Insulin as a probe of mitochondrial metabolism in situ

Our previous studies of insulin action have led us to the finding that insulin acts specifically on the mitochondrial Krebs cycle to stimulate, by 30%, the oxidation of carbons 2 and 3 of pyruvate to CO₂. Insulin also stimulates the oxidation of both carbons of acetate. These carbons can be converted to CO₂ only after passing through all of the reactions of the Krebs cycle more than once. Carboxyl groups, such as number 1 of pyruvate, are oxidized to CO₂ without any effect of insulin, and can be converted to CO₂ by extramitochondrial enzyme. We conclude that insulin must act on the complete intramitochondrial cycle and not on the four enzymes of the Krebs cycle which are present in the cytoplasm. The path taken by those carbons affected by insulin is traced through the complete Krebs cycle, and the necessity for this effect to be mitochondrial has been verified by demonstration of the same specific effect of insulin on the oxidation of the 2 and 3 carbons of succinate. The use of this phenomenon is proposed for the study not only of human diabetes, but of all mitochondrial disorders, by using ¹⁴C specifically labeled tracers in culture or biopsy material, or ¹³C labeled tracer material in vivo. (Mol Cell Biochem 174: 91–96, 1997)     

Deterioration and disappearance of mitochondria during reticulocyte maturation

This study correlates the decreasing aerobic metabolism with the increased deterioration of mitochondria in maturing reticulocytes. The respiration of reticulocytes, separated into four age groups according to their density, was determined manometrically. Their Krebs tricarboxylic acid cycle activity was determined using [2-¹⁴C] acetate as a substrate for [¹⁴C]CO₂ formation. The ability of reticulocytes of various age groups to couple the phosphorylation to oxidation processes was estimated by studying the effect of 2,4-dinitrophenol as an uncoupler.     

The Mechanism of Acetate and Pyruvate Oxidation by Trypanosoma cruzi

The epimastigote or culture form of Trypanosoma cruzi oxidizes [3-¹⁴C] pyruvate and [2-¹⁴C] acetate to ¹⁴CO₂ without an apparent increase in overall respiration. This oxidation takes place through the tricarboxylic acid cycle as shown by (a) the incorporation of substrate ¹⁴C into cycle intermediates; (b) the earlier liberation of acetate carboxyl carbon as CO₂; and (c) the characteristic intramolecular distribution of pyruvate and acetate carbon atoms in the skeletal carbon of aspartic and glutamic acids. Upon oxidation of [3-¹⁴C] pyruvate and [2-¹⁴C] acetate, two of the products, alanine and glutamic acid, are found to account for more than 50% of incorporated ¹⁴C; labeling of alanine predominates with [3-¹⁴C] pyruvate while labeling of glutamic acid predominates with [2-¹⁴C] acetate. Using [1- or 6-¹⁴C] glucose as substrate, the pattern of ¹⁴C distribution in soluble metabolites closely resembles that obtained with [3-¹⁴C] pyruvate, in accordance with the joint operation of the Embden-Meyerhof pathway and Krebs cycle. The cycle operation depends on electron transport through the mitochondrial respiratory chain, since antimycin A, at a relatively low concentration, inhibits the oxidation of [2-¹⁴C] acetate to ¹⁴CO₂, to the same extent as the parasite respiration. Though functional in T. cruzi epimastigotes, the oxidative role of the Krebs’ cycle is apparently limited by the absence of an efficient oxidative apparatus. The cycle operation does, however, constitute an important source of skeletal carbon for the biosynthesis of amino acids and can contribute to the process of glycogenesis.     

The role of the Krebs cycle in the generation of intramitochondrial reducing equivalents for the 11 beta-hydroxylation of deoxycorticosterone in isolated rat adrenal cells

Isolated rat adrenal cells were used to study the possible pathways of intramitochondrial NADPH generation for 11β-hydroxylation of 11-deoxycorticosterone. Pyruvate was efficiently utilized by the mitochondria as shown by evolution of ¹⁴CO₂ from [1-¹⁴C]- and [2-¹⁴C]pyruvate. Citrate, isocitrate, succinate, and malate were not utilized by intact cells due to their inability to permeate the plasma membrane. For every mole of corticosterone formed, 1.9 and 0.8 moles of ¹⁴CO₂ were formed from [1-¹⁴C]- and [2-¹⁴C]pyruvate, respectively, indicating that pyruvate dehydrogenase was quite active and supplied acetyl C?oA to the Krebs cycle. Fluorocitrate and 2,4-dinitrophenol inhibited 11β-hydroxylation of 11-deoxycorticosterone as well as the production of ¹⁴CO₂ from [2-¹⁴C]pyruvate. Comparison of data with the two inhibitors showed that for the same percentage of inhibition of ¹⁴CO₂ production, the inhibition of 11β-hydroxylation was greater with 2,4-dinitrophenol than with fluorocitrate. It is concluded that operation of the Krebs cycle may be essential for 11β-hydroxylation to occur primarily because NADH generated by the cycle provides ATP, via the respiratory chain, as well as the substrate for the energy-linked transhydrogenase that forms NADPH. The NADPH required for 11β-hydroxylation seems to be derived to a large extent via the energy-linked transhydrogenase.     

Changing sources of soil respiration with time since fire in a boreal forest

Radiocarbon signatures (Δ¹⁴C) of carbon dioxide (CO₂) provide a measure of the age of C being decomposed by microbes or respired by living plants. Over a 2‐year period, we measured Δ¹⁴C of soil respiration and soil CO₂ in boreal forest sites in Canada, which varied primarily in the amount of time since the last stand‐replacing fire. Comparing bulk respiration Δ¹⁴C with Δ¹⁴C of CO₂ evolved in incubations of heterotrophic (decomposing organic horizons) and autotrophic (root and moss) components allowed us to estimate the relative contributions of O horizon decomposition vs. plant sources. Although soil respiration fluxes did not vary greatly, differences in Δ¹⁴C of respired CO₂ indicated marked variation in respiration sources in space and time. The ¹⁴C signature of respired CO₂ respired from O horizon decomposition depended on the age of C substrates. These varied with time since fire, but consistently had Δ¹⁴C greater (averaging ～120‰) than autotrophic respiration. The Δ¹⁴C of autotrophically respired CO₂ in young stands equaled those expected for recent photosynthetic products (70‰ in 2003, 64‰ in 2004). CO₂ respired by black spruce roots in stands >40 years old had Δ¹⁴C up to 30‰ higher than recent photosynthates, indicating a significant contribution of C stored at least several years in plants. Decomposition of O horizon organic matter made up 20% or less of soil respiration in the younger (<40 years since fire) stands, increasing to ～50% in mature stands. This is a minimum for total heterotrophic contribution, since mineral soil CO₂ had Δ¹⁴C close to or less than those we have assigned to autotrophic respiration. Decomposition of old organic matter in mineral soils clearly contributed to soil respiration in younger stands in 2003, a very dry year, when Δ¹⁴C of soil respiration in younger successional stands dropped below those of the atmospheric CO₂.     

Conditional lethal mutants of Chinese hamster cells: mutants requiring exogenous carbondioxide for growth

A series of Chinese hamster cell lines were tested and found to be able to proliferate in the absence of added bicarbonate and carbondioxide if hypoxanthine and uridine were present in the medium. Conversely, cells incapable of salvaging one of these precursors, such as hypoxanthine-guanine phosphoribosyltransferase (HGPRT^?) deficient cells did not multiply under these conditions. We describe another variant capable of utilizing hypoxanthine and uridine which has an absolute requirement for exogenous CO₂/NaHCO₃ for growth. These cells appear to be defective in the complete oxidation of pyruvate to carbondioxide, and indications are that the entry of pyruvate into the Krebs cycle is affected.     

The Role of Ethylene Diisothiocyanate (EDI) in the Antifungal Action of Disodium Ethylene Bisdithiocarbamate (Nabam). I. The Mode of Action of EDI on the Metabolism of Yeast

With glucose as a substrate, the oxygen consumption in yeast in inhibited by 2· 10^-5M ethylene diisothiocyanate. The degree of inhibition was only to a small extent dependant on pH. Radiorespirometric experiments with uniformely labelled glucose showed that the CO₂-production from glucose increased, probably due to increased glycolytic activity. Conversion of C-1 to CO₂ was unaffected by the inhibitor, while the evolution of CO₂ from C-6 was strongly inhibited. The same was the case with CO₂ from C-1 in acetate. Respiration of ethanol was more strongly inhibited than that of glucose or acetate. Experiments with dual wavelength spectrophotometry showed the inhibition to be located on the Krebs cycle side of the respiratory flavoproteins. It is concluded that the action of ethylene diisothiocyanate on respiration must be located at the mitochondria.     

Titles of related papers in other sections

Culture of Trypanosoma cruzi (Tulahuen strain) in the presence of ethidium bromide (1–20 μg/ml) resulted in dyskinetoplasty and inhibition of growth, to an extent depending on the dye concentration and the medium composition. The ethidium bromide-induced dyskinetoplasty caused a decrease of (a) the cytochrome content of epimastigotes (a,a₃ and b species); (b) the rate of respiration (endogenous or supported by D-glucose); and (c) the rate of production of ¹⁴CO₂ from [2-¹⁴C]acetate and [1-¹⁴C]glucose. [2-¹⁴C]Acetate oxidation to ¹⁴CO₂ was affected by dyskinetoplasty more than [1-¹⁴C]glucose oxidation, particularly at the exponential growth phase. With dyskinetoplastic epimastigotes, diminution of ¹⁴CO₂ production from [2-¹⁴C]acetate largely exceeded that of oxygen uptake, while with [1-¹⁴C]glucose, ¹⁴CO₂production and respiration were affected to about the same extent. Dyskinetoplasty also decreased the incorporation of [2-¹⁴C]acetate carbon into intermediates of the tricarboxylic acid cycle and related amino acids, and modified the distribution pattern of ¹⁴C in accordance with the decrease of respiration. Reduction of cytochrome content of epimastigotes by restriction of heme compounds during growth decreased ¹⁴CO₂ production from [2-¹⁴C]acetate, like the ethidium-induced dyskinetoplasty. The same occurred after inhibition of electron transfer by antimycin and cyanide, though to a much more significant extent, thus confirming the functional association of electron transport at the mitochondrial cytochrome system of T. cruzi and the enzymatic reactions of the tricarboxylic acid cycle.     

Interrelationships between Water and Cellular Metabolism in Artemia cysts. V. 14CO2 incorporation

The ability of cysts of the brine shrimp, Artemia salina, to incorporate ¹⁴CO₂ into organic compounds soluble in cold-trichloroacetic acid was examined over a broad range of cellular water concentrations. Carbon dioxide was not incorporated by cysts containing less than about 0.3 g H₂O/g dried cysts, the “critical hydration” for CO₂-fixation. This relationship held whether the cysts were hydrated from the liquid or the vapor phase. The incorporation of radioactivity was shown to be due exclusively to metabolic activity in the cellular component of the cyst. Above the critical hydration, the amount of ¹⁴CO₂ incorporated was a function of cyst water content, but the kinds of metabolites labelled with this precursor, and their relative proportions, were found to be similar in cysts of greatly different hydration. Almost all of the radioactivity was associated with amino acids, Krebs cycle intermediates and related acids, and pyrimidine nucleotides. The fact that the pathway involved with CO₂-fixation, and subsequent metabolism of the fixation products are all initiated in cysts containing as little as 0.3 g H₂O/g is particularly noteworthy since this hydration level is well within the range of the amounts of “bound water” described in the literature for a wide array of cells and tissues.     

14C – a tool for separation of autotrophic and heterotrophic soil respiration

We assessed the potential of using ¹⁴C contents of soil respired CO₂ to calculate the contributions of heterotrophic and autotrophic respiration to total soil respiration. The partitioning of these fluxes is of utmost importance to evaluate implications of environmental change on soil carbon cycling and sequestration. At three girdled forest stands in Sweden and Germany, where the tree root (autotrophic) respiration had been eliminated, we measured both flux rates and ¹⁴C contents of soil respired CO₂ in girdled and control plots in the summers of 2001 or 2002. At all stands, CO₂ flux rates were slightly higher in the control plots, whereas the ¹⁴C contents of respired CO₂ tended to be higher in the girdled plots. This was expected and confirmed that heterotrophically respired CO₂ cycles more slowly through the forest ecosystem than autotrophically respired CO₂. On the basis of these data, the contributions of hetero‐ and autotrophic respiration to total soil respiration were calculated using two separate approaches (i.e. based on flux rates or ¹⁴C). Fractions of heterotrophic respiration ranged from 53% to 87%. Values calculated by both approaches did not differ significantly from each other. Finally, we compared the ¹⁴C contents of soil respired CO₂ in the girdled plots with the ¹⁴C contents of heterotrophically respired CO₂ calculated by three different ¹⁴C models. None of the models matched the measured data sufficiently. In addition, we suspect that inherent effects of girdling may cause the ¹⁴C content of CO₂ respired in the girdled plots to be lower than ‘true’ heterotrophically respired CO₂ in an undisturbed plot. Nevertheless, we argue that measurements and modeling of ¹⁴C can be developed into a valuable tool for separating heterotrophic and autotrophic soil respiration (e.g. when girdling cannot be performed).     

PHOTOASSIMILATION OF 14C-ACETATE BY ULVA LACTUCA1

The effect of light on the uptake of ¹⁴C-labeled acetate, glucose, α-ketoglutarate, mannitol, and glycine was investigated in Ulva lactuca var. rigida. Uptake in the light over that in the dark of ¹⁴C-acetale (8-fold) was far higher than that of the other compounds tested. Further study of the phenomenon showed that (1) an increase in light intensity from 60 through 1000 ft-c results in increased ¹⁴C-acetate uptake, the kinetics of which differ from that of ¹⁴CO₂ uptake versus light intensity over the same range: (2) the action spectrum of acetate uptake, between 450 and 730 nm conforms to the action spectrum for photosynthesis in Ulva; (3) the acetate uptake process is sensitive to photosynthetic poisons including N'- (3-4, dichlorophenyl)-N, N-dimethyl urea (DCMU), and phenazine methosulfate (PMS), and inhibitors of the Krebs cycle including sodium monofluoroacetate (MFA) and malonate; (4) uptake of acetate is favored by low CO₂ concentrations; (5) uptake of acetate is not sensitive to 10^?4 M uranyl nitrate; (6) continuous white light, with and without far-red radiation, indicates no significant phytochrome involvement in the process. These results point to an intracellular dependence of the assimilation of acetate on the Krebs cycle in some manner (possibly in cooperation with the glyoxylate cycle) other than simply releasing CO₂ from the acetate moiety with subsequent fixation via the Calvin cycle, and on some product(s) of photo-system 2, eg, NADPH⁺, reduced ferredoxin, or O₂.     

The effect of light on the tricarboxylic Acid cycle in green leaves: I. Relative rates of the cycle in the dark and the light

Excised green leaves of mung bean (Phaseolus aureus L. var. Mungo) were used to determine the effect of light on the rate of endogenous respiration via the tricarboxylic acid cycle. Illumination with white light at an intensity of 0.043 gram calories cm⁻²min⁻¹ (approximately 8600 lux) of visible radiation (400-700 nm) gave a rate of apparent photosynthesis, measured as net CO₂ uptake, of 21 mg CO₂ dm⁻²hr⁻¹ which was about 11-fold greater than the rate of dark respiration. The feeding of ¹⁴CO₂ or ¹⁴C-labeled acids of the tricarboxylic acid cycle in the dark for 2 hours was established as a suitable method for labeling mitochondrial pools of cycle intermediates.     

Substrates for anaerobic CO2-production by the goldfish,Carassius auratus (L.): Decarboxylation of14C-labeled metabolites

Summary Goldfish acclimated to normal oxygen levels and to 20°C were made anoxic and injected i.p. with U-¹⁴C-glucose, 6-¹⁴C-glucose, U-¹⁴C-lactate, 3-¹⁴C-lactate, 1-¹⁴C-acetate or 3,4-¹⁴C-glutamate. Radioactivity released into the water (total¹⁴C and¹⁴CO₂) was monitored over a period of about 12 h. With the exception of 3,4-¹⁴C-glutamate from which only 4% was released, the release of¹⁴C from the other compounds was found to be over 30%. The fraction of the radioactivity released as CO₂ varied with the compound injected but was high during the first 4 h after injection. It is argued that the acid-stable¹⁴C component is ethanol, which arises by the combined action of a modified pyruvate dehydrogenase and of alcohol dehydrogenase in muscle (Shoubridge and Hochachka 1980; Mourik et al. 1982).¹⁴CO₂ release from 3-¹⁴C-lactate, 6-¹⁴C-glucose, 3,4-¹⁴C-glutamate and 1-¹⁴C-acetate cannot be explained by ethanol fermentation. Neither was there a stoichiometric relation between¹⁴CO₂ and¹⁴C-ethanol release after U-¹⁴C-glucose and U-¹⁴C-lactate injection. It is concluded that at least 20% of the CO₂ released is produced by Krebs cycle activity.     

Ammonia inhibits insulin stimulation of the Krebs cycle: Further insight into mechanism of hepatic coma

Oxidation of [2,3-¹⁴C]succinate in the intramitochondrial Krebs cycle was used as a probe to investigate the effect of ammonia on protein incorporation and Krebs cycle oxidation of succinate carbons in isolated rat hepatocytes. At low concentrations of ammonium chloride (0.1 to 0.5 mM) a slight increase in¹⁴CO₂ formation from [2,3-¹⁴C]succinate was observed, however, the stimulatory effect of insulin was significantly reduced. Insulin failed to cause any stimulation of succinate carbons incorporation into hepatocyte protein in the presence of ammonium chloride. Addition of ammonium chloride also depressed the movement of tracer carbons into the gluconeogenesis pathway. The activity of the amphibolic amino acid pool was significantly enhanced by ammonia. The data presented in this paper lend strong support to the Krebs-cycle depletion theory of hepatic coma. They also suggest that reduced mitochondrial Krebs cycle activity caused by increased amphibolic depletion of substrates results in loss of insulin sensitivity in ammonia toxicity.Special issue dedicated to Dr. Santiago Grisolia.     

Root and microbial involvement in the kinetics of 14C-partitioning to rhizosphere respiration after a pulse labelling of maize assimilates

In a previous study, we examined the kinetics of radioactivity evolution from rhizosphere respiration after the pulse labelling of maize shoots with ¹⁴CO₂ (Nguyen et al., 1999). The specific activity of rhizosphere respiration demonstrated two peaks of ¹⁴CO₂ production. The first one occurred a few hours after the pulse of ¹⁴CO₂ and was followed by a second peak, which took place during the night following the labelling. In the present work, we demonstrate that the second phase of activity occurred in both sterile and non sterile plant–soil systems. This was inconsistent with the results obtained for wheat by Warembourg and Billès (1979) who observed the second peak solely in the case of non-sterile cultures. These authors suggested that this second phase of ¹⁴CO₂ production was related to microbial mineralisation of labelled complex compounds. Their synthesis and breakdown into smaller molecules delayed their utilisation by micro-organisms. However, in the present work, we also demonstrate that the second phase of activity was closely related to photoperiod. When plants were transferred from a 16 h to 20 h photoperiod, the second mineralisation of labelled rhizosphere compounds occurred sooner after the initiation of the dark period and it was strongly attenuated. Therefore, we suggest that the second phase of activity resulted from the utilisation by roots and by micro-organisms of stored ¹⁴C-compounds, which accumulated during the previous light period.     

Sul Metabolismo Dell'Alcool Etilico in Tessuti Di Fusto Di « Pisum »

Abstract

On the metabolism of ethanol in the Pea stem tissues. — The average concentration of ethanol in the growing part of the etiolated pea internodes is of the order of 10^-3M. Previous work showed that auxin at growth promoting concentration markedly lowers this level in the excised internodes. This finding prompted a series of investigations on C¹⁴ labeled ethanol utilization in this material.

The capacity of the segments to metabolize ethanol is remarkable: with an external ethanol concentration 5X10^-3M the C¹⁴ labeled CO₂ originated from 1-C¹⁴ ethanol accounted for about 10% of total CO₂ produced during the first hour of treatment. Moreover, an amount of ethanol about 10 fold higher that that dissimilated to CO₂ was metabolized to various yet unidentified compounds. The ratio between the contribution of ethanol to CO₂ and that to other metabolites appeared maximal in the first period after feeding the labeled compound. This ratio was significantly higher then that found for 6-C¹⁴ glucose.

These preliminary results suggest the possibility that ethanol produced in glycolysis could represent an interesting metabolite in an anabolic pathway different from the one leading from pyruvate to the Krebs cycle acids.     

Relationship between the pentose-phosphate pathway and the de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cultures

This study focuses on the activity of the pentose-phosphate pathway and its relationship to de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cell cultures derived from 1-week old rat brain. The proportion of glucose that was metabolized along the pentose-phosphate pathway was estimated by measuring ¹⁴CO₂ production from [1-¹⁴C]-, [2-¹⁴C]- and [6-¹⁴C]glucose, the utilization of glucose and the production of lactate. Incorporation of ¹⁴C from [¹⁴C]glucose and from [3-¹⁴C]acetoacetate into lipids was analysed. The pentose- phosphate pathway produced much more CO₂ from glucose than the Krebs cycle, although it accounted for only a small part of the consumption of glucose (< 3%). The higher ¹⁴CO₂ production from [2-¹⁴C]glucose than from [6-¹⁴C]glucose indicated that recycling of the products of the pentose-phosphate pathway takes place in these cells.Gradual inhibition of the pathway with increasing concentrations of 6-aminonicotinamide resulted in a parallel inhibition of the conversion of acetoacetate and of glucose into fatty acids and into cholesterol. Glycolysis was also strongly inhibited in the presence of 6-aminonicotinamide whereas the activity of the Krebs cycle was not affected.These results suggest that de novo synthesis of fatty acids and cholesterol by oligodendrocytes of neonatal rats is closely geared to the activity of the pentose-phosphate pathway in these cells.     

Non-phototrophic CO 2 fixation by soil microorganisms

Although soils are generally known to be a net source of CO₂ due to microbial respiration, CO₂ fixation may also be an important process. The non-phototrophic fixation of CO₂ was investigated in a tracer experiment with ¹⁴CO₂ in order to obtain information about the extent and the mechanisms of this process. Soils were incubated for up to 91 days in the dark. In three independent incubation experiments, a significant transfer of radioactivity from ¹⁴CO₂ to soil organic matter was observed. The process was related to microbial activity and could be enhanced by the addition of readily available substrates such as acetate. CO₂ fixation exhibited biphasic kinetics and was linearly related to respiration during the first phase of incubation (about 20–40 days). The fixation amounted to 3–5% of the net respiration. After this phase, the CO₂ fixation decreased to 1–2% of the respiration. The amount of carbon fixed by an agricultural soil corresponded to 0.05% of the organic carbon present in the soil at the beginning of the experiment, and virtually all of the fixed CO₂ was converted to organic compounds. Many autotrophic and heterotrophic biochemical processes result in the fixation of CO₂. However, the enhancement of the fixation by addition of readily available substrates and the linear correlation with respiration suggested that the process is mainly driven by aerobic heterotrophic microorganisms. We conclude that heterotrophic CO₂ fixation represents a significant factor of microbial activity in soils.     

Decomposition of old organic matter as a result of deeper active layers in a snow depth manipulation experiment

A snow addition experiment in moist acidic tussock tundra at Toolik Lake, Alaska, increased winter snow depths 2–3 m, and resulted in a doubling of the summer active layer depth. We used radiocarbon (?¹⁴C) to (1) determine the age of C respired in the deep soils under control and deepened active layer conditions (deep snow drifts), and (2) to determine the impact of increased snow and permafrost thawing on surface CO₂ efflux by partitioning respiration into autotrophic and heterotrophic components. ?¹⁴C signatures of surface respiration were higher in the deep snow areas, reflecting a decrease in the proportion of autotrophic respiration. The radiocarbon age of soil pore CO₂ sampled near the maximum mid-July thaw depth was approximately 1,000 years in deep snow treatment plots (45–55 cm thaw depth), while CO₂ from the ambient snow areas was ~100 years old (30-cm thaw depth). Heterotrophic respiration ?¹⁴C signatures from incubations were similar between the two snow depths for the organic horizon and were extremely variable in the mineral horizon, resulting in no significant differences between treatments in either month. Radiocarbon ages of heterotrophically respired C ranged from <50 to 235 years BP in July mineral soil samples and from 1,525 to 8,300 years BP in August samples, suggesting that old soil C in permafrost soils may be metabolized upon thawing. In the surface fluxes, this old C signal is obscured by the organic horizon fluxes, which are significantly higher. Our results indicate that, as permafrost in tussock tundra ecosystems of arctic Alaska thaws, carbon buried up to several thousands of years ago will become an active component of the carbon cycle, potentially accelerating the rise of CO₂ in the atmosphere.     

Carbon fluxes in the rhizosphere of sweet chestnut seedlings (Castanea sativa) grown under two atmospheric CO2 concentrations: 14C partitioning after pulse labelling

Partitioning of ¹⁴C was assessed in sweet chestnut seedlings (Castanea sativa Mill.) grown in ambient and elevated atmospheric [CO₂] environments during two vegetative cycles. The seedlings were exposed to ¹⁴CO₂ atmosphere in both high and low [CO₂] environments for a 6-day pulse period under controlled laboratory conditions. Six days after exposure to ¹⁴CO₂, the plants were harvested, their dry mass and the radioactivity were evaluated. ¹⁴C concentration in plant tissues, root-soil system respiratory outputs and soil residues (rhizodeposition) were measured. Root production and rhizodeposition were increased in plants growing in elevated atmospheric [CO₂]. When measuring total respiration, i.e. CO₂ released from the root/soil system, it is difficult to separate CO₂ originating from roots and that coming from the rhizospheric microflora. For this reason a model accounting for kinetics of exudate mineralization was used to estimate respiration of rhizospheric microflora and roots separately. Root activity (respiration and exudation) was increased at the higher atmospheric CO₂ concentration. The proportion attributed to root respiration accounted for 70 to 90% of the total respiration. Microbial respiration was related to the amount of organic carbon available in the rhizosphere and showed a seasonal variation dependent upon the balance of root exudation and respiration. The increased carbon assimilated by plants grown under elevated atmospheric [CO₂] stayed equally distributed between these increased root activities. ei]H Lambers