around the PSII protein, which coordinates as a cofactor for efficient energy transfer from the light-harvesting antenna to the photosystem core. BN-PAGE and circular dichroism studies indicate changes in macromolecular assembly and thylakoid super-complexes destacking in pgrl1 and pgr5 due to changes in the pigment–protein complexes under high light. Based on this study, we emphasize that this is an excellent aid in understanding the role of CET mutants in thylakoid protein abundances and super-complex organization under high light.

光照与营养物质是光合作用的核心组成成分。激活信号级联反应对于启动响应强光的适应性过程至关重要。本研究选取野生型（wild-type, WT）、循环电子传递（Cyclic Electron Transport, CET）突变体，如质子梯度调节（Proton Gradient Regulation, PGR）相关的PGRL1与PGR5，以阐明强光条件下其在光合色素-蛋白复合物调控与组装中的具体作用。本研究结合生物物理、生物化学与蛋白质组学方法，解析光适应过程中靶向蛋白及类囊体（thylakoid）色素-蛋白复合物的组织模式。蛋白质组学分析显示，相较于对照组，强光条件下共有320种蛋白的表达量发生显著变化，这些蛋白主要参与光合电子传递链、蛋白质合成、代谢过程、糖酵解，以及细胞骨架组装相关蛋白。此外，我们观察到pgr5突变体中细胞色素（Cytochrome, Cyt）b6的表达量上调，以调节类囊体膜两侧的质子动力势与ATP合酶活性。pgr5突变体中Cyt b6功能的增强，可能源于强光下叶绿体（Chloroplast, cp）ATP合酶亚基功能受损，进而影响能量生成与光保护过程。进一步的蛋白质组数据显示，相较于野生型与pgrl1突变体，强光条件下pgr5突变体中光系统II亚基II（Photosystem Subunit II, PSBS）的蛋白亚型（PSBS1与PSBS2）的表达量高于捕光复合物应激相关（Light-Harvesting Complex Stress-Related, LHCSR）蛋白。免疫印迹（immunoblot）实验结果表明，强光条件下pgrl1与pgr5突变体中光系统II的D1、D2蛋白积累量高于野生型。在强光环境中，由于PSII蛋白周围的叶绿素与类胡萝卜素含量发生变化，这些色素作为辅因子协调光能从捕光天线向光系统核心的高效传递，因此pgr5突变体中CP43与CP47的含量出现下降。蓝绿温和电泳（Blue Native Polyacrylamide Gel Electrophoresis, BN-PAGE）与圆二色光谱（circular dichroism）研究显示，强光条件下类囊体色素-蛋白复合物的变化导致pgrl1与pgr5突变体中大分子组装发生改变，类囊体超复合物出现解堆叠现象。基于本研究结果，我们认为该工作可为理解循环电子传递突变体在强光条件下类囊体蛋白丰度与超复合物组织模式中的作用提供重要参考。