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.

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

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.

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.

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.

Far-red light (FR; 700-750 nm) is increasingly incorporated into controlled-environment lighting because it can improve photosynthetic efficiency when combined with comparatively shorter wavelengths. In long-day leafy crops such as spinach, however, FR may also promote the transition from vegetative to reproductive growth and thereby reduce marketable yield. Most studies have evaluated FR fraction, intensity or end-of-day exposure, whereas the developmental timing of FR has rarely been tested, particularly in spinach. Here, we evaluated six commercial spinach cultivars (Amador, Harp, Renegade, Responder, Rubino and Santa Cruz) in an indoor vertical farm under a common red-green-blue background (PPFD 260-264 {micro}mol m-{superscript 2} s-{superscript 1}, 12 h photoperiod, 24 {degrees}C) and four FR timing treatments: no FR (Control), FR throughout production (FullFR), FR during early development only (EarlyFR), and FR during late development only (LateFR). LateFR increased marketable fresh weight relative to Control (244 vs 224 g) and reduced flowering incidence, whereas far-red supplied during early development reduced fresh weight (158 g) and increased flowering. The magnitude of the timing response differed among cultivars: switching from EarlyFR to LateFR recovered 0 % fresh weight in Amador but 107 % in Renegade and Rubino, with the largest penalties occurring in otherwise bolt-resistant cultivars. EarlyFR also increased total chlorophyll and reduced the chlorophyll a:b ratio. These results show that FR response in spinach is strongly conditioned by developmental stage and cultivar. Although LateFR received more total far-red than EarlyFR, it behaved like the Control, indicating that the penalty was set by far-red timing rather than dose. Treatment differences in bolting and yield tracked an estimated phytochrome photostationary-state deficit during early development: a phytochrome-deficit model markedly outperformed a cumulative-dose model ({Delta}AIC = 441), and the deficit x cultivar interaction was strong (p < 0.001), with bolt-resistant cultivars losing most yield when far-red coincided with the early developmental window. We therefore propose that FR should be treated as a genotype-dependent management variable rather than as a fixed spectral input, with late application and bolt-resistant cultivars offering the most favourable combination for vertical-farm spinach production. Framed within the breeders equation, the close match between the trial and production environment and the scope for shorter breeding cycles indoors suggest that genotype and far-red timing can be optimised jointly to accelerate genetic gain.

Microalgal metabolism relies on their chloroplasts, and involves both nucleus and plastidial-encoded proteins of various evolutionary origins. The plastidial ATP synthase complex is a key player in photosynthesis, and has been extensively studied in plants. However, our knowledge in other photosynthetic eukaryotes remains limited, despite their importance in marine environments. Here, we report the characterisation of a novel homologue of the F-type ATP synthase alpha subunit, hereby named xATPA, widespread in microalgae but absent from other photosynthetic organisms. Comparisons of xATPA sequences and predicted structures revealed a specific feature, the bump domain, and highlighted the absence of an ATP-binding site. We assessed xATPA prevalence in microalgae in the global ocean using environmental data from Tara Oceans, with a particular focus on diatoms, and demonstrate that its expression is associated with polar summer conditions. Using a reverse genetic approach in the model diatom Phaeodactylum tricornutum, we show that xATPAP t has a plastidial localisation, and that xATPA KO mutants exhibit growth deficiencies in a combination of low temperature, low salinity and constant light, consistent with environmental analysis. Surprisingly, both RNAseq and physiological assays suggest that xATPA is not involved in ATP synthase functions. On the other hand, xATPA interacts with other F1 ATP synthase subunits in vitro, which we suggest forms transient unassembled complexes. This study hence represents a comprehensive analysis of a novel protein from the environment to the lab, and reveals a new player in the plastidial physiology of eukaryotic microalgae.

Bloom-forming cyanobacteria thrive in highly dynamic light environments, yet the mechanisms enabling rapid acclimation to fluctuating irradiance remain poorly understood. Here, we compared light acclimation in the bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806 and the non-bloom-forming model cyanobacterium Synechocystis sp. PCC 6803 and investigated the role of the cyanobacterial toxin microcystin (MC) and its in vivo binding partner RubisCO in this process. Whereas Synechocystis grew faster under sustained high light, Microcystis performed better under low light and responded to transient high-light exposure with a remarkably rapid increase in photosynthetic activity and glycogen accumulation. These responses were markedly attenuated in an MC-deficient mutant. Although RubisCO from Microcystis exhibited pronounced light-dependent changes in activity, MC had only minor effects on RubisCO catalysis, arguing against a direct role in regulating enzyme function. Instead, extracellular MC elicited a transient transcriptional program characterized by induction of inorganic carbon acquisition systems, including the high-affinity bicarbonate transporter BCT1, consistent with activation of the carbon-concentrating mechanism (CCM) and enhanced carbon fixation in vivo. MC further stimulated the expression of photosynthesis-related genes, and altered carboxysome organization, and promoted extracarboxysomal localization of RubisCO. Together, our findings identify MC as a light-responsive signaling molecule that coordinates CCM activity, carbon acquisition, and photosynthetic acclimation, thereby enhancing adaptation of Microcystis to fluctuating irradiance and potentially contributing to its ecological success in cyanobacterial blooms.

Whether Photosystem I (PSI) core subunits accumulate prior to light exposure in developing angiosperm seedlings remains unresolved, with conflicting reports across species. Here, we investigated the presence and membrane colocalization of the PSI core subunit PsaA in etioplasts of dark-grown angiosperms representing dicot and monocot species. Immunoblotting showed that PsaA accumulates in etioplasts of all three dicot species examined (pea, Arabidopsis, and runner bean), whereas in the monocot oat it was detected only after prolonged etiolation, at substantially lower levels and with an anomalously high apparent molecular weight. Blue-native PAGE analysis reveals that a fraction of PsaA co-migrates with LPOR, PsaB, FNR, and chlorophyll synthase, suggesting co-localization within a shared membrane microdomain rather than stable complex formation. The thylakoid insertase Alb3 was more abundant in dicot etioplasts, consistent with a potential role in the early integration of PsaA into the membrane. Upon illumination, pea reached PSI functionality faster than oat, with P700 oxidation detectable 30 min earlier, linking the dark accumulation of PsaA to an accelerated photosynthetic onset. These findings demonstrate light-independent accumulation of a PSI core subunit in a species-dependent manner and point to early steps in PSI biogenesis that precede full photosynthetic complex assembly. Highlight Contrary to prevailing models, a Photosystem I core subunit PsaA accumulates in dark-grown angiosperm seedlings before light exposure, revealing light-independent early steps in photosynthetic complex biogenesis.

As the worlds oceans change in response to climate change, phytoplankton communities will adapt to warmer, more stratified surface waters via plasticity, evolution, and range shifts. Current global ocean models assume that size structured phytoplankton communities have fixed trait relationships, and as a result generally predict that smaller size classes will become more dominant globally. However, this general expectation fails to consider how intra-species trait tradeoffs may operate orthogonally from large-scale inter-species tradeoffs--allowing for alternative evolutionary pathways given the limits and/or possibilities available to ancestral populations. To identify evolutionary pathways phytoplankton populations might take, we develop a novel modeling framework that combines a trait-based phytoplankton quota model with stochastic evolution (ecoTRACE). EcoTRACE explicitly decouples key phytoplankton traits from interspecific allometric relationships, allowing for novel phenotypes to emerge. We validated ecoTRACE against a long-term artificial size selection experiment on Dunaliella tertiolecta. We show that ecoTRACE captures multi-dimensional evolved phenotypes that quota models based on interspecific relationships fail to reproduce. Under fluctuating multi-stressor growth, model populations evolve phenotypic plasticity that deviates from predicted interspecific allometric relationships. EcoTRACE provides a framework for generating hypotheses as to the evolutionary trajectories that phytoplankton will experience in a warmer, more variable ocean.

Competition for light has driven extensive pigment diversification among phytoplankton species, yet how this diversity shapes their spatiotemporal distribution in the field has been little studied so far. The cyanobacterium Synechococcus is an ideal model for addressing this issue, since this group has colonized most light spectral niches in marine environments. Here, we used an approach based on marker read recruitment from metagenomes to analyze the seasonal succession of Synechococcus pigment types (PTs) at two time-series stations off French coasts exhibiting contrasting oceanic regimes. Marked seasonality was observed at both sites. The shallow, permanently mixed English Channel site SOMLIT-Astan was characterized by an alternation between green-light specialists (PT 3a) peaking in spring, and chromatic acclimaters type A (PT 3dA) accounting for most of the Synechococcus community in winter. In contrast, the pigment diversity was much higher at the deep Mediterranean station BOUSSOLE. In the upper layer, the two main PTs were the blue light specialists (PT 3c), which dominated the community in summer and fall, and PT 3dA cells, which were more abundant in spring. The third most abundant PT was chromatic acclimaters type B (PT 3dB), which accounted for up to 15% of the surface community in late fall. Strikingly, PT 3dA was dominant at depth during most of the year. Multivariate analyses between PT abundances, clade abundances and environmental factors, notably water color indexes, suggested new associations between PTs to specific clades and ecological niches. This study provides novel insights for refining distribution models of Synechococcus PTs and phytoplankton groups in general.

Phycobiliproteins form oligomeric assemblies essential for photosynthetic light harvesting. Here, we engineered phycocyanin (TeCPC) and allophycocyanin (TeAPC) from Thermosynechococcus elongatus to stabilize defined trimers by inhibiting hexamer formation. Structure-guided substitutions at conserved glycine residues (TeCPC G29R, TeAPC G21R) introduce steric hindrance at the hexamer interface. Recombinant expression in Escherichia coli produced holoproteins with native-like chromophorylation. Biophysical and structural analyses confirmed homogeneous trimer formation and absence of higher-order assemblies. Thermal measurements indicated cooperative unfolding, supporting structural uniformity. These engineered trimers provide robust models for studying energy transfer in phycobiliproteins.

Lipid peroxidation (LPO) is widely used as a biomarker of oxidative stress in coral bleaching research, yet its measurement remains poorly standardized across the field. A systematic review of the coral LPO literature reveals substantial variation in methodological approaches, including tissue fraction analysis, lysis protocols, assay choice, and normalization metrics, confounding cross-study comparison and obscuring the biological interpretation of results. We experimentally investigate two key sources of variation: the use of bulk holobiont vs separated host and algal symbiont fractions, and the choice of normalization metric. To do so, we used Montastraea cavernosa (n = 6 colonies) exposed to ambient (28C), heat stress (30.5C), and heat stress + artificial upwelling (AU; heat stress intermitted by daily pulses of cooler water, 30.5/27.5C) conditions in a controlled mesocosm experiment. Using a TBARS-based MDA assay with a lysis buffer optimized for coral tissue, we measured LPO separately in coral host and algal symbiont fractions across four time points throughout the day. Host MDA remained stable across all treatments and time points, consistent with either sufficient antioxidant buffering capacity or thermal acclimation over the experimental period. Algal symbiont MDA, in contrast, exhibited pronounced diel and treatment-specific dynamics, and the two fractions responses were decoupled from one another. Normalizing MDA to coral surface area instead of total protein content produced largely consistent diel and treatment patterns, but the two metrics diverged at specific time points, indicating that normalization choice is not interchangeable and can itself affect interpretation. Together, our literature review and empirical results demonstrate that host and algal symbiont LPO dynamics are not comparable when aggregated and argue for host-symbiont fraction separation and consistent, explicitly reported normalization as minimum standards for interpretable and cross-comparable coral LPO measurement.

Acetate metabolism drives mixotrophic and heterotrophic growth in some microalgae. Acetyl-CoA synthetase (ACS) and acetate kinase (ACK) are often considered the main enzymes involved in acetate catabolism in microalgae; however, their contributions to metabolic flux and carbon allocation are not fully understood. In this study, the functions of cytosolic ACS1 and mitochondrial ACK2 were characterized using two knockout mutants of the model microalga Chlamydomonas reinhardtii. The acs1 mutant exhibited a growth-oriented phenotype, characterized by 29.8% faster cell growth at 96 h and up to a 15.5% higher acetate depletion rate, yet showed a 38.3% lower triacylglycerol (TAG) content at 48 h under heterotrophic conditions. By contrast, the ack2 mutant exhibited an altered carbon-allocation phenotype under heterotrophic conditions. Despite an up to 32.4% lower respiratory oxygen consumption rate and a 27.7% reduction in cell density, ack2 exhibited a 39.3% higher biomass concentration and a 90.4% greater dry weight per cell than the wild type at 96 h. Biochemical analysis revealed that ack2 accumulated 23.3% more carbohydrate than the wild type at 120 h under heterotrophic conditions, whereas its TAG level remained comparable to that of the wild type. These findings suggest that, under heterotrophic conditions, the loss of cytosolic ACS1 facilitates cell growth and division at the expense of TAG biosynthesis, whereas the loss of mitochondrial ACK2 regulates growth by affecting carbon flux toward biomass and carbohydrate accumulation. This work provides insight into acetate catabolism in C. reinhardtii and suggests targets for engineering microalgae for production of biomass and bioproducts.

Reactive nitrogen species (RNS), particularly peroxynitrite generated from the reaction of superoxide and nitric oxide, are implicated in thermally-induced oxidative stress but remain difficult to resolve in live coral cells. We optimized fluorescent dye strategies to directly quantify superoxide, nitric oxide, and peroxynitrite production in thermally stressed Pocillopora acuta cell suspensions. Thermal stress was associated with an increase in intracellular peroxynitrite concentration, but not in its precursors, nitric oxide and superoxide, highlighting challenges with the application of fluorescent probes and their controls to live coral cells. Compounds developed for mammalian systems often translate poorly to non-model systems such as corals: strong endogenous fluorescence and multiple membrane barriers within the coral symbiocyte, for instance, limited the function of the nitric oxide probe, DAF-2DA. Despite these limitations, the detection of peroxynitrite in live, thermally stressed P. acuta cells represents a step forward in understanding the mechanism of coral bleaching. We also outline strategies for improving the performance of commercial dyes in non-model systems, including media optimization with EDTA treatment to preserve both cell viability and probe performance.

Plastids house the biology of eukaryotic photosynthesis. The majority of a plastids proteome is imported after cytosolic translation, but a few dozen proteins on average remain organelle-encoded, translated by the plastids own ribosomes. While 1000s of plastid genomes have been sequenced, the availability of less than ten proteomes and only two species with full 70S plastid ribosomal structures limit our understanding of land plant evolution. To address this, we optimized a protocol for the rapid isolation of Marchantia polymorpha chloroplasts that provides a highly enriched and intact organelle fraction from gradient volumes as little as 2 mL. Our approach was successfully applied to six other species, including Chlamydomonas reinhardtii and Nicotiana tabacum. Focusing on M. polymorpha, we determined the proteome of the chloroplast fraction, identifying 1337 nuclear-encoded proteins with a high confidence, where 83% belong to orthologs shared with angiosperms. We further isolated large protein complexes by RNA affinity purification using poly-lysine and provide the high-resolution structures of the 50S subunit of the chloroplast ribosome and RuBisCO from this bryophyte using cryogenic EM and image reconstruction to 2.23 and 2.12 [A] resolution, respectively, highlighting the structural conservation of both complexes. For chloroplasts, our data show that the genome reduction event experienced by the common ancestor of bryophytes has had little impact on the organelles complexity and that they underscore a high level of structural conservation of core components of plastid biology. Our data provide novel resources and methods to explore the functional evolution of plastid proteomes and major macromolecular complexes of cyanobacterial origin.

Cryogenic-electron microscopy (cryo-EM) has revolutionized the field of protein structural biology. The structures of large membrane proteins are now routinely determined by cryo-EM to near atomic resolution. However, in the medium resolution range of cryo-EM maps (>[~]2 [A]), negatively charged sidechains of acidic residues are not well-resolved due to the negative electrostatic potential of the region. This may lead to incorrect sidechain models for residues like glutamic acid or aspartic acid that are central for proton transfer activity in various respiratory and photosynthetic enzymes. We previously proposed that the acidic residues with weak or non-existent cryo-EM density can be modeled to represent their low proton affinity conformations. Here, we tested this hypothesis on a larger data set of acidic amino acid residues in two high-resolution respiratory complex I structures. By using faster sidechain modeling and proton affinity prediction tools, we created a workflow that generates sidechain conformations of selected amino acid residues. We validated the sidechain conformation predictions by Q-score analysis and atomistic molecular dynamics simulations in different charged states. The proposed workflow provides a way to rapidly obtain sidechain conformations of acidic residues with weak cryo-EM densities and can be integrated into the existing cryo-EM modeling pipelines to speed up sidechain rotamer prediction.

Reconstitution of functional nitrogenase in plants requires the coordinated expression of the [Fe-S] cluster assembly proteins NifU and NifS. However, the extent to which these proteins interact with endogenous Fe-S metabolism and affect plant physiology remains unclear. Here, we compared NifU and NifS homologs from diverse diazotrophs to identify variants compatible with the plant chloroplast environment. Selected variants of Azotobacter vinelandii, Fischerella thermalis, and Marinobacter lutimaris were characterized by transient expression in Nicotiana benthamiana and stable transformation in rice. Plant-produced NifU was largely devoid of [Fe-S] clusters when isolated but retained strong capacity for in vitro [Fe-S] cluster reconstitution and apo-NifH activation in a Ft > Av >Ml gradient, indicating correct folding and function but limited cluster loading or stability in vivo. NifU and NifS expression in transgenic rice resulted in variant-dependent proteome and phenotype effects, with A. vinelandii-expressing lines exhibiting severe defects, F. thermalis lines showing intermediate phenotype, and M. lutimaris lines being indistinguishable from wild type. These results reveal a trade-off between the biochemical activity of NifU and NifS and their compatibility with host metabolism, which must be considered for successful nitrogenase engineering in plants. HighlightNifU/NifS homolog selection determines trade-offs between [Fe-S] cluster assembly activity and plant compatibility, identifying variants that minimize physiological disruption while supporting nitrogenase cofactor assembly in chloroplasts.