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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.

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.