Photosynthesis Research

Biotechnological applications of oxygenic photosynthetic organisms depend on conversion of light energy into chemical energy through photosystems (PS). This energy can then be used to drive engineered metabolic pathways that are designed as strong electron sinks. For optimal performance, the engineered host metabolism must also be balanced with the native photoprotective electron transfer network. This includes the energy-consuming function of flavodiiron (Flv) proteins, which are universal to cyanobacteria and all other oxygenic photosynthetic organisms except angiosperms. In the cyanobacterium Synechocystis sp. PCC 6803, four different Flv proteins have been shown to function in a Mehler-like reaction within two heterodimeric forms (Flv1/Flv3 and Flv2/Flv4), donating electrons to O2 without generating oxidative stress. Previously, deleting Flv3 in the Synechocystis sucrose-producing (S02) strain was shown to cause drastic metabolic changes in S02{Delta}flv3, shifting it from photoautotrophic to mixotrophic growth (Muth-Pawlak, et al., 2024). In this study, we took an opposite approach by complementing S02 with Flv3 overexpression at different levels using RBS tuning. Interestingly, this resulted in S02oeFlv3 strains with significantly increased overall photosynthetic activity and sucrose production, enhanced cell growth, and storage compound accumulation. However, these outcomes are shown not to be due to conventional O2 photoreduction activity catalysed by Flv1/Flv3. Instead, we postulate that the observed changes are linked to the previously unidentified function of homomeric Flv3/Flv3 and the strongly increased sulphate redox metabolism. Based on extensive proteomic and metabolite analyses, we hypothesise that the Flv3 homooligomer uses sulfate metabolites directly or indirectly as the final electron acceptor instead of O2. This would also explain the upregulation of sulfate-related enzymes, as well as SQR, which passes the electrons back to the PQ pool in the Flv3 overexpression strain.

The assimilation of carbon dioxide by plants can be predicted by the Farquhar, von Caemmerer and Berry model of photosynthesis. This largely mechanistic model is central to understanding how plants influence Earths climate. However, it represents the use of light by photosynthesis using an empirical formulation. Johnson and Berry proposed an alternative mechanistic formulation based on the functioning of the cytochrome b6f complex that includes key steps in light harvesting and electron transport. We compared both formulations using photosynthetic light response measurements from 146 C3 species spanning arctic to tropical biomes and implemented them in the terrestrial biosphere model ELM-FATES to simulate global photosynthesis. The Johnson and Berry formulation better fitted the measured response of leaf-level photosynthesis to light, and predicted lower photosynthetic rates at intermediate light levels, which decreased global estimations of terrestrial photosynthesis by 8%. Our findings support adopting the Johnson and Berry formulation to improve model representation of global carbon cycle modeling.

Chilling stress induces photosystem I (PSI) photoinhibition in chilling-sensitive cucumber, in which insufficient activity of the chloroplast NADH dehydrogenase-like complex (NDH) leads to PSI over-reduction and damage. However, it is not yet clear whether these findings can be generalized to other species or what the molecular mechanism underlying impaired NDH function is. In this study, we first examined whether NDH is essential for PSI protection under chilling stress using an NDH-deficient rice mutant. Compared with wild-type plants, the NDH-deficient mutant exhibited enhanced PSI over-reduction and pronounced PSI photoinhibition under chilling stress. In contrast, rice plants expressing flavodiiron protein (FLV), which functions as an alternative electron acceptor downstream of PSI, did not exhibit PSI photoinhibition under chilling stress, demonstrating that electron sink capacity of NDH is important for PSI protection under chilling stress. Furthermore, analysis of the factors responsible for NDH dysfunction under chilling stress in cucumber revealed that chilling stress destabilizes the PSI-NDH supercomplex, leading to NDH monomerization and a consequent loss of NDH activity. This NDH monomerization is likely attributable to chilling-induced damage to the light-harvesting complex Lhca, which mediates the association between PSI and NDH. Together, these results indicate that NDH is essential for protecting PSI from photoinhibition under chilling stress in both rice and cucumber, and that chilling-induced destabilization of the PSI-NDH supercomplex represents a key molecular mechanism underlying PSI over-reduction and photoinhibition.

Photosynthetic membranes undergo structural remodeling in response to environmental stress by altering fatty acid composition and desaturation levels. These changes, mediated by fatty acid desaturases (FADs), are essential for maintaining photosynthetic performance and adaptation. In this study, we demonstrate that copper-deficient Chlamydomonas reinhardtii cells upregulate the expression of the gene encoding stearoyl-ACP desaturase (SAD/FAB2). We propose that this four-fold induction reflects an increased physiological demand for its primary product, oleic acid (18:1{Delta}9), and its subsequent downstream derivatives. The sad mutants exhibit a significant reduction in 18:1{Delta}9 content compared to wild-type cells, which correlates with diminished growth rates. Although SAD abundance increases under Cu deficiency, loss of SAD strongly alters C18 fatty acid composition across Cu conditions, while the growth defect is most apparent under Cu-replete conditions. This suggests that SAD activity may be a limiting factor in copper-depleted environments, leading to slower growth and reduced 18:1{Delta}9 levels in the uncharged galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), both of which are critical for photosynthetic function. The desaturation reaction catalyzed by SAD requires molecular oxygen and electrons supplied by ferredoxin (Fd). Using reciprocal IP-MS, we identified FDX5 as a Cu-deficiency specific SAD interacting protein. However, fdx5 mutants retained wild-type fatty acid profiles, indicating that FDX5 is not strictly required for SAD-dependent lipid desaturation and that another ferredoxin, likely FDX1, can compensate.

O_LIPhytoplankton species display characteristic morphologies that are generally assumed to confer adaptive advantages, yet the functional significance of cell shape remains poorly understood. Here, we investigated whether pleomorphism contributes to acclimation to changing light environments. C_LIO_LIUsing the cyanobacterium Anabaena sp. PCC 7120 as a model system, we combined molecular genetics, microscopy, physiological measurements and biophysical analyses to determine how morphology is regulated and how it affects photosynthetic performance under different light intensities. C_LIO_LIWe show that Anabaena undergoes a reversible light-dependent morphological transition from rod-shaped cells under low light to large globular cells under high light stress. This transition is controlled by the relative activities of the elongasome and class A penicillin-binding proteins and is accompanied by thylakoid reorganization. The globular morphology reduces light absorption and enables cells to maintain photosynthetic activity under photoinhibitory conditions. C_LIO_LIOur findings establish a mechanistic link between cell-wall remodelling, cellular optics and photosynthetic performance, revealing pleomorphism as a dynamic acclimation strategy to high light stress. More broadly, this work provides experimental support for the packaging effect and highlights morphology as an active determinant of phytoplankton fitness. C_LI

Fast cytoplasmic streaming enables extensive chloroplast movements in the giant cells of unicellular, siphonous macroalgae. Here, we studied chloroplast movements in two such algae: the Dasycladalean Acetabularia acetabulum and the Bryopsidales Bryopsis sp.. We hypothesised that chloroplast movements function as a protective avoidance mechanism under excess light, particularly in Bryopsis sp., which lacks capacity for fast induction of photoprotective non-photochemical quenching (NPQ) and state transitions. In addition, we also investigated whether chloroplast movements are involved in responses to wounding and herbivory. The movements were studied by light microscopy, photography and pulse modulated chlorophyll a fluorescence quenching analysis. Chemical inhibitors of actin polymerization and microtubules assembly were used to confirm that the observed effects were active responses controlled by the cytoskeleton. A. acetabulum responded to high light by reversible chloroplast aggregation, probed by macro-imaging; and chemical inhibition of chloroplast movements led to an enhancement of Photosystem II photoinhibition, as probed by the fluorescence parameter FV/FM. No chloroplast movements were observed in Bryopsis sp. in response to high light. In A. acetabulum, wounding caused either by cutting or due to feeding by the sap-sucking sea slug Elysia timida triggered aggregation of chloroplasts within minutes of incurring the damage. Interestingly, the aggregation also occurred in intact cells away from the cutting site. Furthermore, the addition of media collected from the vicinity of cut algae was sufficient to induce chloroplast aggregation in intact algae, suggesting that water-borne cues or signals triggered the aggregation response in A. acetabulum. Bryopsis sp., however, responded to cutting by only local chloroplast aggregation. The relevance of chloroplast movements in protection against both abiotic and biotic stressors in A. acetabulum, and the potential reasons behind the different defence strategies of the algae, are discussed.

Chromosome spatial organization plays critical roles in transcriptional regulation and DNA protection. In cyanobacteria--photosynthetic bacteria that experience dramatic fluctuations in light intensity--chromosome reorganization could facilitate rapid transcriptional reprogramming and protect DNA from photodamage. However, chromosome organization in these polyploid organisms has remained technically challenging to observe, leaving light-dependent responses unexplored. Here, we show that higher-order chromosome organization in Synechocystis sp. PCC 6803 is associated with light intensity, revealing a previously unrecognized light-dependent adaptation in cyanobacteria. We established fluorescence in situ hybridization (FISH) methods for this model cyanobacterium carrying multi-copy genomes, together with a computational pipeline to assign paired FISH signals to individual genome copies. The slope relating genomic and spatial distance was steeper under standard conditions ({beta} = 0.972 nm/kbp, R{superscript 2} = 0.12) than under high-light conditions ({beta} = 0.450 nm/kbp, R{superscript 2} = 0.02), indicating that local chromosome organization is substantially disrupted by elevated light intensity. The spatial distribution of the multiple genome copies also differed between conditions, independently supporting condition-dependent chromosome reorganization. Hi-C analysis corroborated these findings, revealing reduced chromosomal interactions within the 10-100 kbp range under high-light conditions. Together, these results demonstrate that light intensity is a previously unrecognized determinant of higher-order chromosome organization in a photosynthetic bacterium.

Algae require specific acclimation strategies to cope with spectral variability in shallow marine habitats. We investigated how the siphonous green alga Derbesia alters its photosynthetic and metabolic processes under white (WL), blue (BL), green (GL), red (RL), and far-red light (FL) by conducting photobiological and transcriptomic sampling over a 10-day period. Our results show two contrasting photoacclimation strategies: BL and GL promoted metabolic activity associated with growth, whereas FL and RL induced a low-light-like survival strategy characterized by reduced growth and suppression of the core metabolism. Photosynthetic acclimation across all conditions primarily occurs within the light dependent reactions. BL and GL promoted early acclimation marked by the immediate activation of light-harvesting complexes (LHCs) and a key transcriptional regulator MYB, and showed better acclimation marked by the sustained activation of ATPases, ATP transporters, and hormone-signaling components. BL induced a distinct transcriptional shift during the transition to prolonged exposure, including enhanced cyclic electron transport, and key regulators of protein synthesis, DNA replication, and transcriptional regulation. In contrast, FL, and to a lesser extent RL, triggered responses resembling low light acclimation with constrained growth, characterized by inefficient energy utilization, enlarged antenna systems, chloroplast proliferation with aggregations, and reduced growth rates. This study suggests high accumulation of core photopigments and reduction in chlorophyll a/b is an acclimatory response to FL, and consistently higher activation of core metabolic processes under WL likely indicates the evolutionary adaptation of Derbesia to shallow coastal environments where broad-spectrum light predominates. Additionally, our newly sequenced draft genome of the Derbesia strain for this study could serve as a genomic resource for future molecular photobiology research in Bryopsidales algae.

Adaptation of plant photosynthesis to dynamic light conditions experienced in natural environments is achieved through specific protective mechanisms. Energy-dependent non-photochemical quenching (qE), regulated by Photosystem II Subunit S (PsbS), is a key process facilitating acclimation to fluctuating light in C3 plants, which operate conventional photosynthesis. C4 plants, which include some of the world's most productive and agriculturally important crops, have evolved a distinct high-efficiency photosynthetic pathway. Little is known about the role of specific processes, like qE, in acclimation of C4 plants to dynamic light environments. We generated gene-edited lines of the model C4 grass Setaria viridis lacking PsbS, which were found to be deficient in qE. This deficiency resulted in a modest increase in PSII photoinhibition and a CO2 assimilation penalty under light stress in short-term experiments, but photosynthesis and growth under fluctuating light were unaffected. Instead, keeping Photosystem I oxidised through photosynthetic control, negative feedback regulation of the Cytochrome b6f complex, was critical. Therefore, unlike in C3 plants, qE does not provide a significant adaptive advantage to C4 plants under dynamic light conditions. These findings provide important insights into the biology of C4 plants and help prioritise future strategies for improving the productivity and resilience of C4 crops.

Photosymbiosis has evolved multiple times independently in ciliates. However, these associations can be antagonized by shifts in environmental parameters that impose stress on the host, necessitating the evolution of mechanisms to contend with this stress and to control the symbiont population. To investigate whether convergent strategies have evolved among algae-bearing ciliates in the class Oligohymenophorea, we imposed light stress on three host species that represent at least two independent evolutionary origins of photosymbiosis and measured their cellular responses. Under high light, all three species experienced an initial drop in host cell density which recovered to levels commensurate with those under low-light conditions as they decreased their symbiont loads. We then performed a comparative transcriptomic study to investigate whether a core set of genes exists that is involved in this response. Thirty-one gene families possess differentially expressed transcripts across all three species that included the upregulation C1 and S28 class peptidases, genes involved in ROS mitigation, and a gene with potential involvement in mitochondrial remodeling associated with changes in algal symbiont load. We additionally found downregulation in Dicer, which could mitigate the processing of algal transcripts by the hosts RNAi machinery that are freed upon algal digestion, and downregulation of motor proteins that may reflect changes in the hosts swimming behaviors and transport of intracellular vesicles in response to light. The 31 gene families are present and widespread in non-symbiotic oligohymenophoreans, illustrating that a pre-existing genetic toolkit exists in this clade that helps explain how it is predisposed to evolving photosymbioses.

Diurnal changes in light availability are a defining feature of life on Earth. Photoautotrophic organisms therefore store reduced carbon during the day to sustain energy metabolism at night. In cyanobacteria, glycogen is the primary carbon storage compound and supports both energy homeostasis and stress responses. Although glycogen-deficient Synechocystis strains have been studied previously, how these mutants cope with the loss of the major daytime carbon sink and can sustain themselves during the night remains unclear. Using single-cell microfluidics, transcriptomics, and metabolomics, we show that {Delta}glgC mutants exhibit pronounced light sensitivity. At sub-lethal light intensities, daytime transcriptional responses are dominated by downregulation of photosynthesis-related genes, likely preventing NADPH overaccumulation in the absence of a carbon sink. During the night, mutants display severe energy limitation, characterized by reduced ATP levels, altered redox balance, and depletion of central carbon intermediates. In contrast, fumarate and malate accumulate, indicating enhanced respiratory flux through succinate dehydrogenase. These metabolic constraints lead to extended lag phases and delayed cell divisions after the onset of light, demonstrating that glycogen-deficient cells fail to efficiently reinitiate growth after dawn. Overall, our results as a snapshot of the initial response to diurnal regimes highlight glycogen as a central integrator of diurnal physiology in Synechocystis, coordinating energy metabolism, redox balance, and cell division, with implications for metabolic robustness and the evolutionary constraints shaping (endo)symbiosis. Short summaryGlycogen deficiency disrupts day-night energy and redox homeostasis in Synechocystis, revealing constraints on growth, division, and symbiotic potential.

The photosynthesis-transpiration-stomatal conductance (An-E-gs) model framework is widely used for estimating photosynthesis, transpiration, and stomatal conductance in plants. The model equations are solved by numerical iteration, and the converged model values are deemed the solution. However, there has been no general guarantee that the iterative procedure converges to a solution or that the procedure leads to convergence. Building on the recent proof of the existence of a unique set of solutions, we herewith propose a numerical algorithm that is guaranteed to converge to the solution for the An-E-gs model framework. We first analytically prove that the proposed algorithm necessarily converges to a solution. We then demonstrate the convergence across contrasting combinations of leaf temperature, relative humidity, light, atmospheric CO2, and wind speed. We further demonstrate rapid convergence with the algorithm: no more than ca. 10 iterations for approximately 10-3 mol CO2 m-2 s-1 precision in net photosynthesis and no more than ca. 20 iterations for 10-7 mol CO2 m-2 s-1 precision. By guaranteeing convergence to the solution, this algorithm eliminates concerns about nonconvergence in leaf gas-exchange calculations and is expected to serve as a robust foundation for a range of studies from leaf-level gas exchange to global-scale carbon and water cycle dynamics.

Here we introduce OpenEvo, a fully open-source, low-cost turbidostat platform for automated continuous culture and directed evolution experiments. Existing tools are expensive, complex, or lack open-source hardware; OpenEvo addresses this gap. OpenEvo is a complete, fully automated evolution platform with detailed, illustrated construction instructions for beginners, open-source software and firmware, and a single device priced around $300. An optional PC-based version offers enhanced functionality, including remote access, programmable evolution cycles, programmable LED stimulation, and a data visualization tool. OpenEvo can cycle through three types of media for positive, negative, and neutral selection conditions, supporting a wide range of experimental designs. We validate the use of OpenEvo by evolving H. volcanii to grow from 15% to 12% salt over ~150 cycles, ~1,000 hours. Evolved cells grew 36% faster than wild-type at 12% salt. Whole-genome sequencing of adapted cells found SNPs and large deletions. We also demonstrate positive and negative selection using the OpenEvo LEDs to drive optogenetics via a Phytochrome B-based optogenetic tool, with light as the selection stimulus during over 4000 hours of growth. OpenEvo lowers the technical and cost barriers for continuous evolution experiments, serves as a teaching tool, and is designed to grow an open community of users who share modifications.

Microalgae are photosynthetic organisms capable of autotrophic growth. Their applicability in multiple industrial fields has been largely studied, thanks to their ability to fixate CO2 into high added value organic products like fatty acids and carotenoids. However, our understanding of the cellular signaling networks that control carbon flux and acclimation to environmental stress remains incomplete. In this study, we used the Chlamydomonas reinhardtii mutant strain vip1-1, which carries a loss-of-function mutation in the hexakisphosphate kinase re-sponsible for the synthesis of inositol pyrophosphates InsP7 and InsP8 (PP-InsPs), to investi-gate the role of these molecules during high-light acclimation. Our results indicate that PP-InsPs participate in the regulation of carbon storage in the form of starch and their deficiency increases TAGs levels in the algal cells. They also impact chloroplast-specific lipid remodeling by modifying membrane composition and fluidity through fatty acid desaturations and glycer-olipid composition. In addition, our findings suggest that PP-InsPs are involved in chloroplast-nucleus communication, where they coordinate transcriptional repression of photosynthesis associated nuclear genes (PhANGs), fatty acid desaturases and lipid synthases, contributing to cellular acclimation to high light. We also found that PP-InsPs modulating effect extended to protein synthesis and accumulation of Calvin-Benson-Bassham cycle intermediates. Therefore, we propose that PP-InsPs function as integratory molecules that balance carbon allocation between storage and structural pools, in response to environmental cues such as high light. These data uncover a novel function of PP-InsPs in high light acclimation and po-tentially in chloroplast-nucleus communication, providing new insights that may help engineering more resilient and efficient strains.

SummaryBiological growth curves are widely used but inconsistently analyzed due to fragmented workflows and limited quality control. We present growthcurves, a Python package for extracting growth parameters, and two open-source web applications, MicroGrowth and AutoGrowth, that combine automated fitting with interactive, human-in-the-loop inspection, selective refitting and traceable export for microplate reader and mini-bioreactor datasets in batch or turbidostat cultivation mode. Availability and Implementationgrowthcurves is implemented in Python and is freely available to non-commercial users at [https://github.com/biosustain/growthcurves.git] and through PyPI at [https://pypi.org/project/growthcurves/]. MicroGrowth and AutoGrowth are available at [https://biosustain.github.io/growthcurves_app/], and their source code is available at [https://github.com/biosustain/growthcurves_app.git]. Documentation, installation instructions, example datasets and tutorials are available at [https://growthcurves.readthedocs.io/en/latest/]. Contactstefdon@dtu.dk; cargac@dtu.dk Supplementary InformationSupplementary information and Supplementary Methods are available online.

Electrons energised by light energy at photosystem I (PSI) primarily enter the linear photosynthetic electron transfer (LET) or cyclic electron transfer (CET) pathway. The balance between LET and CET activity is a crucial factor in the regulation of photosynthesis, since CET increases the stoichiometry of proton to electron transfer. The additional transmembrane proton gradient ({Delta}pH) triggers feedback control of light harvesting and electron transfer photoprotection via non-photochemical quenching (NPQ) and photosynthetic control (PCON), maintaining the balance between the output of the light reactions and the downstream metabolism. Previously, it was found that Arabidopsis mutants lacking the stromal-facing membrane-extrinsic PSAE subunit of PSI (psae1-3) show enhanced CET activity, decreased LET and lower PSI oxidation in excess light. Here we show that high CET activity in psae1-3 primarily depends on the Proton Gradient Regulation 5 (PGR5)-dependent CET pathway rather than the NDH-dependent pathway. High CET is abolished in the psae1-3 pgr5CAS double mutant. In the psae1-3 ndho double mutant the elevated proton flux and CET are largely maintained; however, CO2 fixation and growth are significantly worsened, indicating that NDH still makes a physiologically meaningful contribution in the psae1-3 background. Biochemical analysis revealed that PGRL1 is redistributed from its normal mixed membrane distribution to become predominantly PSI-associated in psae1-3, providing a physical basis for the enhanced PGR5-dependent CET. These results underscore the primary importance of the PGR5-dependent CET pathway for optimal photosynthesis and CO2 fixation in Arabidopsis and establish the organisation of the PSI acceptor side as a key regulatory determinant of the CET/LET balance. HighlightLoss of the PSI acceptor-side subunit PSAE enhances predominantly PGR5-dependent cyclic electron transfer and redirects PGRL1 to PSI in Arabidopsis, while NDH contributes to maintaining CO2 fixation when the PSI stromal side is disrupted.

C4 photosynthesis evolved from the ancestral C3 pathway through coordinated leaf anatomical and metabolic reorganization that concentrates CO2 to reduce photorespiration. Quantitative understanding of these structure-function relationships remains limited. Here we used anatomy-aware metabolic modeling of a mesophyll-bundle sheath cell system to analyze the interdependence between leaf anatomy and photosynthetic metabolism on the C3-C4 spectrum. Our model faithfully recapitulates the transitory steps from C3 to C4 photosynthesis, reveals a crucial role for plasmodesmata in enabling the C3 to C4 transition, and points at potential pre-C2 metabolic states that provide benefits under conditions that favor elevated photorespiration. Incorporating bundle cell suberisation with our model predicts reduction of PSII activity and dominance of the NADP-ME C4 subtype in leaves with suberized bundle sheath cells and proposes a role for oxygen evolution at PSII as a potential driver for this mechanism. Varying bundle sheath leakage and photorespiratory conditions along the C3-C4 spectrum identify conditions under which C3-C4 intermediate photosynthesis provides energetic benefits and underlines the notion of intermediate photosynthesis as a stable evolutionary state. Overall, our study sheds new light on the quantitative relationship between leaf anatomy and metabolism and its interaction with the environment and suggests targets for climate-adaptation in C3 plants.

Biosynthesis of the linear tetrapyrrole phycocyanobilin (PCB) by the ferredoxin-dependent bilin reductase (FDBRs) PcyA is essential for light-harvesting and regulatory processes in diverse photosynthetic organisms, yet its evolutionary origins are not fully understood. PcyA evolved from pre-PcyA proteins found in diverse bacteria. Three lineages of pre-PcyA proteins were identified: Pre-1, Pre-2 and Pre-3. Using an in vivo co-expression assay, Pre-2 and Pre-3 proteins were shown to be active FDBRs that did not synthesize PCB, whereas Pre-1 activity was apparently low. In refining these results, we noted a discrepancy between phycoerythrobilin populations generated by Pre-3 and by the distantly related FDBR PebS. We therefore examined the properties of pre-PcyA enzymes in vitro, using an updated pre-PcyA phylogeny to select an alternative pre-1 target. Biochemical analyses revealed that Pre-1 and Pre-2 catalyze the two-electron reduction of biliverdin (BV) to 3E-phytochromobilin (3E-P[FE]B), in contrast to the known synthesis of 3Z-phytobilins by other FDBRs. Pre-3 can also carry out an additional two-electron reduction to yield 3E-phycoerythrobilin (3E-PEB), again distinct from the 3Z-PEB produced by PebS. We then used comparative sequence and structure analysis to target candidate catalytic residues for site-directed mutagenesis. Variant Pre-1 exhibited altered product stereochemistry, but no effects on Pre-2 were observed and Pre-3 variants unexpectedly gained the ability to bind cyclic tetrapyrroles. These findings underscore the plasticity and promiscuity of this enzyme family. Together, this work illustrates how the flexible catalytic potential of ancestral enzymes shaped the evolution and diversification of bilin biosynthetic pathways.

Nearly all carbon on Earth today is fixed by the enzyme ribulose-1,5-bisphopshate carboxylase/oxygenase ( rubisco), which converts carbon dioxide (CO2) to sugar phosphates. All rubiscos measured thus far display a kinetic isotope effect (KIE) where 12CO2 is fixed at a faster rate than 13CO2. The relationship between rubiscos KIE and the carbon isotope composition of plants, algae, and organic matter is central to many fields in the Earth sciences, plant biology, and biochemistry. Currently, all applications assume that the KIE does not vary with temperature. Here, we examine this assumption experimentally with in vitro KIE measurements of two rubiscos from phylogenetically distinct host organisms and rubisco protein clades - a Form I rubisco from the plant, Spinacia oleracea (spinach) and a Form II rubisco from the bacterium Rhodosprillium rubrum. We that find that both KIEs decrease linearly by [~]4.5{per thousand} from 10-35{degrees}C with statistically indistinguishable slopes. We place these results into biological and geologic contexts by comparing them to observed variations in the carbon isotope composition of modern terrestrial plants and marine organic carbon, the geologic carbon isotope record, and rubiscos biochemistry. We show that the measured temperature dependencies are sufficiently large to impact our interpretations of the enzymatic processes that drive variations in rubisco KIEs, as well as applications of stable carbon isotopes in the Earth and biological sciences. Significance StatementThe carbon isotope composition of plants, algae, and organic matter are interpreted with models that assume the kinetic isotope effect of the carbon-fixing enzyme rubisco is temperature-independent, even though temperature varies by tens of degrees across the Earth today and in the past. Here, we demonstrate that the kinetic isotope effect of rubisco is temperature-dependent, suggesting that some of this isotopic variation may be due to intrinsic enzyme properties alone. In addition, though the rubiscos we measured are from diverse organisms (plant vs. bacteria), their KIEs show statistically indistinguishable temperature dependencies. This data forms the basis for future thermodynamic models on rubisco biochemistry.

Energy-dependent quenching (qE) represents a critical mechanism for photosynthetic organisms to mitigate photodamage caused by excessive light. In green algae, LHCSR3 protein plays a central role in qE, activated by thylakoid lumen acidification and associated with photosystem II (PSII) to dissipate excess energy. Despite extensive efforts, the assembly and energy dissipation mechanisms of PSII-LHCSR3 have remained unknown. Here, we present the in situ structures of the PSII supercomplex embedded in the native thylakoid membranes of Chlamydomonas reinhardtii in both quenched (LHCSR3-bound) and unquenched (LHCSR3-free) states at near-atomic resolutions. Our results demonstrate that in high-light-acclimated cells, LHCSR3 binds to PSII peripheral antenna CP26 and associates with an extra LHCII trimer (eLHCII). Structural comparison of LHCSR3 with other light-harvesting complexes reveals possible protonation-induced conformational changes in LHCSR3 and rearrangements of its two pigment clusters, which potentially serve as quenching sites to dissipate excess energy transferred from CP26 and eLHCII. Our findings provide a direct visualization of how photoprotection is spatially organized in vivo and have implications for engineering natural and artificial photosynthetic systems with more dynamic photoprotection.