Published studies have reported species-variance between profiles of Calvin-Benson cycle (CBC) intermediates, not only between C4 species and C3 species, but also within C3 species (Arrivault et al., 2019, Borghi et al. 2019). It was proposed that this variance reflects lineage-dependent changes in the balance between different reactions, or poising, of the CBC. These earlier studies investigated phylogenetically-unrelated C3 species. In the current study, CBC intermediates were profiled in five closely-related species from Solanum sect. lycopersicon subsect. Lycopersicum. Levels of individual CBC intermediates showed many significant differences. In a principal component analysis, whilst three species (Solanum lycopersicum, Solanum cheesmaniae, Solanum neorickii) overlapped, Solanum pimpinellifolium and especially Solanum pennellii grouped separately, and were at opposing ends of the distribution. When combined with published data, whilst the separation between Solanum species was retained, they formed a group that was separated from five other C3 species, as well as two C4 species. It is discussed that the observed variation in CBC metabolites profiles within Solanum, together with their separation from other C₃ species, supports the idea that CBC evolution is shaped by phylogenetic relatedness and, by implication, lineage-specific adaptation.
In response to rising CO2 emissions driving global warming, there is an urgent need for a transition toward a sustainable bioeconomy. Photo-biotechnological processes based on oxygenic photosynthesis hold high potential for achieving CO2 neutrality and in this regard, cyanobacteria have emerged as promising biocatalysts. Rational metabolic engineering of cyanobacteria depends on a thorough understanding of regulatory mechanisms governing primary metabolism, because native metabolic flux through specific pathways and, consequently, the formation of target products can be limited. Recent insights have identified a key regulatory node at the 2,3-bisphosphogylcerate-independent phosphoglycerate mutase (PGAM) reaction, where the metabolic flux from newly fixed carbon is redirected from the Calvin-Benson-Bassham (CBB) cycle towards lower glycolysis. This metabolic valve is controlled by the small inhibitor protein PirC, whose binding to PGAM is determined by the central signal transduction protein PII. In this study, we exploit the PirC-PGAM interaction as a novel target for regulatory metabolic engineering in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Chassis strains with engineered control of PGAM, defined as PGAM-ON or PGAM-OFF states, were generated using two complementary approaches: tuning pgam gene expression and modulating PirC abundance to regulate PGAM activity. The effectiveness of this regulatory engineering strategy was demonstrated by redirecting carbon flux toward two representative, naturally occurring products: sucrose, produced via gluconeogenesis fueled by the Calvin-Benson-Bassham (CBB) cycle, and succinate, an intermediate of the tricarboxylic acid (TCA) cycle. Narrowing the PGAM valve resulted in a threefold increase in sucrose accumulation. In contrast, opening the PGAM valve by relieving PGAM inhibition through pirC deletion or separate pgam overexpression resulted in up to an 18-fold increase in succinate excretion. Furthermore, similar genetic configurations were applied to enhance production of a heterologous compound, isoprene, derived from pyruvate. This study establishes the PGAM valve as a tunable control point for the rational re-direction of carbon flux in Synechocystis and highlights small regulatory proteins as powerful targets for metabolic engineering. Together, these findings provide proof of concept for an advanced level of molecular engineering in cyanobacteria and to fully harness their biocatalytic potential in future photosynthesis-driven biotechnological applications.
Strigolactones mitigate Cr(VI) toxicity in Capsicum annuum by limiting chromium uptake and reprogramming photosynthesis and redox metabolism, thereby preserving chloroplast function and cellular integrity. Agricultural soil contamination and its impact on the crops require alternative strategies to reduce associated environmental risks. Strigolactones (SLs) are emerging phytohormones that regulate plant development and stress adaptation; however, their role in hexavalent chromium [Cr(VI)] tolerance remains poorly understood. Here, we investigated the mechanistic basis of SL-mediated mitigation of Cr(VI) toxicity in Capsicum annuum using the synthetic analog rac-GR24. Cr(VI) stress severely impaired photosynthetic performance by disrupting photosystem efficiency, suppressing Calvin-Benson cycle enzyme activities, and reducing ATP and NADPH production, leading to excessive reactive oxygen species accumulation and cellular damage. rac-GR24 markedly decreased Cr uptake and restored photosynthetic carbon metabolism by enhancing the expression of key Calvin cycle enzymes and genes such as RbcS, RbcL, PGK, GAPDH, and PRK. The regulation of fluorescence parameters such as Fv/Fm, ФPSII, and ETR improved PSI and PSII functioning, and rebalanced the adenylate and pyridine nucleotide pools. GR24 reinforced redox homeostasis by activating antioxidant defence, stimulating the ascorbate-glutathione and glyoxalase pathways, resulting in reduced lipid peroxidation and improved cell viability. Ultrastructural observations confirmed improved chloroplast integrity and stomatal behavior under SL treatment. The enhancement of photosynthesis and reduction of stress-related biomarkers are probably the consequences of decreased Cr accumulation in roots and shoots upon rac-GR24 treatment, as indicated by the differential expression of SULTR1.2, SULTR1.3, and SULTR3 genes. These findings demonstrate that GR24 could be highly effective in alleviating Cr-induced toxicity in C. annuum. This protective effect is mediated through the coordinated regulation of photosynthetic carbon metabolism and antioxidant defense system, leading to redox regulation, protection of chloroplasts, and better photosynthetic performance.
Perfluorohexane sulfonate (PFHxS), a widely used alternative to perfluorooctane sulfonate (PFOS), has been prevalent in aquatic environments. However, its toxic mechanisms in algae remain poorly understood. In this study, a 12-day exposure experiment was conducted to systematically investigate the toxicity of PFHxS in Chlorella pyrenoidosa through physiological parameter measurements and transcriptomic analysis. The results revealed that significant growth inhibition (9%) was observed at 1 mg/L PFHxS on day 12, accompanied by altered photosynthetic responses, including decreases in chlorophyll a and carotenoid contents by 11% and 15%, respectively, at the late exposure stage. On day 12, the accumulation of reactive oxygen species (ROS) triggered a compensatory upregulation of antioxidant enzyme activities, with superoxide dismutase (SOD) and catalase (CAT) increasing by 74% and 79%, respectively. This response was insufficient to prevent severe oxidative stress, which resulted in lipid peroxidation and substantial impairment of energy metabolism, marked by a 57% decline in adenosine triphosphate (ATP) and a 55% decline in reduced nicotinamide adenine dinucleotide phosphate (NADPH). Transcriptomic profiling suggested that PFHxS exposure was associated with suppression of the Calvin cycle and glycolysis while inhibiting the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, thereby impairing energy synthesis. Although C. pyrenoidosa appeared to modulate energy metabolism and exhibited transcriptional changes associated with reduced glycogen turnover and altered fatty acid metabolism, severe oxidative damage coupled with impaired energy metabolism ultimately led to metabolic disturbance. This study provides evidence relevant to evaluating the ecological risks of PFHxS to aquatic systems and provides critical evidence for assessing the environmental impacts of PFHxS.
Using the Fast Repetition Rate Fluorometry (FRRF) technique to monitor the variable yield of chlorophyll fluorescence emission (Fv = Fm-Fo), we develop a pulse sequence to measure flux bottlenecks that limit the transport of electrons and protons from their water oxidation source through the photosynthetic electron transport chain (PETC) into CO2 carboxylation and triose phosphate production within the Calvin-Benson-Bassham (CBB) cycle of living organisms. FRRF uses a single source of microsecond flashes to excite photochemistry and produce fluorescence, thus removing slower non-photochemical quenching contributions to total Fv (pFv = total Fv - NPQ). pFv is the fraction of total Fv that originates from net charge separation and recombination in Photosystem II: P ↔ P* ↔ P+QA- ↔. Subsequent charge-transfer steps are monitored, revealing fluxes from water oxidation (reducing P+) into downstream carrier pools of the PETC (oxidizing QA-). As these pools fill in sequence, QA/QA- repopulates at characteristic lag times, as measured by pFv dynamics. Three temporal phases of pFv are observable in various phototrophs performing C3 photosynthesis that reveal five flux bottlenecks between redox carriers in the PETC and CBB cycle. pFv dynamics reveal that the PETC and CBB cycles operate synchronously, governed by the law of mass action and microscopic reversibility, with CO2 accelerating upstream reactions in both linear and cyclic electron pathways. pFv fluxes reliably monitor species-dependent phenotypic changes in PETC/CBB bottlenecks that determine carboxylation rate and influence cellular growth rate. This versatile technique can be applied to monitor the effects of transgene or environmental changes on light-use efficiency in diverse photosynthetic organisms. An animation of the multi-phase kinetics of the photochemical Fv/Fm data is available at this LINK.
Sustainable biomanufacturing in Escherichia coli is advancing through the integration of carbon dioxide (CO2)-fixing modules from algal systems. However, the assimilation efficiency remains limited by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) performance and intracellular CO2 availability. To address these constraints, we introduced carbonic anhydrases from Cyanobacteria aponinum (capoCAs) and repressed the native CA (can) gene using Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) to enhance design specificity. Afterward, capoCAs were overexpressed in E. coli BL21(DE3) and its derivative strains of can deficient (dCA), Calvin-Benson-Bassham (CBB) (RuBisCO-Prk expressing), and dCA+CBB (coupled with dCA and CBB). For functional demonstration, the engineered strains were used to produce a valuable prodrug, 5-aminolevulinic acid (5-ALA). As a result, capoCAs modestly improved 5-ALA titers, with further increments upon CBB coexpression. The optimum dCA+CBB* strain achieved 7.04 g/l (1.46-fold improvement), alongside 9.59% carbon enrichment and a 39.4% reduction in CO2 release. The 13C-isotope confirmed CO2 incorporation during 5-ALA biosynthesis. This work provides tunable carbon-management modules for developing low-footprint microbial cell factories.
Cupriavidus necator strain H16 has emerged as a versatile microbial chassis for sustainable bioproduction due to its metabolic flexibility, enabling growth on a wide range of substrates, including H2/CO2, formate, and organic carbon (waste) sources such as volatile fatty acids. This review first provides a comprehensive overview of recent advances in systems-level understanding and metabolic engineering of C. necator. We next discuss recent advances in omics analyses and genome-scale metabolic modeling that are increasingly used to understand the large genome and wide metabolic portfolio of C. necator. We further discuss the native metabolic network, including autotrophic growth via the Calvin Benson Bassham (CBB) cycle, as well as heterotrophic and mixotrophic capabilities. Engineering strategies to enhance substrate utilization and conversion efficiency particularly for H2/CO2, formate, and mixed feedstocks are discussed alongside efforts to expand the substrate range in this organism. Other than the already industrialized production of the bioplastic polyhydroxybutyrate (PHB) and related polyhydroxyalkanoates in C. necator, we provide an overview of the wide range of products for which proof-of-principles have been shown in C. necator. We also discuss recent advances in bioprocess design for gas fermentation, electromicrobial production, and H2-based biocatalytic reduction using C. necator. Finally, we compare C. necator with other hydrogen and formate-utilizing bacteria to identify key knowledge gaps and outline future directions for advancing C. necator as a host for sustainable industrial biotechnology.
Microbial carbon fixation is central to carbon cycling and carbon sink functioning in coastal aquatic ecosystems. Although carbon fixation pathways have been increasingly investigated across diverse aquatic environments, comparative evidence remains limited for hydrologically connected yet hydrochemically contrasting coastal groundwater and surface water systems. This study aimed to compare carbon-fixation-associated microbial communities and major carbon fixation pathways across groundwater, river water, and reservoir water in the Tianjin coastal region. We integrated metagenomic sequencing with hydrochemical analyses to characterize carbon-fixation-associated microbial communities and six representative carbon fixation pathways. Surface waters were dominated by bacteria and showed relatively stable community composition, whereas groundwater communities comprised both bacteria and archaea and displayed pronounced spatial heterogeneity. The Calvin-Benson-Bassham cycle was prevalent across all water types, and the reductive tricarboxylic acid (rTCA) cycle was also widely distributed. Groundwater showed higher contributions of the Wood-Ljungdahl pathway, the archaeal 3-hydroxypropionate/4-hydroxybutyrate and dicarboxylate/4-hydroxybutyrate cycles, together with the rTCA cycle, indicating coexisting carbon fixation strategies. Pathway abundance and module completeness further suggested differences in pathway integrity among water types. Total dissolved solids, HCO3⁻, CO32⁻, and dissolved organic carbon were key correlates of carbon fixation gene distribution. Carbon-fixation-associated microbial communities, pathway distributions, and pathway integrity differed markedly between coastal groundwater and surface waters. Groundwater exhibited enhanced non-CBB cycle potentials and more diversified carbon fixation strategies, highlighting the importance of groundwater processes in evaluating carbon sequestration potential and carbon cycling in hydrochemically heterogeneous coastal aquatic systems.
Mangrove wetlands are a core component of blue carbon ecosystems worldwide. Elevated suspended particulate matter (SPM) in natural waters is a ubiquitous environmental stressor, yet its effects on the carbon cycling of mangroves remain poorly unelucidated. Here, we conducted a 90-day microcosm experiment using Kandelia obovata and integrated high-resolution chemical and biological profiling to systematically elucidate how SPM loading affects mangrove carbon-cycling processes. As SPM increased from 0 to 30 mg/L, the photosynthetic activity of phytoplankton declined, accompanied by suppressed Calvin-Benson-Bassham-cycle signatures and a relative enrichment of glycolysis- and TCA-related signatures, resulting in a 0.71 mg/L increase in dissolved inorganic carbon (DIC) and a 1.12 mg/L decrease in dissolved organic carbon (DOC) on day 90. Concurrently, suppressed sediment β-glucosidase activity contributed to a 1.76 mg/g increase in sediment organic carbon (SOC). As SPM further increased from 30 to 70 mg/L, strengthened autotrophic carbon fixation and attenuated heterotrophic carbon oxidation in the water column contributed to a 2.45 mg/L decrease in DIC and a 0.30 mg/L increase in DOC. Excessive SPM load compromised the antioxidant defence system of mangroves, leading to oxidative damage in mangrove plants, thereby reducing photosynthetic activity, while sediment β-glucosidase activity increased, collectively resulted in a 1.20 mg/g decrease in SOC. Our results indicate that elevated SPM reorganises carbon composition within mangrove wetlands, with potential implications for global-scale carbon cycling, underscoring the importance of incorporating SPM as a key agent forcing in the management of blue carbon ecosystems.
Glycine betaine (GB) not only plays an important role as an osmotic regulator in the regulation of abiotic stress in plants, but also serves as a methyl donor that affects the methylation level of the plant, which in turn affects its growth, development and response to adversity. Tomato as a major crop cultivated in facilities, facility soil salinization has become an important factor limiting efficient and high qualities tomato production. In contrast, little has been reported on the effect of GB on m6A methylation and its salt tolerance in tomato. In this study, the effects of exogenous GB on chlorophyll synthesis, photosynthesis and m6A methyltransferase in tomato under salt stress were investigated using tomato as experimental material. The results showed that 5 mM GB pretreatment alleviated the inhibitory effect of salt stress on the growth of tomato plants. GB promotes the synthesis and accumulation of Proto IX, Mg-Proto IX and protochlorophyllide in the chlorophyll synthesis pathway under salt stress by protecting the activities of CHLH (Magnesium-binding enzyme H subunit) and POR (protochlorophyllide oxidoreductase) thereby increasing the chlorophyll content. In addition, GB improves photosynthetic capacity of tomato by increasing stomatal opening and protecting Calvin cycle enzyme activity. And GB altered the expression level of m6A methyltransferase, which in turn indirectly affected the salt tolerance of tomato. In summary, GB may enhance tomato salt tolerance by promoting chlorophyll synthesis, enhancing photosynthesis, and regulating the expression levels of m6A methyltransferase genes. These findings suggest that exogenous GB application could serve as an effective strategy to improve tomato productivity in salinized protected cultivation systems.
A mixture of Synechococcus sp., Chroococcus sp., and Synechocystis sp. was immobilized in indole-3-acetic acid (IAA)-supplemented calcium alginate beads and then placed into a four-compartment baffled photo-bioreactor. A 30-day continuous-flow treatment of secondary effluent wastewater using this system achieved removal rates of 74.08-85.12% for COD, 87.52-96.89% for TN, 95.36-99.26% for TP, 84.02-88.36% for cefalexin, 67.15-75.57% for erythromycin, 91.17-96.05% for oxytetracycline, and 74.76-78.87% for norfloxacin. Chroococcus sp. contributed the most to pollutant removal, with its abundance negatively correlated with the concentrations of all pollutants. Bacterial colonization within cyanobacterial beads, upregulated genes involved in signal transduction, quorum sensing, and biofilm formation, as well as correlations between cyanobacteria and seven bacterial genera (Acidovorax, Chitinophaga, Massilia, Algoriphagus, Chryseobacterium, Comamonas, and Candidatus) together confirmed the formation of a cyanobacteria-bacteria consortium. Efficient pollutant removal was attributed to the high cyanobacterial biomass stimulated by IAA and the activation of genes related to stress response, the TCA cycle, oxidative phosphorylation, and pollutant metabolism in bead microorganisms. Reduced abundances of antibiotic resistance genes in the effluent may result from activated mismatch repair pathway and suppressed horizontal gene transfer. Antibiotics, the symbiotic bacterium Azospirillum, and IAA jointly stimulated cyanobacterial growth and lipid accumulation, contributing to a high cyanobacterial lipid productivity of 47.59-51.82 mg/(L·d), mainly through the upregulation of genes involved in the Calvin cycle, pentose phosphate pathway, and fatty acid biosynthesis. Overall, this study provides a sustainable strategy integrating pollutant removal, resistance control, and resource recovery.
Carbon fixation in marine ecosystems is a vital process that contributes to climate regulation, with ocean sediments playing a critical role in carbon sequestration. This process is driven by chemolithoautotrophy in marine sediments, fueled by reduced compounds, such as those containing nitrogen and sulfur. However, the vertical distribution of microbial autotrophs and their energy coupling systems remain poorly understood in many sediments. In this study, we investigated a 750 cm sediment core from the Challenger Deep, the deepest point on Earth, which harbors abundant and diverse microbes under extreme conditions. To explore the autotrophic characteristics across redox conditions in this core, we characterized the microbial community, metagenome, and metagenome-assembled genomes (MAGs), and their potential for carbon fixation processes and associated energy metabolism. The Wood-Ljungdahl (WL) pathway, primarily driven by Planctomycetota and Aerophobota, and the reverse oxidative TCA (roTCA) cycle, primarily driven by Bacteroidota and Gemmatimonadota, were the dominant predicted carbon fixation pathways, with hydrogen as the primary energy source, coupled to nitrogen and sulfur metabolism. Notably, the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle, mediated by Nitrososphaeria, showed the highest abundance in the oxidized environment (15-27 cm below the seafloor), where ammonia oxidation likely served as the primary energy source. Gammaproteobacteria were predicted to utilise sulfur oxidation, whereas Alphaproteobacteria and Chloroflexota used hydrogen to drive the Calvin-Benson-Bassham (CBB), reductive glycine pathway (rGly) in Alphaproteobacteria and the dicarboxylate/4-hydroxybutyrate cycle (DC/4HB) in Chloroflexota, respectively. The abundance of carbon fixation, and nitrogen, sulfur and hydrogen cycling functional genes were significantly correlated with environmental factors (NH4+ and SiO32-) based on Pearson's correlation analysis. This study reveals the vertical distribution of microbial carbon fixation potential and diversity in sediments driven by redox conditions, highlights the crucial role of hydrogen as an energy source, and provides new insights for optimizing global deep-sea carbon cycle models. Collectively, these findings extend the redox tower theory by revealing a hadal-sediment specific distribution of autotrophic genes, characterized by persistent enrichment of energetically efficient pathways and dominant hydrogen-based energy coupling across deep sediment layers.
Phytoplasma-mediated witches' broom disease (WBD) causes abnormal growth and inhibits fruit formation in sour jujube trees, causing significant economic damage. Research on the pathogenic mechanisms of WBD and the disease resistance mechanisms of sour jujube tree remains poorly understood. In this study, these mechanisms were investigated using healthy resistant jujube branches that had been grafted onto WBD-infected trees and grown for seven years. Leaves were collected from both healthy resistant branches (KB) and WBD-affected branches (ZFB) to analyze differences in plant physiological indicators, carbon metabolic processes, and transcriptional and protein regulatory mechanisms. The results indicated that, compared with the ZFB group, the KB group had significantly greater levels of photosynthetic indicators, including the transpiration rate, net photosynthetic rate, stomatal conductance, and photosynthetically active radiation. Nutrient absorption and metabolic indicators, such as soluble proteins, were also higher in KB than in ZFB, whereas soluble sugar and starch concentrations were lower. Carbon metabolism profiling revealed that the KB group showed increased fatty acid synthesis for sustained energy production, whereas the ZFB group presented disrupted central carbon flux through an impaired TCA cycle, glycolysis, and pentose phosphate pathways. In addition, ferredoxin (Fd) was identified as a key resistance determinant in jujube trees against WBD phytoplasma. Genes and proteins associated with the photosynthesis pathway exhibited elevated expression levels in KB leaves, particularly those encoding Fd. High Fd expression enhances NADPH and ATP production, accelerates the Calvin cycle, and promotes carbohydrate synthesis. This enhancement supports various biological pathways, including the assimilation of carbon and nitrogen, chlorophyll metabolism, and the synthesis of photosynthetic pigments and fatty acids. In summary, our study reveals that an intact photosynthetic system and its associated metabolism protect resistant branches against WBD, providing a theoretical basis for breeding resistant cultivars.
Phosphoglucose isomerase (PGI) catalyzes the reversible interconversion of glucose 6-phosphate (G6P) to fructose 6-phosphate (F6P), classically considered a non-regulatory step in the Embden-Meyerhof-Parnas pathway for glycolysis and gluconeogenesis. However, in Synechocystis sp. PCC 6803, 13C flux analysis has identified the "PGI shunt" as a major route under photomixotrophic conditions, in which F6P is funnelled into the regenerative phase of the Calvin-Benson-Bassham (CBB) cycle to enhance CO2 fixation. To elucidate the biochemical basis of this regulation, we characterized the cyanobacterial PGI, SynPGI in detail. SynPGI can reversibly convert G6P and F6P but shows a strong preference for the gluconeogenic reaction (F6P → G6P), with a ∼threefold higher catalytic efficiency for F6P, compared to G6P, in agreement with equilibrium assays, which confirmed that the reaction favors G6P accumulation (75%, Keq = 0.3). Effector studies revealed two potent inhibitors: erythrose 4-phosphate (E4P) and 6-phosphogluconate (6PG). E4P, an intermediate of the regenerative CBB (rCBB) cycle, inhibited SynPGI at μM concentrations and 6PG, a metabolite of the oxidative pentose phosphate pathway, inhibited the enzyme at mM concentrations. Kinetic modeling indicated that a mixed-type inhibition model best describes the inhibitory effects of both metabolites. Enzymatic, structural and phylogenetic analyses reveal that cyanobacterial SynPGI closely resembles plastidic PGIs from plants and suggest that it may function as a metabolic control node, integrating environmental and intracellular signals to fine-tune carbon flux and enhance metabolic efficiency in Synechocystis.
Autotrophic carbon-fixing microbes can assimilate atmospheric carbon dioxide into biomass via the Calvin-Benson-Bassham (CBB) cycle (their primary carbon fixation pathway), thereby reinforcing soil carbon sequestration in the plantation ecosystem; however, the succession of RubisCO-harboring microbial communities across stand ages remains poorly understood. Here, we investigated the community succession of microbes carrying the gene encoding RubisCO, a key enzyme in the CBB cycle, across a stand-age chronosequence in a Picea asperata plantation ecosystem. Our results revealed a progressive decrease in microbial α-diversity and a significant restructuring of community composition with increasing stand age, characterized by an enrichment of Proteobacteria and a concomitant depletion of Actinobacteria. While the Shannon-Wiener index was most strongly correlated with soil total nitrogen content, redundancy analysis identified soil pH as the predominant environmental driver of community turnover, a relationship that was found to be threshold-dependent, with substantial community shifts occurring in response to pH variations of 0.5 to 1.0 units. These findings suggest that sustaining the diversity of RubisCO-harboring microbes in older stands-a process potentially enhanced by soil nitrogen management-provides a viable strategy for augmenting the carbon sequestration capacity of managed forests through targeted microbiome regulation.
King grass is a fast-growing C4 perennial with high biomass potential, yet its productivity is limited by cold stress. WRKY transcription factors (TFs) regulate abiotic stress responses, but their roles in king grass cold adaptation remain unclear. WRKY TFs were identified genome-wide using BLASTP and HMMER, followed by phylogenetic classification, transcriptome profiling, promoter cis-element analysis, weighted gene co-expression network analysis (WGCNA), molecular docking, and yeast heterologous expression assays. Sixty-two PsiWRKY genes were identified and classified into three groups. Time-series transcriptome analysis across nine cold-stress time points revealed four expression patterns, with 29 genes significantly induced (> 2-fold), mainly during late acclimation (48-72 h). qRT-PCR confirmed RNA-seq trends (R² = 0.742). Functional enrichment and WGCNA analyses indicated that cold-responsive PsiWRKYs coordinate abscisic acid-gibberellin antagonism and sugar metabolism. Molecular docking predicted interactions between PsiWRKY proteins and Calvin cycle enzymes, with PsiWRKY16 and PsiWRKY55 showing the highest structural confidence (pTM > 0.75). Network analysis identified PsiWRKY16 and PsiWRKY50 as hub regulators linking GA signaling genes (GID1/DELLA) with sugar metabolism enzymes such as GAPDH and PRK. Yeast assays quantitatively confirmed enhanced cold tolerance, as heterologous expression of PsiWRKY16 reduced doubling time by 0.56 h and increased area under the curve and carrying capacity by 26.20 and 3.39, respectively, while PsiWRKY50 increased these values by 14.45 and 1.43. Promoter analysis showed enrichment of ABRE and DRE elements (p = 0.00061), and integrative target prediction identified 193 genes co-regulated by WRKY and CBF pathways, supporting ABA-CBF signaling convergence. PsiWRKYs exhibit subgroup-specific and time-resolved responses to cold stress. PsiWRKY16, PsiWRKY50, and PsiWRKY55 are implicated in coordinating hormonal signaling and metabolic reprogramming, providing candidate targets for cold-tolerant breeding in king grass.
Chemoautotrophs drive carbon fixation in coastal sediments, but most of them remain uncultured with poorly characterized in situ activities. In this study, a cultivation-independent single-cell approach combining Raman spectroscopy with 13C-stable isotope probing was developed to enable direct identification of active chemoautotrophs in coastal sediments using function-specific spectral biomarkers, targeted metagenomic sequencing and pure culture verification. 13C-induced shifts in cytochrome c (749, 1129, 1312, 1589 cm-1) and phenylalanine (1002 cm-1) Raman bands were systematically evaluated and applied as functional biomarkers through investigations of both representative chemoautotrophic strains and environmental samples. The combined analysis of targeted sorting of active chemoautotrophic cells and metagenomic sequencing revealed dominant species and a complete Calvin-Benson-Bassham (CBB) cycle pathway in sulfur-oxidizing guilds. Remarkably, a novel sulfur-oxidizing chemoautotroph, Guyparkeria sp. TX1, which showed ≥99% gene sequence similarity to contigs recovered from sorted-cell metagenomes, was isolated from enrichment cultures. Its significant carbon fixation capacity provided experimental validation for the effectiveness of Raman-based in situ functional screening. This study establishes Raman-based functional biomarkers applicable to chemoautotrophic carbon fixation, enabling in situ mapping of microbial carbon fluxes. By integrating single-cell phenotypic activity with genomic potential, this work advances the mechanistic understanding of sulfur-driven dark carbon fixation, which sustains coastal blue carbon ecosystems as a keystone process.
Semi-arid tropical soils inherently contain low soil organic carbon (SOC) and limited nutrient reserves, resulting in poor productivity. Intensive cropping with synthetic fertilizers, further deteriorate soil quality and impair ecosystem functioning. In contrast, organic amendments alone or combined with synthetic fertilizers sustain soil biodiversity through microbially mediated processes. However, how long-term nutrient management shapes soil microbiomes and their functional diversity in semi-arid tropical systems remains largely unknown. To address this gap, we investigated a 116-year-old long-term nutrient management experiment using a multi-omic framework. Shotgun metagenomics characterized the total microbiome (bacteria, archaea, and eukaryota) and associated carbon- and nitrogen-cycling genes under four contrasting nutrient management practices: unfertilized control, inorganic fertilizer alone (IC), organic amendment alone (OM), and integrated nutrient management combining organic and inorganic inputs (INM). OM and INM significantly improved soil nutrient stocks, SOC, microbial biomass, and enzyme activities compared with IC and Control. These treatments also enhanced microbial diversity and shifted communities toward copiotrophic and functionally beneficial taxa, whereas IC and Control were dominated by stress-tolerant oligotrophs. Pathway analysis showed that carbon fixation dominated the C-cycling gene pool, with alternative autotrophic pathways prevailing over the Calvin cycle, particularly under OM and INM. These treatments also supported higher abundances of methanogenic and decomposition-associated genes, indicating enhanced carbon turnover. Nitrogen-cycling functions exhibited pathway-specific responses: OM enriched N-fixation and assimilatory nitrate reduction genes, whereas INM enhanced denitrification and dissimilatory nitrate reduction pathways. IC showed increased nitrification potential but the weakest biologically regulated N pathways. Volatomics profiling showed that OM and INM produced more diverse and metabolically active volatile organic compounds that were strongly associated with SOC and key biological attributes. Collectively, our study underscores the importance of carbon-rich organic inputs in rebuilding soil carbon stocks, reinforcing biological processes, and enhancing nutrient cycling for long-term sustainability of agriculture in semi-arid tropical regions.
Antibiotic-containing agricultural wastewater poses a critical challenge for simultaneous pollutant removal and carbon reduction technology. In this study, a visible-light-driven photocatalysis-microalgae (PC-MA) system was constructed by coupling WO3/α-Fe2O3/zeolite (WFZ) heterojunction with Chlorella vulgaris granules to treat simulated wastewater containing ciprofloxacin (CIP). At 20 mg/L CIP concentration, the PC-MA achieved 89% CIP removal over six reuse cycles, and exhibited superior COD, TOC, NH4+ -N, and TP removal compared with the single photocatalytic (PC) and microalgal (MA) systems. Radical quenching and ESR experiments indicated that WFZ generated an intense •OH/O2•⁻ oxidative environment, while LC-MS analysis revealed progressive transformation of CIP into more polar, lower-molecular-weight intermediates that are more amenable to subsequent bioprocessing. The physiological (chlorophyll-a, biomass, and CO2 biofixation rate) and biochemical (EPS fluorescence, oxidative-stress biomarkers, and carbonic anhydrase activity) indicators demonstrated that the PC-MA system relieved CIP-induced oxidative stress and maintained Chlorella in a higher-activity state. Transcriptomic analysis further revealed that the up-regulation of photosynthetic antenna proteins, Calvin cycle enzymes, and carbonic anhydrase-related genes enhanced photosynthetic carbon fixation under CIP exposure. The photocatalysis-biodegradation synergistic relationship offered a sustainable route for the simultaneous antibiotic removal and transformation, nutrient removal, and enhanced carbon assimilation in complex wastewater treatment.
Chemolithoautotrophic bacteria represent a diverse group of microorganisms capable of utilising carbon dioxide (CO2) as their sole carbon source, deriving the necessary energy for growth and metabolism from redox reactions involving inorganic compounds such as hydrogen, sulfur, or iron. These organisms have attracted substantial scientific and industrial interest due to their dual potential to function as biological CO2 sinks, mitigating greenhouse gas accumulation, and as microbial platforms for the sustainable biosynthesis of value-added compounds. Their natural CO2 fixation pathways, including the Calvin-Benson-Bassham cycle, the reductive tricarboxylic acid cycle, the Wood-Ljungdahl pathway, 3-hydroxypropionate bi-cycle, and reductive glycine pathway constitute the biological foundation for carbon assimilation in these organisms. However, these native pathways often exhibit limited efficiency, constraining their broader application. This comprehensive review discusses recent advances and opportunities in the optimization and redesign of CO2 fixation networks in chemolithoautotrophic bacteria. It extends to the design and development of novel, energy-efficient routes for CO2 fixation, which hold significant potential for large-scale implementation aimed at mitigating greenhouse gas emissions. In addition, the review assesses energy generation and utilization within carbon fixation networks, as well as emerging strategies for optimizing energy and redox balance in metabolic pathways. Finally, it highlights the emerging frontier of developing chemolithoautotrophic bacteria as microbial cell factories, capable of coupling carbon capture with sustainable biomanufacturing, thereby positioning these organisms at the forefront of next-generation climate and bioengineering solutions.