Role of the PsbS protein in Chlamydomonas reinhardtii

强光下超额激发能(NPQ)的热耗散是所有光合作用生物中一种重要的光保护机制，其目的是使活性氧物种的光氧化损伤降到最低。在陆地植物中，光系统II(PSII)蛋白PSBS在NPQ调节中起关键作用。PSBS作为管腔pH的感受器，控制PSII天线的构象变化，这是NPQ激活所必需的。在莱茵衣藻中，LHCSR蛋白作为pH感受器激活NPQ。在这里，NPQ只有在细胞的HL驯化几个小时后才可能激活，这启动了LHCSR蛋白质的合成。铜绿假单胞菌在莱茵梭菌中的作用尚不清楚。在过去的资助期间，我们发现PSB只在HL暴露的前4-8小时内瞬时表达，并在大约24小时的高氧驯化后迅速降解。PSB定位在类囊体膜上，并与PSII相互作用。PSB的积累与LHCSR的积累和NPQ的激活是平行的。PSB的下调导致NPQ容量和LHCSR积累的减少。在缺乏LHCSR蛋白的情况下，PSB不能补偿LHCSR在NPQ激活中的作用，PSB水平的增加也不能增加NPQ的容量。这表明PSB对NPQ不是必需的，但可能是在HL驯化过程中建立高NPQ能力所必需的。我们进一步积累了PSB具有NPQ非依赖光保护功能的证据。计划中的工作旨在破译PSB在C.reinhardtii中的详细功能。为此，将解决以下核心问题：1)通过CRISPRCas9获得稳定的PSB基因敲除系，并对其NPQ能力和HL驯化反应进行表征。到目前为止，我们只分析了PSB的下跌线。2)PSB和LHCSR蛋白相互作用伙伴的确定和化学计量。3)PSBs降解过程的表征及其对HL驯化的影响。4)在QE非依赖光保护机制中影响突变的PsbS动力学和HL驯化反应的特征。5)测定PSBs动态和HL对较自然波动(高)光照条件的驯化。以前对莱茵梭子藻HL驯化的研究大多是在非自然永久光照下进行的，没有间歇性的暗期。暗周期的包含和波动光强度的应用使人们有理由期待自然生境方面的令人兴奋的信息，特别是关于PSB和LHCSR积累的动态。对具有NPQ非依赖光保护机制缺陷的突变体的鉴定将进一步提供有关PsbS整合到莱茵梭菌光保护网络中的信息。

Heat dissipation of excess excitation energy (NPQ) under high light(HL) is an important photoprotective mechanism active in allphotosynthetic organisms, which aims at the minimization of photooxidativedamage by reactive oxygen species. In land plants, thephotosystem II (PSII) protein PsbS plays a key role in NPQ regulation.PsbS acts as sensor of the lumen pH and by that controlsconformational changes in the PSII antenna, which are required forNPQ activation. In the green alga Chlamydomonas reinhardtii,LHCSR proteins act as pH-sensor and trigger NPQ activation. Here,NPQ activation is only possible after several hours of HL acclimationof the cells, which initiates the synthesis of LHCSR proteins. The roleof PsbS in C. reinhardtii is still unclear. During the past funding periodwe showed that PsbS is only transiently expressed during the first 4-8h of HL exposure and is rapidly degraded after about 24h of HLacclimation. PsbS localizes to thylakoid membranes and interacts withPSII. PsbS accumulation occurs in parallel with LHCSR accumulationand NPQ activation. Down-regulation of PsbS leads to reduction ofboth NPQ capacity and LHCSR accumulation. In the absence ofLHCSR protein, PsbS cannot compensate for the role of LHCSR inNPQ activation, and increased PsbS levels fail to increase the NPQcapacity. This indicates that PsbS is not essential for NPQ, butpossibly required for the establishment of a high NPQ capacity duringHL acclimation. We further accumulated evidence for a NPQindependentphotoprotective function of PsbS. The planned workaims at the goal to decipher the detailed function of PsbS in C.reinhardtii. To this end, the following central issues will be addressed:1) Generation of stable PSBS knockout lines by means of CRISPRCas9and characterization of their NPQ capacity and HL acclimationresponse. So far, we have analyzed only PsbS knock-down lines. 2)Determination of interaction partners and stoichiometry of PsbS andof LHCSR proteins. 3) Characterization of the PsbS degradationprocess and its impact on HL acclimation. 4) Characterization of thePsbS dynamics and of the HL acclimation response in mutantsaffected in qE-independent photoprotective mechanisms. 5)Determination of the PsbS dynamics and of HL acclimation inresponse to more natural fluctuating (high) light conditions. Most ofthe former studies on HL acclimation in C. reinhardtii have beenconducted with cells grown under non-natural permanent illuminationwithout intermittent dark periods. Inclusion of dark periods andapplication of fluctuating light intensities give reason to expect exciting information in terms of natural habitats, particularly regarding thedynamic of PsbS and LHCSR accumulation. The characterization ofmutants with defects in NPQ-independent photoprotectivemechanisms will further provide information about the integration ofPsbS into the photoprotective network of C. reinhardtii.