Search

Meta-analyses and theory show that with rising atmospheric [CO 2 ], Rubisco has become the greatest limitation to light-saturated leaf CO 2 assimilation rates ( A sat ) in C 4 crops. So would transgenically increasing Rubisco increase A sat and result in increased productivity in the field? Here, we successfully overexpressed the Rubisco small subunit ( RbcS ) with Rubisco accumulation factor 1 ( Raf1 ) in both sorghum and sugarcane, resulting in significant increases in Rubisco content of 13 to 25% and up to 90% respectively. A sat increased 12 to 15% and Rubisco enzyme activity ~40% in three independent transgenic events of both species. Sorghum plants also showed increased speeds of photosynthetic induction and decreased bundle sheath leakiness. These improvements translated into average increases of 15.5% in biomass in field-grown sorghum and a 37 to 81% increase in greenhouse-grown sugarcane. This suggests a potential opportunity to achieve substantial increases in productivity of this key economically important clade of C 4 crops, future proofing their value under global atmospheric change., Competing Interests: Competing interests statement:The authors declare no competing interest.

Several microalgae, including marine diatoms, significantly contribute to the global primary production and play a vital role in the food webs of benthic and planktonic ecosystems. Diatoms of the genus Cocconeis frequently inhabit benthic substrates, including the leaves of seagrasses. They are seasonally dominant in the leaf epiphytic layer of the Mediterranean seagrass Posidonia oceanica L. Delile, and have been proposed as model organisms for chemical ecology studies. However, the genome of Cocconeis spp. has not been sequenced. Consequently, their low-level molecular identification is currently impossible, besides a few examples. To address this gap, a polyphasic identification of C. neothumensis has been employed, combining ultra-morphological data with DNA barcoding markers. A strain of diatoms was isolated from P. oceanica leaves. It has been cultured in the laboratory and examined under Scanning Electron Microscopy (SEM). The 18S ribosomal RNA gene (18S rRNA, nrDNA) and the ribulose 1,5-biphosphate carboxylase (rbcL, cpDNA) gene were analysed for DNA barcoding characterisation. Since ultra-morphology data unambiguously identified the isolated strain as C. neothumensis Krammer, 1991, the molecular sequences herein reported will facilitate its rapid and accurate identification. In addition, our comparative analyses will facilitate the evaluation of these molecular markers for identification of closely related benthic diatoms., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Somma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The chloroplast genome encodes key components of the photosynthetic light reaction machinery as well as the large subunit of the enzyme central for carbon fixation, Ribulose-1,5-bisphosphat-carboxylase/-oxygenase (RuBisCo). Its expression is predominantly regulated posttranscriptionally, with nuclear-encoded RNA-binding proteins (RBPs) playing a key role. Mutants of chloroplast gene expression factors often exhibit impaired chloroplast biogenesis, especially in cold conditions. Low temperatures pose a challenge for plants as this leads to electron imbalances and oxidative damage. A well-known response of plants to this problem is to increase the production of RuBisCo and other Calvin Cycle enzymes in the cold, but how this is achieved is unclear. The chloroplast RBP CP29A has been shown to be essential for cold resistance in growing leaf tissue of Arabidopsis thaliana . Here, we examined CP29A-RNA interaction sites at nucleotide resolution. We found that CP29A preferentially binds to the 5'-untranslated region of rbcL , downstream of the binding site of the pentatricopeptide repeat protein MATURATION OF RBCL 1 (MRL1). MRL1 is an RBP known to be necessary for the accumulation of rbcL . In Arabidopsis mutants lacking CP29A, we were unable to observe significant effects on rbcL , possibly due to CP29A's restricted role in a limited number of cells at the base of leaves. In contrast, CRISPR/Cas9-induced mutants of tobacco NtCP29A exhibit cold-dependent photosynthetic deficiencies throughout the entire leaf blade. This is associated with a parallel reduction in rbcL mRNA and RbcL protein accumulation. Our work indicates that a chloroplast RNA-binding protein contributes to cold acclimation of RbcL production., Competing Interests: Competing interests statement:The authors declare no competing interest.

The biodiversity of CO 2 -assimilating bacterial communities is pivotal for carbon sequestration in agricultural systems. Changes in the diversity, structure, and activity of the soil chemolithoautotrophic bacteria were examined in four agricultural areas, Dulan (DL), Gonghe (GH), Huzhu (HZ), and Datong (DT) counties in Qinghai Province, where wheat, oilseed rape, and barley were planted. This process was performed using Illumina amplicon sequencing of the ribulose-1,5-bisphosphatecarboxylase/oxygenase (RubisCO) gene ( cbbL Form I) and activity data. The diversity, community, and activity of soil autotrophic CO 2 -fixing bacteria differed significantly across soil sites, whereas cbbL -bearing bacterial diversity and activity were similar across different crop types. RubisCO activity in the HZ region was significantly greater than in the other three regions ( P < 0.001). The overall relative abundance trend of the bacterial taxa was similar among the three crop samples. Moreover, 31, 27, 10, and 8 significant linear discriminant analysis effect sizes were identified in the four regions collected from HZ, DL, DT, and GH, respectively. No significant biomarkers were detected in any of the crop groups. Some soil properties had significant relationships with the autotrophic bacterial community composition., Importance: Agricultural soil plays important roles in carbon fixation during carbon capture and storage. Autotrophic bacteria that utilize inorganic compounds as electron donors for growth fix CO 2 photosynthetically or chemo-autotrophically in diverse ecosystems and affect soil organic carbon sequestration. Soil properties, agronomic management measures, and environmental factors can influence the community composition, abundance, and activity of CO 2 -assimilating bacteria. This study aims at evaluating the effects of different regions and crop types on the abundance, composition, and activity of CO 2 -fixing bacteria in agricultural soil., Competing Interests: The authors declare no conflict of interest.

Rubisco is the primary CO 2 -fixing enzyme of the biosphere 1 , yet it has slow kinetics 2 . The roles of evolution and chemical mechanism in constraining its biochemical function remain debated 3,4 . Engineering efforts aimed at adjusting the biochemical parameters of rubisco have largely failed 5 , although recent results indicate that the functional potential of rubisco has a wider scope than previously known 6 . Here we developed a massively parallel assay, using an engineered Escherichia coli 7 in which enzyme activity is coupled to growth, to systematically map the sequence-function landscape of rubisco. Composite assay of more than 99% of single-amino acid mutants versus CO 2 concentration enabled inference of enzyme velocity and apparent CO 2 affinity parameters for thousands of substitutions. This approach identified many highly conserved positions that tolerate mutation and rare mutations that improve CO 2 affinity. These data indicate that non-trivial biochemical changes are readily accessible and that the functional distance between rubiscos from diverse organisms can be traversed, laying the groundwork for further enzyme engineering efforts., Competing Interests: Competing interests: D.F.S. is a co-founder and scientific advisory board member of Scribe Therapeutics. The other authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Glyphosate is an extensively employed herbicide in agriculture, specifically for sugarcane cultivation. The situation is different with the extensive physiological and genetic effects exerted by this herbicide on a range of plant species, including sugarcane, whose model basis is still poorly characterized, although its primary mode of action, which acts on the EPSPS enzyme in the shikimic acid pathway, is completely elucidated. The current study was aimed at investigating the stability of glyphosate formulation, molecular interactions of glyphosate formulation with rbcL enzyme associated with chlorophyll metabolism, and its effects on varieties of sugarcane., Methods and Results: The stability of a ground-up glyphosate formulation was assessed under accelerated storage conditions. Molecular docking was performed to analyze interactions between glyphosate derivatives and rbcL. Two sugarcane varieties (G84-47 and GT54-9) were treated with increasing glyphosate concentrations (0.2, 0.4, and 0.8 mg L -1 ) to evaluate effects on chlorophyll content, plant height, herbicide tolerance, and rbcL expression. The formulation showed good stability with minor degradation (47.55-47.14%). N-Nitrosoglyphosate and 2-(phosphonomethylamino)acetic acid exhibited favorable binding affinities with rbcL. Glyphosate treatment reduced chlorophyll content and plant height dose-dependently, with G84-47 showing higher sensitivity. GT54-9 demonstrated higher herbicide tolerance in survival analysis. rbcL expression remained stable in G84-47 but was significantly upregulated in GT54-9 under high herbicide stress., Conclusions: This study reveals genetic variability in sugarcane responses to glyphosate, with variety-specific mechanisms underlying stress adaptation. The inducible rbcL expression in tolerant varieties provides insights into herbicide resistance mechanisms. These findings can inform marker-assisted breeding and improve agronomic practices for enhanced sugarcane cultivation resilience and sustainability., Competing Interests: Declarations. Ethics approval and consent to participate: There is no ethical issue in this research. Competing interests: The authors declare no competing interests. Conflict of interest: There is no conflict of interest among the authors., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an ancient protein critical for CO2-fixation and global biogeochemistry. Form-I RuBisCO complexes uniquely harbor small subunits that form a hexadecameric complex together with their large subunits. The small subunit protein is thought to have significantly contributed to RuBisCO's response to the atmospheric rise of O2 ∼2.5 billion years ago, marking a pivotal point in the enzyme's evolutionary history. Here, we performed a comprehensive evolutionary analysis of extant and ancestral RuBisCO sequences and structures to explore the impact of the small subunit's earliest integration on the molecular dynamics of the overall complex. Our simulations suggest that the small subunit restricted the conformational flexibility of the large subunit early in its history, impacting the evolutionary trajectory of the Form-I RuBisCO complex. Molecular dynamics investigations of CO2 and O2 gas distribution around predicted ancient RuBisCO complexes suggest that a proposed "CO2-reservoir" role for the small subunit is not conserved throughout the enzyme's evolutionary history. The evolutionary and biophysical response of RuBisCO to changing atmospheric conditions on ancient Earth showcase multi-level and trackable responses of enzymes to environmental shifts over long timescales., Competing Interests: Conflict of interest: The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The major limiting factor of photosynthesis in C3 plants is the enzyme, rubisco which inadequately distinguishes between carbon dioxide and oxygen. To overcome catalytic deficiencies of Rubisco, cyanobacteria utilize advanced protein microcompartments, called the carboxysomes which envelopes the enzymes, Rubisco and Carbonic Anhydrase (CA). These microcompartments facilitate the diffusion of bicarbonate ions which are converted to CO 2 by CA, following in an increase in carbon flux near Rubisco boosting CO 2 fixation process. Inspired by this mechanism, our study aims to improve photosynthetic efficiency in the C 3 model crop, rice (Oryza sativa), by stably engineering the genetic components of the β-carboxysome of Synechococcus elongatus PCC 7942 (hereafter, Syn7942) in the rice genome. We demonstrated this proof of concept by developing two types of transgenic rice plants. The first type involved targeting the chloroplasts with three key carboxysome structural proteins (ccmL, ccmO, and ccmK) and a chimeric protein (ccmC), which integrates domains from four distinct carboxysome proteins. The second type combined these proteins with the introduction of cyanobacterial Rubisco targeted to chloroplasts. Additionally, in the second transgenic background, RNA interference was employed to silence the endogenous rice Rubisco along with stromal carbonic anhydrase gene. The transgenic plants exhibited the assembly of carboxysome-like compartments and aggregated proteins in the chloroplasts and the second type demonstrated reduced plant growth and yield. We have followed a bottom-up approach for targeting the cyanobacterial CCM in rice chloroplast which would help in stacking up the components further required for increasing the photosynthetic efficiency in future., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Protein complexes composed of strictly essential subunits are abundant in nature and often arise through the gradual complexification of ancestral precursor proteins. Essentiality can arise through the accumulation of changes that are tolerated in the complex state but would be deleterious for the standalone complex components. While this theoretical framework to explain how essentiality arises has been proposed long ago, it is unclear which factors cause essentiality to persist over evolutionary timescales. In this work we show that the central enzyme of photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), can easily start to depend on a newly recruited interaction partner through multiple, genetically distinct mechanisms that affect stability, solubility, and catalysis. We demonstrate that layering multiple mechanisms of essentiality can lead to its persistence, even if any given mechanism reverts. More broadly, our work highlights that new interaction partners can drastically re-shape which substitutions are tolerated in the proteins they are recruited into. This can lead to the evolution of multilayered essentiality through the exploration of areas of sequence space that are only accessible in the complex state., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Chilling stress is an important environmental factor that affects rice ( Oryza sativa L.) growth and yield, and the booting stage is the most sensitive stage of rice to chilling stress. In this study, we focused on OsRBCS3 , a rice gene related to chilling tolerance at the booting stage, which encodes the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit in photosynthesis. The aim of this study was to elucidate the role and mechanism of OsRBCS3 in rice chilling tolerance at the booting stage. The expression levels of OsRBCS3 under chilling stress were compared in two japonica rice cultivars with different chilling tolerances: Kongyu131 (KY131) and Longjing11 (LJ11). A positive correlation was found between OsRBCS3 expression and chilling tolerance. Over-expression (OE) and knock-out (KO) lines of OsRBCS3 were constructed using over-expression and CRISPR/Cas9 technology, respectively, and their chilling tolerance was evaluated at the seedling and booting stages. The results showed that OE lines exhibited higher chilling tolerance than wild-type (WT) lines at both seedling and booting stages, while KO lines showed lower chilling tolerance than WT lines. Furthermore, the antioxidant enzyme activities, malondialdehyde (MDA) content and Rubisco activity of four rice lines under chilling stress were measured, and it was found that OE lines had stronger antioxidant and photosynthetic capacities, while KO lines had the opposite effects. This study validated that OsRBCS3 plays an important role in rice chilling tolerance at the booting stage, providing new molecular tools and a theoretical basis for rice chilling tolerance breeding.

As an important yield component, rice tiller number controls panicle number and determines grain yield. Regulation of rice tiller number by chloroplast pentatricopeptide repeat (PPR) proteins has not been reported previously. Here, we report the rice reduced culm number22 (rcn22) mutant, which produces few tillers owing to suppressed tiller bud elongation. Map-based cloning revealed that RCN22 encodes a chloroplast-localized P-type PPR protein. We found that RCN22 specifically binds to the 5' UTR of RbcL mRNA (encoding the large subunit of Rubisco) and enhances its stability. The reduced abundance of RbcL mRNA in rcn22 leads to a lower photosynthetic rate and decreased sugar levels. Consequently, transcript levels of DWARF3 (D3) and TEOSINTE BRANCHED1 (TB1) (which encode negative regulators of tiller bud elongation) are increased, whereas protein levels of the positive regulator DWARF53 (D53) are decreased. Furthermore, high concentrations of sucrose can rescue the tiller bud growth defect of the rcn22 mutant. On the other hand, TB1 directly binds to the RCN22 promoter and downregulates its expression. The tb1/rcn22 double mutant shows a tillering phenotype similar to that of rcn22. Our results suggest that the TB1-RCN22-RbcL module plays a vital role in rice tiller bud elongation by affecting sugar levels., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cyanobacteria are highly abundant in the marine photic zone and primary drivers of the conversion of inorganic carbon into biomass. To date, all studied cyanobacterial lineages encode carbon fixation machinery relying upon form I Rubiscos within a CO 2 -concentrating carboxysome. Here, we report that the uncultivated anoxic marine zone (AMZ) IB lineage of Prochlorococcus from pelagic oxygen-deficient zones (ODZs) harbors both form I and form II Rubiscos, the latter of which are typically noncarboxysomal and possess biochemical properties tuned toward low-oxygen environments. We demonstrate that these cyanobacterial form II enzymes are functional in vitro and were likely acquired from proteobacteria. Metagenomic analysis reveals that AMZ IB are essentially restricted to ODZs in the Eastern Pacific, suggesting that form II acquisition may confer an advantage under low-O 2 conditions. AMZ IB populations express both forms of Rubisco in situ, with the highest form II expression at depths where oxygen and light are low, possibly as a mechanism to increase the efficiency of photoautotrophy under energy limitation. Our findings expand the diversity of carbon fixation configurations in the microbial world and may have implications for carbon sequestration in natural and engineered systems., Competing Interests: Competing interests statement:The authors declare no competing interest.

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2-concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid membranes called pyrenoid tubules, which are proposed to deliver CO2. In the model alga Chlamydomonas (Chlamydomonas reinhardtii), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules, and heterologous expression of BST4 in Arabidopsis (Arabidopsis thaliana) did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutants did not show impaired growth under continuous light at air level CO2 but were impaired in their growth under fluctuating light. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we propose that bst4 has a transiently lower thylakoid lumenal pH during dark-to-light transition compared to control strains. We conclude that BST4 is not a tethering protein but is most likely a pyrenoid tubule ion channel involved in the ion homeostasis of the lumen with particular importance during light fluctuations., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Some bacteria possess microcompartments that function as protein-based organelles. Bacterial microcompartments (BMCs) sequester enzymes to optimize metabolic reactions. Several BMCs have been characterized to date, including carboxysomes and metabolosomes. Genomic analysis has identified novel BMCs and their loci, often including genes for signature enzymes critical to their function, but further characterization is needed to confirm their roles. Among the various BMCs, carboxysomes, which are found in cyanobacteria and some chemoautotrophic bacteria, and are most extensively investigated. These self-assembling polyhedral proteinaceous BMCs are essential for carbon fixation. Carboxysomes encapsulate the enzymes RuBisCo and carbonic anhydrase, which increase the carbon fixation rate in the cell and decrease the oxygenation rate by RuBisCo. The ability of carboxysomes to concentrate carbon dioxide in crops and industrially relevant microorganisms renders them attractive targets for carbon assimilation bioengineering. Thus, carboxysome characterization is the first step toward developing carboxysome-based applications. Therefore, this review comprehensively explores carboxysome morphology, physiology, and biochemistry. It also discusses recent advances in microscopy and complementary techniques for isolating and characterizing this versatile class of prokaryotic organelles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

CO 2 fixation in most unicellular algae relies on the pyrenoid, a biomolecular condensate, which sequesters the cell's carboxylase Rubisco. In the marine diatom Phaeodactylum tricornutum, the pyrenoid tandem repeat protein Pyrenoid Component 1 (PYCO1) multivalently binds Rubisco to form a heterotypic Rubisco condensate. PYCO1 contains prion-like domains and can phase-separate homotypically in a salt-dependent manner. Here we dissect PYCO1 homotypic liquid-liquid phase separation (LLPS) by evaluating protein fragments and the effect of site-directed mutagenesis. Two of PYCO1's six repeats are required for homotypic LLPS. Mutagenesis of a minimal phase-separating fragment reveals tremendous sensitivity to the substitution of aromatic residues. Removing positively charged lysines and arginines instead enhances the propensity of the fragment to condense. We conclude that PYCO1 homotypic LLPS is mostly driven by π-π interactions mediated by tyrosine and tryptophan stickers. In contrast π-cation interactions involving arginine or lysine are not significant drivers of LLPS in this system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The carboxysome is a natural proteinaceous organelle for carbon fixation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble to form a polyhedral shell structure to sequester cargo enzymes, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), and carbonic anhydrases. How these protein components assemble to construct a functional carboxysome is a central question in not only understanding carboxysome structure and function but also synthetic engineering of carboxysomes for biotechnological applications. Here, we determined the structure of the chaperone protein CcmS, which has recently been identified to be involved in β-carboxysome assembly, and its interactions with β-carboxysome proteins. The crystal structure at 1.99 Å resolution reveals CcmS from Nostoc sp. PCC 7120 forms a homodimer, and each CcmS monomer consists of five α-helices and four β-sheets. Biochemical assays indicate that CcmS specifically interacts with the C-terminal extension of the carboxysome shell protein CcmK1, but not the shell protein homolog CcmK2 or the carboxysome scaffolding protein CcmM. Moreover, we solved the structure of a stable complex of CcmS and the C-terminus of CcmK1 at 1.67 Å resolution and unveiled how the CcmS dimer interacts with the C-terminus of CcmK1. These findings allowed us to propose a model to illustrate CcmS-mediated β-carboxysome assembly by interacting with CcmK1 at the outer shell surface. Collectively, our study provides detailed insights into the accessory factors that drive and regulate carboxysome assembly, thereby improving our knowledge of carboxysome structure, function, and bioengineering., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

We have previously suggested that in rice (Oryza sativa L.) leaves of different ages and N nutrition statuses, photosystems II and I (PSII and PSI, respectively) are regulated depending on N partitioning to Rubisco, which can determine the magnitude of unutilized light energy. The robustness of this mechanism was tested using Rubisco-antisense transgenic rice plants, in which reduced N partitioning to Rubisco markedly increases unutilized light energy. In wild-type plants, N partitioning to Rubisco tended to be smaller in the leaves at lower positions owing to leaf senescence. In the transgenic plants, N partitioning to Rubisco was generally smaller than in the wild-type plants and was relatively constant among leaf positions. The quantum efficiency of PSII [Y(II)] and quantum yield of non-photochemical quenching [Y(NPQ)] correlated positively and negatively, respectively, with N partitioning to Rubisco irrespective of leaf position or genotype. The oxidation levels of the reaction center chlorophyll of PSI (P700) [Y(ND)] negatively correlated with N partitioning to Rubisco. However, in mature and early senescent leaves of the transgenic plants, Y(ND) was markedly lower than expected from N partitioning to Rubisco. These results suggest that in the transgenic plants, the regulation depending on N partitioning to Rubisco is robust for PSII but fails for PSI in mature and early senescing leaves. In these leaves, the magnitudes of P700 oxidation were found to be less than expected from the Y(II) and Y(NPQ) values. The mechanistic reasons and physiological implications of these phenomena are discussed., (© 2024. The Author(s) under exclusive licence to The Botanical Society of Japan.)

Laizhou Bay, a major breeding ground for economic marine organisms in the northern waters of China, is facing rapid environmental degradation. In this study, field surveys in this area were conducted in the spring, summer, and autumn of 2020. Microscopic observation and RuBisCO large subunit (rbcL) gene analysis were employed to understand the community structure and temporal dynamics of phytoplankton. The phytoplankton community structures detected by the two methods showed significant differences. Microscopic observation revealed the dominance of dinoflagellates in spring that shifted to the dominance of diatoms in summer and autumn. However, rbcL gene sequencing consistently identified diatoms as dominant throughout all three seasons, with their relative abundance showing an increasing trend. Conversely, the relative abundance of the second- and third-most abundant taxa, namely, haptophytes and ochrophytes, decreased as the seasons transitioned. rbcL gene sequencing annotated more species than microscopy. It could detect haptophytes and cryptophytes, which were overlooked by microscopy. In addition, rbcL gene sequencing detected a remarkable amount of Thalassiosira profunda, which was previously unidentified in this sea area. However, it appeared to underestimate the contribution of dinoflagellates considerably, with most taxa being only identified through microscopic identification. The two methods jointly identified 28 harmful algal bloom taxa with similar detection quantities but substantial differences in species composition. Phytoplankton communities were influenced by temperature, salinity, and nutrients. The results of this work suggest that a combination of multiple techniques is necessary for a comprehensive understanding of phytoplankton., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Rising temperatures due to the current climate crisis will soon have devastating impacts on crop performance and resilience. In particular, CO2 assimilation is dramatically limited at high temperatures. CO2 assimilation is accomplished by rubisco, which is inhibited by the binding of inhibitory sugar phosphates to its active site. Plants therefore utilize the essential chaperone rubisco activase (RCA) to remove these inhibitors and enable continued CO2 fixation. However, RCA does not function at moderately high temperatures (42°C), resulting in impaired rubisco activity and reduced CO2 assimilation. We set out to understand temperature-dependent RCA regulation in four different C4 plants, with a focus on the crop plants maize (two cultivars) and sorghum, as well as the model grass Setaria viridis (setaria) using gas exchange measurements, which confirm that CO2 assimilation is limited by carboxylation in these organisms at high temperatures (42°C). All three species express distinct complements of RCA isoforms and each species alters the isoform and proteoform abundances in response to heat; however, the changes are species-specific. We also examine whether the heat-mediated inactivation of RCA is due to biochemical regulation rather than simple thermal denaturation. We reveal that biochemical regulation affects RCA function differently in different C4 species, and differences are apparent even between different cultivars of the same species. Our results suggest that each grass evolved different strategies to maintain RCA function during stress and we conclude that a successful engineering approach aimed at improving carbon capture in C4 grasses will need to accommodate these individual regulatory mechanisms., (© 2024 The Author(s).)

Pyrenoids are subcompartments of algal chloroplasts that increase the efficiency of Rubisco-driven CO 2 fixation. Diatoms fix up to 20% of global CO 2 , but their pyrenoids remain poorly characterized. Here, we used in vivo photo-crosslinking to identify pyrenoid shell (PyShell) proteins, which we localized to the pyrenoid periphery of model pennate and centric diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana. In situ cryo-electron tomography revealed that pyrenoids of both diatom species are encased in a lattice-like protein sheath. Single-particle cryo-EM yielded a 2.4-Å-resolution structure of an in vitro TpPyShell1 lattice, which showed how protein subunits interlock. T. pseudonana TpPyShell1/2 knockout mutants had no PyShell sheath, altered pyrenoid morphology, and a high-CO 2 requiring phenotype, with reduced photosynthetic efficiency and impaired growth under standard atmospheric conditions. The structure and function of the diatom PyShell provide a molecular view of how CO 2 is assimilated in the ocean, a critical ecosystem undergoing rapid change., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Carboxysomes are proteinaceous organelles featuring icosahedral protein shells that enclose the carbon-fixing enzymes, ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), along with carbonic anhydrase. The intrinsically disordered scaffolding protein CsoS2 plays a vital role in the construction of α-carboxysomes through bridging the shell and cargo enzymes. The N-terminal domain of CsoS2 binds Rubisco and facilitates Rubisco packaging within the α-carboxysome, whereas the C-terminal domain of CsoS2 (CsoS2-C) anchors to the shell and promotes shell assembly. However, the role of the middle region of CsoS2 (CsoS2-M) has remained elusive. Here, we conducted in-depth examinations on the function of CsoS2-M in the assembly of the α-carboxysome shell by generating a series of recombinant shell variants in the absence of cargos. Our results reveal that CsoS2-M assists CsoS2-C in the assembly of the α-carboxysome shell and plays an important role in shaping the α-carboxysome shell through enhancing the association of shell proteins on both the facet-facet interfaces and flat shell facets. Moreover, CsoS2-M is responsible for recruiting the C-terminal truncated isoform of CsoS2, CsoS2A, into α-carboxysomes, which is crucial for Rubisco encapsulation and packaging. This study not only deepens our knowledge of how the carboxysome shell is constructed and regulated but also lays the groundwork for engineering and repurposing carboxysome-based nanostructures for diverse biotechnological purposes., Importance: Carboxysomes are a paradigm of organelle-like structures in cyanobacteria and many proteobacteria. These nanoscale compartments enclose Rubisco and carbonic anhydrase within an icosahedral virus-like shell to improve CO 2 fixation, playing a vital role in the global carbon cycle. Understanding how the carboxysomes are formed is not only important for basic research studies but also holds promise for repurposing carboxysomes in bioengineering applications. In this study, we focuses on a specific scaffolding protein called CsoS2, which is involved in facilitating the assembly of α-type carboxysomes. By deciphering the functions of different parts of CsoS2, especially its middle region, we provide new insights into how CsoS2 drives the stepwise assembly of the carboxysome at the molecular level. This knowledge will guide the rational design and reprogramming of carboxysome nanostructures for many biotechnological applications., Competing Interests: The authors declare no conflict of interest.

RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (Rubisco) produces pyruvate in the chloroplast through β-elimination of the aci-carbanion intermediate 1 . Here we show that this side reaction supplies pyruvate for isoprenoid, fatty acid and branched-chain amino acid biosynthesis in photosynthetically active tissue. 13 C labelling studies of intact Arabidopsis plants demonstrate that the total carbon commitment to pyruvate is too large for phosphoenolpyruvate to serve as a precursor. Low oxygen stimulates Rubisco carboxylase activity and increases pyruvate production and flux through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which supplies the precursors for plastidic isoprenoid biosynthesis 2,3 . Metabolome analysis of mutants defective in phosphoenolpyruvate or pyruvate import and biochemical characterization of isolated chloroplasts further support Rubisco as the main source of pyruvate in chloroplasts. Seedlings incorporated exogenous, 13 C-labelled pyruvate into MEP pathway intermediates, while adult plants did not, underscoring the developmental transition in pyruvate sourcing. Rubisco β-elimination leading to pyruvate constituted 0.7% of the product profile in in vitro assays, which translates to 2% of the total carbon leaving the Calvin-Benson-Bassham cycle. These insights solve the "pyruvate paradox" 4 , improve the fit of metabolic models for central metabolism and connect the MEP pathway directly to carbon assimilation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

With the rising demand for sustainable renewable resources, microorganisms capable of producing bioproducts such as bioplastics are attractive. While many bioproduction systems are well-studied in model organisms, investigating non-model organisms is essential to expand the field and utilize metabolically versatile strains. This investigation centers on Rhodopseudomonas palustris TIE-1, a purple non-sulfur bacterium capable of producing bioplastics. To increase bioplastic production, genes encoding the putative regulatory protein PhaR and the depolymerase PhaZ of the polyhydroxyalkanoate (PHA) biosynthesis pathway were deleted. Genes associated with pathways that might compete with PHA production, specifically those linked to glycogen production and nitrogen fixation, were deleted. Additionally, RuBisCO form I and II genes were integrated into TIE-1's genome by a phage integration system, developed in this study. Our results show that deletion of phaR increases PHA production when TIE-1 is grown photoheterotrophically with butyrate and ammonium chloride (NH 4 Cl). Mutants unable to produce glycogen or fix nitrogen show increased PHA production under photoautotrophic growth with hydrogen and NH 4 Cl. The most significant increase in PHA production was observed when RuBisCO form I and form I & II genes were overexpressed, five times under photoheterotrophy with butyrate, two times with hydrogen and NH 4 Cl, and two times under photoelectrotrophic growth with N 2 . In summary, inserting copies of RuBisCO genes into the TIE-1 genome is a more effective strategy than deleting competing pathways to increase PHA production in TIE-1. The successful use of the phage integration system opens numerous opportunities for synthetic biology in TIE-1.IMPORTANCEOur planet has been burdened by pollution resulting from the extensive use of petroleum-derived plastics for the last few decades. Since the discovery of biodegradable plastic alternatives, concerted efforts have been made to enhance their bioproduction. The versatile microorganism Rhodopseudomonas palustris TIE-1 (TIE-1) stands out as a promising candidate for bioplastic synthesis, owing to its ability to use multiple electron sources, fix the greenhouse gas CO 2 , and use light as an energy source. Two categories of strains were meticulously designed from the TIE-1 wild-type to augment the production of polyhydroxyalkanoate (PHA), one such bioplastic produced. The first group includes mutants carrying a deletion of the phaR or phaZ genes in the PHA pathway, and those lacking potential competitive carbon and energy sinks to the PHA pathway (namely, glycogen biosynthesis and nitrogen fixation). The second group comprises TIE-1 strains that overexpress RuBisCO form I or form I & II genes inserted via a phage integration system. By studying numerous metabolic mutants and overexpression strains, we conclude that genetic modifications in the environmental microbe TIE-1 can improve PHA production. When combined with other approaches (such as reactor design, use of microbial consortia, and different feedstocks), genetic and metabolic manipulations of purple nonsulfur bacteria like TIE-1 are essential for replacing petroleum-derived plastics with biodegradable plastics like PHA., Competing Interests: The authors declare no conflict of interest.

Improved understanding of mosquito-plant feeding interactions can reveal insights into the ecological dynamics of pathogen transmission. In wild malaria vectors Anopheles gambiae s.l. and An. funestus group surveyed in selected dryland ecosystems of Kenya, we found a low level of plant feeding (2.8%) using biochemical cold anthrone test but uncovered 14-fold (41%) higher rate via DNA barcoding targeting the chloroplast rbcL gene. Plasmodium falciparum positivity was associated with either reduced or increased total sugar levels and varied by mosquito species. Gut analysis revealed the mosquitoes to frequently feed on acacia plants (~ 89%) (mainly Vachellia tortilis) in the family Fabaceae. Chemical analysis revealed 1-octen-3-ol (29.9%) as the dominant mosquito attractant, and the sugars glucose, sucrose, fructose, talose and inositol enriched in the vegetative parts, of acacia plants. Nutritional analysis of An. longipalpis C with high plant feeding rates detected fewer sugars (glucose, talose, fructose) compared to acacia plants. These results demonstrate (i) the sensitivity of DNA barcoding to detect plant feeding in malaria vectors, (ii) Plasmodium infection status affects energetic reserves of wild anopheline vectors and (iii) nutrient content and olfactory cues likely represent potent correlates of acacia preferred as a host plant by diverse malaria vectors. The results have relevance in the development of odor-bait control strategies including attractive targeted sugar-baits., (© 2024. The Author(s).)

Photosynthetic and chemosynthetic extremophiles have evolved adaptations to thrive in challenging environments by finely adjusting their metabolic pathways through evolutionary processes. A prime adaptation target to allow autotrophy in extreme conditions is the enzyme Rubisco, which plays a central role in the conversion of inorganic to organic carbon. Here, we present an extensive compilation of Rubisco kinetic traits from a wide range of species of bacteria, archaea, algae, and plants, sorted by phylogenetic group, Rubisco type, and extremophile type. Our results show that Rubisco kinetics for the few extremophile organisms reported up to date are placed at the margins of the enzyme's natural variability. Form ID Rubisco from thermoacidophile rhodophytes and form IB Rubisco from halophile terrestrial plants exhibit higher specificity and affinity for CO 2 than their non-extremophilic counterparts, as well as higher carboxylation efficiency, whereas form ID Rubisco from psychrophile organisms possess lower affinity for O 2 . Additionally, form IB Rubisco from thermophile cyanobacteria shows enhanced CO 2 specificity when compared to form IB non-extremophilic cyanobacteria. Overall, these findings highlight the unique characteristics of extremophile Rubisco enzymes and provide useful clues to guide next explorations aimed at finding more efficient Rubiscos., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48-72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Rubisco small subunit (RbcS), a core component with crucial effects on the structure and kinetic properties of the Rubisco enzyme, plays an important role in response to plant growth, development, and various stresses. Although Rbcs genes have been characterized in many plants, their muti-functions in soybeans remain elusive. In this study, a total of 11 GmRbcS genes were identified and subsequently divided into three subgroups based on a phylogenetic relationship. The evolutionary analysis revealed that whole-genome duplication has a profound effect on GmRbcSs . The cis-acting elements responsive to plant hormones, development, and stress-related were widely found in the promoter region. Expression patterns based on the RT-qPCR assay exhibited that GmRbcS genes are expressed in multiple tissues, and notably Glyma.19G046600 ( GmRbcS8 ) exhibited the highest expression level compared to other members, especially in leaves. Moreover, differential expressions of GmRbcS genes were found to be significantly regulated by exogenous plant hormones, demonstrating their potential functions in diverse biology processes. Finally, the function of GmRbcS8 in enhancing soybean resistance to soybean mosaic virus (SMV) was further determined through the virus-induced gene silencing (VIGS) assay. All these findings establish a strong basis for further elucidating the biological functions of RbcS genes in soybeans.

Rubisco (D-ribulose 1,5-bisphosphate carboxylase/oxygenase) is the most abundant enzyme in the world, constituting up to half of the soluble protein content in plant leaves. Such is its ubiquity that its chemical fingerprint can be detected in the geological record spanning billions of years. Rubisco catalyses the conversion of inorganic CO 2 into organic sugars, which underpin almost all of the biosphere, including our entire food chain. Due to its central role in the global carbon cycle, rubisco has been the subject of intense research for over 50 years. Rubisco is often considered inefficient due to its slow rate of carboxylation compared with other central metabolism enzymes, and its promiscuous oxygenase activity, which competes with the productive carboxylation reaction. It is hoped that engineering improved CO 2 fixation will have significant advantages in agriculture and climate change mitigation. However, rubisco has proven difficult to engineer, with decades of efforts yielding limited results. Recent research has focused on reconstructing the evolutionary trajectory of rubisco to help elucidate its cryptic origins. Such evolutionary studies have led to a better understanding of both the origins of more complex rubisco forms and the broader relationship between rubisco's structure and function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Carbon fixation is a key metabolic function shaping marine life, but the underlying taxonomic and functional diversity involved is only partially understood. Using metagenomic resources targeted at marine piconanoplankton, we provide a reproducible machine learning framework to derive the potential biogeography of genomic functions through the multi-output regression of gene read counts on environmental climatologies. Leveraging the Marine Atlas of Tara Oceans Unigenes, we investigate the genomic potential of primary production in the global ocean. The latter is performed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and is often associated with carbon concentration mechanisms in piconanoplankton, major marine unicellular photosynthetic organisms. We show that the genomic potential supporting C 4 enzymes and RUBISCO exhibits strong functional redundancy and important affinity toward tropical oligotrophic waters. This redundancy is taxonomically structured by the dominance of Mamiellophyceae and Prymnesiophyceae in mid and high latitudes. These findings enhance our understanding of the relationship between functional and taxonomic diversity of microorganisms and environmental drivers of key biogeochemical cycles.

Enhancing crop photosynthesis through genetic engineering technologies offers numerous opportunities to increase plant productivity. Key approaches include optimizing light utilization, increasing cytochrome b 6 f complex levels, and improving carbon fixation. Modifications to Rubisco and the photosynthetic electron transport chain are central to these strategies. Introducing alternative photorespiratory pathways and enhancing carbonic anhydrase activity can further increase the internal CO 2 concentration, thereby improving photosynthetic efficiency. The efficient translocation of photosynthetically produced sugars, which are managed by sucrose transporters, is also critical for plant growth. Additionally, incorporating genes from C 4 plants, such as phosphoenolpyruvate carboxylase and NADP-malic enzymes, enhances the CO 2 concentration around Rubisco, reducing photorespiration. Targeting microRNAs and transcription factors is vital for increasing photosynthesis and plant productivity, especially under stress conditions. This review highlights potential biological targets, the genetic modifications of which are aimed at improving photosynthesis and increasing plant productivity, thereby determining key areas for future research and development.

All cyanobacteria and some chemoautotrophic bacteria fix CO 2 into sugars using specialized proteinaceous compartments called carboxysomes. Carboxysomes enclose the enzymes Rubisco and carbonic anhydrase inside a layer of shell proteins to increase the CO 2 concentration for efficient carbon fixation by Rubisco. In the ⍺-carboxysome lineage, a disordered and highly repetitive protein named CsoS2 is essential for carboxysome formation and function. Without it, the bacteria require high CO 2 to grow. How does a protein predicted to be lacking structure serve as the architectural scaffold for such a vital cellular compartment? In this study, we identify key residues present in the repeats of CsoS2, VTG and Y, which are necessary for building functional ⍺-carboxysomes in vivo. These highly conserved and repetitive residues contribute to the multivalent binding interaction and phase separation behavior between CsoS2 and shell proteins. We also demonstrate 3-component reconstitution of CsoS2, Rubisco, and shell proteins into spherical condensates and show the utility of reconstitution as a biochemical tool to study carboxysome biogenesis. The precise self-assembly of thousands of proteins is crucial for carboxysome formation, and understanding this process could enable their use in alternative biological hosts or industrial processes as effective tools to fix carbon., Competing Interests: Conflict of interest D. F. S. is a co-founder and scientific advisory board member of Scribe Therapeutics. All other authors declare that thye have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Heterosis is a crucial factor in enhancing crop yield, particularly in sorghum (Sorghum bicolor ). This research utilised six sorghum restorer lines, six sorghum sterile lines, and 36 hybrid combinations created through the NCII incomplete double-row hybridisation method. We evaluated the performance of F1 generation hybrids for leaf photosynthesis-related parameters, carbon metabolism-related enzymes, and their correlation with yield traits during the flowering stage. Results showed that hybrid sorghum exhibited significant high-parent heterosis in net photosynthetic rate (P n ), transpiration rate (T r ), stomatal conductance (G s ), apparent leaf meat conductance (AMC), ribulose-1,5-bisphosphate (RuBP) carboxylase, phosphoenolpyruvate (PEP) carboxylase, and sucrose phosphate synthase (SPS). Conversely, inter-cellular carbon dioxide concentration (C i ), instantaneous water uses efficiency (WUE), and sucrose synthase (SuSy) displayed mostly negative heterosis. Traits such as 1000-grain weight (TGW), grain weight per spike (GWPS), and dry matter content (DMC) exhibited significant high-parent heterosis, with TGW reaching the highest value of 82.54%. P n demonstrated positive correlations with T r , C i , G s , RuBP carboxylase, PEP carboxylase, GWPS, TGW, and DMC, suggesting that T r , C i , and G s could aid in identifying high-photosynthesis sorghum varieties. Concurrently, P n could help select carbon-efficient sorghum varieties due to its close relationship with yield. Overall, the F1 generation of sorghum hybrids displayed notable heterosis during anthesis. Combined with field performance, P n at athesis can serve as a valuable indicator for early prediction of the yield potential of the F1 generation of sorghum hybrids and for screening carbon-efficient sorghum varieties.

The coordinated interspecific variation in leaf traits and leaf lifespan is known as the leaf economic spectrum (LES). The limitation of CO 2 diffusion to chloroplasts within the lamina is significant in C 3 photosynthesis, resulting in a shortage of CO 2 for Rubisco. Although Rubisco CO 2 /O 2 specificity (S C/O ) should be adaptively adjusted in response to the interspecific variation in CO 2 concentrations [CO 2 ] associated with Rubisco, S C/O variations across species along the LES remain unknown. We investigated the coordination among leaf traits, including S C/O , CO 2 conductance, leaf protein content, and leaf mass area, across 23 woody C 3 species coexisting on an oceanic island through phylogenetic correlation analyses. A high S C/O indicates a high CO 2 specificity of Rubisco. S C/O was negatively correlated with [CO 2 ] at Rubisco and total CO 2 conductance within lamina, while it was positively correlated with leaf protein across species, regardless of phylogenetic constraint. A simulation analysis shows that the optimal S C/O for maximizing photosynthesis depends on both [CO 2 ] at Rubisco sites and leaf protein per unit leaf area. S C/O is a key parameter along the LES axis and is crucial for maximizing photosynthesis across species and the adaptation of woody plants., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Carbon assimilation by Rubisco is often a limitation to photosynthesis and therefore plant productivity. We have previously shown that transgenic co-expression of the Rubisco large (LS) and small (SS) subunits along with an essential Rubisco accumulation factor, Raf1, leads to faster growth, increased photosynthesis, and enhanced chilling tolerance in maize (Zea mays). Maize also requires Rubisco accumulation factor2 (Raf2) for full accumulation of Rubisco. Here we have analyzed transgenic maize lines with increased expression of Raf2 or Raf2 plus LS and SS. We show that increasing Raf2 expression alone had minor effects on photosynthesis, whereas expressing Raf2 with Rubisco subunits led to increased Rubisco content, more rapid carbon assimilation, and greater plant height, most notably in plants at least 6 weeks of age. The magnitude of the effects was similar to what was observed previously for expression of Raf1 together with Rubisco subunits. Taken together, this suggests that increasing the amount of either assembly factor with Rubisco subunits can independently enhance Rubisco abundance and some aspects of plant performance. These results could also imply either synergy or a degree of functional redundancy for Raf1 and Raf2, the latter of whose precise role in Rubisco assembly is currently unknown., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Autotrophy is the basis for complex life on Earth. Central to this process is rubisco-the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 bacterial form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubisco's carboxylation velocity. We show that the presence of a carboxysome CO 2 concentrating mechanism correlates with faster rubiscos with a median fivefold higher rate. In contrast to prior studies, we find that rubiscos originating from α-cyanobacteria exhibit the highest carboxylation rates among form I enzymes (≈10 s -1 median versus <7 s -1 in other groups). Our study systematically reveals biological and environmental properties associated with kinetic variation across rubiscos from nature., (© 2024. The Author(s).)

Light management strategy is crucial for improving microalgal production in terms of higher biomass and economically valuable bioactive molecules. However, green light has received less attention in developing light managements for algae and higher plant due to its low absorption rate by chlorophyll. In this study, the effects of green light supplementation, in the combination with red and blue light were investigated in Haematococcus pluvialis. 10% and 20% of green light supplementations were applied in 3:2 ratios of red and blue LED light combinations as an expense of red-light. Growth rates, chlorophyll concentration, and dry weight were measured to assess the growth kinetics of H. pluvialis along with the relative transcript accumulations of four mRNAs: Rubisco, PTOX 2 , PsaB, and PsbS. Growth rates, chlorophyll concentrations and dry weight were found significantly higher in presence of 10% green light supplementation compared to red and blue light combinations. The relative transcript accumulations of Rubisco and PsbS genes showed significant upregulation at the end of the experiments (with the fold change of 42.91 ± 12.08 and 98.57 ± 27.38, respectively, relative to the beginning of the experiments) compared to combinations of red and blue light (fold change of 19.09 ± 3.0 and 47.77 ± 14.21, respectively, relative to beginning of the experiments). PsaB and PTOX 2 transcripts did not show significant accumulation differences between treatments. It seems that green light has a dose dependent additive effect on the growth rate of H. pluvialis. The upregulation of Rubisco and PsbS may indicate green light dependent carbon assimilation and light-harvesting response in H. pluvialis., (© 2024 American Institute of Chemical Engineers.)

Cyanobacterial CO 2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO 2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.

The introduction of the carboxysome-based CO 2 concentrating mechanism (CCM) into crop plants has been modelled to significantly increase crop yields. This projection serves as motivation for pursuing this strategy to contribute to global food security. The successful implementation of this engineering challenge is reliant upon the transfer of a microcompartment that encapsulates cyanobacterial Rubisco, known as the carboxysome, alongside active bicarbonate transporters. To date, significant progress has been achieved with respect to understanding various aspects of the cyanobacterial CCM, and more recently, different components of the carboxysome have been successfully introduced into plant chloroplasts. In this Perspective piece, we summarise recent findings and offer new research avenues that will accelerate research in this field to ultimately and successfully introduce the carboxysome into crop plants for increased crop yields., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis , an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Rubisco activase (RCA) is a pivotal enzyme that can catalyse the activation of Rubisco in carbon assimilation pathway. Many studies have shown that RCA may be a potential target for genetic manipulation aimed at enhancing photosynthetic efficiency and crop yield., Objective: To understand the biological function of the GhRCAβ2 gene in upland cotton, we cloned the coding sequence (CDS) of the GhRCAβ2 gene and investigated its sequence features, evolutionary relationship, subcellular localization, promoter sequence and expression pattern., Methods: The bioinformatics tools were used to analyze the sequence features of GhRCAβ2 protein. Transient transformation of Arabidopsis mesophyll protoplasts was performed to determine the subcellular localization of the GhRCAβ2 protein. The expression pattern of the GhRCAβ2 gene was examined by analyzing transcriptome data and using the quantitative real-time PCR (qRT-PCR)., Results: The full-length CDS of GhRCAβ2 was 1317 bp, and it encoded a protein with a chloroplast transit peptide. The GhRCAβ2 had two conserved ATP-binding domains, and did not have the C-terminal extension (CTE) domain that was unique to the RCA α-isoform in plants. Evolutionarily, GhRCAβ2 was clustered in Group A, and had a close evolutionary relationship with the soybean RCA. Western blot analysis demonstrated that GhRCAβ2 was immunoreactive to the RCA antibody displaying a molecular weight similar to that of the RCA β-isoform. The GhRCAβ2 protein was found in chloroplast, aligning with its role as a vital enzyme in the process of photosynthesis. The GhRCAβ2 gene had a leaf tissue-specific expression pattern, and the yellow-green leaf mutant exhibited a decreased expression of GhRCAβ2 in comparison to the wild-type cotton plants. The GhRCAβ2 promoter contained several cis-acting elements that respond to light, phytohormones and stress, suggesting that the expression of GhRCAβ2 may be regulated by these factors. An additional examination of stress response indicated that GhRCAβ2 expression was influenced by cold, heat, salt, and drought stress. Notably, diverse expression pattern was observed across different stress conditions. Additionally, low phosphorus and low potassium stress may result in a notable reduction in the expression of GhRCAβ2 gene., Conclusion: Our findings will establish a basis for further understanding the function of the GhRCAβ2 gene, as well as providing valuable genetic knowledge to improve cotton photosynthetic efficiency and yield under challenging environmental circumstances., (© 2024. The Author(s) under exclusive licence to The Genetics Society of Korea.)

Rubisco large-subunit methyltransferase (LSMT), a SET-domain protein lysine methyltransferase, catalyzes the formation of trimethyl-lysine in the large subunit of Rubisco or in fructose-1,6-bisphosphate aldolases (FBAs). Rubisco and FBAs are both vital proteins involved in CO 2 fixation in chloroplasts; however, the physiological effect of their trimethylation remains unknown. In Nannochloropsis oceanica , a homolog of LSMT (NoLSMT) is found. Phylogenetic analysis indicates that NoLSMT and other algae LSMTs are clustered in a basal position, suggesting that algal species are the origin of LSMT. As NoLSMT lacks the His-Ala/ProTrp triad, it is predicted to have FBAs as its substrate instead of Rubisco. The 18-20% reduced abundance of FBA methylation in NoLSMT-defective mutants further confirms this observation. Moreover, this gene ( nolsmt ) can be induced by low-CO 2 conditions. Intriguingly, NoLSMT-knockout N. oceanica mutants exhibit a 9.7-13.8% increase in dry weight and enhanced growth, which is attributed to the alleviation of photoinhibition under high-light stress. This suggests that the elimination of FBA trimethylation facilitates carbon fixation under high-light stress conditions. These findings have implications in engineering carbon fixation to improve microalgae biomass production.

Rubisco is the primary entry point for carbon into the biosphere. However, rubisco is widely regarded as inefficient leading many to question whether the enzyme can adapt to become a better catalyst. Through a phylogenetic investigation of the molecular and kinetic evolution of Form I rubisco we uncover the evolutionary trajectory of rubisco kinetic evolution in angiosperms. We show that rbcL is among the 1% of slowest-evolving genes and enzymes on Earth, accumulating one nucleotide substitution every 0.9 My and one amino acid mutation every 7.2 My. Despite this, rubisco catalysis has been continually evolving toward improved CO 2 /O 2 specificity, carboxylase turnover, and carboxylation efficiency. Consistent with this kinetic adaptation, increased rubisco evolution has led to a concomitant improvement in leaf-level CO 2 assimilation. Thus, rubisco has been slowly but continually evolving toward improved catalytic efficiency and CO 2 assimilation in plants., Competing Interests: Competing interests statement:S.K. is co-founder of Wild Bioscience Ltd.

Pyrenoids are microcompartments that are universally found in the photosynthetic plastids of various eukaryotic algae. They contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and play a pivotal role in facilitating CO 2 assimilation via CO 2 -concentrating mechanisms (CCMs). Recent investigations involving model algae have revealed that pyrenoid-associated proteins participate in pyrenoid biogenesis and CCMs. However, these organisms represent only a small part of algal lineages, which limits our comprehensive understanding of the diversity and evolution of pyrenoid-based CCMs. Here we report a pyrenoid proteome of the chlorarachniophyte alga Amorphochlora amoebiformis , which possesses complex plastids acquired through secondary endosymbiosis with green algae. Proteomic analysis using mass spectrometry resulted in the identification of 154 potential pyrenoid components. Subsequent localization experiments demonstrated the specific targeting of eight proteins to pyrenoids. These included a putative Rubisco-binding linker, carbonic anhydrase, membrane transporter, and uncharacterized GTPase proteins. Notably, most of these proteins were unique to this algal lineage. We suggest a plausible scenario in which pyrenoids in chlorarachniophytes have evolved independently, as their components are not inherited from green algal pyrenoids., Competing Interests: Competing interests statement:The authors declare no competing interest.

Rubisco catalyses the entry of almost all CO 2 into the biosphere and is often the rate-limiting step in plant photosynthesis and growth. Its notoriety as the most abundant protein on Earth stems from the slow and error-prone catalytic properties that require plants, cyanobacteria, algae and photosynthetic bacteria to produce it in high amounts. Efforts to improve the CO 2 -fixing properties of plant Rubisco has been spurred on by the discovery of more effective isoforms in some algae with the potential to significantly improve crop productivity. Incompatibilities between the protein folding machinery of leaf and algae chloroplasts have, so far, prevented efforts to transplant these more effective Rubisco variants into plants. There is therefore increasing interest in improving Rubisco catalysis by directed (laboratory) evolution. Here we review the advances being made in, and the ongoing challenges with, improving the solubility and/or carboxylation activity of differing non-plant Rubisco lineages. We provide perspectives on new opportunities for the directed evolution of crop Rubiscos and the existing plant transformation capabilities available to evaluate the extent to which Rubisco activity improvements can benefit agricultural productivity., Competing Interests: Declarations of interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO 2 -concentrating mechanism that improves the efficiency of CO 2 capture. Engineering a pyrenoid-based CO 2 -concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO 2 diffusion. Recently, we have reconstituted a phase-separated "proto-pyrenoid" Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well-studied pyrenoid, Chlamydomonas reinhardtii [N. Atkinson, Y. Mao, K. X. Chan, A. J. McCormick, Nat. Commun. 11 , 6303 (2020)]. Here, we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesized as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, while also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and broadening the avenues for engineering starch morphology., Competing Interests: Competing interests statement:The authors declare no competing interest.

In plants, complex signaling networks monitor and respond to environmental cues to determine the optimal time for the transition from the vegetative to reproductive phase. Understanding these networks requires robust tools to examine the levels and subcellular localization of key factors. The florigen FLOWERING LOCUS T (FT) is a crucial regulator of flowering time and occurs in soluble and membrane-bound forms. At low ambient temperatures, the ratio of these forms of FT undergoes a significant shift, which leads to a delay in the onset of flowering. To investigate these changes in FT localization, epitope-tagged FT protein can be isolated from plants by subcellular fractionation and its localization examined by immunoblot analysis of the resulting fractions. However, the highly abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) can interfere with methods to detect and characterize low-abundance proteins such as FT. In this chapter, we present a method for analyzing the ratio of HA-tagged FT (HA:FT) in different subcellular fractions while mitigating the interference from RuBisCO by using protamine sulfate (PS) to deplete RuBisCO during protein purification, thereby enhancing HA:FT detection in fractionated samples., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Form I rubisco underpins life on Earth, but the origins of its anomalous and highly complex multimeric structure have long proven elusive. New research helps to unravel this enigma by uncovering key missing links in the enzyme's evolutionary history., Competing Interests: Declaration of interests The author declares no competing interests., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

The enzyme rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the majority of biological carbon fixation on Earth. Although the vast majority of rubiscos across the tree of life assemble as homo-oligomers, the globally predominant form I enzyme-found in plants, algae, and cyanobacteria-forms a unique hetero-oligomeric complex. The recent discovery of a homo-oligomeric sister group to form I rubisco (named form I') has filled a key gap in our understanding of the enigmatic origins of the form I clade. However, to elucidate the series of molecular events leading to the evolution of form I rubisco, we must examine more distantly related sibling clades to contextualize the molecular features distinguishing form I and form I' rubiscos. Here, we present a comparative structural study retracing the evolutionary history of rubisco that reveals a complex structural trajectory leading to the ultimate hetero-oligomerization of the form I clade. We structurally characterize the oligomeric states of deep-branching form Iα and I'' rubiscos recently discovered from metagenomes, which represent key evolutionary intermediates preceding the form I clade. We further solve the structure of form I'' rubisco, revealing the molecular determinants that likely primed the enzyme core for the transition from a homo-oligomer to a hetero-oligomer. Our findings yield new insight into the evolutionary trajectory underpinning the adoption and entrenchment of the prevalent assembly of form I rubisco, providing additional context when viewing the enzyme family through the broader lens of protein evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae. Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions. Functional expression of heterologous phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes enables yeast cells to couple a net oxidation of NADH to the conversion of glucose to ethanol. In another strategy, NADH-dependent reduction of exogenous acetate to ethanol is enabled by introduction of a heterologous acetylating acetaldehyde dehydrogenase (A-ALD). This study explores potential advantages of co-cultivating engineered PRK-RuBisCO-based and A-ALD-based strains in anaerobic bioreactor batch cultures. Co-cultivation of these strains, which in monocultures showed reduced glycerol yields and improved ethanol yields, strongly reduced the formation of acetaldehyde and acetate, two byproducts that were formed in anaerobic monocultures of a PRK-RuBisCO-based strain. In addition, co-cultures on medium with low acetate-to-glucose ratios that mimicked those in industrial feedstocks completely removed acetate from the medium. Kinetics of co-cultivation processes and glycerol production could be optimized by tuning the relative inoculum sizes of the two strains. Co-cultivation of a PRK-RuBisCO strain with a Δgpd1 Δgpd2 A-ALD strain, which was unable to grow in the absence of acetate and evolved for faster anaerobic growth in acetate-supplemented batch cultures, further reduced glycerol formation but led to extended fermentation times. These results demonstrate the potential of using defined consortia of engineered S. cerevisiae strains for high-yield, minimal-waste ethanol production., Competing Interests: Declaration of competing interest The PhD project of AA is funded by DSM Bio-based Products & Services B.V. (Delft, The Netherlands). Royal DSM owns intellectual property rights of technology discussed in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Rubisco is the primary entry point for carbon into the biosphere. It has been widely proposed that rubisco is highly constrained by catalytic trade-offs due to correlations between the enzyme's kinetic traits across species. In previous work, we have shown that the strength of these correlations, and thus the strength of catalytic trade-offs, have been overestimated due to the presence of phylogenetic signal in the kinetic trait data (Bouvier et al., 2021). We demonstrated that only the trade-offs between the Michaelis constant for CO 2 and carboxylase turnover, and between the Michaelis constants for CO 2 and O 2 were robust to phylogenetic effects. We further demonstrated that phylogenetic constraints have limited rubisco adaptation to a greater extent than the combined action of catalytic trade-offs. Recently, however, our claims have been contested by Tcherkez and Farquhar (2021), who have argued that the phylogenetic signal we detect in rubisco kinetic traits is an artefact of species sampling, the use of rbcL-based trees for phylogenetic inference, laboratory-to-laboratory variability in kinetic measurements, and homoplasy of the C 4 trait. In the present article, we respond to these criticisms on a point-by-point basis and conclusively show that all are unfounded. As such, we stand by our original conclusions. Namely, although rubisco kinetic evolution has been limited by biochemical trade-offs, these are not absolute and have been previously overestimated due to phylogenetic biases. Instead, rubisco adaptation has in fact been more limited by phylogenetic constraint., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SK is co-founder of Wild Bioscience Ltd., (Copyright © 2023 Elsevier GmbH. All rights reserved.)