Role of the PsbS protein in Chlamydomonas reinhardtii

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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.