EPR Spin-Trapping for Monitoring Temporal Dynamics of Singlet Oxygen during Photoprotection in Photosynthesis
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Homosalate has been found to exhibit favourable photophysics for inclusion in sunscreens, using a combination of spectroscopic and computational approaches.
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SUMMARY Photosynthetic organisms must cope with rapid fluctuations in light intensity. Nonphotochemical quenching (NPQ) enables the dissipation of excess light energy as heat under high light conditions, whereas its relaxation under low light maximizes photosynthetic productivity. We quantified variation in NPQ kinetics across a large sorghum ( Sorghum bicolor ) association panel in four environments, uncovering significant genetic control for NPQ. A genome‐wide association study (GWAS) confidently identified three unique regions in the sorghum genome associated with NPQ and suggestive associations in an additional 61 regions. We detected strong signals from the sorghum ortholog of Arabidopsis thaliana Suppressor Of Variegation 3 ( SVR3 ) involved in plastid–nucleus signaling. By integrating GWAS results for NPQ across maize ( Zea mays ) and sorghum‐association panels, we identified a second gene, Non‐yellowing 1 ( NYE1 ), originally studied by Gregor Mendel in pea ( Pisum sativum ) and involved in the degradation of photosynthetic pigments in light‐harvesting complexes. Analysis of nye1 insertion alleles in A. thaliana confirmed the effect of this gene on NPQ kinetics in eudicots. We extended our comparative genomics GWAS framework across the entire maize and sorghum genomes, identifying four additional loci involved in NPQ kinetics. These results provide a baseline for increasing the accuracy and speed of candidate gene identification for GWAS in species with high linkage disequilibrium.
The long-term goal of this research is to elaborate the catalysis of thiol-disulfide transactions in the thylakoid lumen, a compartment required for transducing energy via photophosphorylation. The molecular identity of the redox players catalyzing thiol-disulfide exchanges, their relevant targets of action in vivo and how thiol-disulfide chemistry in general controls photosynthesis are the experimental questions this research aims to address. Through previous work, we documented the requirement of catalyzed disulfide formation and reduction for the biogenesis of two photosynthetic enzymes, namely Photosystem II and the cytochrome b6f complex. We established that dedicated trans-thylakoid pathways operate in these processes by delivering reducing and oxidizing power to cysteine-containing subunits of the photosynthetic complexes, which are localized in the lumen compartment. Here we focused on CCDA, CCS4 and CCS5/HCF164, the components of the disulfide-reducing pathway which maintains the heme binding cysteines of apoforms of plastid cytochrome c in the reduced form prior to covalent heme ligation. We showed that CCS4 and CCS5 operate in functionally redundant pathways for the supply of reducing power and postulate a role for CCS4 in recruiting the source of reductants to CCDA or controlling the CCDA-dependent transduction of reducing equivalents. We also demonstrate the disulfide reducing pathway operates to counter thiol oxidation of the heme-binding cysteines by disulfide forming enzyme LTO1 in the thylakoid lumen.
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Abstract A group of vascular plants called homoiochlorophyllous resurrection plants evolved unique capabilities to protect their photosynthetic machinery against desiccation-induced damage. This study examined whether the ontogenetic status of the resurrection plant Craterostigma pumilum has an impact on how the plant responds to dehydration at the thylakoid membrane level to prepare cells for the desiccated state. Thus, younger plants (<4 months) were compared with their older (>6 months) counterparts. Ultrastructural analysis provided evidence that younger plants suppressed senescence-like programs that are realized in older plants. During dehydration, older plants degrade specific subunits of the photosynthetic apparatus such as the D1 subunit of PSII and subunits of the cytochrome b6f complex. The latter leads to a controlled down-regulation of linear electron transport. In contrast, younger plants increased photoprotective high-energy quenching mechanisms and maintained a high capability to replace damaged D1 subunits. It follows that depending on the ontogenetic state, either more degradation-based or more photoprotective mechanisms are employed during dehydration of Craterostigma pumilum.
While photoactive metal oxides such as TiO 2 find widespread use in paints and coatings as well as cosmetics and suncare products, they also generate reactive oxygen species (ROS), which degrade materials and are associated with human-health pathologies. Here, we demonstrate a robust and potent catalytic antioxidant consisting of earth-abundant cerium carbonate nanoparticles and micron-size Ce 2 (CO 3 ) 3 . 8H 2 O, which are characterized by powder X-ray diffraction and scanning nanobeam electron diffraction. When dispersed with photoactive metal oxides, these cerium carbonate catalysts decrease the photodecomposition rate of organic dyes and commercial pigment colorants in aqueous media by up to 820-fold, as well as in acrylic coatings. X-ray photoelectron spectroscopy and kinetic experiments support the same tandem catalysis mechanism of photoprotection when using both micron-size Ce 2 (CO 3 ) 3 . 8H 2 O and cerium carbonate nanoparticles. This mechanism involves ROS disproportionation (catalyzed by cerium carbonate) and H 2 O 2 decomposition (partially catalyzed by TiO 2 ) pathways, both of which cerium carbonate also catalyzes on its own, crudely mimicking the function of the cascade system of superoxide dismutase and catalase enzymes. When cerium carbonate nanoparticles were dispersed at 2 wt % in polymethylmethacrylate, the transparency of the polymer film was preserved and the photo-oxidative degradation of the polymer was prevented following UV irradiation at 254 nm, which otherwise resulted in the loss of optical properties and hydroxylation as characterized by ATR-FTIR spectroscopy, in the control polymer lacking cerium carbonate. Similar observations were made regarding color preservation in paint films comprising dye and insoluble commercial colorant pigments. The material chemistry associated with this photoprotection catalysis is subtle and emphasizes the importance of both Ce(III) and carbonate together, as both CePO 4 and Na 2 CO 3 are inactive. In conclusion, this emphasis is also apparent in comparisons of photoprotection catalysis with previously reported cerium oxide nanoparticles, which are significantly less active compared with Ce 2 (CO 3 ) 3 . 8H 2 O under the same conditions.
In oxygenic photosynthesis, high light leads to a set of photoprotective processes, known as nonphotochemical quenching, that are required for fitness. In moss and algae, the pigment−protein complex, light-harvesting stress-related (LHCSR), is crucial for photoprotection. Acidification of the thylakoid lumen under high light triggers the activation of LHCSR and the conversion of the xanthophyll violaxanthin into zeaxanthin, which is found within LHCSR. These interrelated molecular components combine to turn on a safety valve that dissipates excess energy as heat. Previous studies of detergent-solubilized LHCSR1 found that pH and zeaxanthin regulate distinct quenching sites via the protein conformation. However, protein function in the native membrane environment can be significantly different. In this work, we applied single-molecule fluorescence spectroscopy to LHCSR1 in membrane nanodiscs. The membrane environment enhanced total quenching, regardless of pH or zeaxanthin-binding. pH-dependent quenching was still observed whereas zeaxanthin-dependent quenching was suppressed. Conformational changes also increased in the membrane, establishing that the local environment can change the nature of the equilibrium between light harvesting and photoprotection.
Photosynthetic organisms have evolved multiple non-photochemical quenching (NPQ) processes, providing photoprotection by safely dissipating excess excitation energy. These processes involve various molecular players functioning on overlapping timescales from seconds to days, making it challenging to isolate and quantify their individual kinetics. In this study, we perform whole-leaf chlorophyll fluorescence lifetime and xanthophyll concentration measurements on wild-type and various newly characterized NPQ mutants of Nicotiana benthamiana, a vascular land plant. Based on these measurements, we construct a fluorescence lifetime-based quantitative kinetic model that disentangles individual photoprotection components and, when integrated additively, accurately predicts wild-type and mutant NPQ behaviors under various light-dark regimes. Additionally, the model quantifies the per-molecule quenching effectiveness of various xanthophylls and the contributions of six quenching components (qE V , qE A , qE Z , qE L , qZ, and qI) across different genotypes. It also suggests improved overall quenching efficiency at specific VDE:ZEP:PsbS overexpression stoichiometries, aligning with previous studies and supporting translational efforts to optimize photoprotection and enhance crop yields under dynamic light environments.
Abstract The D1 polypeptide of the photosystem II (PSII) reaction center complex contains domains that regulate primary photochemical yield and charge recombination rate. Many prokaryotic oxygenic phototrophs express two or more D1 isoforms differentially in response to environmental light needs, a capability absent in flowering plants and algae. We report that tobacco (Nicotiana tabacum) plants carrying the Synechococcus (Synechococcus elongatus PCC 7942) low-light mutation (LL-E130Q) in the D1 polypeptide (NtLL) acquire the cyanobacterial photochemical phenotype: faster photodamage in high light and significantly more charge separations in productive linear electron flow in low light. This flux increase produces 16.5% more (dry) biomass under continuous low-light illumination (100 μE m−2 s−1, 24 h). This gain is offset by the predicted lower photoprotection at high light. By contrast, the introduction of the Synechococcus high-light mutation (HL-A152S) into tobacco D1 (NtHL) has slightly increased photoprotection, achieved by photochemical quenching, but no apparent impact on biomass yield compared to wild type under the tested conditions. The universal design principle of all PSII reaction centers trades off energy conversion for photoprotection in different proportions across all phototrophs and provides a useful guidance for testing in crop plants. The observed biomass advantage under continuous low light can be transferred between evolutionarily isolated lineages to benefit growth under artificial lighting conditions. However, removal of the selective marker gene was essential to observe the growth phenotype, indicating growth penalty imposed by use of the particular spectinomycin-resistance gene.
Abstract Cold stress is a key environmental constraint that dramatically affects the growth, productivity, and quality of tomato (Solanum lycopersicum); however, the underlying molecular mechanisms of cold tolerance remain poorly understood. In this study, we identified REDUCED CHLOROPLAST COVERAGE 2 (SlREC2) encoding a tetratricopeptide repeat protein that positively regulates tomato cold tolerance. Disruption of SlREC2 largely reduced abscisic acid (ABA) levels, photoprotection, and the expression of C-REPEAT BINDING FACTOR (CBF)-pathway genes in tomato plants under cold stress. ABA deficiency in the notabilis (not) mutant, which carries a mutation in 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (SlNCED1), strongly inhibited the cold tolerance of SlREC2-silenced plants and empty vector control plants and resulted in a similar phenotype. In addition, foliar application of ABA rescued the cold tolerance of SlREC2-silenced plants, which confirms that SlNCED1-mediated ABA accumulation is required for SlREC2-regulated cold tolerance. Strikingly, SlREC2 physically interacted with β-RING CAROTENE HYDROXYLASE 1b (SlBCH1b), a key regulatory enzyme in the xanthophyll cycle. Disruption of SlBCH1b severely impaired photoprotection, ABA accumulation, and CBF-pathway gene expression in tomato plants under cold stress. Taken together, this study reveals that SlREC2 interacts with SlBCH1b to enhance cold tolerance in tomato via integration of SlNCED1-mediated ABA accumulation, photoprotection, and the CBF-pathway, thus providing further genetic knowledge for breeding cold-resistant tomato varieties.
Chlamydomonas acclimates to repeated low (LL) or high light (HL) days by changing the abundance of photosynthetic complexes and the ultrastructure of its thylakoid membranes. These phenotypes persist through the night phases, suggesting a readiness for the daylight environment that is routinely experienced despite the intervening dark periods [S. Dupuis et al., Plant Cell 37, koaf086 (2025), 10.1093/plcell/koaf086]. Here, we investigate how prior acclimation impacts algal fitness upon a change in daylight intensity and how quickly Chlamydomonas can reprogram its photoprotective strategy in a diurnal context. We performed a systems analysis of synchronized populations acclimated to diurnal LL when subjected to HL days and of populations acclimated to diurnal HL when subjected to LL days. In the latter case, diurnal photoacclimation decreased fitness during the first day at a new light intensity: HL-acclimated cells barely increased in size over the first LL period, and they failed to complete a cell cycle. However, although LL-acclimated cells showed severe photodamage after 6 h of HL, they recovered chloroplast form and function later that afternoon and successfully divided at nightfall. These cells rapidly altered their thylakoid membrane ultrastructure, increased their photoprotective quenching capacity, and decreased their inventory of photosystem and antenna proteins by the end of the first HL day. Transcriptomic and proteomic analyses revealed rapid induction of thousands of genes, including those encoding proteases, chaperones, and other proteins involved in the chloroplast unfolded protein response. These results show that the alga is highly flexible and competent to rapidly acclimate to changes in diurnal light intensity.
The phycobilisome (PBS) captures light energy and transfers it to photosystem I (PSI) and photosystem II (PSII). Which and how many copies of protein subunits in PBSs, PSI, and PSII remain unbound in thylakoids are unknown. Here, quantitative mass spectrometry (QMS) was used to quantify substantial pools of free extrinsic subunits of PSII and PSI. Interestingly, the membrane intrinsic PsaL is 3-fold higher than PsaA/B. This scenario complements the static structures of these complexes as revealed by X-ray crystallography and cryo-EM. Furthermore, the ratios of ApcG and photoprotective OCP over PBS indicate a pool of extra ApcG. The 2.5 ratio of CpcG-PBS over CpcL-PBS improves our understanding of these light-harvesting complexes involved in energy capture and photoprotection in cyanobacteria.
Abstract Photoprotection mechanisms are ubiquitous among photosynthetic organisms. The photoprotection capacity of the green alga Chlamydomonas reinhardtii is correlated with protein levels of stress-related light-harvesting complex (LHCSR) proteins, which are strongly induced by high light (HL). However, the dynamic response of overall thylakoid structure during acclimation to growth in HL has not been fully understood. Here, we combined live-cell super-resolution microscopy and analytical membrane subfractionation to investigate macroscale structural changes of thylakoid membranes during HL acclimation in Chlamydomonas . Subdiffraction-resolution live-cell imaging revealed that the overall thylakoid structures became thinned and shrunken during HL acclimation. The stromal space around the pyrenoid also became enlarged. Analytical density-dependent membrane fractionation indicated that the structural changes were partly a consequence of membrane unstacking. The analysis of both an LHCSR loss-of-function mutant , npq4 lhcsr1 , and a regulatory mutant that over-expresses LHCSR, spa1-1 , showed that structural changes occurred independently of LHCSR protein levels, demonstrating that LHCSR was neither necessary nor sufficient to induce the thylakoid structural changes associated with HL acclimation. In contrast, stt7-9 , a mutant lacking a kinase of major light-harvesting antenna proteins, had a slower thylakoid structural response to HL relative to all other lines tested but still showed membrane unstacking. These results indicate that neither LHCSR- nor antenna-phosphorylation-dependent HL acclimation are required for the observed macroscale structural changes of thylakoid membranes in HL conditions.
Photosystem II (PSII) has the unique ability to perform water-splitting. With light-harvesting complexes, it forms the PSII supercomplex (PSII-SC) which is a functional unit that can perform efficient energy conversion, as well as photoprotection, allowing photosynthetic organisms to adapt to the naturally fluctuating sunlight intensity. Achieving these functions requires a collaborative energy transfer network between all subunits of the PSII-SC. In this work, we perform kinetic analyses and characterise the energy landscape of the PSII-SC with a structure-based energy transfer model. With first passage time analyses and kinetic Monte Carlo simulations, we are able to map out the overall energy transfer network. We also investigate how energy transfer pathways are affected when individual protein complexes are removed from the network, revealing the functional roles of the subunits of the PSII-SC. In addition, we provide a quantitative description of the flat energy landscape of the PSII-SC. We show that it is a unique landscape that produces multiple kinetically relevant pathways, corresponding to a high pathway entropy. These design principles are crucial for balancing efficient energy conversion and photoprotection.
Abstract Photorespiration consumes substantial amounts of energy in the forms of adenosine triphosphate (ATP) and reductant making the pathway an important component in leaf energetics. Because of this high reductant demand, photorespiration is proposed to act as a photoprotective electron sink. However, photorespiration consumes more ATP relative to reductant than the C3 cycle meaning increased flux disproportionally increases ATP demand relative to reductant. Here we explore how energetic consumption from photorespiration impacts the flexibility of the light reactions in nicotiana tabacum . Specifically, we demonstrate that decreased photosynthetic efficiency (ϕ II ) at low photorespiratory flux was related to feedback regulation at the chloroplast ATP synthase. Additionally, decreased ϕ II at high photorespiratory flux resulted in the accumulation of photoinhibition at photosystem II centers. These results are contrary to the proposed role of photorespiration as a photoprotective electron sink. Instead, our results suggest a novel role of ATP consumption from photorespiration in maintaining ATP synthase activity, with implications for maintaining energy balance and preventing photodamage that will be critical for plant engineering strategies.