Photodamaged PSII does not accumulate in the non-appressed thylakoid membranes in the absence of PSII repair.

Photosystem II (PSII) is highly sensitive to light-induced damage. Photoinhibition, the light-dependent inactivation of PSII, is associated with an increase of excitation-energy quenching. Recovery from photoinhibition involves migration of PSII complexes from the appressed to the non-appressed region of the thylakoid membrane, where D1 (the PSII core protein most sensitive to photodamage) is degraded and repair occurs. However, it remains unclear whether damaged PSII core complexes accumulate in the stroma lamellae when repair is blocked. Here, we combined confocal Fluorescence Lifetime Imaging Microscopy (FLIM) with biochemical fractionation of the thylakoid membrane to investigate the localization of damaged PSII following photoinhibition in the presence of lincomycin, an inhibitor of D1 synthesis. This condition mimics natural stress scenarios such as heat, where D1 synthesis is impaired. FLIM analysis of structured, intact thylakoid membranes, segmented into grana- and stroma-lamellae-enriched regions, revealed a decrease in PSII fluorescence lifetime upon photoinhibition, consistent with increased excitation-energy quenching. Surprisingly, no significant difference in fluorescence lifetime components was observed between membrane domains, suggesting that damaged, quenched PSII does not accumulate in the stroma lamellae under these conditions. Western blot analysis of biochemically isolated membrane fractions confirmed a uniform decrease in D1 levels across grana and stroma lamellae upon photoinhibition. Our results indicate that when D1 synthesis is blocked, the relocation and degradation of photodamaged PSII proceed efficiently enough to prevent its accumulation in the stroma lamellae. This reveals new aspects of PSII repair and demonstrates the strength of FLIM for spatially resolved analysis of the thylakoid membrane.