δ13C of CO2 respired in the dark in relation to δ13C of leaf metabolites: comparison between Nicotiana sylvestris and Helianthus annuus under drought

The variations of δ¹³C in leaf metabolites (lipids, organic acids, starch and soluble sugars), leaf organic matter and CO₂ respired in the dark from leaves of Nicotiana sylvestris and Helianthus annuus were investigated during a progressive drought. Under well‐watered conditions, CO₂ respired in the dark was ¹³C‐enriched compared to sucrose by about 4‰ in N. sylvestris and by about 3‰ and 6‰ in two different sets of experiments in H. annuus plants. In a previous work on cotyledonary leaves of Phaseolus vulgaris, we observed a constant ¹³C‐enrichment by about 6‰ in respired CO₂ compared to sucrose, suggesting a constant fractionation during dark respiration, whatever the leaf age and relative water content. In contrast, the ¹³C‐enrichment in respired CO₂ increased in dehydrated N. sylvestris and decreased in dehydrated H. annuus in comparison with control plants. We conclude that (i) carbon isotope fractionation during dark respiration is a widespread phenomenon occurring in C₃ plants, but that (ii) this fractionation is not constant and varies among species and (iii) it also varies with environmental conditions (water deficit in the present work) but differently among species. We also conclude that (iv) a discrimination during dark respiration processes occurred, releasing CO₂ enriched in ¹³C compared to several major leaf reserves (carbohydrates, lipids and organic acids) and whole leaf organic matter.     

Disentangling CO2 fluxes: direct measurements of mesocosm‐scale natural abundance 13CO2/12CO2 gas exchange, 13C discrimination,and labelling of CO2 exchange flux components in controlled environments

A ¹³C/¹²C mass spectrometer was interfaced with a open gas exchange system including four growth chambers to investigate CO₂ exchange components of perennial ryegrass (Lolium perenne L.) stands. Chambers were fed with air containing CO₂ with known δ¹³C (δ_CΟ2?2.6 or ?46.8‰). The system did not fractionate C isotopes and no extraneous CO₂ leaked into chambers. The on‐line ¹³C discrimination (Δ) of ryegrass stands in light was independent of δ_CΟ2 when δ_CΟ2 was constant. The δ of CO₂ exchanged by the stands in light (δ_Nd) and darkness (δ_Rn) differed by 0.7‰, suggesting some Δ in dark respiration at the stand‐level. However, Δ decreased by ～ 10‰ when δ_CΟ2 was switched from ?46.8 to ?2.5‰, and increased by ～ 10‰ following a shift from ?2.6 to ?46.7‰ due to isotopic disequilibria between photosynthetic and respiratory fluxes. Isotopic imbalances were used to assess (non‐photorespiratory) respiration in light and the replacement of the respiratory substrate pool(s) by new photosynthate. Respiration was partially inhibited by light, but increased during the light period and decreased in darkness, in association with temperature changes. The labelling kinetics of respiratory CO₂ indicated the existence of two major respiratory substrate pools: a fast pool which was exchanged within hours, and a slow pool accounting for ～ 60% of total respiration and having a mean residence time of 3.6 d.     

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.     

Diffusive fractionation complicates isotopic partitioning of autotrophic and heterotrophic sources of soil respiration

Carbon isotope ratios (δ¹³C) of heterotrophic and rhizospheric sources of soil respiration under deciduous trees were evaluated over two growing seasons. Fluxes and δ¹³C of soil respiratory CO₂ on trenched and untrenched plots were calculated from closed chambers, profiles of soil CO₂ mole fraction and δ¹³C and continuous open chambers. δ¹³C of respired CO₂ and bulk carbon were measured from excised leaves and roots and sieved soil cores. Large diel variations (>5‰) in δ¹³C of soil respiration were observed when diel flux variability was large relative to average daily fluxes, independent of trenching. Soil gas transport modelling supported the conclusion that diel surface flux δ¹³C variation was driven by non‐steady state gas transport effects. Active roots were associated with high summertime soil respiration rates and around 1‰ enrichment in the daily average δ¹³C of the soil surface CO₂ flux. Seasonal δ¹³C variability of about 4‰ (most enriched in summer) was observed on all plots and attributed to the heterotrophic CO₂ source.     

Atmospheric CO2 mole fraction affects stand‐scale carbon use efficiency of sunflower by stimulating respiration in light

Plant carbon‐use‐efficiency (CUE), a key parameter in carbon cycle and plant growth models, quantifies the fraction of fixed carbon that is converted into net primary production rather than respired. CUE has not been directly measured, partly because of the difficulty of measuring respiration in light. Here, we explore if CUE is affected by atmospheric CO₂. Sunflower stands were grown at low (200 μmol mol^?1) or high CO₂ (1000 μmol mol^?1) in controlled environment mesocosms. CUE of stands was measured by dynamic stand‐scale ¹³C labelling and partitioning of photosynthesis and respiration. At the same plant age, growth at high CO₂ (compared with low CO₂) led to 91% higher rates of apparent photosynthesis, 97% higher respiration in the dark, yet 143% higher respiration in light. Thus, CUE was significantly lower at high (0.65) than at low CO₂ (0.71). Compartmental analysis of isotopic tracer kinetics demonstrated a greater commitment of carbon reserves in stand‐scale respiratory metabolism at high CO₂. Two main processes contributed to the reduction of CUE at high CO₂: a reduced inhibition of leaf respiration by light and a diminished leaf mass ratio. This work highlights the relevance of measuring respiration in light and assessment of the CUE response to environment conditions.     

Diel variations in carbon isotopic composition and concentration of organic acids and their impact on plant dark respiration in different species

Leaf respiration in the dark and its C isotopic composition (δ¹³C_R) contain information about internal metabolic processes and respiratory substrates. δ¹³C_R is known to be less negative compared to potential respiratory substrates, in particular shortly after darkening during light enhanced dark respiration (LEDR). This phenomenon might be driven by respiration of accumulated ¹³C‐enriched organic acids, however, studies simultaneously measuring δ¹³C_R during LEDR and potential respiratory substrates are rare. We determined δ¹³C_R and respiration rates (R) during LEDR, as well as δ¹³C and concentrations of potential respiratory substrates using compound‐specific isotope analyses. The measurements were conducted throughout the diel cycle in several plant species under different environmental conditions. δ¹³C_R and R patterns during LEDR were strongly species‐specific and showed an initial peak, which was followed by a progressive decrease in both values. The species‐specific differences in δ¹³C_R and R during LEDR may be partially explained by the isotopic composition of organic acids (e.g., oxalate, isocitrate, quinate, shikimate, malate), which were ¹³C‐enriched compared to other respiratory substrates (e.g., sugars and amino acids). However, the diel variations in both δ¹³C and concentrations of the organic acids were generally low. Thus, additional factors such as the heterogeneous isotope distribution in organic acids and the relative contribution of the organic acids to respiration are required to explain the strong ¹³C enrichment in leaf dark‐respired CO₂.     

Estimating the contribution of leaf litter decomposition to soil CO2 efflux in a beech forest using 13C-depleted litter

The contribution of leaf litter decomposition to total soil CO₂ efflux (F_L/F) was evaluated in a beech (Fagus sylvatica L.) forest in eastern France. The Keeling‐plot approach was applied to estimate the isotopic composition of respired soil CO₂ from soil covered with either control (?30.32‰) or ¹³C‐depleted leaf litter (?49.96‰). The δ¹³C of respired soil CO₂ ranged from ?25.50‰ to ?22.60‰ and from ?24.95‰ to ?20.77‰, respectively, with depleted or control litter above the soil. The F_L/F ratio was calculated by a single isotope linear mixing model based on mass conservation equations. It showed seasonal variations, increasing from 2.8% in early spring to about 11.4% in mid summer, and decreasing to 4.2% just after leaf fall. Between December 2001 and December 2002, cumulated F and F_L reached 0.98 and 0.08 kg_C m^?2, respectively. On an annual basis, decomposition of fresh leaf litter accounted for 8% of soil respiration and 80% of total C loss from fresh leaf litter. The other fraction of carbon loss during leaf litter decomposition that is assumed to have entered the soil organic matter pool (i.e. 20%) represents only 0.02 kg_C m^?2.     

Short‐term effects of CO2 and O2 on citrate metabolism in illuminated leaves

Although there is now a considerable literature on the inhibition of leaf respiration (CO₂ evolution) by light, little is known about the effect of other environmental conditions on day respiratory metabolism. In particular, CO₂ and O₂ mole fractions are assumed to cause changes in the tricarboxylic acid pathway (TCAP) but the amplitude and even the direction of such changes are still a matter of debate. Here, we took advantage of isotopic techniques, new simple equations and instant freeze sampling to follow respiratory metabolism in illuminated cocklebur leaves (Xanthium strumarium L.) under different CO₂/O₂ conditions. Gas exchange coupled to online isotopic analysis showed that CO₂ evolved by leaves in the light came from ‘old’ carbon skeletons and there was a slight decrease in ¹³C natural abundance when [CO₂] increased. This suggested the involvement of enzymatic steps fractionating more strongly against ¹³C and thus increasingly limiting for the metabolic respiratory flux as [CO₂] increased. Isotopic labelling with ¹³C₂‐2,4‐citrate lead to ¹³C‐enriched Glu and 2‐oxoglutarate (2OG), clearly demonstrating poor metabolism of citrate by the TCAP. There was a clear relationship between the ribulose‐1,5‐bisphosphate oxygenation‐to‐carboxylation ratio (v_o/v_c) and the ¹³C commitment to 2OG, demonstrating that 2OG and Glu synthesis via the TCAP is positively influenced by photorespiration.     

13CO2/12CO2 exchange fluxes in a clamp‐on leaf cuvette: disentangling artefacts and flux components

Leaks and isotopic disequilibria represent potential errors and artefacts during combined measurements of gas exchange and carbon isotope discrimination (Δ). This paper presents new protocols to quantify, minimize, and correct such phenomena. We performed experiments with gradients of CO₂ concentration (up to ±250 μmol mol^?1) and δ¹³C_CO2 (34‰), between a clamp‐on leaf cuvette (LI‐6400) and surrounding air, to assess (1) leak coefficients for CO₂, ¹²CO₂, and ¹³CO₂ with the empty cuvette and with intact leaves of Holcus lanatus (C₃) or Sorghum bicolor (C₄) in the cuvette; and (2) isotopic disequilibria between net photosynthesis and dark respiration in light. Leak coefficients were virtually identical for ¹²CO₂ and ¹³CO₂, but ～8 times higher with leaves in the cuvette. Leaks generated errors on Δ up to 6‰ for H. lanatus and 2‰ for S. bicolor in full light; isotopic disequilibria produced similar variation of Δ. Leak errors in Δ in darkness were much larger due to small biological : leak flux ratios. Leak artefacts were fully corrected with leak coefficients determined on the same leaves as Δ measurements. Analysis of isotopic disequilibria enabled partitioning of net photosynthesis and dark respiration, and indicated inhibitions of dark respiration in full light (H. lanatus: 14%, S. bicolor: 58%).     

Environmental and physiological controls on the carbon isotope composition of CO2 respired by leaves and roots of a C3 woody legume (Prosopis velutina) and a C4 perennial grass (Sporobolus wrightii)

Accurate estimates of the δ¹³C value of CO₂ respired from roots (δ¹³C_{R_root}) and leaves (δ¹³C_{R_leaf}) are important for tracing and understanding changes in C fluxes at the ecosystem scale. Yet the mechanisms underlying temporal variation in these isotopic signals are not fully resolved. We measured δ¹³C_{R_leaf}, δ¹³C_{R_root}, and the δ¹³C values and concentrations of glucose and sucrose in leaves and roots in the C₄ grass Sporobolus wrightii and the C₃ tree Prosopis velutina in a savanna ecosystem in southeastern Arizona, USA. Night‐time variation in δ¹³C_{R_leaf} of up to 4.6 ± 0.6‰ in S. wrightii and 3.0 ± 0.6‰ in P. velutina were correlated with shifts in leaf sucrose concentration, but not with changes in δ¹³C values of these respiratory substrates. Strong positive correlations between δ¹³C_{R_root} and root glucose δ¹³C values in P. velutina suggest large diel changes in δ¹³C_{R_root} (were up to 3.9‰) influenced by short‐term changes in δ¹³C of leaf‐derived phloem C. No diel variation in δ¹³C_{R_root} was observed in S. wrightii. Our findings show that short‐term changes in δ¹³C_{R_leaf} and δ¹³C_{R_root} were both related to substrate isotope composition and concentration. Changes in substrate limitation or demand for biosynthesis may largely control short‐term variation in the δ¹³C of respired CO₂ in these species.     

Malate Metabolism in the Dark After CO(2) Fixation in the Crassulacean Plant Kalanchoë tubiflora

The metabolism of [¹³C]malate was studied in the Crassulacean plant Kalanchoë tubiflora following exposure to ¹³CO₂ for 2 hour intervals during a 16 hour dark cycle. Nuclear magnetic resonance spectroscopy of [¹³C]malate extracted from labeled tissue revealed that the transient flux of malate to the mitochondria, estimated by the randomization of [4-¹³C]malate to [1- ¹³C]malate by fumarase, varied substantially during the dark period. At both 15 and 25°C, the extent of malate label randomization in the mitochondria was greatest during the early and late parts of the dark period and was least during the middle of the night, when the rate of ¹³CO₂ uptake was highest. Randomization of labeled malate continued for many hours after malate synthesis had initially occurred. Internally respired ¹²CO₂ also served as a source of carbon for malate formation. At 15°C, 15% of the total malate was formed from respired ¹²CO₂, while at 25°C, 49% of the accumulated malate was derived from respired ¹²CO₂. Some of the malate synthesized from external ¹³CO₂ was also respired during the night. The proportion of the total [¹³C]malate respired during the dark period was similar at 15 and 25°C, and respiration of newly formed [¹³C]malate increased as the night period progressed. These data are discussed with regard to the relative fluxes of malate to the mitochondria and the vacuole during dark CO₂ fixation.     

Partitioning sources of soil respiration in boreal black spruce forest using radiocarbon

Separating ecosystem and soil respiration into autotrophic and heterotrophic component sources is necessary for understanding how the net ecosystem exchange of carbon (C) will respond to current and future changes in climate and vegetation. Here, we use an isotope mass balance method based on radiocarbon to partition respiration sources in three mature black spruce forest stands in Alaska. Radiocarbon (Δ¹⁴C) signatures of respired C reflect the age of substrate C and can be used to differentiate source pools within ecosystems. Recently‐fixed C that fuels plant or microbial metabolism has Δ¹⁴C values close to that of current atmospheric CO₂, while C respired from litter and soil organic matter decomposition will reflect the longer residence time of C in plant and soil C pools. Contrary to our expectations, the Δ¹⁴C of C respired by recently excised black spruce roots averaged 14‰ greater than expected for recently fixed photosynthetic products, indicating that some portion of the C fueling root metabolism was derived from C storage pools with turnover times of at least several years. The Δ¹⁴C values of C respired by heterotrophs in laboratory incubations of soil organic matter averaged 60‰ higher than the contemporary atmosphere Δ¹⁴CO₂, indicating that the major contributors to decomposition are derived from a combination of sources consistent with a mean residence time of up to a decade. Comparing autotrophic and heterotrophic Δ¹⁴C end members with measurements of the Δ¹⁴C of total soil respiration, we calculated that 47–63% of soil CO₂ emissions were derived from heterotrophic respiration across all three sites. Our limited temporal sampling also observed no significant differences in the partitioning of soil respiration in the early season compared with the late season. Future work is needed to address the reasons for high Δ¹⁴C values in root respiration and issues of whether this method fully captures the contribution of rhizosphere respiration.     

13C isotope fractionation during rhizosphere respiration of C3 and C4 plants

Stable carbon isotopes are used extensively to partition total soil CO₂ efflux into root-derived rhizosphere respiration or autotrophic respiration and soil-derived heterotrophic respiration. However, it remains unclear whether CO₂ from rhizosphere respiration has the same δ¹³C value as root biomass. Here we investigated the magnitude of ¹³C isotope fractionation during rhizosphere respiration relative to root biomass in six plant species. Plants were grown in a carbon-free sand-perlite medium inoculated with microorganisms from a farm soil for 62 days inside a greenhouse. We measured the δ¹³C value of rhizosphere respiration using a closed-circulation 48-hour CO₂ trapping method during 40~42 and 60~62 days after sowing. We found a consistent depletion in ¹³C (0.9~1.7‰) of CO₂ from rhizosphere respiration relative to root biomass in three C₃ species (Glycine max L. Merr., Helianthus annuus L. and Triticum aestivum L.), but a relatively large depletion in ¹³C (3.7~7.0‰) in three C₄ species (Amaranthus tricolor L., Sorghum bicolor (L.) Moench and Zea mays L. ssp. mays). Overall, our results indicate that CO₂ from rhizosphere respiration is more ¹³C-depleted than root biomass. Therefore, accounting for this ¹³C fractionation is required for accurately partitioning total soil CO₂ efflux into root-derived and soil-derived components using natural abundance stable carbon isotope methods.     

Respiration of blue-green algae in the light

The CO₂ evolution in the light of Anabaena as well as several other blue-green algae is below 10% of the dark control. Addition of DCMU restores CO₂ evolution in the light almost to the dark level. Furthermore, by adding unlabeled NaHCO₃, a ¹⁴CO₂ release is observed with prelabeled algal cells attaining 15 to 100% of dark control. Analysis by double-reciprocal plots exhibits a competitive relationship between added and endogenously released carbon dioxide. We conclude that CO₂ evolved by respiration is immediately refixed in the light without being liberated.The degree of ¹⁴CO₂ release induced by unlabeled bicarbonate in the light allows to determine true photoinhibition of respiration. Anabaena variabilis Kütz. exhibits almost no inhibition while in eight other species respiration is light-inhibited between 50 and 85% of the dark control.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - TCA trichloroacetic acid     

Evidence for Photorespiration in Tropical Grasses

Sugarcane leaves respired in full light and the CO₂ evolved could be detected in sorghum or miaze photosynthesizing in the same closed system. A combination of radiometric and infra-red gas analysis techniques allowed the estimation of photorespiration (total CO₂ evolution in light) and photosynthesis at increasing light intensities and of dark respiration. Rates of CO₂ evolution approaching those of temperate zone plants occurred at lower light intensities but rapidly decreased with higher light. Smaller but significant quantities of ¹⁴CO₂ were released even at intensities approximating full sunlight in leaves of maize, sorghum and sugarcane. Highly efficient CO₂ capture may explain the low rates of photorespiration at high light intensities.     

Short-term variation in the isotopic composition of organic matter allocated from the leaves to the stem of Pinus sylvestris: effects of photosynthetic and postphotosynthetic carbon isotope fractionation

We aimed to quantify the separate effects of photosynthetic and postphotosynthetic carbon isotope discrimination on δ¹³C of the fast‐turn‐over carbon pool (water soluble organic carbon and CO₂ emitted from heterotrophic tissues), including their diel variation, along the pathway of carbon transport from the foliage to the base of the stem. For that purpose, we determined δ¹³C in total and water‐soluble organic matter of the foliage plus δ¹³C and δ¹⁸O in phloem organic matter of twigs and at three heights along the stem of Pinus sylvestris over a nine‐day period, including four measurements per day. These data were related to meteorological and photosynthesis parameters and to the δ¹³C of stem‐emitted CO₂. In the canopy (foliage and twigs), the δ¹³C of soluble organic matter varied diurnally with amplitudes of up to 1.9‰. The greatest ¹³C enrichment was recorded during the night/early morning, indicating a strong influence of starch storage and remobilization on the carbon isotope signatures of sugars exported from the leaves. ¹³C enrichment of soluble organic matter from the leaves to the twig phloem and further on to the phloem of the stem was supposed to be a result of carbon isotope fractionation associated with metabolic processes in the source and sink tissues. CO₂ emitted from the stem was enriched by 2.3–5.2‰ compared with phloem organic matter. When day‐to‐day variation was addressed, water‐soluble leaf δ¹³C and twig phloem δ¹⁸O were strongly influenced by c_i/c_a and stomatal conductance (G_s), respectively. These results show that both photosynthetic and postphotosynthetic carbon isotope fractionation influence δ¹³C of organic matter over time, and over the length of the basipetal transport pathway. Clearly, these influences on the δ¹³C of respired CO₂ must be considered when using the latter for partitioning of ecosystem CO₂ fluxes or when the assessment of δ¹³C in organic matter is applied to estimate environmental effects in c_i/c_a.     

Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest

The stable C isotope composition (δ¹³C) of CO₂ respired by trunks was examined in a mature temperate deciduous oak forest (Quercus petraea). Month-to-month, day-to-day and diurnal, measurements were made to determine the range of variations at different temporal scales. Trunk growth and respiration rates were assessed. Phloem tissue was sampled and was analysed for total organic matter and soluble sugar ¹³C composition. The CO₂ respired by trunk was always enriched in ¹³C relative to the total organic matter, sometimes by as much as 5‰. The δ¹³C of respired CO₂ exhibited a large seasonal variation (3.3‰), with a relative maximum at the beginning of the growth period. The lowest values occurred in summer when the respiration rates were maximal. After the cessation of radial trunk growth, the respired CO₂ δ¹³C values showed a progressive increase, which was linked to a parallel increase in soluble sugar content in the phloem tissue (R = 0.95; P < 0.01). At the same time, the respiration rates declined. This limited use of the substrate pool might allow the discrimination during respiration to be more strongly expressed. The late-season increase in CO₂ δ¹³C might also be linked to a shift from recently assimilated C to reserves. At the seasonal scale, CO₂ δ¹³C was negatively correlated with air temperature (R = −0.80; P < 0.01). The diurnal variation sometimes reached 3‰, but the range and the pattern depended on the period within the growing season. Contrary to expectations, diurnal variations were maximal in winter and spring when the leaves were missing or not totally functional. By contrast to the seasonal scale, these diurnal variations were not related to air temperature or sugar content. Our study shows that seasonal and diurnal variations of respired ¹³C exhibited a similar large range but were probably explained by different mechanisms.     

C-isotope composition of CO2 respired by shoots and roots: fractionation during dark respiration?

The CO₂ respired by leaves is ¹³C-enriched relative to leaf biomass and putative respiratory substrates (Ghashghaie et al., Phytochemistry Reviews 2, 145–161, 2003), but how this relates to the ¹³C content of root, or whole plant respiratory CO₂ is unknown. The C isotope composition of respiratory CO₂ (δ_R) from shoots and roots of sunflower (Helianthus annuus L.), alfalfa (Medicago sativa L.), and perennial ryegrass (Lolium perenne L.) growing in a range of conditions was analysed. In all instances plants were grown in controlled environments with CO₂ of constant concentration and δ¹³C. Respiration of roots and shoots of individual plants was measured with an open CO₂ exchange system interfaced with a mass spectrometer. Respiratory CO₂ from shoots was always ¹³C-enriched relative to that of roots. Conversely, shoot biomass was always ¹³C-depleted relative to root biomass. The δ-difference between shoot and root respiratory CO₂ was variable, and negatively correlated with the δ-difference between shoot and root biomass (r² = 0.52, P = 0.023), suggesting isotope effects during biosynthesis. ¹³C discrimination in respiration (R) of shoots, roots and whole plants (e_Shoot, e_Root, e_Plant) was assessed as e = (δ_Substrate − δ_R)/(1 + δ_R/1000), where root and shoot substrate is defined as imported C, and plant substrate is total photosynthate. Estimates were obtained from C isotope balances of shoots, roots and whole plants of sunflower and alfalfa using growth and respiration data collected at intervals of 1 to 2 weeks. e_plant and e_Shoot differed significantly from zero. e_plant ranged between −0.4 and −0.9‰, whereas e_Shoot was much greater (−0.6 to −1.9‰). e_Root was not significantly different from zero. The present results help to resolve the apparent conflict between leaf- and ecosystem-level ¹³C discrimination in respiration.     

Partitioning net ecosystem carbon exchange with isotopic fluxes of CO2

Because biological and physical processes alter the stable isotopic composition of atmospheric CO₂, variations in isotopic content can be used to investigate those processes. Isotopic flux measurements of ¹³CO₂ above terrestrial ecosystems can potentially be used to separate net ecosystem CO₂ exchange (NEE) into its component fluxes, net photosynthetic assimilation (F_A) and ecosystem respiration (F_R). In this paper theory is developed to partition measured NEE into F_A and F_R, using measurements of fluxes of CO₂ and ¹³CO₂, and isotopic composition of respired CO₂ and forest air. The theory is then applied to fluxes measured (or estimated, for ¹³CO₂) in a temperate deciduous forest in eastern Tennessee (Walker Branch Watershed). It appears that there is indeed enough additional information in ¹³CO₂ fluxes to partition NEE into its photosynthetic and respiratory components. Diurnal patterns in F_A and F_R were obtained, which are consistent in magnitude and shape with patterns obtained from NEE measurements and an exponential regression between night‐time NEE and temperature (a standard technique which provides alternate estimates of F_R and F_A). The light response curve for photosynthesis (F_A vs. PAR) was weakly nonlinear, indicating potential for saturation at high light intensities. Assimilation‐weighted discrimination against ¹³CO₂ for this forest during July 1999 was 16.8–17.1‰, depending on canopy conductance. The greatest uncertainties in this approach lie in the evaluation of canopy conductance and its effect on whole‐canopy photosynthetic discrimination, and thus the indirect methods used to estimate isotopic fluxes. Direct eddy covariance measurements of ¹³CO₂ flux are needed to assess the validity of the assumptions used and provide defensible isotope‐based estimates of the component fluxes of net ecosystem exchange.     

Assimilate partitioning affects 13C fractionation of recently assimilated carbon in maize

Coupling ¹³C natural abundance and ¹⁴C pulse labelling enabled us to investigate the dependence of ¹³C fractionation on assimilate partitioning between shoots, roots, exudates, and CO₂ respired by maize roots. The amount of recently assimilated C in these four pools was controlled by three levels of nutrient supply: full nutrient supply (NS), 10 times diluted nutrient supply (DNS), and deionised water (DW). After pulse labelling of maize shoots in a ¹⁴CO₂ atmosphere, ¹⁴C was traced to determine the amounts of recently assimilated C in the four pools and the δ¹³C values of the four pools were measured. Increasing amounts of recently assimilated C in the roots (from 8% to 10% of recovered ¹⁴C in NS and DNS treatments) led to a 0.3‰ ¹³C enrichment from NS to DNS treatments. A further increase of C allocation in the roots (from 10% to 13% of recovered ¹⁴C in DNS and DW treatments) resulted in an additional enrichment of the roots from DNS to DW treatments by 0.3‰. These findings support the hypothesis that ¹³C enrichment in a pool increases with an increasing amount of C transferred into that pool. δ¹³C of CO₂ evolved by root respiration was similar to that of the roots in DNS and DW treatments. However, if the amount of recently assimilated C in root respiration was reduced (NS treatment), the respired CO₂ became 0.7‰ ¹³C depleted compared to roots. Increasing amounts of recently assimilated C in the CO₂ from NS via DNS to DW treatments resulted in a 1.6‰ δ¹³C increase of root respired CO₂ from NS to DW treatments. Thus, for both pools, i.e. roots and root respiration, increasing amounts of recently assimilated C in the pool led to a δ¹³C increase. In DW and DNS plants there was no ¹³C fractionation between roots and exudates. However, high nutrient supply decreased the amount of recently assimilated C in exudates compared to the other two treatments and led to a 5.3‰ ¹³C enrichment in exudates compared to roots. We conclude that ¹³C discrimination between plant pools and within processes such as exudation and root respiration is not constant but strongly depends on the amount of C in the respective pool and on partitioning of recently assimilated C between plant pools. Section Editor: H. Lambers