Carbon isotope fractionation by ribulose-1,5-bisophosphate carboxylase from various organisms

Carbon isotope fractionation by structurally and catalytically distinct ribulose-1,5-bisphosphate carboxylases from one eucaryotic and four procaryotic organisms has been measured under nitrogen. The average fractionation for 40 experiments was −34.1 ‰ with respect to the δ¹³C of the dissolved CO₂ used, although average fractionations for each enzyme varied slightly: spinach carboxylase, −36.5 ‰; Hydrogenomonas eutropha, −38.7 ‰; Agmenellum quadruplicatum, −32.2 ‰; Rhodospirillum rubrum, −32.1 ‰; Rhodopseudomonas sphaeroides peak I carboxylase, −31.4 ‰; and R. sphaeroides peak II carboxylase, −28.3 ‰. The carbon isotope fractionation value was largely independent of method of enzyme preparation, purity, or reaction temperature, but in the case of spinach ribulose-1,5-bisphosphate carboxylase fractionation, changing the metal cofactor used for enzyme activation had a distinct effect on the fractionation value. The fractionation value of −36.5 ‰ with Mg²⁺ as activator shifted to −29.9 ‰ with Ni²⁺ as activator and to −41.7 ‰ with Mn²⁺ as activator. These dramatic metal effects on carbon isotope fractionation may be useful in examining the catalytic site of the enzyme.     

Effect of Varying CO(2) Partial Pressure on Photosynthesis and on Carbon Isotope Composition of Carbon-4 of Malate from the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana Hamet et Perr

Intact leaves of Kalanchoë daigremontiana were exposed to CO₂ partial pressures of 100, 300, and 1000 microbars. Malic acid was extracted, purified, and degraded in order to obtain isotopic composition of carbon-1 and carbon-4. From these data, it is possible to calculate the carbon isotope composition of newly fixed carbon in malate. In all three treatments, the isotopic composition of newly introduced carbon is the same as that of the CO₂ source and is independent of CO₂ partial pressures over the range tested. Comparison with numerical models described previously (O'Leary 1981 Phytochemistry 20: 553-567) indicates that we would expect carbon 4 of malate to be 4‰ more negative than source CO₂ if diffusion is totally limiting or 7‰ more positive than source CO₂ if carboxylation is totally limiting. Our results demonstrate that stomatal aperture adjusts to changing CO₂ partial pressures and maintains the ratio of diffusion resistance to carboxylation resistance approximately constant. In this study, carboxylation and diffusion resistances balance so that essentially no fractionation occurs during malate synthesis. Gas exchange studies of the same leaves from which malate was extracted show that the extent of malate synthesis over the whole night is nearly independent of CO₂ partial pressure, although there are small variations in CO₂ uptake rate. Both the gas exchange and the isotope studies indicate that the ratio of external to internal CO₂ partial pressure is the same in all three treatments. Inasmuch as a constant ratio will result in constant isotope fractionation, this observation may explain why plants in general have fairly invariable ¹³C contents, despite growing under a variety of environmental conditions.     

Isotope Fractionation in Photosynthetic Bacteria during Carbon Dioxide Assimilation

The δ _PDB¹³C values have been determined for the cellular constituents and metabolic intermediates of autotrophically grown Chromatium vinosum. The isotopic composition of the HCO₃^- in the medium and the carbon isotopic composition of the bacterial cells change with the growth of the culture. The δ _PDB¹³C value of the HCO₃^- in the media changes from an initial value of −6.6‰ to +8.1‰ after 10 days of bacterial growth and the δ _PDB¹³C value of the bacterial cells change from −37.5‰ to −29.2‰ in the same period. The amount of carbon isotope fractionation during the synthesis of hexoses by the photoassimilation of CO₂ has a range of −15.5‰ at time zero to −22.0‰ after 10 days. This range of fractionation compares to the range of carbon isotope fractionation for the synthesis of sugars from CO₂ by ribulose 1,5-diphosphate carboxylase and the Calvin cycle.     

Temperature Dependence of Carbon Isotope Fractionation in CAM Plants

The carbon isotope fractionation associated with nocturnal malic acid synthesis in Kalanchoë daigremontiana and Bryophyllum tubiflorum was calculated from the isotopic composition of carbon-4 of malic acid, after appropriate corrections. In the lowest temperature treatment (17°C nights, 23°C days), the isotope fractionation for both plants is −4‰ (that is, malate is enriched in ¹³C relative to the atmosphere). For K. daigremontiana, the isotope fractionation decreases with increasing temperature, becoming approximately 0‰ at 27°C/33°C. Detailed analysis of temperature effects on the isotope fractionation indicates that stomatal aperture decreases with increasing temperature and carboxylation capacity increases. For B. tubiflorum, the temperature dependence of the isotope fractionation is smaller and is principally attributed to the normal temperature dependences of the rates of diffusion and carboxylation steps. The small change in the isotopic composition of remaining malic acid in both species which is observed during deacidification indicates that malate release, rather than decarboxylation, is rate limiting in the deacidification process.     

Carbon Isotope Fractionation of 11 Acetogenic Strains Grown on H2 and CO2

Acetogenic bacteria are able to grow autotrophically on hydrogen and carbon dioxide by using the acetyl coenzyme A (acetyl-CoA) pathway. Acetate is the end product of this reaction. In contrast to the fermentative route of acetate production, which shows almost no fractionation of carbon isotopes, the acetyl-CoA pathway has been reported to exhibit a preference for light carbon. In Acetobacterium woodii the isotope fractionation factor (ε) for ¹³C and ¹²C has previously been reported to be ε = −58.6‰. To investigate whether such a strong fractionation is a general feature of acetogenic bacteria, we measured the stable carbon isotope fractionation factor of 10 acetogenic strains grown on H₂ and CO₂. The average fractionation factor was ε_TIC = −57.2‰ for utilization of total inorganic carbon and ε_acetate = −54.6‰ for the production of acetate. The strongest fractionation was found for Sporomusa sphaeroides (ε_TIC = −68.3‰), the lowest fractionation for Morella thermoacetica (ε_TIC = −38.2‰). To investigate the reproducibility of our measurements, we determined the fractionation factor of 21 biological replicates of Thermoanaerobacter kivui. In general, our study confirmed the strong fractionation of stable carbon during chemolithotrophic acetate formation in acetogenic bacteria. However, the specific characteristics of the bacterial strain, as well as the cultural conditions, may have a moderate influence on the overall fractionation.     

Stable Carbon Isotope Fractionation by Sulfate-Reducing Bacteria

Biogeochemical transformations occurring in the anoxic zones of stratified sedimentary microbial communities can profoundly influence the isotopic and organic signatures preserved in the fossil record. Accordingly, we have determined carbon isotope discrimination that is associated with both heterotrophic and lithotrophic growth of pure cultures of sulfate-reducing bacteria (SRB). For heterotrophic-growth experiments, substrate consumption was monitored to completion. Sealed vessels containing SRB cultures were harvested at different time intervals, and δ¹³C values were determined for gaseous CO₂, organic substrates, and products such as biomass. For three of the four SRB, carbon isotope effects between the substrates, acetate or lactate and CO₂, and the cell biomass were small, ranging from 0 to 2‰. However, for Desulfotomaculum acetoxidans, the carbon incorporated into biomass was isotopically heavier than the available substrates by 8 to 9‰. SRB grown lithoautotrophically consumed less than 3% of the available CO₂ and exhibited substantial discrimination (calculated as isotope fractionation factors [α]), as follows: for Desulfobacterium autotrophicum, α values ranged from 1.0100 to 1.0123; for Desulfobacter hydrogenophilus, the α value was 0.0138, and for Desulfotomaculum acetoxidans, the α value was 1.0310. Mixotrophic growth of Desulfovibrio desulfuricans on acetate and CO₂ resulted in biomass with a δ¹³C composition intermediate to that of the substrates. The extent of fractionation depended on which enzymatic pathways were used, the direction in which the pathways operated, and the growth rate, but fractionation was not dependent on the growth phase. To the extent that environmental conditions affect the availability of organic substrates (e.g., acetate) and reducing power (e.g., H₂), ecological forces can also influence carbon isotope discrimination by SRB.     

Anapleurotic CO(2) Fixation by Phosphoenolpyruvate Carboxylase in C(3) Plants

The role of phosphoenolpyruvate carboxylase in photosynthesis in the C₃ plant Nicotiana tabacum has been probed by measurement of the ¹³C content of various materials. Whole leaf and purified ribulose bisphosphate carboxylase are within the range expected for C₃ plants. Aspartic acid purified following acid hydrolysis of this ribulose bisphosphate carboxylase is enriched in ¹³C compared to whole protein. Carbons 1-3 of this aspartic acid are in the normal C₃ range, but carbon-4 (obtained by treatment of the aspartic acid with aspartate β-decarboxylase) has an isotopic composition in the range expected for products of C₄ photosynthesis (−5‰), and it appears that more than half of the aspartic acid is synthesized by phosphoenolpyruvate carboxylase using atmospheric CO₂/HCO₃⁻. Thus, a primary role of phosphoenolpyruvate carboxylase in C₃ plants appears to be the anapleurotic synthesis of four-carbon acids.     

Photosynthetic carbon metabolism of a marine grass

The δ¹³C value of a tropical marine grass Thalassia testudinum is −9.04‰. This value is similar to the δ¹³C value of terrestrial tropical grasses. The δ¹³C values of the organic acid fraction, the amino acid fraction, the sugar fraction, malic acid, and glucose are: −11.2‰, −13.1‰, −10.1‰, −11.1‰, and −11.5‰, respectively. The δ¹³C values of malic acid and glucose of Thalassia are similar to the δ¹³C values of these intermediates in sorghum leaves and attest to the presence of the photosynthetic C₄-dicarboxylic acid pathway in this marine grass. The inorganic HCO₃⁻ for the growth of the grass fluctuates between −6.7 to −2.7‰ during the day. If CO₂ fixation in Thalassia is catalyzed by phosphoenolpyruvate carboxylase (which would result in a −3‰ fractionation between HCO₃⁻ and malic acid), the predicted δ¹³C value for Thalassia would be −9.7 to −5.7‰. This range is close to the observed range of −12.6 to −7.8‰ for Thalassia and agree with the operation of the C₄-dicarboxylic acid pathway in this plant. The early products of the fixation of HCO₃⁻ in the leaf sections are malic acid and aspartic acid which are similar to the early products of CO₂ fixation in C₄ terrestrial plants.     

Carbon isotope ratios in crassulacean Acid metabolism plants: seasonal patterns from plants in natural stands

A year round study of photosynthesis and carbon isotope fractionation was conducted with plants of Opuntia phaeacantha Engelm. and Yucca baccata Torr. occurring in natural stands at elevations of 525, 970, 1450 and 1900 m. Plant water potentials and the daytime pattern of ¹⁴CO₂ photosynthesis were similar for all cacti along the elevational gradient, despite significant differences in temperature regime and soil water status. Carbon isotope ratios of total tissue and soluble extract fractions were relatively constant throughtout the entire year. Additionally, the σ¹³C values were similar in all plants of the same species along the elevational gradient, i.e. −12.5 ± 0.86 ‰ for O. phaeacantha and −15.7 ± 0.95 ‰ for Y. baccata. The results of this study indicate Crassulacean acid metabolism predominates as the major carbon pathway of these plants, which do not facultatively utilize the reductive pentose phosphate cycle of photosynthesis as the primary carboxylation reaction.     

Effects of Ontogeny on δ13C of Plant- and Soil-Respired CO2 and on Respiratory Carbon Fractionation in C3 Herbaceous Species

Knowledge gaps regarding potential ontogeny and plant species identity effects on carbon isotope fractionation might lead to misinterpretations of carbon isotope composition (δ¹³C) of respired CO₂, a widely-used integrator of environmental conditions. In monospecific mesocosms grown under controlled conditions, the δ¹³C of C pools and fluxes and leaf ecophysiological parameters of seven herbaceous species belonging to three functional groups (crops, forage grasses and legumes) were investigated at three ontogenetic stages of their vegetative cycle (young foliage, maximum growth rate, early senescence). Ontogeny-related changes in δ¹³C of leaf- and soil-respired CO₂ and ¹³C/¹²C fractionation in respiration (Δ_R) were species-dependent and up to 7‰, a magnitude similar to that commonly measured in response to environmental factors. At plant and soil levels, changes in δ¹³C of respired CO₂ and Δ_R with ontogeny were related to changes in plant physiological status, likely through ontogeny-driven changes in the C sink to source strength ratio in the aboveground plant compartment. Our data further showed that lower Δ_R values (i.e. respired CO₂ relatively less depleted in ¹³C) were observed with decreasing net assimilation. Our findings highlight the importance of accounting for ontogenetic stage and plant community composition in ecological studies using stable carbon isotopes.     

Carbon isotope ratios demonstrate carbon flux from c(4) host to c(3) parasite

Carbon isotope ratios of mature leaves from the C₃ angiosperm root hemiparasites Striga hermonthica (Del.) Benth (−26.7‰) and S. asiatica (L.) Kuntze (−25.6‰) were more negative than their C₄ host, sorghum (Sorghum bicolor [L.] Moench cv CSH1), (−13.5‰). However, in young photosynthetically incompetent plants of S. hermonthica this difference was reduced to less than 1‰. Differences between the carbon isotope ratios of two C₃-C₃ associations, S. gesnerioides (Willd.) Vatke—Vigna unguiculata (L.) Walp. and Oryza sativa L.—Rhamphicarpa fistulosa (Hochst.) Benth differed by less than 1‰. Theoretical carbon isotope ratios for mature leaves of S. hermonthica and S. asiatica, calculated from foliar gas exchange measurements, were −31.8 and −32.0‰, respectively. This difference between the measured and theoretical δ¹³C-values of 5 to 6‰ suggests that even in mature, photosynthetically active plants, there is substantial input of carbon from the C₄ host. We estimate this to be approximately 28% of the total carbon in S. hermonthica and 35% in S. asiatica. This level of carbon transfer contributes to the host's growth reductions observed in Striga-infected sorghum.     

Carbon Isotope Composition of Nighttime Leaf-Respired CO2 in the Agricultural-Pastoral Zone of the Songnen Plain,Northeast China

Variations in the carbon isotope signature of leaf dark-respired CO₂ (δ¹³C_R) within a single night is a widely observed phenomenon. However, it is unclear whether there are plant functional type differences with regard to the amplitude of the nighttime variation in δ¹³C_R. These differences, if present, would be important for interpreting the short-term variations in the stable carbon signature of ecosystem respiration and the partitioning of carbon fluxes. To assess the plant functional type differences relating to the magnitude of the nighttime variation in δ¹³C_R and the respiratory apparent fractionation, we measured the δ¹³C_R, the leaf gas exchange, and the δ¹³C of the respiratory substrates of 22 species present in the agricultural-pastoral zone of the Songnen Plain, northeast China. The species studied were grouped into C₃ and C₄ plants, trees, grasses, and herbs. A significant nocturnal shift in δ¹³C_R was detected in 20 of the studied species, with the magnitude of the shift ranging from 1‰ to 5.8‰. The magnitude of the nighttime variation in δ¹³C_R was strongly correlated with the daytime cumulative carbon assimilation, which suggests that variation in δ¹³C_R were influenced, to some extent, by changes in the contribution of malate decarboxylation to total respiratory CO₂ flux. There were no differences in the magnitude of the nighttime variation in δ¹³C_R between the C₃ and C₄ plants, as well as among the woody plants, herbs and graminoids. Leaf respired CO₂ was enriched in ¹³C compared to biomass, soluble carbohydrates and lipids; however the magnitude of enrichment differed between 8 pm and 4 am, which were mainly caused by the changes in δ¹³C_R. We also detected the plant functional type differences in respiratory apparent fractionation relative to biomass at 4 am, which suggests that caution should be exercised when using the δ¹³C of bulk leaf material as a proxy for the δ¹³C of leaf-respired CO₂.     

Consumption of Methane and CO2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH₄ and CO₂ assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO₂ reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average δ¹³C carbon isotopic signature of −67.1‰, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (−66.4‰ ± 3.9 ‰ [mean ± standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (−72.9‰ ± 2.2 ‰; n = 7). Incorporation of ¹⁴C from radiolabeled CH₄ or CO₂ revealed one hot spot for methanotrophy and CO₂ fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with ¹⁴CH₄ or ¹⁴CO₂ revealed that there was interconversion of CH₄ and CO_2. The level of CO₂ reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.     

Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves

The Pereskia are morphologically primitive, leafed members of the Cactaceae. Gas exchange characteristics using a dual isotope porometer to monitor ¹⁴CO₂ and tritiated water uptake, diurnal malic acid fluctuations, phosphoenolpyruvate carboxylase, and malate dehydrogenase activities were examined in two species of the genus Pereskia, Pereskia grandifolia and Pereskia aculeata. Investigations were done on well watered (control) and water-stressed plants. Nonstressed plants showed a CO₂ uptake pattern indicating C₃ carbon metabolism. However, diurnal fluctuations in titratable acidity were observed similar to Crassulacean acid metabolism. Plants exposed to 10 days of water stress exhibited stomatal opening only during an early morning period. Titratable acidity, phosphoenolpyruvate carboxylase activity, and malate dehydrogenase activity fluctuations were magnified in the stressed plants, but showed the same diurnal pattern as controls. Water stress causes these cacti to shift to an internal CO₂ recycling (“idling”) that has all attributes of Crassulacean acid metabolism except nocturnal stomata opening and CO₂ uptake. The consequences of this shift, which has been observed in other succulents, are unknown, and some possibilities are suggested.     

C/C ratio changes in crassulacean Acid metabolism plants

¹³C/¹²C ratios have been found in totally combusted leaves of Crassulacean acid metabolism plants to range from −14 to −33 δ ¹³C‰ compared with a limestone standard. Crassulacean acid metabolism plants apparently utilize both ribulose-1, 5-diphosphate carboxylase and phosphoenolpyruvate carboxylase to assimilate atmospheric CO₂ and, depending on environmental conditions, have ¹³C/¹²C ratios indicative of either carboxylase or to any intermediate value. The degree of discrimination against ¹³C and the resultant ¹³C/¹²C ratio from the photosynthetically fixed CO₂ is influenced by environmental conditions and is not a specific and fixed characteristic of a Crassulacean acid metabolism plant. Certain Crassulacean acid metabolism plants may shift their ratios as much as 17 δ ¹³C‰ in specific environments.     

Effect of Substrate Concentration on Carbon Isotope Fractionation during Acetoclastic Methanogenesis by Methanosarcina barkeri and M. acetivorans and in Rice Field Soil

Methanosarcina is the only acetate-consuming genus of methanogenic archaea other than Methanosaeta and thus is important in methanogenic environments for the formation of the greenhouse gases methane and carbon dioxide. However, little is known about isotopic discrimination during acetoclastic CH₄ production. Therefore, we studied two species of the Methanosarcinaceae family, Methanosarcina barkeri and Methanosarcina acetivorans, and a methanogenic rice field soil amended with acetate. The values of the isotope enrichment factor (ɛ) associated with consumption of total acetate (ɛ_ac), consumption of acetate-methyl (ɛ_ac-methyl) and production of CH₄ (ɛ_CH4) were an ɛ_ac of −30.5‰, an ɛ_ac-methyl of −25.6‰, and an ɛ_CH4 of −27.4‰ for M. barkeri and an ɛ_ac of −35.3‰, an ɛ_ac-methyl of −24.8‰, and an ɛ_CH4 of −23.8‰ for M. acetivorans. Terminal restriction fragment length polymorphism of archaeal 16S rRNA genes indicated that acetoclastic methanogenic populations in rice field soil were dominated by Methanosarcina spp. Isotope fractionation determined during acetoclastic methanogenesis in rice field soil resulted in an ɛ_ac of −18.7‰, an ɛ_ac-methyl of −16.9‰, and an ɛ_CH4 of −20.8‰. However, in rice field soil as well as in the pure cultures, values of ɛ_ac and ɛ_ac-methyl decreased as acetate concentrations decreased, eventually approaching zero. Thus, isotope fractionation of acetate carbon was apparently affected by substrate concentration. The ɛ values determined in pure cultures were consistent with those in rice field soil if the concentration of acetate was taken into account.Methane (CH₄) is the most abundant reduced gas in the earth''s atmosphere and is an important greenhouse gas with a high global-warming potential (7). It is presently a matter of discussion whether the contribution of CH₄ to the greenhouse effect will increase in the future (3, 23). This has made it necessary and more urgent to understand natural processes that lead to the production of CH₄.Methanogenesis, the microbial formation of CH₄, is the final step in the degradation of organic matter in anoxic environments like natural wetlands, lake sediments, and flooded rice fields. The most important precursors for the production of CH₄ are acetate (equation 1) and CO₂ (equation 2) with the following reactions (8): (1) (2)Acetate is the most important substrate since it contributes more than 67% to microbial methanogenesis during anoxic degradation of polysaccharides. In methanogenic environments only two genera of archaea, Methanosaeta and Methanosarcina, are capable of using acetate (2). While Methanosaeta can be considered a specialist that uses only acetate, Methanosarcina can use a wide range of substrates besides acetate, for example, H₂/CO₂, methanol, methylamines, and methylated sulfides. Among methanogens, Methanosarcinaceae also display the largest environmental distribution. They can be found in freshwater sediments and soil, marine habitats, landfills, and animal gastrointestinal tracts (46).Additionally, differences between Methanosarcina and Methanosaeta were found for isotope fractionation of stable carbon. The fractionation factor (α) or, equivalently, the enrichment factor (ɛ) during acetoclastic methanogenesis in Methanosarcina barkeri strains typically ranges from an α of 1.021 to 1.027 or an ɛ of −27‰ to −21‰ (14, 27, 48), whereas isotope fractionation in Methanosaeta spp. is weaker, i.e., an α of 1.007 (ɛ = −7‰) for Methanosaeta thermophila (43) and an α of 1.010 (ɛ = −10‰) for Methanosaeta concilii (34). It is suggested that the two archaeal genera differ in isotope fractionation due to differences in their biochemical activation of acetate to acetyl-coenzyme A (acetyl-CoA) (34). However, isotopic data for acetoclastic methanogens are rare. For instance, all data for Methanosarcina refer to only one species, namely M. barkeri.Hence, in this study we investigated whether differences in carbon isotope fractionation within the genus Methanosarcina occur. Therefore, we determined isotope ratios of stable carbon in cultures of the acetoclastic species M. barkeri and Methanosarcina acetivorans. Second, we were interested if these data, obtained from pure cultures, could also be applied to understand natural environments. For that reason, we determined isotope fractionation during acetoclastic methanogenesis in the model system rice field soil. Furthermore, we discuss the effect of substrate concentration on carbon isotope fractionation and the importance of monitoring isotope fractionation during the course of acetate consumption.     

Bacteria and Fungi Respond Differently to Multifactorial Climate Change in a Temperate Heathland,Traced with 13C-Glycine and FACE CO2

It is vital to understand responses of soil microorganisms to predicted climate changes, as these directly control soil carbon (C) dynamics. The rate of turnover of soil organic carbon is mediated by soil microorganisms whose activity may be affected by climate change. After one year of multifactorial climate change treatments, at an undisturbed temperate heathland, soil microbial community dynamics were investigated by injection of a very small concentration (5.12 µg C g⁻¹ soil) of ¹³C-labeled glycine (¹³C₂, 99 atom %) to soils in situ. Plots were treated with elevated temperature (+1°C, T), summer drought (D) and elevated atmospheric carbon dioxide (510 ppm [CO2]), as well as combined treatments (TD, TCO2, DCO2 and TDCO2). The ¹³C enrichment of respired CO₂ and of phospholipid fatty acids (PLFAs) was determined after 24 h. ¹³C-glycine incorporation into the biomarker PLFAs for specific microbial groups (Gram positive bacteria, Gram negative bacteria, actinobacteria and fungi) was quantified using gas chromatography-combustion-stable isotope ratio mass spectrometry (GC-C-IRMS).Gram positive bacteria opportunistically utilized the freshly added glycine substrate, i.e. incorporated ¹³C in all treatments, whereas fungi had minor or no glycine derived ¹³C-enrichment, hence slowly reacting to a new substrate. The effects of elevated CO₂ did suggest increased direct incorporation of glycine in microbial biomass, in particular in G⁺ bacteria, in an ecosystem subjected to elevated CO₂. Warming decreased the concentration of PLFAs in general. The FACE CO₂ was ¹³C-depleted (δ¹³C = 12.2‰) compared to ambient (δ¹³C = ∼−8‰), and this enabled observation of the integrated longer term responses of soil microorganisms to the FACE over one year. All together, the bacterial (and not fungal) utilization of glycine indicates substrate preference and resource partitioning in the microbial community, and therefore suggests a diversified response pattern to future changes in substrate availability and climatic factors.     

Equilibration of Label in Malate during Dark Fixation of CO(2) in Kalanchoë fedtschenkoi

In vitro studies of dark ¹⁴CO₂ fixation with isolated cell aggregates of Kalanchoë fedtschenkoi showed that malate synthesized after 20 sec is predominantly (85 to 92%) labeled at carbon 4, while after 20 min only 65 to 69% of the radioactivity was located in this position. The intramolecular labeling pattern of malate could not be changed by supplementing the cells with carboxylation reaction substrates such as ribulose diphosphate or phosphoenolpyruvate. The kinetic decline of label at carbon 4 of malate occurs independently of CO₂ fixation, since 4-¹⁴C-labeled aspartate fed to the cells gave rise to malate labeled 62% at carbon 4 after 20 min. Furthermore, the cells were capable of converting fed malate to fumarate. It is concluded that synthesis of malate during dark CO₂ fixation is accomplished by a single carboxylation step via phosphoenolpyruvate carboxylase and labeling patterns observed in malate are a consequence of the action of fumarase.     

The mechanism of carbon isotope fractionation in photosynthesis and carbon dioxide component of the greenhouse effect

The relationship between the global climate warming, which is largely induced by increased CO₂ atmospheric concentration, and the changes in carbon isotope fractionation in plants was explained in terms of the previously proposed oscillatory mechanism of photosynthesis, according to which CO₂ assimilation and photorespiration are two reciprocally coupled oscillating mechanisms controlled by ribulose bisphosphate carboxylase/oxygenase switching. This explanation is confirmed by the changes in carbon isotope fractionation in the annual rings of trees and demonstrates that the light carbon isotope ¹²C enrichment before 1990s resulted from increased photosynthetic assimilation of CO₂. The subsequent sharp ¹³C enrichment of the tree ring carbon until the present time suggests that the compensatory role of photosynthesis in boreal forests has been lost for the global climate.     

Use of Carbon Isotopes to Estimate Incorporation of Added CO(2) by Greenhouse-Grown Tomato Plants

A method is presented which uses the ¹³C and ¹⁴C isotope abundance in CO₂-enriched greenhouse crops to determine the percentage of plant organic carbon derived from artificially added CO₂. In a greenhouse experiment with CO₂ concentrations elevated to 1100 ± 100 microliters per liter during part of the daylight hours and maintained at normal atmospheric concentrations (340 microliters per liter) during the rest of the time, it was shown by ¹⁴C analysis that between 41% and 42% of the carbon in tomato plants (Lycopersicon esculentum var 4884) came from the artificially added CO₂. Similar results were obtained from ¹³C analyses when the CO₂ pressure-dependent isotope separation was taken into account.