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Photosynthetic light-harvesting complexes mediate light absorption and energy dissipation. By modulating the photosystems' absorption cross-section, they affect both photosynthetic activity and non-photochemical quenching (NPQ). These processes are often studied by spectrally integrated chlorophyll fluorescence, masking their associated spectral information. We explore in Aspen and Arabidopsis npq mutants how qE affects the development of NPQ spectra under two contrasting conditions: in the absence and the presence of photoinhibition. We introduce a new parameter, the development of new emitting species (NESD), during time- and spectrally resolved NPQ inductions, and develop a pipeline to resolve PSII energy-partitioning heterogeneity. LHCII, PsbS, and zeaxanthin are required for NESD. Combining gas exchange, P700 oxidation, and spectrally resolved kinetics, we show that under photoinhibitory conditions, NES can develop even without PsbS or zeaxanthin, producing sustained quenching independent of photoinhibition of PSII or PSI. Furthermore, the absence of LHCII and CURVATURE THYLAKOID 1 leads to increased photoinhibition, indicating that long-term photoprotection relies on LHCII and thylakoid plasticity, whereas PsbS and zeaxanthin mainly facilitate LHCII-dependent quenching. Finally, we show the limitations of traditional parameters in discriminating between photoinhibition and photoprotective sustained quenching and propose time-resolved monitoring of CO₂ assimilation and Y(II) for their accurate assessment.

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.

Chlorophyll fluorescence is a ubiquitous tool in basic and applied plant science research. Various standard commercial instruments are available for characterization of photosynthetic material like leaves or microalgae, most of which integrate the overall fluorescence signals above a certain cut-off wavelength. However, wavelength-resolved (fluorescence signals appearing at different wavelengths having different time dependent decay) signals contain vast information required to decompose complex signals and processes into their underlying components that can untangle the photo-physiological process of photosynthesis. Hence, to address this we describe an advanced chlorophyll fluorescence spectrometer - ChloroSpec - allowing three-dimensional simultaneous detection of fluorescence intensities at different wavelengths in a time-resolved manner. We demonstrate for a variety of typical examples that most of the generally used fluorescence parameters are strongly wavelength dependent. This indicates a pronounced heterogeneity and a highly dynamic nature of the thylakoid and the photosynthetic apparatus under actinic illumination. Furthermore, we provide examples of advanced global analysis procedures integrating this three-dimensional signal and relevant information extracted from them that relate to the physiological properties of the organism. This conveniently obtained broad range of data can make ChloroSpec a new standard tool in photosynthesis research.

Green organisms evolve oxygen (O2) via photosynthesis and consume it by respiration. Generally, net O2 consumption only becomes dominant when photosynthesis is suppressed at night. Here, we show that green thylakoid membranes of Scots pine (Pinus sylvestris L) and Norway spruce (Picea abies) needles display strong O2 consumption even in the presence of light when extremely low temperatures coincide with high solar irradiation during early spring (ES). By employing different electron transport chain inhibitors, we show that this unusual light-induced O2 consumption occurs around photosystem (PS) I and correlates with higher abundance of flavodiiron (Flv) A protein in ES thylakoids. With P700 absorption changes, we demonstrate that electron scavenging from the acceptor-side of PSI via O2 photoreduction is a major alternative pathway in ES. This photoprotection mechanism in vascular plants indicates that conifers have developed an adaptative evolution trajectory for growing in harsh environments.

Photosynthesis is a biological process which converts light energy into chemical energy that is used in the Calvin–Benson cycle to produce organic compounds. An excess of light can induce damage to the photosynthetic machinery. Therefore, plants have evolved photoprotective mechanisms such as non-photochemical quenching (NPQ). To focus molecular insights on slowly relaxing NPQ processes in Arabidopsis thaliana, previously, a qE-deficient line—the PsbS mutant—was mutagenized and a mutant with high and slowly relaxing NPQ was isolated. The mutated gene was named suppressor of quenching 1, or SOQ1, to describe its function. Indeed, when present, SOQ1 negatively regulates or suppresses a form of antenna NPQ that is slow to relax and is photoprotective. We have now termed this component qH and identified the plastid lipocalin, LCNP, as the effector for this energy dissipation mode to occur. Recently, we found that the relaxation of qH1, ROQH1, protein is required to turn off qH. The aim of this study is to identify new molecular players involved in photoprotection qH by a whole genome sequencing approach of chemically mutagenized Arabidopsis thaliana. We conducted an EMS-mutagenesis on the soq1 npq4 double mutant and used chlorophyll fluorescence imaging to screen for suppressors and enhancers of qH. Out of 22,000 mutagenized plants screened, the molecular players cited above were found using a mapping-by-sequencing approach. Here, we describe the phenotypic characterization of the other mutants isolated from this genetic screen and an additional 8000 plants screened. We have classified them in several classes based on their fluorescence parameters, NPQ kinetics, and pigment content. A high-throughput whole genome sequencing approach on 65 mutants will identify the causal mutations thanks to allelic mutations from having reached saturation of the genetic screen. The candidate genes could be involved in the formation or maintenance of quenching sites for qH, in the regulation of qH at the transcriptional level, or be part of the quenching site itself.