Photosynthesis Research

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.

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.

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.

Cyanobacteria are leading biomass producers of the ocean whose ecological success relies on their ability to respond to dynamic availability of nutrients like CO2 and nitrogen, which require distinct adaptive mechanisms. To survive nitrogen deprivation, cyanobacteria undergo a reversible transition to a dormant mode. Under low CO2 levels, a CO2 concentrating mechanism (CCM) supports their CO2 fixation. While the CCM and nitrogen assimilation have been shown to share some regulatory pathways, how the CCM impacts the response to nitrogen deprivation remains underexplored. In this study, by using mutants of the coastal cyanobacteria Synechococcus sp. PCC 7002 lacking a CCM component, we show that the high rate of carbon fixation mediated by the CCM tunes the speed of the nitrogen deprivation response in {beta}-cyanobacteria. We first show that CCM mutants are deficient in inducing their typical nitrogen deprivation response under atmospheric CO2. However, at higher CO2 concentrations, the CCM mutants induce the nitrogen deprivation response. By combining Rubisco kinetics modeling with measurement of the response speed to nitrogen in various CO2 concentrations, we show that the speed of the nitrogen deprivation response increases linearly with Rubiscos carboxylation rate. We further reveal that the regulation of nitrogen response by the CCM is also present in the distantly related freshwater cyanobacteria Synechococcus elongatus PCC 7942, suggesting a widespread role of this regulation across {beta}-cyanobacteria. This study demonstrates that CO2 fixation by the cyanobacterial CCM is a key regulator of the nitrogen deprivation response, favoring a rapid response to dynamic environments.

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.

Chlorophyll is the central cofactor in oxygenic photosynthesis, responsible for both light capture in antenna complexes and light-driven charge separation in photosystems. The tetraprenyl tail of chlorophyll is attached to the chlorophyllide macrocycle by the transmembrane enzyme chlorophyll synthase (ChlG). In the cyanobacterium Synechocystis sp. PCC 6803, ChlG forms a complex with the LHC-like high-light-inducible protein HliD. To understand the substrate specificity and catalytic mechanism of ChlG, and how it is photoprotected by HliD, we determined the structure of a ChlG2HliD2 complex in both substrate-free and geranylgeranyl pyrophosphate (GGPP)-bound states using cryogenic electron microscopy (cryo-EM). A homodimer of HliD, which binds four chlorophylls and two zeaxanthin molecules in a quenched state, is flanked by two ChlG monomers. AlphaFold modelling placed chlorophyllide adjacent to the structurally resolved GGPP bound to the active site of ChlG. Cryo-EM data, site-directed mutagenesis and molecular dynamics were used to formulate a molecular mechanism for ChlG catalysis. The structure of the ChlG2HliD2 complex shows how Hlips bind to the synthase, reveals the arrangements of carotenoids and chlorophylls that mediate energy dissipation, and sheds light on the evolution of eukaryotic LHC antennae from their cyanobacterial ancestors.

Plant photosynthesis operates under naturally fluctuating light, yet its dynamic responses across timescales remain incompletely understood. Here, we apply sinusoidal light modulation as a controlled periodic input and analyze the response in the frequency domain, enabling quantitative system identification of photosynthetic dynamics. Using a minimal biochemical model of photosynthetic electron transport and regulation, we show that the system exhibits distinct dynamic regimes separated by a characteristic timescale of approximately 10 s. In the high-frequency domain, the response is governed by constitutive processes and reflects steady-state properties such as the plastoquinone redox state. In the low-frequency domain, regulatory feedback dominates, particularly non-photochemical quenching (NPQ), which modulates both the amplitude and phase of the response. For small-amplitude perturbations, the system behaves linearly and can be characterized using transfer functions and Bode plots. We show that key physiological parameters, including relaxation times and regulatory gains, can be extracted directly from frequency-response features such as phase maxima and gain transitions. In the nonlinear regime, large-amplitude oscillations generate higher-harmonic structure and alter time-averaged photosynthetic performance relative to constant illumination. We further introduce the concept of regulation fingerprints, defined as ratios of transfer functions between regulated and unregulated systems. These fingerprints reveal distinct spectral signatures of fast (PsbS-mediated) and slow (zeaxanthin-dependent) NPQ processes, enabling their quantitative separation. Together, these results establish frequency-domain analysis as a framework for probing and identifying the dynamic regulation of photosynthesis under fluctuating light, with direct applicability to non-invasive measurements in laboratory and field conditions.

Photoswitchable ligands enable photocontrol of biomolecular activity by binding to targets in an isomer-dependent, light-responsive manner. Recent developments in ionizable photoswitchable ligands greatly expand their applications but introduce a major design challenge: light-responsive binding can depend on isomeric form, chemical substitution, and binding-induced shifts in protonation equilibria. These effects are tightly coupled, subtle in magnitude, and difficult to predict. Consequently, few computational methods have been developed and systematically benchmarked for quantitatively predicting them. Here, we establish a multiscale free-energy method and benchmark it against experimental data for a series of recently developed photoswitchable inhibitors of Escherichia coli dihydrofolate reductase (eDHFR), a crucial target in photopharmacology. Constant pH replica-exchange molecular dynamics and quantum mechanics/molecular mechanics umbrella sampling quantitatively characterize the ligands protonation-state change upon binding to the eDHFR active site. Thermodynamic integration simulations using alternative alchemical pathways, thermodynamic cycles, and protonation-state assignments were evaluated for predicting light-responsive affinity differentials and substituent effects. Direct cis-to-trans transformations with explicit treatment of environment-dependent protonation states best reproduce experimental trends. Compound-to-compound pathways are less reliable because force-field inaccuracies introduce large pK errors that are difficult to correct when protonation/deprotonation processes implicitly enter the thermodynamic cycle. TI simulations that ignore binding-induced protonation-state changes fail to consistently reproduce experimental trends. Protein-ligand and ligand-water interaction analyses further reveal the energetic and structural origins of isomer-dependent binding. This study establishes a systematic free-energy method for designing ionizable photoswitches in photopharmacology.

In the plant phyllosphere, many bacteria produce pigments to mitigate oxidative stress, including pink-pigmented facultative methylotrophs (PPFMs). PPFMs are prominent members of the phyllosphere microbiota, yet their secondary metabolism remains underexplored. This limits our understanding of how these bacteria interact with other phyllosphere constituents at the molecular level. Here, we develop a screen for PPFM biosynthetic gene clusters and identify listianol, a previously undescribed metabolite that inhibits the pigmentation and growth of diverse bacteria. Listianol blocks carotenoid biosynthesis by targeting the desaturases CrtN and CrtI, revealing a previously unrecognized inhibitory mechanism for a secondary metabolite. This activity sensitizes normally pigmented bacteria to UVB radiation in vitro, and a listianol-producing strain reshapes the composition of a synthetic bacterial community on Arabidopsis thaliana under UVB exposure. Together, these findings establish a platform for exploring PPFM biosynthetic potential and uncover a new metabolite with potential to impact the phyllosphere microbiota composition.

Snow algae darken snowpacks and accelerate melt worldwide. Although elevation strongly structures the physical conditions of mountain snowfields, its influence on snow algal traits and their effects on snowpack reflectance remains unclear. Here, we investigated snow algal composition, cellular traits, and optical properties in summer blooms across an elevational range of 1,059-3,423 m a.s.l. in the western United States, spanning two elevational gradients in the Cascade Range (CA, OR, WA) and the Rocky Mountains (UT, WY, MT). Across all samples (n = 294), snow albedo declined strongly with increasing algal cell density, indicating that total biomass, rather than pigment composition, is the dominant driver of albedo reduction. However, within Sanguina-dominated blooms (117 of 206 samples bloom samples identified across the dataset), neither relative abundance nor algal cell density varied systematically with elevation. Instead, mean cell size increased with elevation, while per-cell pigment concentrations declined, leading to higher astaxanthin:chlorophyll-a ratios driven primarily by reductions in chlorophyll-a per cell. These elevation-dependent shifts in cell size and pigment balance were consistent across both mountain ranges, indicating phenotypic acclimation to increasing environmental stress with elevation. Together, these findings link cellular-scale acclimation of a widespread snow alga to radiative processes shaping mountain snowpacks.

The standard set of 20 genetically-encoded amino acids (C20) exhibits a statistically non-random distribution in primarily two structurally-relevant physicochemical properties: hydrophobicity and molecular volume, and to a lesser extent charge. It remains an open question, however, whether evolutionary pressures similarly optimized the same alphabet for the distribution of functionally-relevant properties, such as reactivity. In this study, we used semi-empirical quantum chemistry simulations to calculate the highest occupied molecular orbital and lowest unoccupied molecular orbital (HOMO-LUMO) gaps for 84 xeno amino acids and constructed 10 million random 20-mer amino acid alphabets to determine where C20 fit amongst this background. The HOMO-LUMO gap measurements demonstrated that C20, similar to hydrophobicity and volume, also exhibits a non-random distribution. However, unlike hydrophobicity and volume, this distribution is non-random across an unevenly broad range. The results expand upon previous theory and suggest HOMO-LUMO gap energies as one synthetic biologists may consider when developing novel protein design tools or designing functional xeno amino acid alphabets. HighlightsO_LILifes amino acid alphabet is non-randomly distributed within an expanded computationally-generated chemistry space generated from large-scale quantum chemistry simulations. C_LIO_LIAmino acid alphabet coverage theory applies beyond structurally-relevant physicochemical descriptors to include functionally-relevant properties like reactivity as measured by frontier molecular orbitals C_LIO_LIFindings here provide a theoretical framework to guide the design of novel proteins and development of synthetic amino acid alphabets. C_LI

Invertebrate vision relies on bistable visual pigments flipping upon photon absorption between rhodopsin and metarhodopsin states. In living butterflies, the UV-VIS absorption spectra of rhodopsin and metarhodopsin, respectively with 11-cis and all-trans isomers of 3-hydroxy-retinal (A3) chromophore, can be conveniently recorded from the eyeshine, the light reflected from the compound eye after passing twice through the light-guiding rhabdoms. * Here, a microscope coupled with a broadband LED source and a microspectrometer was used to record photorelaxations reported in eyeshine reflection spectra. Fitting temporal exponential relaxations to log-reflectance arrays yielded transient and baseline spectra that are analogous to absorbance difference and sum, respectively. Both types of spectra were subjected to singular value decomposition and to fitting of templated visual pigment absorption spectra. * The compound eye of the high brown fritillary Fabriciana adippe was exposed to a series of second-long broadband light pulses, causing photorelaxations with time constants between 40 and 120 ms that led to 80% metarhodopsin in equilibrium. The transient and baseline spectra were fitted with pigment templates, estimating the alpha peak wavelength 547-552 nm for rhodopsin and 496-501 nm for metarhodopsin. The metarhodopsin to rhodopsin alpha peak absorbance ratio 1.25-1.35 is consistent with the isosbestic wavelength at 530 nm. The second isosbestic wavelength indicates that rhodopsin beta (UV) peak absorbs more strongly than metarhodopsin below 405 nm. * Baseline spectra, which were not explicitly analysed in previous studies, enable concatenation of exposures, monitor long-term changes of pigment, and enhance the estimation of beta peak parameters. * The method can be directly used in many butterflies and could be adapted to other insects, particularly fruitflies, facilitating studies of the relation between the visual pigment spectra and the opsin sequences. Spectroscopic results can be complemented with physiologically measured photoreceptor spectral sensitivity datasets and analysed with the same global fitting procedure.

A global nuclear war could inject soot into the stratosphere, blocking sunlight and causing rapid cooling. Assessments of the resulting agricultural collapse rely on crop models never validated under such conditions. We grew wheat, canola, and potato in growth chambers simulating the light and temperature of an extreme nuclear winter at tropical and temperate sites. In the tropical chamber (18-20 {degrees}C, 200 mol m-2 s-1 PAR), all three crops produced viable yields. Wheat yielded 2.1-2.3 t/ha (n=3 well-watered, n=3 water-stressed pots), 60% of the global average, and single-pot observations of canola and potato suggested biological yields comparable to global averages. In the temperate chamber simulating nuclear winter irradiance (60-360 mol m-2 s-1), wheat stems collapsed under their own weight. Although hand-harvesting recovered 0.6-2.8 t/ha of grain, mechanical field harvest of a flat canopy would recover substantially less. This failure mode was not observed in a higher-light control chamber and is not captured by existing crop models, which may therefore overestimate temperate cereal production under nuclear winter. Canola produced comparable yields under both temperate light regimes without lodging. Empirical screening of additional staples is needed to identify which remain viable under nuclear winter.

Avian phototransduction and magnetoreception have been proposed to involve shared retinal proteins, including interactions between long-wavelength opsin (LWO), the cone-specific heterotrimeric G protein (Gt), and cryptochrome 4a (Cry4a), yet structural information on avian phototransduction complexes is lacking. Here we present and critically assess two atomistic models of the European robin LWO-Gt complex generated by distinct modelling strategies. A full-complex prediction using AlphaFold3 yields a tightly packed, structurally stable interface but exhibits pronounced activation-like conformational features of the Gt-subunit that persist in simulations of the isolated protein, revealing a strong bias toward the active state. In contrast, a template-guided assembly based on single-chain predictions and an experimental rhodopsin-Gt reference structure forms a weaker interface and shows no intrinsic activation bias, while still displaying subtle activation-related dynamics. These results demonstrate that machine-learned complex prediction can encode functional states independently of the local interaction environment, thereby limiting its interpretability for signalling mechanisms that hinge on activation equilibria. Our findings highlight the need for explicit assessment of conformational-state bias when modelling regulatory protein assemblies and provide a structural framework for future studies of Cry4a-dependent modulation of retinal G-protein signalling in avian magnetoreception.

Understanding plant growth dynamics requires imaging across day-and-night cycles to quantify growth, movement and development in the aerial plant body and to capture the rhythmic nature of these processes. This requires imaging in light during the day and in darkness at night without perturbing plant physiology. Nighttime imaging has typically depended on infrared (IR) illumination, producing monochrome datasets that require specialised hardware and separate analysis pipelines when combined with daytime RGB imaging. Here, we evaluated very low-intensity green (dimG) illumination from standard LEDs as a practical alternative for colour-consistent nighttime imaging and assessed its physiological impact in Arabidopsis thaliana and Lactuca sativa (lettuce). We show that high resolution colour images can be obtained under dimG using low- cost cameras, with sufficient consistency between full-spectrum and dimG images to allow direct comparison and unified image analysis. We show that very low-fluence green light (<0.5 mol m-2 s-1) does not sustain circadian oscillations of gene activity under continuous exposure and does not perturb rhythms when applied during the dark phase of diel cycles. DimG imaging enabled accurate detection of diel leaf movement profiles in Arabidopsis circadian mutants, revealing genotype-specific phase differences under varying photoperiods. In lettuce, dimG pulses and continuous dimG enabled accurate quantification of diel leaf movement without affecting growth, stomatal opening, electron transport rate or chlorophyll content. Motion profiles under continuous dimG mirrored those under darkness. Our findings establish dim green illumination as a cost-effective solution for night-time imaging, simplifying phenotyping workflows with minimal impact on physiology.

Brown algae evolved independently from animals, land plants and other algae, and we know very little about the spatio-temporal dynamics of their embryogenesis. Here, we used time-lapse, bright-field microscopy to study cell division and lineage development during early embryogenesis in the kelp Saccharina latissima, a large brown alga. We discovered a radical change of cell identity as early as the 4-cell stage: after fertilization, the zygote underwent two or three unequal cell divisions before the basal cell - that closest to the maternal tissue - stopped dividing and radically differentiated into a hyperpolarized cell, the rhizoid, which anchors the embryo to the substrate. RNA-seq analysis showed that differentiation of rhizoid cells was preceded by expression of 130 basal cell-specific genes. Phylostratigraphic analysis further revealed that more than 40% of these basal cell-specific genes appeared after the emergence of the brown algae group, and their functions are largely unknown. By contrast, the apical cell predominantly expressed more ancestral, metabolism-related genes, and it continued to divide to produce the long, blade-shaped thallus of the alga. The early and radical nature of cell differentiation in Saccharina embryos, combined with differential gene expression from various evolutionary periods, highlights the unique mechanisms of embryogenesis of this alga.

KaiC, a clock protein in cyanobacteria, cycles between dephosphorylated and phosphorylated states in a 24-hour period in the presence of KaiA and KaiB. We identified the 322nd residue of KaiC as a third example of period-tuning sites. 322nd-site-directed saturation mutagenesis resulted in a variety of KaiC mutants exhibiting either shortened or lengthened cycles. The tunable range of the periods was from approximately 11 to 78 h without significantly compromising temperature compensation. We conducted biochemical analyses of the 322nd variants and examined their predicted structural models. In contrast to another known period-tuning site, where the period decreases sharply as the side-chain volume increases due to mutations, the cycle lengths correlate only modestly with bulkiness at the 322nd residues. The 322nd residue is located in a C-terminal domain of KaiC and influences ATPase cycles in both the C-terminal domain and an N-terminal domain through its interaction with a flexible loop connecting the two domains. The structural models predict that placing less bulky but polar side chains, such as serine and threonine, at the 322nd position leads to the formation of a hydrogen-bonding network between that site and the loop. This reduces the mobility of the loop, resulting in the longer cycles due to decreases in the ATPase activity of the N-terminal domain. Conversely, placing bulky residues such as phenylalanine at the 322nd position appears to alter the loop structure, shortening the periods by enhancing the ATP activities of both the domains. The third period-tuning mechanism is distinct from other known mechanisms. Significance StatementA Kai-protein clock system serves as a model for studying how long circadian rhythms are achieved. We identified the 322nd residue of KaiC as a third example of period-tuning sites that allow tuning of the period in either long- and short-period directions. The third period-tuning mechanism differs from the two previously known types in several respects. Previous studies have suggested that the ATPase activity in an N-terminal domain of KaiC is the primary regulator of the period. On the other hand, the 322nd residues of KaiC can affect the period by activating the ATPase cycle in its C-terminal domain. Our findings will stimulate future studies on the period-tuning mechanism mediated by the ATPase activity in the C-terminal domain of KaiC.

Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of few cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of{Delta} sodB mutant further confirmed its targeted deletion. Overall, CRISRPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. Key PointsO_LIRapid and efficient CRISPR/Cas12a editing established in Cyanothece PCC 7425. C_LIO_LIFully segregated knockout mutants obtained after single selection round. C_LIO_LIPlatform for nuclear waste bioremediation and other biotechnological applications. C_LI