Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2.

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO2. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO2. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.

Biosynthesis of polyhydroxybutyrate from methane and carbon dioxide using type II methanotrophs.

Methane (CH4) and carbon dioxide (CO2) are the dominant greenhouse gases (GHGs) that are increasing at an alarming rate. Methanotrophs have emerged as potential CH4 and CO2 biorefineries. This study demonstrated the synchronous incorporation of CH4 and CO2 into polyhydroxybutyrate (PHB) for the first time using 13C-labeling experiments in methanotrophs. By supplying substantial amounts of CO2, PHB content was enhanced in all investigated type II methanotrophic strains by 140 %, 146 %, and 162 %. The highest content of PHB from CH4 and CO2 in flask-scale cultivation reached 38 % dry cell weight in Methylocystis sp. MJC1, in which carbon percentage in PHB from CO2 was 45 %. Flux balance analysis predicted the critical roles of crotonyl-CoA carboxylase/reductase and phosphoenolpyruvate carboxylase in CO2 recycling. This study provided proof of the conversion of GHGs into a valuable and practical product using methanotrophic bacteria, contributing to addressing GHG emissions.

A non-methanogenic archaeon within the order Methanocellales.

Serpentinization, a geochemical process found on modern and ancient Earth, provides an ultra-reducing environment that can support microbial methanogenesis and acetogenesis. Several groups of archaea, such as the order Methanocellales, are characterized by their ability to produce methane. Here, we generate metagenomic sequences from serpentinized springs in The Cedars, California, and construct a circularized metagenome-assembled genome of a Methanocellales archaeon, termed Met12, that lacks essential methanogenesis genes. The genome includes genes for an acetyl-CoA pathway, but lacks genes encoding methanogenesis enzymes such as methyl-coenzyme M reductase, heterodisulfide reductases and hydrogenases. In situ transcriptomic analyses reveal high expression of a multi-heme c-type cytochrome, and heterologous expression of this protein in a model bacterium demonstrates that it is capable of accepting electrons. Our results suggest that Met12, within the order Methanocellales, is not a methanogen but a CO2-reducing, electron-fueled acetogen without electron bifurcation.

Alpha Carbonic Anhydrase from Nitratiruptor tergarcus Engineered for Increased Activity and Thermostability.

The development of carbon capture and storage technologies has resulted in a rising interest in the use of carbonic anhydrases (CAs) for CO2 fixation at elevated temperatures. In this study, we chose to rationally engineer the α-CA (NtCA) from the thermophilic bacterium Nitratiruptor tergarcus, which has been previously suggested to be thermostable by in silico studies. Using a combination of analyses with the DEEPDDG software and available structural knowledge, we selected residues in three regions, namely, the catalytic pocket, the dimeric interface and the surface, in order to increase thermostability and CO2 hydration activity. A total of 13 specific mutations, affecting seven amino acids, were assessed. Single, double and quadruple mutants were produced in Escherichia coli and analyzed. The best-performing mutations that led to improvements in both activity and stability were D168K, a surface mutation, and R210L, a mutation in the dimeric interface. Apart from these, most mutants showed improved thermostability, with mutants R210K and N88K_R210L showing substantial improvements in activity, up to 11-fold. Molecular dynamics simulations, focusing particularly on residue fluctuations, conformational changes and hydrogen bond analysis, elucidated the structural changes imposed by the mutations. Successful engineering of NtCA provided valuable lessons for further engineering of α-CAs.

Molecular Basis of CO2 Sensing in Hyphantria cunea.

Carbon dioxide (CO2) released by plants can serve as a cue for regulating insect behaviors. Hyphantria cunea is a widely distributed forestry pest that may use CO2 as a cue for foraging and oviposition. However, the molecular mechanism underlying its ability to sense CO2 has not been elucidated. Our initial study showed that CO2 is significantly attractive to H. cunea adults. Subsequently, 44 H. cunea gustatory receptors (GRs) were identified using transcriptome data, and 3 candidate CO2 receptors that are specifically expressed in the labial palps were identified. In vivo electrophysiological assays revealed that the labial palp is the primary organ for CO2 perception in H. cunea, which is similar to findings in other lepidopteran species. By using the Xenopus oocyte expression system, we showed that the HcunGR1 and HcunGR3 co-expressions produced a robust response to CO2, but HcunGR2 had an inhibitory effect on CO2 perception. Finally, immunohistochemical staining revealed sexual dimorphism in the CO2-sensitive labial pit organ glomerulus (LPOG). Taken together, our results clarified the mechanism by which H. cunea sense CO2, laying the foundation for further investigations into the role of CO2 in the rapid spread of H. cunea.

Bomb radiocarbon evidence for strong global carbon uptake and turnover in terrestrial vegetation.

Vegetation and soils are taking up approximately 30% of anthropogenic carbon dioxide emissions because of small imbalances in large gross carbon exchanges from productivity and turnover that are poorly constrained. We combined a new budget of radiocarbon produced by nuclear bomb testing in the 1960s with model simulations to evaluate carbon cycling in terrestrial vegetation. We found that most state-of-the-art vegetation models used in the Coupled Model Intercomparison Project underestimated the radiocarbon accumulation in vegetation biomass. Our findings, combined with constraints on vegetation carbon stocks and productivity trends, imply that net primary productivity is likely at least 80 petagrams of carbon per year presently, compared with the 43 to 76 petagrams per year predicted by current models. Storage of anthropogenic carbon in terrestrial vegetation is likely more short-lived and vulnerable than previously predicted.

Assessing the impact of climate variability on maize yields in the different regions of Ghana-A machine learning perspective.

Climate variability has become one of the most pressing issues of our time, affecting various aspects of the environment, including the agriculture sector. This study examines the impact of climate variability on Ghana's maize yield for all agro-ecological zones and administrative regions in Ghana using annual data from 1992 to 2019. The study also employs the stacking ensemble learning model (SELM) in predicting the maize yield in the different regions taking random forest (RF), support vector machine (SVM), gradient boosting (GB), decision tree (DT), and linear regression (LR) as base models. The findings of the study reveal that maize production in the regions of Ghana is inconsistent, with some regions having high variability. All the climate variables considered have positive impact on maize yield, with a lesser variability of temperature in the Guinea savanna zones and a higher temperature variability in the Volta Region. Carbon dioxide (CO2) also plays a significant role in predicting maize yield across all regions of Ghana. Among the machine learning models utilized, the stacking ensemble model consistently performed better in many regions such as in the Western, Upper East, Upper West, and Greater Accra regions. These findings are important in understanding the impact of climate variability on the yield of maize in Ghana, highlighting regional disparities in maize yield in the country, and highlighting the need for advanced techniques for forecasting, which are important for further investigation and interventions for agricultural planning and decision-making on food security in Ghana.

Hydrogen isotope fractionation is controlled by CO2 in coccolithophore lipids.

Hydrogen isotope ratios (δ2H) 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 2H/1H fractionation in algal lipids by systematically manipulating temperature, light, and CO2(aq) in continuous cultures of the haptophyte Gephyrocapsa oceanica. We analyze the hydrogen isotope fractionation in alkenones (αalkenone), a class of acyl lipids specific to this species and other haptophyte algae. We find a strong decrease in the αalkenone with increasing CO2(aq) and confirm αalkenone correlates with temperature and light. Based on the known biosynthesis pathways, we develop a cellular model of the δ2H 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 2H-richer intracellular water, increasing αalkenone. Higher chloroplast CO2(aq) and temperature shorten NADPH residence time by enhancing the carbon fixation and lipid synthesis rates. The inverse correlation of αalkenone to CO2(aq) in our cultures suggests that carbon concentrating mechanisms (CCM) do not achieve a constant saturation of CO2 at the Rubisco site, but rather that chloroplast CO2 varies with external CO2(aq). The pervasive inverse correlation of αalkenone with CO2(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, αalkenone may be a powerful tool to elucidate the carbon limitation of photosynthesis.

Multi-year aboveground data of minirhizotron facilities in Selhausen.

Improved understanding of crops' response to soil water stress is important to advance soil-plant system models and to support crop breeding, crop and varietal selection, and management decisions to minimize negative impacts. Studies on eco-physiological crop characteristics from leaf to canopy for different soil water conditions and crops are often carried out at controlled conditions. In-field measurements under realistic field conditions and data of plant water potential, its links with CO2 and H2O gas fluxes, and crop growth processes are rare. Here, we presented a comprehensive data set collected from leaf to canopy using sophisticated and comprehensive sensing techniques (leaf chlorophyll, stomatal conductance and photosynthesis, canopy CO2 exchange, sap flow, and canopy temperature) including detailed crop growth characteristics based on destructive methods (crop height, leaf area index, aboveground biomass, and yield). Data were acquired under field conditions with contrasting soil types, water treatments, and different cultivars of wheat and maize. The data from 2016 up to now will be made available for studying soil/water-plant relations and improving soil-plant-atmospheric continuum models.

Protocol to study the photosynthetic response of maize to the CO2-temperature coupling effect at ear stage using a specialized designed gradient.

Global warming will change the photosynthesis and transpiration of plants greatly and ultimately affect water use efficiency (WUE). Here, we present a protocol to investigate the response of maize WUE to the coupling effect of CO2 and temperature at ear stage using a specialized designed gradient. We describe steps for plant culture, parameter measurements, model fitting, and statistical analysis. This protocol holds potential for studying the response of WUE and CO2 adaptation across various plant species. For complete details on the use and execution of this protocol, please refer to Sun et al.1.

Development of a digital droplet PCR approach for the quantification of soil micro-organisms involved in atmospheric CO2 fixation.

Carbon-fixing micro-organisms (CFMs) play a pivotal role in soil carbon cycling, contributing to carbon uptake and sequestration through various metabolic pathways. Despite their importance, accurately quantifying the absolute abundance of these micro-organisms in soils has been challenging. This study used a digital droplet polymerase chain reaction (ddPCR) approach to measure the abundance of key and emerging CFMs pathways in fen and bog soils at different depths, ranging from 0 to 15 cm. We targeted total prokaryotes, oxygenic phototrophs, aerobic anoxygenic phototrophic bacteria and chemoautotrophs, optimizing the conditions to achieve absolute quantification of these genes. Our results revealed that oxygenic phototrophs were the most abundant CFMs, making up 15% of the total prokaryotic abundance. They were followed by chemoautotrophs at 10% and aerobic anoxygenic phototrophic bacteria at 9%. We observed higher gene concentrations in fen than in bog. There were also variations in depth, which differed between fen and bog for all genes. Our findings underscore the abundance of oxygenic phototrophs and chemoautotrophs in peatlands, challenging previous estimates that relied solely on oxygenic phototrophs for microbial carbon dioxide fixation assessments. Incorporating absolute gene quantification is essential for a comprehensive understanding of microbial contributions to soil processes. This approach sheds light on the complex mechanisms of soil functioning in peatlands.

Efficient carbon recycling between calcification and photosynthesis in red coralline algae.

Red coralline algae create abundant, spatially vast, reef ecosystems throughout our coastal oceans with significant ecosystem service provision, but our understanding of their basic physiology is lacking. In particular, the balance and linkages between carbon-producing and carbon-sequestering processes remain poorly constrained, with significant implications for understanding their role in carbon sequestration and storage. Using dual radioisotope tracing, we provide evidence for coupling between photosynthesis (which requires CO2) and calcification (which releases CO2) in the red coralline alga Boreolithothamnion soriferum (previously Lithothamnion soriferum)-a marine ecosystem engineer widely distributed across Atlantic mid-high latitudes. Of the sequestered HCO3 -, 38 ± 22% was deposited as carbonate skeleton while 39 ± 14% was incorporated into organic matter via photosynthesis. Only 38 ± 2% of the sequestered HCO3 - was transformed into CO2, and almost 40% of that was internally recycled as photosynthetic substrate, reducing the net release of carbon to 23 ± 3% of the total uptake. The calcification rate was strongly dependent on photosynthetic substrate production, supporting the presence of photosynthetically enhanced calcification. The efficient carbon-recycling physiology reported here suggests that calcifying algae may not contribute as much to marine CO2 release as is currently assumed, supporting a reassessment of their role in blue carbon accounting.

Measuring and Quantifying Characteristics of the Post-illumination Burst.

Leaf-level gas exchange enables accurate measurements of net CO2 assimilation in the light, as well as CO2 respiration in the dark. Net positive CO2 assimilation in the light indicates that the gain of carbon by photosynthesis offsets the photorespiratory loss of CO2 and respiration of CO2 in the light (RL), while the CO2 respired in the dark is mainly attributed to respiration in the dark (RD). Measuring the CO2 release specifically from photorespiration in the light is challenging since net CO2 assimilation involves three concurrent processes (the velocity of rubisco carboxylation; vc, velocity of rubisco oxygenation; vo, and RL). However, by employing a rapid light-dark transient, it is possible to transiently measure some of the CO2 release from photorespiration without the background of vc-based assimilation in the dark. This method is commonly known as the post-illumination CO2 burst (PIB) and results in a "burst" of CO2 immediately after the transition to the dark. This burst can be quantitatively characterized using several approaches. Here, we describe how to set up a PIB measurement and provide some guidelines on how to analyze and interpret the data obtained using a PIB analysis application developed in R.

Measurement of Photorespiratory Oxygen Exchange by Membrane Inlet Mass Spectrometry.

Photosynthesis and metabolism in plants involve oxygen as both a product and substrate. Oxygen is taken up during photorespiration and respiration and produced through water splitting during photosynthesis. To distinguish between processes that produce or consume O2 in leaves, isotope mass separation and detection by mass spectrometry allows measurement of evolution and uptake of O2 as well as CO2 uptake. This chapter describes how to calculate the rate of Rubisco oxygenation and carboxylation from in vivo gas exchange of stable isotopes of 16O2 and 18O2 with a closed cuvette system for leaf discs and membrane inlet mass spectrometry.

Low-Cost System for Maintaining Low Atmospheric CO2 Levels During Arabidopsis Cultivation.

Photosynthesis requires CO2 as the carbon source, and the levels of ambient CO2 determine the oxygenation or carboxylation of Ribulose-1,5-bisphosphate (RuBP) by RuBP carboxylase/oxygenase (Rubisco). Low CO2 levels lead to oxygenation and result in photorespiration, which ultimately causes a reduction in net carbon assimilation through photosynthesis. Therefore, an increased understanding of plant responses to low CO2 contributes to the knowledge of how plants circumvent the harmful effects of photorespiration. Methods for elevating CO2 above ambient concentrations are often achieved by external sources of CO2, but reducing CO2 below the ambient value is much more difficult as CO2 gas needs to be scrubbed from the atmosphere rather than added to it. Here, we describe a low-cost method of achieving low CO2 conditions for Arabidopsis growth.

Using Dynamic Gas Exchange Measurements During Oxygen Transients to Study Nonsteady-State Photorespiration.

Leaf-level gas exchange is widely used to investigate the largest carbon fluxes in illuminated leaves, offering a nondestructive way to investigate the impact of photorespiration on plant carbon balance. Modern commercial gas exchange systems allow high temporal resolution measurements under changing environments, aiding the development of nonsteady-state approaches for measuring dynamic photosynthetic responses. Here, we describe a nonsteady-state technique for acquiring the dynamic response of net CO2 assimilation to changes in photorespiratory fluxes manipulated by O2 mole fractions. This technique allows for the screening of plant genotypes with variations in their efficiencies of photorespiration under nonsteady-state conditions.

Analysis of Photorespiratory Intermediates Under Transient Conditions by Mass Spectrometry.

Photorespiration is an essential process of phototropic organisms caused by the limited ability of rubisco to distinguish between CO2 and O2. To understand the metabolic flux through the photorespiratory pathway, we combined a mass spectrometry-based approach with a shift experiment from elevated CO2 (3000 ppm) to ambient CO2 (390 ppm). Here, we describe a protocol for quantifying photorespiratory intermediates, starting from plant cultivation through extraction and evaluation.

Combining Gas Exchange and Rapid Quenching of Leaf Tissue for Mass Spectrometry Analysis Directly in Gas Exchange Cuvette.

Isotopically nonstationary metabolic flux analysis (INST-MFA) is a powerful technique for studying plant central metabolism, which involves introducing a 13CO2 tracer to plant leaves and sampling the labeled metabolic intermediates during the transient period before reaching an isotopic steady state. The metabolic intermediates involved in the C3 cycle have exceptionally fast turnover rates, with some intermediates turning over many times a second. As a result, it is necessary to rapidly introduce the label and then rapidly quench the plant tissue to determine concentrations in the light or capture the labeling kinetics of these intermediates at early labeling time points. Here, we describe a rapid quenching (0.1-0.5 s) system for 13CO2 labeling experiments in plant leaves to minimize metabolic changes during labeling and quenching experiments. This system is integrated into a commercially available gas exchange analyzer to measure initial rates of gas exchange, precisely control ambient conditions, and monitor the conversion from 12CO2 to 13CO2.

Using Gas Exchange to Study CO2 Release During Photosynthesis with Steady- and Nonsteady-State Approaches.

Measures of respiration in the light and Ci* are crucial to the modeling of photorespiration and photosynthesis. This chapter provides background on the equations used to model C3 photosynthesis and the history of the incorporation of the effects of rubisco oxygenation into these models. It then describes three methods used to determine two key parameters necessary to incorporate photorespiratory effects into C3 photosynthesis models: respiration in the light (RL) and Ci*. These methods include the Laisk, Yin, and isotopic methods. For the Laisk method, we also introduce a new rapid measurement technique.

Reaction-Kinetic Modeling of Photorespiration Using Modelbase.

Plant science has become more and more complex. With the introduction of new experimental techniques and technologies, it is now possible to explore the fine details of plant metabolism. Besides steady-state measurements often applied in gas-exchange or metabolomic analyses, new approaches, e.g., based on 13C labeling, are now available to understand the changes in metabolic concentrations under fluctuating environmental conditions in the field or laboratory. To explore those transient phenomena of metabolite concentrations, kinetic models are a valuable tool. In this chapter, we describe ways to implement and build kinetic models of plant metabolism with the Python software package modelbase. As an example, we use a part of the photorespiratory pathway. Moreover, we show additional functionalities of modelbase that help to explore kinetic models and thus can reveal information about a biological system that is not easily accessible to experiments. In addition, we will point to extra information on the mathematical background of kinetic models to give an impetus for further self-study.