Distinct features of PsbS essential for mediating plant photoprotection.

Abstract

For optimum photosynthetic productivity, it is crucial for plants to swiftly transition between light-harvesting and photoprotective states as light conditions change in the field. The PsbS protein plays a pivotal role in this process by switching the light-harvesting antenna, light-harvesting complex II (LHCII), into the photoprotective state, energy-dependent chlorophyll fluorescence quenching (qE), to avoid photoinhibition in high-light environments. However, the molecular mechanism by which PsbS acts upon LHCII has remained unclear. In our study, we identified the specific amino acid domains that are essential for PsbS function. Using amino-acid point mutagenesis of PsbS in vivo, we found that the activation of photoprotection involves dynamic changes in the oligomeric state and conformation of PsbS, with two residues, E67 and E173, playing a key role in this process. Further, the replacement of hydrophobic phenylalanine residues in transmembrane helixes II (F83, F84, F87) and IV (F191, F193, F194) with tyrosine revealed that phenylalanine localized in helix IV can play a significant role in hydrophobic interactions of PsbS with LHCII. Removal of the 3₁₀ helix (H3) amino acids I74, Y75, and E76 did not affect the amplitude but strongly delayed the recovery of qE in darkness. Moreover, an AI-assisted protein-folding evolutionary scale model approach (ESMFold) was adopted to intelligently manipulate protein functions in silico and thus streamline and evaluate experimental point mutagenesis strategies. This provides new insights into the molecular architecture of PsbS that are essential for regulating light harvesting in higher plants.