Photosynthesis Research

In the next years, several space missions will search for evidence of life on exoplanets, focusing on robust biosignatures associated with oxygenic photosynthesis, including atmospheric oxygen accumulation and the Vegetation Red-Edge in surface reflectance spectra. Many potentially habitable rocky exoplanets orbit M-dwarf stars, whose spectral energy distribution may challenge oxygenic photosynthesis. Differently from the Sun, M-dwarf stars emit predominantly far-red (700- 750 nm) and infrared (750-1000 nm) light, and relatively little visible (400-700 nm) radiation, which constitutes photosynthetically active radiation. Some organisms have been found to photosynthesize under such spectrum but less efficiently than under solar light, as their photosynthetic apparatus evolved to harvest visible light emitted by the Sun. Around M-dwarfs, such different irradiation might have selected adaptations optimized for harvesting far-red / infra-red light. On Earth, similar selection can be found in Acaryochloris marina strains, constitutively presenting high chlorophyll d content in photosystem II & I, with in vivo absorption peaks beyond 700 nm. Here we tested the Moss Beach strain under a simulated M-dwarf spectrum and a simulated primeval atmosphere - anoxic and enriched in carbon dioxide. Results underline how this permanently red-shifted photosynthetic apparatus does not require acclimation to the stellar spectrum and enables for a strong growth and oxygen production, higher than under simulated solar light. Moreover, cells reflectance spectrum highlights a shift of the canonical red-edge toward longer wavelengths, resulting in a Chl d-near-infrared edge, suggesting a similar metabolism on exoplanets orbiting M-dwarfs could successfully produce both a gaseous biosignature and a characteristic surface biosignature. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/719884v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@7f91bdorg.highwire.dtl.DTLVardef@1391bdborg.highwire.dtl.DTLVardef@53f7b4org.highwire.dtl.DTLVardef@ab59fa_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Liistro, E. (2026) https://BioRender.com/j2de4ay

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.

O_LIAquatic algae are key primary producers in the Arctic and Antarctic, yet how cold-water species respond to environmental change is poorly understood. The Polar Regions are increasingly exposed to frequent heat waves, leading to declining ice cover, increased light availability, and decreasing salinity in polar waters. We compared three phylogenetically related but geographically distant polar Chlamydomonas species to test how habitat history shapes algal responses to light, salinity, and temperature stress. C_LIO_LIWe assessed the growth, morphology, and photochemistry of psychrophilic Chlamydomonas acclimated to native-like (lower light, higher salinity) and climate-shifted conditions (higher light, lower salinity). Next, we exposed acclimated cultures to a lethal heat shock and observed how acclimation affects algal temperature stress resilience. C_LIO_LIAll three species acclimated to climate-shifted conditions grew rapidly but showed the greatest sensitivity to temperature stress, with rapid loss of viability and photosynthetic efficiency. In contrast, slow-growing cultures acclimated to native-like conditions exhibited significantly greater resilience to temperature stress. C_LIO_LIOur work is the first to directly link light and salinity acclimation with temperature resilience in psychrophilic algae, suggesting that fast-growing polar green algae may be particularly vulnerable to increasingly frequent heat waves, with major implications for primary productivity in polar environments. C_LI

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.

1.Space weather exerts profound effects on Earths technological systems, yet its influence on the terrestrial biosphere remains largely unexplored at the global scale. Despite decades of research on solar-terrestrial interactions, most studies have focused on technological and atmospheric effects, while potential influences on biological regulation remain largely unexplored. While local experiments suggest magnetic sensitivity in plants (Galland and Pazur 2005; Belyavskaya 2004), observational evidence for a planetary-scale vegetative response to geomagnetic disturbances is lacking. In particular, it is unclear whether weak and intermittent geomagnetic disturbances can leave detectable signatures in ecosystem-scale physiological processes. Here, we analyze a decade of satellite-derived solar-induced chlorophyll fluorescence (SIF) data alongside geomagnetic indices to isolate non-seasonal physiological anomalies. Using temperature-stratified cumulative correlation analysis and multivariate models controlling for radiative and hydrological drivers, we identify a robust, cumulative, and thermally gated association between geomagnetic activity and vegetation fluorescence. We report a global-scale coherent modulation of photosystem balance, potentially inferred from the SIF757/SIF771 ratio, with recurrent geomagnetic disturbances, exhibiting maximal coherence under cold and moderate thermal conditions and weakening under Optimum and Warm Stress regimes. This response intensifies with increasing integration window length, indicating progressive physiological integration of repeated perturbations. Comparative analyses demonstrate that geomagnetic forcing is frequently comparable to or exceeds major climatic drivers in explaining fluorescence variability within biologically active regimes. We propose a mechanism consistent with magnetic modulation of radical pair spin dynamics in iron-sulfur clusters and cryptochromes, potentially influencing reactive oxygen species generation and redox-regulatory adaptation. Our findings suggest that plants have evolutionarily co-opted geomagnetic variability as an informational signal, integrating it into existing redox-regulatory networks. Rather than a passive mechanical perturbation, the observed response reflects an evolved sensitivity that operates near physiological criticality--a hypothesis that opens new frontiers in understanding magnetosphere-biosphere coupling.

The unicellular green alga Chlamydomonas reinhardtii provides a tractable model for investigating how carbon availability influences metabolic organization and cell-cycle control in photosynthetic eukaryotes. Its capacity for autotrophic (light, CO2), mixotrophic (light, CO2, acetate), and heterotrophic (acetate, dark) growth enables systematic analysis of trophic-state-dependent regulation. We performed comparative transcriptomic analyses of strain 21gr grown under these three regimes at 30 {degrees}C. Mixotrophy resulted in the highest biomass accumulation and was associated with earlier cell-cycle commitment compared with autotrophy, whereas heterotrophy displayed delayed commitment and reduced growth. Transcriptomic profiling revealed coordinated upregulation of central carbon metabolic pathways under mixotrophy, including photorespiration, glycolysis, the oxidative pentose phosphate pathway, and tricarboxylic acid cycle functions, consistent with enhanced carbon flux and biosynthetic capacity. In contrast, heterotrophy preferentially induced acetate assimilation and glyoxylate cycle genes and was accompanied by elevated expression of cell-cycle regulators, including the CDK-inhibitory kinase WEE1. Together, these findings indicate that trophic mode modulates the coupling between carbon metabolism and cell-cycle progression, with mixotrophy supporting integrated metabolic and proliferative activity, whereas heterotrophy is associated with delayed cell-cycle timing and transcriptional signatures of metabolic adjustment.

Knock-out mutants are often used to study gene function by disrupting a specific gene and comparing the mutant to a wild-type strain. Reliable interpretation, however, requires that the two strains differ only by the intended mutation and that the observed phenotype is caused specifically by the deleted gene. In the highly polyploid cyanobacterium Synechocystis sp. PCC 6803, this is particularly challenging because incomplete segregation can mask genetic heterogeneity or secondary suppressor mutations. The genetic variation among laboratory wild-type lines can further confound phenotypic analyses. We show that these challenges can be addressed by routine strain validation via whole-genome sequencing (WGS). To this end, we developed and tested user friendly workflows for short-read (Illumina), long-read (Oxford Nanopore Technologies; ONT), and hybrid data, providing standardized quality control, variant calling, and structural variant detection. We benchmarked their performance in detecting single-nucleotide polymorphisms (SNPs), small indels, and structural variants using simulated datasets across different coverages and mixed populations. Applying the workflows to three Synechocystis sp. PCC 6803 wild-type lines revealed multiple sequence and structural differences relative to the reference genome, including previously undescribed genetic variants, underscoring the importance of documenting the strain background and the value of long-read sequencing. Characterization of two independent 6-phosphogluconate dehydrogenase (gnd) knock-out mutants and their complemented strains highlighted how a failed rescue can reveal a phenotype unrelated to the intended knock-out. An automated literature analysis revealed that only a minority of the investigated Synechocystis studies that used knock-out mutants included complementation as a control (39%), whereas this practice is more common in studies involving Escherichia coli (63%) and Saccharomyces cerevisiae (55%). Based on these results, we propose a practical guide for standardizing knock-out phenotyping in Synechocystis PCC 6803. Combined with accessible workflows for routine whole-genome validation, this framework aims to support more robust and reproducible knock-out studies in the future.

In C4 photosynthesis, incoming CO2 is incorporated in mesophyll cells (MC) into 4-carbon acids that diffuse to bundle sheath cells (BSC) and decarboxylated to generate a high CO2 concentration that suppresses the oxygenation reaction of Rubisco. Decarboxylation can occur by NADP-malic enzyme, (NADP-ME), NAD-malic enzyme (NAD-ME) or phosphoenolpyruvate carboxykinase (PEPCK). NADP-ME generates NADPH in the BSC chloroplast and species that use it as the major route for decarboxylation typically have dimorphic BSC chloroplasts with little or no photosystem II. They operate an energy shuttle: much of the 3-phosphoglycerate formed in the Calvin-Benson cycle diffuses to the MC, enters the chloroplasts and is reduced to triose phosphates that return to the BSC. In species where carboxylation occurs mainly via NAD-ME or PEPCK, BSC chloroplasts possess photosystem II. Indirect evidence indicates they nevertheless have the capacity to operate an energy shuttle. We show here that NAD-ME and PEPCK species possess large pools of 3PGA and triose phosphates and, for two examples of each subtype, opposed concentration gradients of 3-phosphoglycerate and triose phosphates to drive rapid exchange between the BSC and MC. Reasons for and consequences of the widespread operation of the intercellular energy shuttle in C4 plants are discussed. Highlight StatementAn intercellular energy shuttle in which 3-phosphoglycerate moves from the bundle sheath to the mesophyll and triose phosphates return to the bundle sheath is a general feature of C4 photosynthesis.

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.

When three cyanobacterial proteins--KaiA, KaiB, and KaiC--are incubated with ATP in vitro, the phosphorylation level of KaiC exhibits stable circadian oscillations. Biochemical and structural analyses have shown that KaiCs ATPase activity is crucial for these oscillations, leading to the hypothesis that ATP-consuming dynamics function as a molecular clock, determining the oscillation period of individual molecules. Moreover, these molecular clocks synchronize with one another, resulting in collective oscillations at the ensemble level. In this study, we develop a theoretical model to test this molecular clockwork hypothesis. Our model clarifies the relationship between the oscillation period and ATPase activity, explaining the significant changes in the period induced by amino-acid substitutions near the CI-CII domain boundary of the KaiC hexamer. Furthermore, the model addresses the physical basis for temperature compensation concerning both the oscillation period and ATPase activity. Thus, the molecular clockwork perspective provides a framework for understanding the atomic design behind collective oscillations.

Under natural growth conditions, plants are not usually exposed to the high-energy ultraviolet C range (UV-C, 100-280 nm) of the solar spectrum, as this is absorbed by the ozone layer. However, low doses of UV-C radiation can trigger stress responses in plants. Nevertheless, it is not yet fully understood how UV-C light affects plant development at the hormonal level. Here we show that a single one-min UV-C light pulse (20 W/m2) alters gibberellin (GA) homeostasis in Arabidopsis in two phases: initially, the level of GA12 - a key precursor of the final part of gibberellin biosynthesis - is reduced. Consistent with this, the transcript levels of the CPS, KS and KAO2 genes, which encode enzymes involved in the initial parts of gibberellin biosynthesis, decrease. The level of the plant hormone GA4 also decreases initially, probably due to the reduced GA12 precursor levels. However, in a second phase, the endogenous GA4 levels rise in UV-C treated plants relative to control plants. This increase leads to an early onset of flowering, as well as increased growth and fertility, in UV-C-treated Arabidopsis plants. The GA signalling mutant gdella does not exibit wild-type phenotypic responses to UV-C treatment, indicating that GA signalling is essential for the UV-C response. To further narrow down the responsible steps in the GA-signalling pathway, we tested the kao1 and kao2 mutants, which are both impaired in early gibberellin biosynthesis. Neither mutant displays phenotypic responses to the UV-C treatment, indicating that both genes are required for mediating the UV-C response. In contrast, the quintuple 2-oxidase mutant C19--2oxqM exhibits responses to UV-C treatment similar to the wild-type, suggesting that the five catabolic 2-oxidases that act on C19-GAs play a negligible role in regulation GA-hormone levels for growth and development in this case. HighlightUV-C pulse triggers biphasic gibberellin dynamics, delaying early development but ultimately enhancing growth and fertility in Arabidopsis thaliana.

Photosynthetic electron transport is mediated by several protein supercomplexes that are spatially arranged in the thylakoid membranes of chloroplasts. The chloroplast NADH dehydrogenase-like (NDH) complex is part of the photosynthetic alternative electron transport (AET) chain, which reduces the plastoquinone (PQ) pool using reduced ferredoxin as a substrate. This NDH complex is associated with photosystem I (PSI) and mediates a portion of AET in stroma lamellae, whereas photosystem II (PSII) is concentrated in grana stacks. This study presents the findings regarding post-illumination chlorophyll fluorescence increase (PIFI), a protein crucial for regulating AET via the NDH pathway. A marked increase in NDH activity and a reduction in the PQ pool in the dark were observed in PIFI-deficient mutant strains (g-pifi) generated by genome editing. Blue native PAGE analysis indicated that PIFI was associated with the NDH-PSI supercomplex in the wild type, and the NDH complex was dissociated from PSI in the g-pifi mutants. Additionally, the g-pifi mutants exhibited a decrease in the maximum quantum yield of PSII (Fv/Fm). Notably, Fv/Fm was restored in a double mutant harboring both g-pifi and NDH-deficient pnsl1 mutations, demonstrating that deregulated NDH activity in g-pifi causes downregulation of PSII efficiency. However, the lower Fv/Fm was not observed in a mutant lacking thioredoxin m4 (trxm4), which showed deregulated NDH activity but maintained the NDH-PSI supercomplex. These data suggest that PIFI stabilizes the NDH-PSI supercomplex and maintains the spatial localization of PQ reduction via AET in thylakoid membranes, which is essential for the proper functioning of PSII.

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.

Cyclic electron transport (CET) around photosystem I (PSI) is essential for maintaining photosynthetic efficiency by balancing ATP/NADPH production and protecting PSI from photoinhibition. Although the PROTON GRADIENT REGULATION 5 (PGR5)-dependent CET pathway is known to be critical under high or fluctuating light conditions, its role under fluctuating low light remains poorly understood. In natural environments, plants frequently experience prolonged low irradiance interspersed with brief sunflecks, making fluctuating low light a physiologically relevant condition. Here, we investigated Arabidopsis thaliana lines with graded PGR5 expression levels to evaluate the dose-dependent contribution of PGR5 to CET activity, photosynthetic regulation, and growth performance under both low light and fluctuating low light conditions. Moderate increase in the PGR5 protein level enhanced CET activity, accelerated photosynthetic induction, improved PSI protection and increased biomass accumulation under fluctuating low light. In contrast, excessive PGR5 accumulation impaired photosynthetic performance and reduced plant growth, indicating that optimal CET capacity requires precise tuning of PGR5 abundance. These results reveal a non-linear relationship between PGR5 protein levels and photosynthetic performance and demonstrate that moderate enhancement of CET improves plant productivity under fluctuating low light. Our findings highlight the importance of optimizing CET capacity to match dynamic light environments and suggest that fine-tuning PGR5 expression could be a promising strategy for improving crop performance under natural canopy conditions. Significance statementModerate increase in the PGR5 improves plant productivity, whereas excessive PGR5 accumulation impaired photosynthetic performance and reduced plant growth. Therefore, optimizing CET capacity by the fine-tuning PGR5 expression is important for improving crop productivity.

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.

Ferredoxins are central to cellular metabolism by mediating electron flow in energy conversion reactions. The focus of this study was to systematically examine twelve ferredoxin and ferredoxin-like proteins from Synechocystis sp. PCC 6803 to identify their properties, activities, and functions in electron transfer. Using electron paramagnetic resonance spectroscopy, we detected cluster types consistent with major ferredoxin families including plant-type [2Fe-2S], adrenodoxin, thioredoxin, and bacterial-type [4Fe- 4S] ferredoxins. In addition, we found that the ssr3184 ferredoxin-like protein exchanged between a [3Fe-4S] or a [4Fe-4S] cluster, pointing to a possible functional change in response to changes in oxygen or cellular redox poise. Electrochemical measurements demonstrated that these ferredoxins constitute a broad potential window, from -243 mV to -520 mV vs SHE. Investigations on their capacity to support electron-transfer focused on reactions with two major redox hubs: Photosystem I and pyruvate:ferredoxin oxidoreductase and included testing of binding interactions with nitrite reductase. Expression profiling under multiple environmental conditions was also used to predict function and revealed distinct regulatory patterns. Collectively, these findings identified a group of core ferredoxins that directly support photosynthetic electron transfer, and more specialized ones that may serve other functions. In summary, Synechocystis utilizes a suite of ferredoxins to maintain cellular redox homeostasis under dynamic environmental conditions.

In 2016, the glycolytic Entner-Doudoroff (ED) pathway was reported in cyanobacteria and plants (1). The claim was based on the biochemical characterization of its key enzyme the 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase EDA (ED aldolase), on protein sequence alignments, physiological data from cyanobacterial mutants, and the in vivo detection of an ED pathway specific metabolite (1). However, two enzymes 6-phoshogluconate (6PG) dehydratase (EDD) and EDA are unique to this route. A recent study suggests that EDD (Slr0452) from Synechocystis sp. PCC 6803 most likely encodes an enzyme involved exclusively in amino acid synthesis, indicating that a complete ED pathway would be missing (2). To answer the presence or absence of the ED pathway in Synechocystis, we conducted extended biochemical and physiological studies, revisited old data and resolved contradictions. These investigations reveal that Synechocystis lacks both an ED pathway and a glucose dehydrogenase/glucokinase (GDH/GK) bypass but contains a promiscuous aldolase EDA. EDA prefers KDPG as substrate but also decarboxylates oxaloacetate (OAA) and cleaves 2-keto-4-hydroxyglutarate (KHG). Synthesis of KDPG from pyruvate and glyceraldehyde 3-phosphate (GAP) is catalyzed with very low efficiency. These in vitro data suggest that EDA might be involved in the phosphoenolpyruvate (PEP)-pyruvate-OAA node and proline catabolism, which requires further clarification. The previous misconception was based on missing enzymatic characterizations, the oversight of a secondary mutation in a deletion strain, and an outdated view on carbohydrate fluxes. We conclude with a list of lessons and provide a solid foundation for future investigations into the role of EDA in cyanobacteria and other photoautotrophs. Significance statementThis study provides a retrospective on why, for many years, it was mistakenly assumed that the glycolytic Enter-Doudoroff (ED) pathway exists in the cyanobacterium Synechocystis sp. PCC 6803. It shows that the first enzyme of this pathway, ED dehydratase EDD, is absent, while the second enzyme, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase EDA, is present but is promiscuous, cleaving KDPG in addition to 2-keto-4-hydroxyglutarate (KHG) and decarboxylating oxaloacetate (OAA) in vitro. Finally, valuable lessons are drawn from prior misconceptions and experimental limitations. This study provides a solid foundation for future studies on the role of the ED aldolase in absence of the ED pathway in cyanobacteria and other photoautotrophs.

Understanding the regulation of enzyme activity involved in photosynthesis is essential for engineering enhanced carbon fixation in crops. In C4 plants, the enzyme phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is one of the most abundant leaf enzymes and plays an essential role in photosynthetic carbon dioxide (CO2) fixation. The enzyme also plays a key role in central metabolism (e.g., providing intermediates to the citric acid cycle) and therefore must be highly regulated to coordinate its activity. The regulation of PEPC activity can occur allosterically by glucose 6-phosphate activation and malate inhibition, which is in part influenced by reversible phosphorylation. A specific light-dependent phosphorylation of PEPC at an N-terminal serine residue by the PEPC-protein kinase (PEPC-PK) can regulate its sensitivity to this allosteric regulation. However, the impact of this PEPC phosphorylation has not been tested in a C4 crop. Therefore, we created PEPC-PK mutant lines in Zea mays to assess the impact of PEPC phosphorylation on its allosteric regulation, photosynthesis, and growth. While the maximum PEPC activity was unchanged, PEPC in the PEPC-PK mutant plants was not phosphorylated under light and was more sensitive to malate inhibition. However, gas exchange, electron transport, and field biomass analyses showed no differences in the PEPC-PK mutant plants. These results demonstrate that in Z. mays PEPC phosphorylation affects enzyme sensitivity to malate in vitro but does not substantially alert photosynthetic performance or growth under field conditions suggesting additional regulation of PEPC activity in planta.

The photosynthetic thylakoid membrane systems of many oxygenic photosynthesizers form elaborate three-dimensional structures. Curvature Thylakoid (CurT/CURT1) proteins are central regulators of thylakoid architecture in cyanobacteria and plants, driving both grana stacking and the formation of thylakoid convergence zones (TCZs) by promoting membrane curvature while also contributing to cyanobacterial cell and green-algal chloroplast division. While the functional role of grana stacks in terrestrial photosynthesis is well established, the physiological significance of cyanobacterial TCZs remains unclear. Here, we investigated the role of TCZs in Synechocystis sp. PCC 6803 by quantitatively assessing how CurT protein abundance shapes thylakoid membrane organization, TCZ frequency, and thylakoid layering, and how these architectural features relate to photosynthetic efficiency in a set of curT expression mutants. By correlating defined structural parameters of the thylakoid system with cellular CurT levels, we defined for the first time the quantitative relationship between CurT abundance, thylakoid architecture, and photosystem II (PSII) performance. Our results reveal a non-linear, logarithmic relationship: minimal CurT suffices to restore TCZ formation and sustain WT-like growth, while overexpression further enhances PSII activity and culture yield. Together, these findings identify CurT as a dose-dependent key determinant of thylakoid structure and underscore the functional importance of thylakoid membrane architecture for efficient photosynthesis. Significance StatementThis study assesses how the membrane-shaping protein CurT quantitatively regulates thylakoid architecture and photosynthetic performance in the model cyanobacterium Synechocystis sp. PCC 6803. By relating CurT abundance to the formation of thylakoid convergence zones, photosystem II efficiency, and growth rate, we provide evidence of CurT acting as a key regulator of cyanobacterial membrane organization in a dosage-dependent manner. Our findings provide insight into the structural basis and physiological relevance of thylakoid convergence zones and suggest CurT as a potential target for improving cyanobacterial light-energy conversion.