The Response to High CO2 Levels Requires the Neuropeptide Secretion Component HID-1 to Promote Pumping Inhibition

Carbon dioxide (CO₂) is a key molecule in many biological processes; however, mechanisms by which organisms sense and respond to high CO₂ levels remain largely unknown. Here we report that acute CO₂ exposure leads to a rapid cessation in the contraction of the pharynx muscles in Caenorhabditis elegans. To uncover the molecular mechanisms underlying this response, we performed a forward genetic screen and found that hid-1, a key component in neuropeptide signaling, regulates this inhibition in muscle contraction. Surprisingly, we found that this hid-1-mediated pathway is independent of any previously known pathways controlling CO₂ avoidance and oxygen sensing. In addition, animals with mutations in unc-31 and egl-21 (neuropeptide secretion and maturation components) show impaired inhibition of muscle contraction following acute exposure to high CO₂ levels, in further support of our findings. Interestingly, the observed response in the pharynx muscle requires the BAG neurons, which also mediate CO₂ avoidance. This novel hid-1-mediated pathway sheds new light on the physiological effects of high CO₂ levels on animals at the organism-wide level.     

Sequencing and Modification of the Gene Encoding the 42-Kilodalton Protein in the Cytoplasmic Membrane of Synechococcus PCC 7942

A 42-kilodalton cytoplasmic membrane protein is synthesized when high CO₂-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) are exposed to low CO₂. The structural gene for this protein (cmpA) has been cloned and sequenced and shown to encode a 450 amino acid polypeptide with a molecular mass of 49 kilodalton. A deletion mutant lacking the 42-kilodalton protein was obtained by transformation of Synechococcus PCC 7942 following in vitro mutagenesis of the cloned gene. There were no significant differences between the mutant and wild-type cells in their growth rates under either low or high CO₂ conditions. The activity of inorganic carbon (C_i) transport in the mutant was as high as that in the wild-type strain. In both types of cells, CO₂ was the main species of C_i transported and the activities of CO₂ and HCO₃⁻ transport increased when high CO₂-grown cells were exposed to low CO₂. We conclude that the 42-kilodalton protein is not directly involved in the C_i-accumulating mechanism of Synechococcus PCC 7942.     

Interactions of Carbon Dioxide and Food Odours in Drosophila: Olfactory Hedonics and Sensory Neuron Properties

Behavioural responses of animals to volatiles in their environment are generally dependent on context. Most natural odours are mixtures of components that can each induce different behaviours when presented on their own. We have investigated how a complex of two olfactory stimuli is evaluated by Drosophila flies in a free-flying two-trap choice assay and how these stimuli are encoded in olfactory receptor neurons. We first observed that volatiles from apple cider vinegar attracted flies while carbon dioxide (CO₂) was avoided, confirming their inherent positive and negative values. In contradiction with previous results obtained from walking flies in a four-field olfactometer, in the present assay the addition of CO₂ to vinegar increased rather than decreased the attractiveness of vinegar. This effect was female-specific even though males and females responded similarly to CO₂ and vinegar on their own. To test whether the female-specific behavioural response to the mixture correlated with a sexual dimorphism at the peripheral level we recorded from olfactory receptor neurons stimulated with vinegar, CO₂ and their combination. Responses to vinegar were obtained from three neuron classes, two of them housed with the CO₂-responsive neuron in ab1 sensilla. Sensitivity of these neurons to both CO₂ and vinegar per se did not differ between males and females and responses from female neurons did not change when CO₂ and vinegar were presented simultaneously. We also found that CO₂-sensitive neurons are particularly well adapted to respond rapidly to small concentration changes irrespective of background CO₂ levels. The ability to encode temporal properties of stimulations differs considerably between CO₂- and vinegar-sensitive neurons. These properties may have important implications for in-flight navigation when rapid responses to fragmented odour plumes are crucial to locate odour sources. However, the flies’ sex-specific response to the CO₂-vinegar combination and the context-dependent hedonics most likely originate from central rather than peripheral processing.     

A Microbial Link between Elevated CO2 and Methane Emissions that is Plant Species-Specific

Rising atmospheric CO₂ levels alter the physiology of many plant species, but little is known of changes to root dynamics that may impact soil microbial mediation of greenhouse gas emissions from wetlands. We grew co-occurring wetland plant species that included an invasive reed canary grass (Phalaris arundinacea L.) and a native woolgrass (Scirpus cyperinus L.) in a controlled greenhouse facility under ambient (380 ppm) and elevated atmospheric CO₂ (700 ppm). We hypothesized that elevated atmospheric CO₂ would increase the abundance of both archaeal methanogen and bacterial methanotroph populations through stimulation of plant root and shoot biomass. We found that methane levels emitted from S. cyperinus shoots increased 1.5-fold under elevated CO₂, while no changes in methane levels were detected from P. arundincea. The increase in methane emissions was not explained by enhanced root or shoot growth of S. cyperinus. Principal components analysis of the total phospholipid fatty acid (PLFA) recovered from microbial cell membranes revealed that elevated CO₂ levels shifted the composition of the microbial community under S. cyperinus, while no changes were detected under P. arundinacea. More detailed analysis of microbial abundance showed no impact of elevated CO₂ on a fatty acid indicative of methanotrophic bacteria (18:2ω6c), and no changes were detected in the terminal restriction fragment length polymorphism (T-RFLP) relative abundance profiles of acetate-utilizing archaeal methanogens. Plant carbon depleted in ¹³C was traced into the PLFAs of soil microorganisms as a measure of the plant contribution to microbial PLFA. The relative contribution of plant-derived carbon to PLFA carbon was larger in S. cyperinus compared with P. arundinacea in four PLFAs (i14:0, i15:0, a15:0, and 18:1ω9t). The δ¹³C isotopic values indicate that the contribution of plant-derived carbon to microbial lipids could differ in rhizospheres of CO₂-responsive plant species, such as S. cyperinus in this study. The results from this study show that the CO₂–methane link found in S. cyperinus can occur without a corresponding change in methanogen and methanotroph relative abundances, but PLFA analysis indicated shifts in the community profile of bacteria and fungi that were unique to rhizospheres under elevated CO₂.     

Mutants in Drosophila TRPC Channels Reduce Olfactory Sensitivity to Carbon Dioxide

Background

Members of the canonical Transient Receptor Potential (TRPC) class of cationic channels function downstream of Gαq and PLCβ in Drosophila photoreceptors for transducing visual stimuli. Gαq has recently been implicated in olfactory sensing of carbon dioxide (CO₂) and other odorants. Here we investigated the role of PLCβ and TRPC channels for sensing CO₂ in Drosophila.

Methodology/Principal Findings

Through behavioral assays it was demonstrated that Drosophila mutants for plc21c, trp and trpl have a reduced sensitivity for CO₂. Immuno-histochemical staining for TRP, TRPL and TRPγ indicates that all three channels are expressed in Drosophila antennae including the sensory neurons that express CO₂ receptors. Electrophysiological recordings obtained from the antennae of protein null alleles of TRP (trp³⁴³) and TRPL (trpl³⁰²), showed that the sensory response to multiple concentrations of CO₂ was reduced. However, trpl³⁰²; trp³⁴³ double mutants still have a residual response to CO₂. Down-regulation of TRPC channels specifically in CO₂ sensing olfactory neurons reduced the response to CO₂ and this reduction was obtained even upon down-regulation of the TRPCs in adult olfactory sensory neurons. Thus the reduced response to CO₂ obtained from the antennae of TRPC RNAi strains is not due to a developmental defect.

Conclusion

These observations show that reduction in TRPC channel function significantly reduces the sensitivity of the olfactory response to CO₂ concentrations of 5% or less in adult Drosophila. It is possible that the CO₂ receptors Gr63a and Gr21a activate the TRPC channels through Gαq and PLC21C.     

Effects of elevated CO2 on the vasculature and phenolic secondary metabolism of Plantago maritima

We have examined the effect of elevated CO₂ on the vasculature and phenolic secondary metabolism on clones of the maritime plant Plantago maritima (L.). Plants were exposed to either ambient (360 μmol CO₂ mol⁻¹) or elevated (600 μmol CO₂ mol⁻¹) atmospheric CO₂ within a Solardome facility and harvested after 12 months' growth. Histochemical analysis of the leaves identified increases in the diameter of the minor leaf vein and associated lignified vessels in plants exposed to elevated CO₂. In the roots the number of lignified root vessels and stele width were also increased, but overall the lignified vessel-wall thickness was reduced in plants exposed to elevated CO₂, compared to those grown under ambient CO₂. To investigate whether or not these subtle changes in lignification were associated with perturbations in phenolic metabolism, aromatic natural products were analysed by HPLC-MS after treatment with cellulase to hydrolyse the respective glycosidic conjugates. The phenylpropanoids p-coumaric acid, caffeic acid, ferulic acid and the flavone luteolin were identified, together with the caffeoyl phenylethanoid glycosides, verbascoside and plantamajoside which were resistant to enzymatic digestion. Exposure to enhanced CO₂ resulted in subtle changes in the levels of individual metabolites. In the foliage a one-year exposure to enhanced CO₂ resulted in an increased accumulation of caffeic acid, whilst in the roots p-coumaric acid and verbascoside were enhanced. Our results suggest that significant changes in the vasculature of P. maritima on exposure to increased CO₂ are associated with only minor changes in the leaves of specific lignin-related metabolites.     

Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

An in vitro system of autotropic synthesis of activated acetic acid from¹⁴CO₂ inMethanobacterium thermoautotrophicum was developed.

1. A recognized¹⁴CO₂-fixation product in vitro was activated [¹⁴C] acetic acid. It could be trapped enzymatically into citrate and released again as [¹⁴C] acetate by citrate synthase and citrate lyase, respectively.
2. The synthesis of both activated acetic acid and methane from CO₂ proceeded in parallel under a variety of conditions. Both of these processes were stimulated greatly and to the same extent by the addition of methyl coenzyme M to the assay.
3. Various inhibitors of methanogenesis tested also inhibited acetate synthesis, e.g. CH₂Cl₂, CHCl₃, CCl₄, N₂O, and bromoethane sulfonic acid. Cyanide specifically inhibited the synthesis of activated acetic acid, whereas methane formation was unaffected. Cyanide inhibition was relieved by adding CO, whereas the inhibition by the other compounds was not.

The data suggest: The product studied in vitro was acetyl CoA. Its synthesis involves intermediates of CO₂ reduction to methane. In addition, a cyanide-sensitive reaction is required which does not participate in CO₂ reduction to methane.     

Aspartyl Protease-Mediated Cleavage of BAG6 Is Necessary for Autophagy and Fungal Resistance in Plants

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved group of cochaperones that modulate numerous cellular processes. Previously we found that Arabidopsis thaliana BAG6 is required for basal immunity against the fungal phytopathogen Botrytis cinerea. However, the mechanisms by which BAG6 controls immunity are obscure. Here, we address this important question by determining the molecular mechanisms responsible for BAG6-mediated basal resistance. We show that Arabidopsis BAG6 is cleaved in vivo in a caspase-1-like-dependent manner and via a combination of pull-downs, mass spectrometry, yeast two-hybrid assays, and chemical genomics, we demonstrate that BAG6 interacts with a C2 GRAM domain protein (BAGP1) and an aspartyl protease (APCB1), both of which are required for BAG6 processing. Furthermore, fluorescence and transmission electron microscopy established that BAG6 cleavage triggers autophagy in the host that coincides with disease resistance. Targeted inactivation of BAGP1 or APCB1 results in the blocking of BAG6 processing and loss of resistance. Mutation of the cleavage site blocks cleavage and inhibits autophagy in plants; disease resistance is also compromised. Taken together, these results identify a mechanism that couples an aspartyl protease with a molecular cochaperone to trigger autophagy and plant defense, providing a key link between fungal recognition and the induction of cell death and resistance.     

Long-term acclimation to elevated pCO2 alters carbon metabolism and reduces growth in the Antarctic diatom Nitzschia lecointei

Increasing atmospheric CO₂ levels are driving changes in the seawater carbonate system, resulting in higher pCO₂ and reduced pH (ocean acidification). Many studies on marine organisms have focused on short-term physiological responses to increased pCO₂, and few on slow-growing polar organisms with a relative low adaptation potential. In order to recognize the consequences of climate change in biological systems, acclimation and adaptation to new environments are crucial to address. In this study, physiological responses to long-term acclimation (194 days, approx. 60 asexual generations) of three pCO₂ levels (280, 390 and 960 µatm) were investigated in the psychrophilic sea ice diatom Nitzschia lecointei. After 147 days, a small reduction in growth was detected at 960 µatm pCO₂. Previous short-term experiments have failed to detect altered growth in N. lecointei at high pCO₂, which illustrates the importance of experimental duration in studies of climate change. In addition, carbon metabolism was significantly affected by the long-term treatments, resulting in higher cellular release of dissolved organic carbon (DOC). In turn, the release of labile organic carbon stimulated bacterial productivity in this system. We conclude that long-term acclimation to ocean acidification is important for N. lecointei and that carbon overconsumption and DOC exudation may increase in a high-CO₂ world.     

The Influence of 0.03% Carbon Dioxide on Protein Metabolism of Etiolated Avena sativa Coleoptiles

The influence of indoleacetic acid, 0.03% CO₂, and malate on protein metabolism of etiolated Avena sativa coleoptile sections has been investigated. All three were found to elevate both the rate of incorporation of labeled leucine into protein, and the level of soluble protein. The combination of indoleacetic acid and CO₂ stimulated these values in an additive or weakly synergistic manner, in contrast to the nonadditive influence of malate and CO₂. Evidence is presented that cyclo-heximide inhibited the stimulation of protein synthesis by CO₂, and that indoleacetic acid increased the incorporation of ¹⁴C-bicarbonate into protein. These data are discussed in the context of CO₂-stimulated growth of etiolated tissue, and proposals that CO₂-stimulated growth involves dark CO₂ fixation.     

CO2- and Anaerobiosis-Induced Changes in Physiology and Gene Expression of Different Listeria monocytogenes Strains

Although carbon dioxide (CO₂) is known to inhibit growth of most bacteria, very little is known about the cellular response. The food-borne pathogen Listeria monocytogenes is characterized by its ability to grow in high CO₂ concentrations at refrigeration temperatures. We examined the listerial responses of different strains to growth in air, 100% N₂, and 100% CO₂. The CO₂-induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent. The acid-tolerant L. monocytogenes LO28 responded in the same manner to CO₂ as to other anaerobic, slightly acidic environments (100% N₂, pH 5.7). An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres. In contrast, the acid-sensitive L. monocytogenes strain EGD responded differently to CO₂ and N₂ at the same pH. In a separate experiment with L. monocytogenes 412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO₂-containing atmospheres. Together, our findings demonstrate that the CO₂-response is a partly strain-dependent complex mechanism. The possible links between the CO₂-dependent changes in isocitrate dehydrogenase activity, glutamate metabolism and branched fatty acid biosynthesis are discussed.     

Functional validation of the carbon dioxide receptor in labial palps of Helicoverpa armigera moths

Adult moths possess an organ in their labial palps, the labial-palp pit organ, which is specialized for sensing carbon dioxide (CO₂). They use CO₂ as a cue to detect healthy plants and find food or lay eggs on them. The molecular bases of the CO₂ receptor in Drosophila melanogaster and Aedes aegypti have been reported, but the molecular mechanisms of the CO₂ receptor in Lepidoptera remains elusive. In this study, we first re-examined three putative Helicoverpa armigera CO₂ gustatory receptor genes (HarmGr1, HarmGr2, and HarmGr3), and then analyzed expression patterns of them. RT-PCR results verified they were predominantly expressed in the labial palps of H. armigera. Thus, we used in situ hybridization to localize the expression of three genes in the labial palps. We found that all three genes were co-expressed in the same cells of the labial palps. Next, we employed the Xenopus laevis oocyte expression system and the two-electrode voltage-clamp recording to study the function of the three genes. Results showed that only oocytes co-expressing HarmGr1 and HarmGr3 or co-expressing HarmGr1, HarmGr2 and HarmGr3 gave robust responses to NaHCO₃. Finally, we confirmed that the sensory cells in labial palps of both females and males show dose dependent responses to CO₂ stimuli by using single sensillum recording. Our work uncovers that HarmGr1 and HarmGr3 are indispensable and sufficient for CO₂ sensing in labial palps of H. armigera.     

Carbon Dioxide and Fruit Odor Transduction in Drosophila Olfactory Neurons. What Controls their Dynamic Properties?

We measured frequency response functions between odorants and action potentials in two types of neurons in Drosophila antennal basiconic sensilla. CO₂ was used to stimulate ab1C neurons, and the fruit odor ethyl butyrate was used to stimulate ab3A neurons. We also measured frequency response functions for light-induced action potential responses from transgenic flies expressing H134R-channelrhodopsin-2 (ChR2) in the ab1C and ab3A neurons. Frequency response functions for all stimulation methods were well-fitted by a band-pass filter function with two time constants that determined the lower and upper frequency limits of the response. Low frequency time constants were the same in each type of neuron, independent of stimulus method, but varied between neuron types. High frequency time constants were significantly slower with ethyl butyrate stimulation than light or CO₂ stimulation. In spite of these quantitative differences, there were strong similarities in the form and frequency ranges of all responses. Since light-activated ChR2 depolarizes neurons directly, rather than through a chemoreceptor mechanism, these data suggest that low frequency dynamic properties of Drosophila olfactory sensilla are dominated by neuron-specific ionic processes during action potential production. In contrast, high frequency dynamics are limited by processes associated with earlier steps in odor transduction, and CO₂ is detected more rapidly than fruit odor.     

Dissimilation of Carbon Monoxide to Acetic Acid by Glucose-Limited Cultures of Clostridium thermoaceticum

Clostridium thermoaceticum was cultivated in glucose-limited media, and the dissimilation of CO to acetic acid was evaluated. We found that cultures catalyzed the rapid dissimilation of CO to acetic acid and CO₂, with the stoichiometry obtained for conversion approximating that predicted from the following reaction: 4CO + 2H₂O → CH₃CO₂H + 2CO₂. Growing cultures formed approximately 50 mmol (3 g) of CO-derived acetic acid per liter of culture, with the rate of maximal consumption approximating 9.1 mmol of CO consumed/h per liter of culture. In contrast, resting cells were found not to dissimilate CO to acetic acid. ¹⁴CO was incorporated, with equal distribution between the carboxyl and methyl carbons of acetic acid when the initial cultivation gas phase was 100% CO, whereas ¹⁴CO₂ preferentially entered the carboxyl carbon when the initial gas phase was 100% CO₂. Significantly, in the presence of saturating levels of CO, ¹⁴CO₂ preferentially entered the methyl carbon, whereas saturating levels of CO₂ yielded ¹⁴CO-derived labeling predominantly in the carboxyl carbon. These findings are discussed in relation to the path of carbon flow to acetic acid.     

Ectomycorrhizal exudates and pre-exposure to elevated CO2 affects soil bacterial growth and community structure

Ectomycorrhizal fungi produce low molecular weight organic compounds, supporting diverse microbial communities. To link mycorrhizal root exudation directly to bacterial responses, we used Scots pine exudates with (Suillus variegatus and Piloderma fallax) and without mycorrhiza as substrata for forest soil bacteria. Bacterial growth and vitality was monitored, and community composition determined using T-RFLP, cloning and sequencing. We investigated if the amount of organic acids in exudates explained bacterial growth, and whether bacterial communities were influenced by pre-exposure to elevated atmospheric CO₂. We demonstrated functional differences in bacterial growth rates related to CO₂. There was a shift in the bacterial community (e.g. Burkholderia sp. and gamma-proteobacteria) toward organisms better able to rapidly utilize exudates when pine microcosms were pre-exposed to elevated CO₂. Soil bacteria from all treatments tended to grow more abundantly and rapidly in exudates from Piloderma-colonized seedlings, suggesting that the organic acids and/or unidentified compounds present supported greater growth.     

Stable Photosymbiotic Relationship under CO2-Induced Acidification in the Acoel Worm Symsagittifera Roscoffensis

As a consequence of anthropogenic CO₂ emissions, oceans are becoming more acidic, a phenomenon known as ocean acidification. Many marine species predicted to be sensitive to this stressor are photosymbiotic, including corals and foraminifera. However, the direct impact of ocean acidification on the relationship between the photosynthetic and nonphotosynthetic organism remains unclear and is complicated by other physiological processes known to be sensitive to ocean acidification (e.g. calcification and feeding). We have studied the impact of extreme pH decrease/pCO₂ increase on the complete life cycle of the photosymbiotic, non-calcifying and pure autotrophic acoel worm, Symsagittifera roscoffensis. Our results show that this species is resistant to high pCO₂ with no negative or even positive effects on fitness (survival, growth, fertility) and/or photosymbiotic relationship till pCO₂ up to 54 K µatm. Some sub-lethal bleaching is only observed at pCO₂ up to 270 K µatm when seawater is saturated by CO₂. This indicates that photosymbiosis can be resistant to high pCO₂. If such a finding would be confirmed in other photosymbiotic species, we could then hypothesize that negative impact of high pCO₂ observed on other photosymbiotic species such as corals and foraminifera could occur through indirect impacts at other levels (calcification, feeding).     

Kinetics and Apparent K(m) of Oxygen Cycle under Conditions of Limiting Carbon Dioxide Fixation

A mass spectrometer with a membrane inlet was used to monitor light-driven O₂ evolution, O₂ uptake, and CO₂ uptake in suspensions of algae (Scenedesmus obliquus). We observed the following. (a) The rate of O₂ uptake, which, in the presence of iodoacetamide, replaces the uptake of CO₂, showed a distinct plateau (V_max) beyond ~30% O₂ and was half-maximal at ~8% O₂. We concluded that this light-driven O₂ uptake process, which does not involve carbon compounds, is saturated at lower O₂ concentrations than are photorespiration and glycolate formation. (b) In the absence of inhibitor, O₂ evolution was relatively unaffected by the presence or absence of CO₂. During the course of CO₂ depletion, electron flow to CO₂ was replaced by an equivalent flow to O₂. (c) There was a distinct delay between the cessation of CO₂ uptake and the increase in O₂ uptake. We ascribe this delay to the transient utilization of another electron acceptor—possibly bicarbonate or another bound form of CO₂.