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Photoprotection mechanisms in plants play a crucial role in maintaining photosystem integrity and preventing photooxidative damage. In this study, we aimed to identify new molecular players involved in the qH process, a sustained photoprotective mechanism. We performed a forward genetic screen to select mutants with altered phenotype related to non-photochemical quenching (NPQ) and photosynthetic parameters. The mutants were classified into three main classes based on their NPQ phenotype, further subcategorized by photosynthetic parameters and chlorophyll content. Whole genome sequencing and mapping-by-sequencing approaches were employed to identify putative causative genes for the observed phenotypes. Potential causative mutations were retrieved by direct allelic comparison, gene ontology analysis, or mapping-by-sequencing. Sanger sequencing was utilized to validate the identified mutations and immunoblot analysis to confirm the protein accumulation disruptions in the mutants.

We found in the genetic screen multiple putative genes altering qH. We investigated the role in qH of two of them, low photosystem II accumulation 1 (LPA1) and photosynthesis affected mutant 68 (PAM68). Mutants lacking LPA1 or PAM68 exhibit lower NPQ compared to the parental line soq1 npq4. We show that photosystem II (PSII) integrity is important for qH to occur.

We also used a reverse genetic screen to decrypt the involvement of the different PSII antennae in qH. We demonstrated that qH can occur in the trimeric antennae, independent of a specific major antenna. Surprisingly, mutants with decreased or without major antennae accumulation still exhibited active qH to a high level. We found that qH can occur in the minor antennae in these mutants. However, qH is decreased in the soq1 lhcb6 mutant. Our findings provide novel insights into the molecular players and mechanism underlying photoprotective qH. 

This thesis investigates non-photochemical quenching (NPQ), emphasizing molecular mechanisms, thylakoid organisation and photosynthetic variability in plants. Spectro-kinetic analysis using ChloroSpec enabled detection of direct energy transfer from photosystem II (PSII) to photosystem I (PSI) - “spillover” - and the dissection of a unified NPQ mechanism, revealing photosystem II subunit S (PsbS) and zeaxanthin as critical regulators. PsbS facilitates light harvesting complex II (LHCII) quenching and spillover, while zeaxanthin accelerates spillover formation, ensuring rapid energy dissipation. The absence of these components severely affected the occurrence of spillover, underscoring their synergistic roles in photoprotection. Hybrid aspen mutants highlighted conserved functions of PsbS and zeaxanthin in angiosperms, with plant species-specific differences in NPQ kinetics. Aspen exhibited faster spillover occurrence and superior spillover characteristics compared to Arabidopsis, reflecting its enhanced photoprotective capacity. Transmission electron microscopy (TEM) linked NPQ to changes in thylakoid ultrastructure. Light-induced NPQ decreased grana layers per stack and increased stack numbers in wild-type Arabidopsis. Zeaxanthin levels affected the trends in thylakoid reorganisation. The Swedish aspen collection (SwAsp) study explored photosynthetic variation between genotypes and across latitudes, showing limited geographic influence but robust photoprotection via rapid NPQ induction and relaxation processes. These findings provide mechanistic insights into NPQ, its evolutionary conservation and genetic underpinnings, with implications for enhancing photosynthetic efficiency in plants under light stress.