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Background and Aims: To better understand C4 evolution in monocots, we characterized C3-C4 intermediate phenotypes in the grass genus Homolepis (subtribe Arthropogoninae)., Methods: Carbon isotope ratio (δ13C), leaf gas exchange, mesophyll (M) to bundle sheath (BS) tissue characteristics, organelle size and numbers in M and BS tissue, and tissue distribution of the P-subunit of glycine decarboxylase (GLDP) were determined for five Homolepis species and the C4 grass Mesosetum loliiforme from a phylogenetic sister clade. We generated a transcriptome-based phylogeny for Homolepis and Mesosetum species to interpret physiological and anatomical patterns in an evolutionary context, and to test for hybridization., Key Results: Homolepis contains two C3 (H. glutinosa, H. villaricensis), one weaker form of C2 termed sub-C2 (H. isocalycia), and two C2 species (H. longispicula, H. aturensis). Homolepis longispicula and H. aturensis express over 85% of leaf GDC in centripetal mitochondria within the BS, and have increased fractions of leaf chloroplasts, mitochondria and peroxisomes within the BS relative to H. glutinosa. Analysis of leaf gas exchange, cell ultrastructural, and transcript expression show M. loliiforme is a C4 plant of the NADP-malic enzyme subtype. Homolepis is comprised of two sister clades, one containing H. glutinosa and H. villaricensis and the second H. longispicula and H. aturensis. Homolepis isocalycia is of hybrid origin, with parents being H. aturensis and a common ancestor of the C3  Homolepis clade and H. longispicula., Conclusions: Photosynthetic activation of BS tissue in the sub-C2 and C2 species of Homolepis is similar to patterns observed in C3-C4 intermediate eudicots, indicating common evolutionary pathways from C3 to C4 photosynthesis in these disparate clades. Hybridization can diversify the C3-C4 intermediate character state and should be considered in reconstructing putative ancestral states using phylogenetic analyses., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company.)

Algae play critical roles in the carbon dioxide (CO 2 ) exchange between the water bodies and the atmosphere. However, the effects of prokaryotic and eukaryotic algae on carbon utilization, CO 2 flux, and the underlying mechanisms remain poorly understood. Therefore, this study investigated the differences in carbon preferences and CO 2 fluxes under different algal dominance days. Our research revealed that dissolved inorganic carbon (DIC) concentration fluctuations had a limited effect on the relative abundance of algae. However, shifts in dominant algal phyla induced changes in DIC, with Cyanobacteria preferring HCO 3 - and Chlorophyta preferring CO 2 . Analysis of the water chemistry balance indicated that the growth of Chlorophyta had a 15.59 times greater effect on CO 2 sinks compared with that of Cyanobacteria. During the Cyanobacteria dominance days, the lower DIC concentration did not result in a reduction in CO 2 emissions. However, increases in the dissolved organic carbon concentration provided a favorable environment for Cyanobacteria, which promoted CO 2 emissions. The CCM model indicated that the growth of Chlorophyta resulted in CO 2 uptake rates at least 3.57 times higher and CO 2 leakage rates up to 0.97 times lower compared to Cyanobacteria, accelerating CO 2 transport into the cell. Overall, CO 2 sink was stronger on Chlorophyta dominance days than on Cyanobacteria dominance days. This study emphasized the influence of algal phyla on CO 2 fluxes, revealing the significant CO 2 sink associated with Chlorophyta. Further research should investigate how to manipulate environmental factors to favor Chlorophyta growth and effectively reduce CO 2 emissions., 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.)

Photosynthetic eukaryotes have metabolic pathways that occur in distinct subcellular compartments. However, because metabolites synthesized in one compartment, including fixed carbon compounds and reductant generated by photosynthetic electron flows, may be integral to processes in other compartments, the cells must efficiently move metabolites among the different compartments. This review examines the various photosynthetic electron flows used to generate ATP and fixed carbon and the trafficking of metabolites in the green alga Chlamydomomas reinhardtii ; information on other algae and plants is provided to add depth and nuance to the discussion. We emphasized the trafficking of metabolites across the envelope membranes of the two energy powerhouse organelles of the cell, the chloroplast and mitochondrion, the nature and roles of the major mobile metabolites that move among these compartments, and the specific or presumed transporters involved in that trafficking. These transporters include sugar-phosphate (sugar-P)/inorganic phosphate (Pi) transporters and dicarboxylate transporters, although, in many cases, we know little about the substrate specificities of these transporters, how their activities are regulated/coordinated, compensatory responses among transporters when specific transporters are compromised, associations between transporters and other cellular proteins, and the possibilities for forming specific 'megacomplexes' involving interactions between enzymes of central metabolism with specific transport proteins. Finally, we discuss metabolite trafficking associated with specific biological processes that occur under various environmental conditions to help to maintain the cell's fitness. These processes include C4 metabolism in plants and the carbon concentrating mechanism, photorespiration, and fermentation metabolism in algae.

Hydrogen isotope ratios (δ 2 H) represent an important natural tracer of metabolic processes, but quantitative models of processes controlling H-fractionation in aquatic photosynthetic organisms are lacking. Here, we elucidate the underlying physiological controls of 2 H/ 1 H fractionation in algal lipids by systematically manipulating temperature, light, and CO 2 (aq) in continuous cultures of the haptophyte Gephyrocapsa oceanica . We analyze the hydrogen isotope fractionation in alkenones (α alkenone ), a class of acyl lipids specific to this species and other haptophyte algae. We find a strong decrease in the α alkenone with increasing CO 2 (aq) and confirm α alkenone correlates with temperature and light. Based on the known biosynthesis pathways, we develop a cellular model of the δ 2 H of algal acyl lipids to evaluate processes contributing to these controls on fractionation. Simulations show that longer residence times of NADPH in the chloroplast favor a greater exchange of NADPH with 2 H-richer intracellular water, increasing α alkenone . Higher chloroplast CO 2 (aq) and temperature shorten NADPH residence time by enhancing the carbon fixation and lipid synthesis rates. The inverse correlation of α alkenone to CO 2 (aq) in our cultures suggests that carbon concentrating mechanisms (CCM) do not achieve a constant saturation of CO 2 at the Rubisco site, but rather that chloroplast CO 2 varies with external CO 2 (aq). The pervasive inverse correlation of α alkenone with CO 2 (aq) in the modern and preindustrial ocean also suggests that natural populations may not attain a constant saturation of Rubisco with the CCM. Rather than reconstructing growth water, α alkenone may be a powerful tool to elucidate the carbon limitation of photosynthesis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Allometric relationships among the dimensions of leaves and their cells hold across diverse eudicotyledons, but have remained untested in the leaves of grasses. We hypothesised that geometric (proportional) allometries of cell sizes across tissues and of leaf dimensions would arise due to the coordination of cell development and that of cell functions such as water, nutrient and energy transport, and that cell sizes across tissues would be associated with light-saturated photosynthetic rate. We tested predictions across 27 globally distributed C 3 and C 4 grass species grown in a common garden. We found positive relationships among average cell sizes within and across tissues, and of cell sizes with leaf dimensions. Grass leaf anatomical allometries were similar to those of eudicots, with exceptions consistent with the fewer cell layers and narrower form of grass leaves, and the specialised roles of epidermis and bundle sheath in storage and leaf movement. Across species, mean cell sizes in each tissue were associated with light-saturated photosynthetic rate per leaf mass, supporting the functional coordination of cell sizes. These findings highlight the generality of evolutionary allometries within the grass lineage and their interlinkage with coordinated development and function., (© 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Increasing carbon dioxide (CO 2 ) emission has already become a dire threat to the human race and Earth's ecology. Microalgae are recommended to be engineered as CO 2 fixers in biorefinery, which play crucial roles in responding climate change and accelerating the transition to a sustainable future. This review sorted through each segment of microalgal biorefinery to explore the potential for its practical implementation and commercialization, offering valuable insights into research trends and identifies challenges that needed to be addressed in the development process. Firstly, the known mechanisms of microalgal photosynthetic CO 2 fixation and the approaches for strain improvement were summarized. The significance of process regulation for strengthening fixation efficiency and augmenting competitiveness was emphasized, with a specific focus on CO 2 and light optimization strategies. Thereafter, the massive potential of microalgal refineries for various bioresource production was discussed in detail, and the integration with contaminant reclamation was mentioned for economic and ecological benefits. Subsequently, economic and environmental impacts of microalgal biorefinery were evaluated via life cycle assessment (LCA) and techno-economic analysis (TEA) to lit up commercial feasibility. Finally, the current obstacles and future perspectives were discussed objectively to offer an impartial reference for future researchers and investors., 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 © 2023. Published by Elsevier B.V.)

The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is co-transcribed with the conserved PII-like protein, SbtB, to augment intracellular inorganic carbon levels for efficient CO 2 fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cyclic AMP (cAMP) and c-di-AMP. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge of potential redox-regulatory function, which we call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of hydrolysis reactions from ATP over ADP to AMP in crystallographic snapshots and unravel the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin, suggesting that it is generally linked to cellular metabolism, which is supported by physiological alterations in site-specific mutants of the SbtB protein. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding but also from the day/night status reported by the C-terminal redox switch.

Incorporating C 4 photosynthetic traits into C 3 crops is a rational approach for sustaining future demands for crop productivity. Using classical plant breeding, engineering this complex trait is unlikely to achieve its target. Therefore, it is critical and timely to implement novel biotechnological crop improvement strategies to accomplish this goal. However, a fundamental understanding of C 3 , C 4 , and C 3 -C 4 intermediate metabolism is crucial for the targeted use of biotechnological tools. This review assesses recent progress towards engineering C 4 photosynthetic traits in C 3 crops. We also discuss lessons learned from successes and failures of recent genetic engineering attempts in C 3 crops, highlighting the pros and cons of using rice as a model plant for short-, medium- and long-term goals of genetic engineering. This review provides an integrated approach towards engineering improved photosynthetic efficiency in C 3 crops for sustaining food, fibre and fuel production around the globe., (© 2022 German Society for Plant Sciences, Royal Botanical Society of the Netherlands.)

Microalgae are a diverse group of photosynthetic organisms that can be exploited for the production of different compounds, ranging from crude biomass and biofuels to high value-added biochemicals and synthetic proteins. Traditionally, algal biotechnology relies on bioprospecting to identify new highly productive strains and more recently, on forward genetics to further enhance productivity. However, it has become clear that further improvements in algal productivity for biotechnology is impossible without combining traditional tools with the arising molecular genetics toolkit. We review recent advantages in developing high throughput screening methods, preparing genome-wide mutant libraries, and establishing genome editing techniques. We discuss how algae can be improved in terms of photosynthetic efficiency, biofuel and high value-added compound production. Finally, we critically evaluate developments over recent years and explore future potential in the field., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The availability of inorganic carbon (C i ) as the source for photosynthesis is fluctuating in aquatic environments. Despite the involvement of transcriptional regulators CmpR and NdhR in regulating genes encoding C i transporters at limiting CO 2 , the C i -sensing mechanism is largely unknown among cyanobacteria. Here we report that a cAMP-dependent transcription factor SyCRP1 mediates C i response in Synechocystis. The mutant ∆sycrp1 exhibited a slow-growth phenotype and reduced maximum rate of bicarbonate-dependent photosynthetic electron transport (V max ) compared to wild-type at the scarcity of CO 2 . The number of carboxysomes was decreased significantly in the ∆sycrp1 at low CO 2 consistent with its reduced V max . The DNA microarray analysis revealed the upregulation of genes encoding C i transporters in ∆sycrp1. The membrane-localized SyCRP1 was released into the cytosol in wild-type cells shifted from low to high CO 2 or upon cAMP treatment. Soluble His-tagged SyCRP1 was shown to target DNA-binding sites upstream of the C i -regulated genes sbtA and ccmK3. In addition, cAMP enhanced the binding of SyCRP1 to its target sites. Our data collectively suggest that the C i is sensed through the second messenger cAMP releasing membrane-bound SyCRP1 into cytoplasm under sufficient CO 2 conditions. Hence, SyCRP1 is a possible regulator of carbon concentrating mechanism, and such a regulation might be mediated via sensing C i levels through cAMP in Synechocystis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In reef-building corals (order Scleractinia) and giant clams (phylum Molluca), V-type H + -ATPase (VHA) in host cells is part of a carbon concentrating mechanism (CCM) that regulates photosynthetic rates of their symbiotic algae. Here, we show that VHA plays a similar role in the sea anemone Anemonia majano , a member of the order Actinaria and sister group to the Scleractinia, which in contrast to their colonial calcifying coral relatives is a solitary, soft-bodied taxa. Western blotting and immunofluorescence revealed that VHA was abundantly present in the host-derived symbiosome membrane surrounding the photosymbionts. Pharmacological inhibition of VHA activity in individual anemones resulted in an approximately 80% decrease of photosynthetic O 2 production. These results extend the presence of a host-controlled VHA-dependent CCM to non-calcifying cnidarians of the order Actiniaria, suggesting it is widespread among photosymbiosis between aquatic invertebrates and Symbiodiniaceae algae., (© 2022 The Authors.)

In algae, it is well established that the pyrenoid, a component of the carbon-concentrating mechanism (CCM), is essential for efficient photosynthesis at low CO 2 . However, the signal that triggers the formation of the pyrenoid has remained elusive. Here, we show that, in Chlamydomonas reinhardtii , the pyrenoid is strongly induced by hyperoxia, even at high CO 2 or bicarbonate levels. These results suggest that the pyrenoid can be induced by a common product of photosynthesis specific to low CO 2 or hyperoxia. Consistent with this view, the photorespiratory by-product, H 2 O 2 , induced the pyrenoid, suggesting that it acts as a signal. Finally, we show evidence for linkages between genetic variations in hyperoxia tolerance, H 2 O 2 signaling, and pyrenoid morphologies., Competing Interests: PN, JT, OT, JB, NN, EP, BL, SW, AW, BS, GM, MF, DH No competing interests declared, JW Joseph Weissman is affiliated with ExxonMobil. The author has no financial interests to declare, DK Reviewing editor, eLife, (© 2021, Neofotis et al.)

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) is the carbon-fixing enzyme present in most photosynthetic organisms, converting CO 2 into organic matter. Globally, photosynthetic efficiency in terrestrial plants has become increasingly challenged in recent decades due to a rapid increase in atmospheric CO 2 and associated changes toward warmer and dryer environments. Well adapted for these new climatic conditions, the C 4 photosynthetic pathway utilizes carbon concentrating mechanisms to increase CO 2 concentrations surrounding RuBisCO, suppressing photorespiration from the oxygenase catalyzed reaction with O 2 . The energy efficiency of C 3 photosynthesis, from which the C 4 pathway evolved, is thought to rely critically on an uninterrupted supply of chloroplast CO 2 . Part of the homeostatic mechanism that maintains this constancy of supply involves the CO 2 produced as a byproduct of photorespiration in a negative feedback loop. Analyzing the database of RuBisCO kinetic parameters, we suggest that in genera ( Flaveria and Panicum ) for which both C 3 and C 4 examples are available, the C 4 pathway evolved only from C 3 ancestors possessing much lower than the average carboxylase specificity relative to that of the oxygenase reaction ( S C/O = S C / S O ), and hence, the higher CO 2 levels required for development of the photorespiratory CO 2 pump (C 2 photosynthesis) essential in the initial stages of C 4 evolution, while in the later stage (final optimization phase in the Flaveria model) increased CO 2 turnover may have occurred, which would have been supported by the higher CO 2 levels. Otherwise, C 4 RuBisCO kinetic traits remain little changed from the ancestral C 3 species. At the opposite end of the spectrum, C 3 plants (from Limonium ) with higher than average S C/O , which may be associated with the ability of increased CO 2 , relative to O 2 , affinity to offset reduced photorespiration and chloroplast CO 2 levels, can tolerate high stress environments. It is suggested that, instead of inherently constrained by its kinetic mechanism, RuBisCO possesses the extensive kinetic plasticity necessary for adaptation to changes in photorespiration that occur in the homeostatic regulation of CO 2 supply under a broad range of abiotic environmental conditions., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cummins.)

Hygrophila polysperma is a heterophyllous amphibious plant. The growth of H. polysperma in submerged conditions is challenging due to the low CO 2 environment, increased resistance to gas diffusion, and bicarbonate ion (HCO 3 - ) being the dominant dissolved inorganic carbon source. The submerged leaves of H. polysperma have significantly higher rates of underwater photosynthesis compared with the terrestrial leaves. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonate (DIDS), an anion exchanger protein inhibitor, and ethoxyzolamide (EZ), an inhibitor of internal carbonic anhydrase, repressed underwater photosynthesis by the submerged leaves. These results suggested that H. polysperma acclimates to the submerged condition by using HCO 3 - for photosynthesis. H. polysperma transports HCO 3 - into the leaf by a DIDS-sensitive HCO 3 - transporter and converted to CO 2 by carbonic anhydrase. Additionally, proteome analysis revealed that submerged leaves accumulated fewer proteins associated with C4 photosynthesis compared with terrestrial leaves. This finding suggested that H. polysperma is capable of C4 and C3 photosynthesis in the terrestrial and submerged leaves, respectively. The ratio of phosphoenol pyruvate carboxylase to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the submerged leaves was less than that in the terrestrial leaves. Upon anatomical observation, the terrestrial leaves exhibited a phenotype similar to the Kranz anatomy found among C4 plants; however, chloroplasts in the bundle sheath cells were not located adjacent to the vascular bundles, and the typical Kranz anatomy was absent in submerged leaves. These results suggest that H. polysperma performs proto-Kranz type photosynthesis in a terrestrial environment and shifts from a proto-Kranz type in terrestrial leaves to a HCO 3 - use photosynthesis in the submerged environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Horiguchi, Matsumoto, Nemoto, Inokuchi and Hirotsu.)

C 2 photosynthesis is a carbon concentrating mechanism that can increase net CO 2 assimilation by capturing, concentrating and re-assimilating CO 2 released by photorespiration. Empirical and modelling studies indicate that C 2 plants assimilate more carbon than C 3 plants under high temperature, bright light, and low CO 2 conditions. I argue that engineering C 2 photosynthesis into C 3 crops is a promising approach to improve photosynthetic performance under these - and temporally heterogeneous - environments, and review the modifications that may re-create a C 2 phenotype in C 3 plants. Although a C 2 engineering program would encounter many of the same challenges faced by C 4 engineering programmes, the simpler leaf anatomical requirements make C 2 engineering a feasible approach to improve crops in the medium term., (© 2020 The Author New Phytologist © 2020 New Phytologist Foundation.)

Membrane permeabilities to CO 2 and HCO 3 - constrain the function of CO 2 concentrating mechanisms that algae use to supply inorganic carbon for photosynthesis. In diatoms and green algae, plasma membranes are moderately to highly permeable to CO 2 but effectively impermeable to HCO 3 - . Here, CO 2 and HCO 3 - membrane permeabilities were measured using an 18 O-exchange technique on two species of haptophyte algae, Emiliania huxleyi and Calcidiscus leptoporus, which showed that the plasma membranes of these species are also highly permeable to CO 2 (0.006-0.02 cm · s -1 ) but minimally permeable to HCO 3 - . Increased temperature and CO 2 generally increased CO 2 membrane permeabilities in both species, possibly due to changes in lipid composition or CO 2 channel proteins. Changes in CO 2 membrane permeabilities showed no association with the density of calcium carbonate coccoliths surrounding the cell, which could potentially impede passage of compounds. Haptophyte plasma-membrane permeabilities to CO 2 were somewhat lower than those of diatoms but generally higher than membrane permeabilities of green algae. One caveat of these measurements is that the model used to interpret 18 O-exchange data assumes that carbonic anhydrase, which catalyzes 18 O-exchange, is homogeneously distributed in the cell. The implications of this assumption were tested using a two-compartment model with an inhomogeneous distribution of carbonic anhydrase to simulate 18 O-exchange data and then inferring plasma-membrane CO 2 permeabilities from the simulated data. This analysis showed that the inferred plasma-membrane CO 2 permeabilities are minimal estimates but should be quite accurate under most conditions., (© 2020 Phycological Society of America.)

Background: Pyropia yezoensis (Rhodophyta) is widely cultivated in East Asia and plays important economic, ecological and research roles. Although inorganic carbon utilization of P. yezoensis has been investigated from a physiological aspect, the carbon concentration mechanism (CCM) of P. yezoensis remains unclear. To explore the CCM of P. yezoensis, especially during its different life stages, we tracked changes in the transcriptome, photosynthetic efficiency and in key enzyme activities under different inorganic carbon concentrations., Results: Photosynthetic efficiency demonstrated that sporophytes were more sensitive to low carbon (LC) than gametophytes, with increased photosynthesis rate during both life stages under high carbon (HC) compared to normal carbon (NC) conditions. The amount of starch and number of plastoglobuli in cells corresponded with the growth reaction to different inorganic carbon (Ci) concentrations. We constructed 18 cDNA libraries from 18 samples (three biological replicates per Ci treatment at two life cycles stages) and sequenced these using the Illumina platform. De novo assembly generated 182,564 unigenes, including approximately 275 unigenes related to CCM. Most genes encoding internal carbonic anhydrase (CA) and bicarbonate transporters involved in the biophysical CCM pathway were induced under LC in comparison with NC, with transcript abundance of some PyCAs in gametophytes typically higher than that in sporophytes. We identified all key genes participating in the C4 pathway and showed that their RNA abundances changed with varying Ci conditions. High decarboxylating activity of PEPCKase and low PEPCase activity were observed in P. yezoensis. Activities of other key enzymes involved in the C4-like pathway were higher under HC than under the other two conditions. Pyruvate carboxylase (PYC) showed higher carboxylation activity than PEPC under these Ci conditions. Isocitrate lyase (ICL) showed high activity, but the activity of malate synthase (MS) was very low., Conclusion: We elucidated the CCM of P. yezoensis from transcriptome and enzyme activity levels. All results indicated at least two types of CCM in P. yezoensis, one involving CA and an anion exchanger (transporter), and a second, C4-like pathway belonging to the PEPCK subtype. PYC may play the main carboxylation role in this C4-like pathway, which functions in both the sporophyte and gametophyte life cycles.

Raphidiopsis raciborskii is a widely distributed, potentially toxic cyanobacterium described as a tropical-subtropical species. However, its occurrence in temperate regions has been expanding. Understanding the environmental factors underlying the expansion and colonization success of Raphidiopsis has been the object of numerous studies. However, less is known regarding its responses to pH and inorganic carbon in water. Thus, the aim of the present study was to investigate the effects of changes in pH and dissolved inorganic carbon on growth and saxitoxins production in the strain R. raciborskii ITEP-A1. We incubated batch cultures with different unbuffered and buffered pH (neutral-acid and alkaline) and inorganic carbon availability (CO 2 -rich air bubbling and the addition of NaHCO 3 ) to assess the effect of these factors on the growth, toxin production as well as saxitoxins composition of the cyanobacterium. The carbon concentrating mechanism (CCM) system of ITEP-A1 was also characterized by an in silico analysis of its previously sequenced genome. The growth and saxitoxins production of R. raciborskii were affected. The addition of sodium bicarbonate and air bubbling enhanced the growth of the cyanobacterium in alkaline pH. In contrast, saxitoxins production and relative toxicity were decreased. Moreover, significant changes in the cellular composition of saxitoxins were strongly related to pH changes. ITEP-A1 potentially expresses the low-flux bicarbonate transporter BicA, an efficient CCM which uptakes most of its carbon from HCO 3 - . Hence, increasing the diffusion of CO 2 in alkaline eutrophic lakes is likely to increase R. raciborskii dominance, but produce less toxic blooms., 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 © 2020 Elsevier B.V. All rights reserved.)

Heterosigma akashiwo is a raphidophyte known for forming ichthyotoxic blooms. In order to predict the potential impacts of rising CO 2 on H. akashiwo it is necessary to understand the factors influencing growth rates over a range of CO 2 concentrations. Here we examined the physiology and gene expression response of H. akashiwo to concentrations from 200 to 1000ppm CO 2 . Growth rate data were combined from this and previous studies and fit with a CO 2 limitation-inhibition model that revealed an apparent growth optimum around 600-800ppm CO 2 . Physiological changes included a significant increase in C:N ratio at ∼800ppm CO 2 and a significant decrease in hydrogen peroxide concentration at ∼1000ppm. Whole transcriptome sequencing of H. akashiwo revealed sharp distinctions in metabolic pathway gene expression between ∼600 and ∼800ppm CO 2 . Hierarchical clustering by co-expression identified groups of genes with significant correlations to CO 2 and growth rate. Genes with significant differential expression with CO 2 included carbon concentrating mechanism genes such as beta-carbonic anhydrases and a bicarbonate transporter, which may underpin shifts in physiology. Genes involved in cell motility were significantly changed by both elevated CO 2 and growth rate, suggesting that future ocean conditions could modify swimming behavior in this species., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Carbon concentrating mechanism (CCM) and photorespiration (PR) are interlinked and co-regulated in Chlamydomonas reinhardtii, but conditions where co-regulation alters are not sufficiently explored. Here, we uncover that PR gene transcripts, like CCM transcripts, are induced even in the dark when both processes are not active. Such diurnal cycles show that transcript levels peak in the middle of 12 h day, decline by early part of 12-h dark followed by their onset again at mid-dark. Interestingly, the onset in the mid-dark phase is sensitive to high CO 2 , implying that the active carbon sensing mechanism operates even in the dark. The rhythmic alterations of both CCM and PR transcript levels are unlinked to circadian clock: the "free-running state" reveals no discernible rhythmicity in transcript changes. Only continuous light leads to high transcript levels but no detectable transcripts were observed in continuous dark. Asynchronous continuous light cultures, upon shifting to low from high CO 2 exhibit only transient induction of PR transcripts/proteins while CCM transcript induction is stable, indicating the loss of co-regulation between PR and CCM gene transcription. Lastly, we also describe that both CCM and PR transcripts/proteins are induced in low CO 2 even in mixotrophic cultures, but only in high light, the same being attenuated in high CO 2 , implying that high light is a mandatory "trigger" for CCM and PR induction in low CO 2 mixotrophy. Our study provides comprehensive analyses of conditions where CCM and PR were differently regulated, setting a paradigm for a detailed mechanistic probing of these responses.

Red coralline algae are projected to be sensitive to ocean acidification, particularly in polar oceans. As important ecosystem engineers, their potential sensitivity has broad implications, and understanding their carbon acquisition mechanisms is necessary for making reliable predictions. Therefore, we investigated the localized carbonate chemistry at the surface of Arctic coralline algae using microsensors. We report for the first time carbonate ion concentration and pH measurements ([CO 3 2- ]) at and above the algal surface in the microenvironment. We show that surface pH and [CO 3 2- ] are higher than the bulk seawater in the light, and even after hours of darkness. We further show that three species of Arctic coralline algae have efficient carbon concentrating mechanisms including direct bicarbonate uptake and indirect bicarbonate use via a carbonic anhydrase enzyme. Our results suggest that Arctic corallines have strong biological control over their surface chemistry, where active calcification occurs, and that net dissolution in the dark does not occur. We suggest that the elevated pH and [CO 3 2- ] in the dark could be explained by a high rate of light independent carbon fixation that reduces respiratory CO 2 release. This mechanism could provide a potential adaptation to ocean acidification in Arctic coralline algae, which has important implications for future Arctic marine ecosystems.

The response of marine phytoplankton to the ongoing increase in atmospheric pCO 2 reflects the consequences of both increased CO 2 concentration and decreased pH in surface seawater. In the model diatom Thalassiosira weissflogii, we explored the effects of varying pCO 2 and pH, independently and in concert, on photosynthesis and respiration by incubating samples in water enriched in H 2 18 on growth rate, photosynthesis or respiration. This absence of a measurable response reflects the very small change in energy used by the carbon concentrating mechanism (CCM) compared to the energy used in carbon fixation. In short-term experiments (~3 min), we also observed no effects of pCO 2 on growth rate, photosynthesis or respiration. This absence of a measurable response reflects the very small change in energy used by the carbon concentrating mechanism (CCM) compared to the energy used in carbon fixation. In short-term experiments (~3 min), we also observed no effects of pCO 2 or pH, even under limiting light intensity. We surmise that in T. weissflogii, it is the photosynthetic production of NADPH and ATP, rather than the CO 2 -saturation of Rubisco that controls the rate of photosynthesis at low irradiance. In short-term experiments, we observed a slightly higher respiration rate at low pH at the onset of the dark period, possibly reflecting the energy used for exporting H + and maintaining pH homeostasis. Based on what is known of the biochemistry of marine phytoplankton, our results are likely generalizable to other diatoms and a number of other eukaryotic species. The direct effects of ocean acidification on growth, photosynthesis and respiration in these organisms should be small over the range of atmospheric pCO 2 predicted for the twenty-first century.

Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO 2 + HCO 3 - + CO 3 2- ) with CO 2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotroph Thiomicrospira crunogena has a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes of T. crunogena cultivated under low- and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, were at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded by Tcr_0854 ; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853 , required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853 -encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla. IMPORTANCE DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among the Bacteria and Archaea In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotroph T. crunogena were identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla in Bacteria and also in one phylum of Archaea , the Euryarchaeota Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genus Thiomicrospira ., (Copyright © 2017 American Society for Microbiology.)

Autotrophic bacteria rely on various mechanisms to increase intracellular concentrations of inorganic forms of carbon (i.e., bicarbonate and CO 2 ) in order to improve the efficiency with which they can be converted to organic forms. Transmembrane bicarbonate transporters and carboxysomes play key roles in accumulating bicarbonate and CO 2 , but other regulatory elements of carbon concentration mechanisms in bacteria are less understood. In this study, after analyzing the genomic regions around α-type carboxysome operons, we characterize a protein that is conserved across these operons but has not been previously studied. On the basis of a series of apo- and ligand-bound crystal structures and supporting biochemical data, we show that this protein, which we refer to as the carboxysome-associated PII protein (CPII), represents a new and distinct subfamily within the broad superfamily of previously studied PII regulatory proteins, which are generally involved in regulating nitrogen metabolism in bacteria. CPII undergoes dramatic conformational changes in response to ADP binding, and the affinity for nucleotide binding is strongly enhanced by the presence of bicarbonate. CPII therefore appears to be a unique type of PII protein that senses bicarbonate availability, consistent with its apparent genomic association with the carboxysome and its constituents., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Bicarbonate species in the aqueous phase is the primary source for CO 2 for the growth of microalgae. The potential of carbon dioxide (CO 2 ) fixation by Chlorella pyrenoidosa in enriched bicarbonate medium was evaluated. In the present study, effects of parameters such as pH, sodium bicarbonate concentration and inoculum size were assessed for the removal of CO 2 by C. pyrenoidosa under mixotrophic condition. Central composite design tool from response surface methodology was used to validate statistical methods in order to study the influence of these parameters. The obtained results reveal that the maximum removal of CO 2 was attained at pH 8 with sodium bicarbonate concentration of 3.33 g/l, and inoculum size of 30 %. The experimental results were statistically significant with R 2 value of 0.9527 and 0.960 for CO 2 removal and accumulation of chlorophyll content, respectively. Among the various interactions, interactive effects between the parameters pH and inoculum size was statistically significant (P < 0.05) for CO 2 removal and chlorophyll accumulation. Based on the studies, the application of C. pyrenoidosa as a potential source for carbon dioxide removal at alkaline pH from bicarbonate source is highlighted.

Photorespiration and oxygenic photosynthesis are intimately linked processes. It has been shown that under the present day atmospheric conditions cyanobacteria and all eukaryotic phototrophs need functional photorespiration to grow autotrophically. The question arises as to when this essential partnership evolved, i.e. can we assume a coevolution of both processes from the beginning or did photorespiration evolve later to compensate for the generation of 2-phosphoglycolate (2PG) due to Rubisco's oxygenase reaction? This question is mainly discussed here using phylogenetic analysis of proteins involved in the 2PG metabolism and the acquisition of different carbon concentrating mechanisms (CCMs). The phylogenies revealed that the enzymes involved in the photorespiration of vascular plants have diverse origins, with some proteins acquired from cyanobacteria as ancestors of the chloroplasts and others from heterotrophic bacteria as ancestors of mitochondria in the plant cell. Only phosphoglycolate phosphatase was found to originate from Archaea. Notably glaucophyte algae, the earliest branching lineage of Archaeplastida, contain more photorespiratory enzymes of cyanobacterial origin than other algal lineages or land plants indicating a larger initial contribution of cyanobacterial-derived proteins to eukaryotic photorespiration. The acquisition of CCMs is discussed as a proxy for assessing the timing of periods when photorespiratory activity may have been enhanced. The existence of CCMs also had marked influence on the structure and function of photorespiration. Here, we discuss evidence for an early and continuous coevolution of photorespiration, CCMs and photosynthesis starting from cyanobacteria via algae, to land plants., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The gammaproteobacterium Thiomicrospira crunogena XCL-2 is an aerobic sulfur-oxidizing hydrothermal vent chemolithoautotroph that has a CO2 concentrating mechanism (CCM), which generates intracellular dissolved inorganic carbon (DIC) concentrations much higher than extracellular, thereby providing substrate for carbon fixation at sufficient rate. This CCM presumably requires at least one active DIC transporter to generate the elevated intracellular concentrations of DIC measured in this organism. In this study, the half-saturation constant (K CO2) for purified carboxysomal RubisCO was measured (276 ± 18 µM) which was much greater than the K CO2 of whole cells (1.03 µM), highlighting the degree to which the CCM facilitates CO2 fixation under low CO2 conditions. To clarify the bioenergetics powering active DIC uptake, cells were incubated in the presence of inhibitors targeting ATP synthesis (DCCD) or proton potential (CCCP). Incubations with each of these inhibitors resulted in diminished intracellular ATP, DIC, and fixed carbon, despite an absence of an inhibitory effect on proton potential in the DCCD-incubated cells. Electron transport complexes NADH dehydrogenase and the bc 1 complex were found to be insensitive to DCCD, suggesting that ATP synthase was the primary target of DCCD. Given the correlation of DIC uptake to the intracellular ATP concentration, the ABC transporter genes were targeted by qRT-PCR, but were not upregulated under low-DIC conditions. As the T. crunogena genome does not include orthologs of any genes encoding known DIC uptake systems, these data suggest that a novel, yet to be identified, ATP- and proton potential-dependent DIC transporter is active in this bacterium. This transporter serves to facilitate growth by T. crunogena and other Thiomicrospiras in the many habitats where they are found.

The transition of C3 , via C2 towards C4 photosynthesis is an important example of stepwise evolution of a complex genetic trait. A common feature that was gradually emphasized during this trajectory is the evolution of a CO2 concentration mechanism around Rubisco. In C2 plants, this mechanism is based on tissue-specific accumulation of glycine decarboxylase (GDC) in bundle sheath (BS) cells, relative to global expression in the cells of C3 leaves. This limits photorespiratory CO2 release to BS cells. Because BS cells are surrounded by photosynthetically active mesophyll cells, this arrangement enhances the probability of re-fixation of CO2 . The restriction of GDC to BS cells was mainly achieved by confinement of its P-subunit (GLDP). Here, we provide a mechanism for the establishment of C2 -type gene expression by studying the upstream sequences of C3 Gldp genes in Arabidopsis thaliana. Deletion of 59 bp in the upstream region of AtGldp1 restricted expression of a reporter gene to BS cells and the vasculature without affecting diurnal variation. This region was named the 'M box'. Similar results were obtained for the AtGldp2 gene. Fusion of the M box to endogenous or exogenous promoters supported mesophyll expression. Nucleosome densities at the M box were low, suggesting an open chromatin structure facilitating transcription factor binding. In silico analysis defined a possible consensus for the element that was conserved across the Brassicaceae, but not in Moricandia nitens, a C2 plant. Collective results provide evidence that a simple mutation is sufficient for establishment of C2 -specific gene expression in a C3 plant., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Inorganic carbon (Ci) is the major sink for photosynthetic reductant in organisms capable of oxygenic photosynthesis. In the absence of abundant Ci, the cyanobacterium Synechocystis sp. strain PCC6803 expresses a high affinity Ci acquisition system, the CO2-concentrating mechanisms (CCM), controlled by the transcriptional regulator CcmR and the metabolites NADP+ and α-ketoglutarate, which act as co-repressors of CcmR by modulating its DNA binding. The CCM thus responds to internal cellular redox changes during the transition from Ci-replete to Ci-limited conditions. However, the actual changes in the metabolic state of the NADPH/NADP+ system that occur during the transition to Ci-limited conditions remain ill-defined. Analysis of changes in the redox state of cells experiencing Ci limitation reveals systematic changes associated with physiological adjustments and a trend towards the quinone and NADP pools becoming highly reduced. A rapid and persistent increase in F0 was observed in cells reaching the Ci-limited state, as was the induction of photoprotective fluorescence quenching. Systematic changes in the fluorescence induction transients were also observed. As with Chl fluorescence, a transient reduction of the NADPH pool ('M' peak), is assigned to State 2→State 1 transition associated with increased electron flow to NADP+. This was followed by a characteristic decline, which was abolished by Ci limitation or inhibition of the Calvin-Benson-Bassham (CBB) cycle and is thus assigned to the activation of the CBB cycle. The results are consistent with the proposed regulation of the CCM and provide new information on the nature of the Chl and NADPH fluorescence induction curves., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H(+)-ATPase (VHA), which acidifies the symbiosome space down to pH ∼ 4. Inhibition of VHA results in a significant decrease in average H(+) activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts.

As all plastids that have been investigated so far can be traced back to endosymbiotic uptake of cyanobacteria by heterotrophic host cells, they accordingly show a high similarity regarding photosynthesis, which includes both the photosystems and the biochemical reactions around the CO2 fixation via the Calvin-Bassham cycle. Major differences between the different algal and plant groups may include the presence or absence of carbon concentrating mechanisms, pyrenoids, Rubisco activases, carbonic anhydrases as well as differences in the regulation of the Calvin-Bassham cycle. This review describes the diversity of primary carbon fixation steps in algae and plants and the respective regulatory mechanisms., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

We investigated the catalytic activity and inhibition of the β-class carbonic anhydrase (CA, EC 4.2.1.1) CahB1, from the relict cyanobacterium Coleofasciculus chthonoplastes (previously denominated Microcoleus chthonoplastes). The enzyme showed good activity as a catalyst for the CO2 hydration, with a kcat of 2.4 × 10(5)s(-1) and a kcat/Km of 6.3 × 10(7)M(-1)s(-1). A range of inorganic anions and small molecules were investigated as inhibitors of CahB1. Perchlorate and tetrafluoroborate did not inhibit the enzyme (KIs >200 mM) whereas selenate and selenocyanide were ineffective inhibitors too, with KIs of 29.9-48.61 mM. The halides, pseudohalides, carbonate, bicarbonate, trithiocarbonate and a range of heavy metal ions-containing anions were submillimolar-millimolar inhibitors (KIs in the range of 0.15-0.90 mM). The best CahB1 inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with KIs in the range of 8-75 μM, whereas acetazolamide inhibited the enzyme with a KI of 76 nM. This is the first kinetic and inhibition study of a cyanobacterial CA. As these enzymes are widespread in many cyanobacteria, being crucial for the carbon concentrating mechanism which assures substrate to RubisCO for the CO2 fixation by these organisms, a detailed kinetic/inhibition study may be essential for a better understanding of this superfamily of metalloenzymes and for potential biotechnological applications in biomimetic CO2 capture processes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)