Soil 13C–15N dynamics in an N2-fixing clover system under long-term exposure to elevated atmospheric CO2

Reduced soil N availability under elevated CO₂ may limit the plant's capacity to increase photosynthesis and thus the potential for increased soil C input. Plant productivity and soil C input should be less constrained by available soil N in an N₂‐fixing system. We studied the effects of Trifolium repens (an N₂‐fixing legume) and Lolium perenne on soil N and C sequestration in response to 9 years of elevated CO₂ under FACE conditions. ¹⁵N‐labeled fertilizer was applied at a rate of 140 and 560 kg N ha^?1 yr^?1 and the CO₂ concentration was increased to 60 Pa pCO₂ using ¹³C‐depleted CO₂. The total soil C content was unaffected by elevated CO₂, species and rate of ¹⁵N fertilization. However, under elevated CO₂, the total amount of newly sequestered soil C was significantly higher under T. repens than under L. perenne. The fraction of fertilizer‐N (f_N) of the total soil N pool was significantly lower under T. repens than under L. perenne. The rate of N fertilization, but not elevated CO₂, had a significant effect on f_N values of the total soil N pool. The fractions of newly sequestered C (f_C) differed strongly among intra‐aggregate soil organic matter fractions, but were unaffected by plant species and the rate of N fertilization. Under elevated CO₂, the ratio of fertilizer‐N per unit of new C decreased under T. repens compared with L. perenne. The L. perenne system sequestered more ¹⁵N fertilizer than T. repens: 179 vs. 101 kg N ha^?1 for the low rate of N fertilization and 393 vs. 319 kg N ha^?1 for the high N‐fertilization rate. As the loss of fertilizer‐¹⁵N contributed to the ¹⁵N‐isotope dilution under T. repens, the input of fixed N into the soil could not be estimated. Although N₂ fixation was an important source of N in the T. repens system, there was no significant increase in total soil C compared with a non‐N₂‐fixing L. perenne system. This suggests that N₂ fixation and the availability of N are not the main factors controlling soil C sequestration in a T. repens system.     

Natural 13C and 15N abundance of field-collected fungi and their ecological implications

The natural abundance of ¹³C and ¹⁵N was measured in basidiocarps of at least 115 species in 88 genera of ectomycorrhizal, wood-decomposing and litter-decomposing fungi from Japan and Malaysia. The natural abundance of ¹³C and ¹⁵N was also measured in leaves, litter, soil and wood from three different sites. ¹⁵N and ¹³C were enriched in ectomycorrhizal and wood-decomposing fungi, respectively, relative to their substrates. Ectomycorrhizal and wood-decomposing fungi could be distinguished on the basis of their δ¹³C and δ¹⁵N signatures. Although there was high variability in the isotopic composition of fungi, the following isotope- enrichment factors (ε, mean±SD) of the fungi relative to substrates were observed:
ε_{ectomycorrhizal fungi/litter} = 6.1±0.4‰¹⁵N
ε_{ectomycorrhizal fungi/wood} = 1.4±0.8‰¹³C
ε_{wood-decomposing fungi/wood} = −0.6±0.7‰¹⁵N
ε_{wood-decomposing fungi/wood} = 3.5±0.9‰¹³C
The basis of isotope fractionation in C metabolism from wood to wood-decomposing fungus is discussed.     

Acquisition and within-plant allocation of 13C and 15N in CO2-enriched Quercus robur plants

We assessed the effects of doubling atmospheric CO₂ concentration, [CO₂], on C and N allocation within pedunculate oak plants (Quercus robur L.) grown in containers under optimal water supply. A short-term dual ¹³CO₂ and ¹⁵NO₃^? labelling experiment was carried out when the plants had formed their third growing flush. The 22-week exposure to 700 μl l^?1 [CO₂] stimulated plant growth and biomass accumulation (+53% as compared with the 350 μl l^?1 [CO₂] treatment) but decreased the root/shoot biomass ratio (-23%) and specific leaf area (-18%). Moreover, there was an increase in net CO₂ assimilation rate (+37% on a leaf dry weight basis; +71% on a leaf area basis), and a decrease in both above- and below-ground CO₂ respiration rates (-32 and -26%, respectively, on a dry mass basis) under elevated [CO₂]. ¹³C acquisition, expressed on a plant mass basis or on a plant leaf area basis, was also markedly stimulated under elevated [CO₂] both after the 12-h ¹³CO₂ pulse phase and after the 60-h chase phase. Plant N content was increased under elevated CO₂ (+36%), but not enough to compensate for the increase in plant C content (+53%). Thus, the plant C/N ratio was increased (+13%) and plant N concentration was decreased (-11%). There was no effect of elevated [CO₂] on fine root-specific ¹⁵N uptake (amount of recently assimilated ¹⁵N per unit fine root dry mass), suggesting that modifications of plant N pools were merely linked to root size and not to root function. N concentration was decreased in the leaves of the first and second growing flushes and in the coarse roots, whereas it was unaffected by [CO₂] in the stem and in the actively growing organs (fine roots and leaves of the third growth flush). Furthermore, leaf N content per unit area was unaffected by [CO₂]. These results are consistent with the short-term optimization of N distribution within the plants with respect to growth and photosynthesis. Such an optimization might be achieved at the expense of the N pools in storage compartments (coarse roots, leaves of the first and second growth flushes). After the 60-h ¹³C chase phase, leaves of the first and second growth flushes were almost completely depleted in recent ¹³C under ambient [CO₂], whereas these leaves retained important amounts of recently assimilated ¹³C (carbohydrate reserves?) under elevated [CO₂].     

15N natural abundance of vascular rainforest epiphytes: implications for nitrogen source and acquisition

The foliar natural abundance of ¹⁵N was analysed to compare the potential nitrogen sources of vascular rainforest epiphytes and associated soil-rooted trees. Leaves of epiphytes collected from six rainforest communities in Brazil, Australia and the Solomon Islands were depleted in ¹⁵N relative to the trees at each site. Epiphyte δ¹⁵N was as low as -6.4%_o, while trees were generally enriched in ¹⁵N (0.7 to 3.5%_o). These results indicate either that epiphytes use nitrogen sources depleted in ¹⁵N or that discrimination against ¹⁵N is an intrinsic function of epiphyte physiology. At three sites, epiphytes could be grouped into those having both low δ¹⁵N and low leaf-nitrogen content and those possessing both high δ¹⁵N and high leaf-nitrogen content. The second group had δ¹⁵N values in the range sometimes attributable to N₂ fixation (-2 to 0%o). There was no correlation between growth form and δ¹⁵N. It is concluded that epiphytes may utilize ¹⁵N-depleted nitrogen from atmospheric deposition and N₂ fixation.     

Effects of the ecto- and VA-mycorrhizal fungi Hydnagium carneum and Glomus clarum on the δ15N and δ13C values of Eucalyptus globulus and Ricinus communis

Glasshouse experiments with Ricinus communis showed that the presence/absence of a VA mycorrhizal fungus (Glomus clarum) changed the δ¹⁵N value of the host by as much as 2‰ when the plants were given urea (released as NH₄⁺) as their only N-source. This small change in Δ¹⁵N would create a large error in calculating sources of plant N. In particular, these results throw into doubt any models of N-cycling which assume that soil N can be treated as a single source. The correct N-source value for VAM-infected NH₄^? -using plants may be the δ¹⁵N of soil NH₄⁺+ 2‰. Treatment effects were also found in the distribution of δ¹⁵N and % N among plant organs. Plants with VAM had a lower N:P atom ratio and were larger in total biomass. Carbon discrimination (δ¹³C) was greater in the VA-infected plants. The measured effects of VAM infection suggest that for some plants the fungus may be the primary site of N assimilation. A parallel experiment with Eucalyptus globulus and the ectomycorrhizal fungus Hydnangium carneum resulted in no significant differences in any of the variables measured for this host-fungus pair when the sole N-sources were inorganic (NO₃^? and NH₄⁺ released from urea). Ectomycorrhizal fungi are diverse in their physiological behaviour, and these data should not be taken as being representative of the whole group. More work is required with other types of mycorrhiza and more complex sources of N. Future work will include a water balance to partition the effects of water use and nutrient supply in determining δ¹³C. An on-line combustion-ANCA-MS method is described for fully automated measurement of natural abundance levels of ^15/14N and ^13/12C for plant materials. This method achieves the required precision while dramatically increasing sample throughout.     

Nitrate and ammonium influxes in soybean (Glycine max) roots: direct comparison of 13N and 15N tracing

We compared influxes and internal transport in soybean plants (Glycine max cv. Kingsoy) of labelled N from external solutions where either ammonium or nitrate was labelled with the stable isotope¹⁵N and the radioactive isotope¹³N. The objective was to see whether mass spectrometric determinations of tissue ¹⁵N content were sufficiently sensitive to measure influxes accurately over short time periods. Our findings were as follows. (1) There was a close quantitative correspondence between estimates of N influx of individual plants using ¹⁵N or ¹³N measurements with either NO_3/^? or NH₄⁺ at 4 or 2 mol^?3, respectively in the external solution. (2) Transport to the shoot of N from NO₃ absorbed over a 5–15 min period could be monitored when the external NO₃^? concentration ranged from 0–05 to 4 mol m^?3. NH₄⁺ as the N source labelled shoot tissue more slowly, and estimates of the transport between root and shoot could be made only with ¹³N. (3) Influx of NO₃^? into root tissue could be measured by ¹⁵N enrichment after 5–10 min at concentrations approaching the probable K_M of the high-affinity transport system. (4) There was some indication of isotope discrimination, especially with respect to the movement of labelled N to the shoot, when NO₃^? is the N source. For many purposes, ¹⁵N tracing can be used satisfactorily to estimate influxes of both NO₃^? and NH₄⁺ in soybean roots. Use of the short-lived radio nuclide ¹³N remains the method of choice for more refined measurements of internal distribution and assimilation.     

Kinetics and relative significance of remobilized and current C and N incorporation in leaf and root growth zones of Lolium perenne after defoliation: assessment by 13C and 15N steady-state labelling

The contribution of pre-defoliation reserves and current assimilates to leaf and root growth was examined in Lolium perenne L. during regrowth after defoliation. Differential steady-state labelling with ¹³C (CO₂ with δ¹³C = -0.0281 and -0.0088) and ¹⁵N (NO₃^? with 1.0 and 0.368 atom percentage, i.e. δ¹⁵N = 1.742 and 0.0052, respectively) was applied for 2 weeks after defoliation. Rapidly growing tissues were isolated, i.e. the basal elongation and maturation zones of the most rapidly expanding leaves and young root tips, with a biomass turnover rate > 1 d^?1. C and N weights of the elongation zone showed a transient decline. The dry matter and C concentration in fresh biomass of leaf growth zones transiently decreased by up to 25% 2 d after defoliation, while the N concentration remained constant. This ‘dilution’ of growth zone C indicates a decreased net influx of carbohydrates relative to growth-related influx of water and N in expanding cells, immediately after defoliation. Recovery of the total C and N weights of the leaf elongation zone coincided with net incorporation of currently absorbed C and N, as shown by the kinetics of δ¹³C and atom percentage ¹⁵N in the growth zones after defoliation. C isotope discrimination (Δ¹³C) in leaf growth zones was about 23‰, 1–2‰ higher than the Δ in root tips. Δ¹⁵N in the leaf and root growth zones was 10±3‰. The leaf elongation zones (at 0–0.03 m from the tiller base) and the distant root tips (about 0.2 m from the base) exhibited similar kinetics of current C and N incorporation. The amount of pre-defoliation C and N in the growth zones, expressed as a fraction of total C and N, decreased from 1.0 to 0.5 at 3 (C) and 5 (N) d after defoliation, and to 0.1 at 5 (C) and 14 (N) d after defoliation. Thus, the dependence of growth zones on current assimilate supply was significant, and stronger for C than for N. The important roles of current assimilates (as compared to pre-defoliation reserves) and ‘dilution’ of dry matter in regrowth after defoliation are discussed in relation to the method of labelling and the functional and morphological heterogeneity of shoot tissues.     

13C and 15N in microarthropods reveal little response of Douglas-fir ecosystems to climate change

Understanding ecosystem carbon (C) and nitrogen (N) cycling under global change requires experiments maintaining natural interactions among soil structure, soil communities, nutrient availability, and plant growth. In model Douglas-fir ecosystems maintained for five growing seasons, elevated temperature and carbon dioxide (CO₂) increased photosynthesis and increased C storage belowground but not aboveground. We hypothesized that interactions between N cycling and C fluxes through two main groups of microbes, mycorrhizal fungi (symbiotic with plants) and saprotrophic fungi (free-living), mediated ecosystem C storage. To quantify proportions of mycorrhizal and saprotrophic fungi, we measured stable isotopes in fungivorous microarthropods that efficiently censused the fungal community. Fungivorous microarthropods consumed on average 35% mycorrhizal fungi and 65% saprotrophic fungi. Elevated temperature decreased C flux through mycorrhizal fungi by 7%, whereas elevated CO₂ increased it by 4%. The dietary proportion of mycorrhizal fungi correlated across treatments with total plant biomass (n= 4, r²= 0.96, P= 0.021), but not with root biomass. This suggests that belowground allocation increased with increasing plant biomass, but that mycorrhizal fungi were stronger sinks for recent photosynthate than roots. Low N content of needles (0.8–1.1%) and A horizon soil (0.11%) coupled with high C : N ratios of A horizon soil (25–26) and litter (36–48) indicated severe N limitation. Elevated temperature treatments increased the saprotrophic decomposition of litter and lowered litter C : N ratios. Because of low N availability of this litter, its decomposition presumably increased N immobilization belowground, thereby restricting soil N availability for both mycorrhizal fungi and plant growth. Although increased photosynthesis with elevated CO₂ increased allocation of C to ectomycorrhizal fungi, it did not benefit plant N status. Most N for plants and soil storage was derived from litter decomposition. N sequestration by mycorrhizal fungi and limited N release during litter decomposition by saprotrophic fungi restricted N supply to plants, thereby constraining plant growth response to the different treatments.     

Natural 15N enrichment of amide-exporting legume nodules

The natural ¹⁵N abundance of amide-exporting nodules was compared to that of shoots in 12 plant species. Nodules were statistically less abundant in ¹⁵N than shoots in one of three cultivars of Pisum sativum L., in Vicia faba L. and in Medicago sativa L., but the ¹⁵N depletion of nodules was very samall. Nodules were statistically more abundant in ¹⁵N than shoots in Trifolium pratense L., depending on time during the growing season, Cyamopsis tetragonaloba L. Taub. and 7 Lupinus species, but the enrichment was small except for C. tetragonalova and 6 Lupinus species. Nodules of 3 Lupinus species infected with Rhizobium lupini isolated from Lupinus subcarnosa Hook, were only slightly enriched in ¹⁵N, but nodules of two of these species were substantially enriched in ¹⁵N when infected with a mix of other Rhizobium lupini strains. The third species, L. texensis Hook., was not infected by this mix of strains. Differences in ¹⁵N abundance between nodules and other tissues of amide-exporting and ureide-exporting nodules from several studies are tabulated. All ureide-exporting nodules in this tabulation are enriched in ¹⁵N. Amide-exporting nodules are considerably more variable in this regard. These results confirm that events associated with ureide synthesis or transport cannot be the sole cause of the substantial ¹⁵N enrichment seen in nodules.     

Linking sequestration of 13C and 15N in aggregates in a pasture soil following 8 years of elevated atmospheric CO2

The influence of N availability on C sequestration under prolonged elevated CO₂ in terrestrial ecosystems remains unclear. We studied the relationships between C and N dynamics in a pasture seeded to Lolium perenne after 8 years of elevated atmospheric CO₂ concentration (FACE) conditions. Fertilizer‐¹⁵N was applied at a rate of 140 and 560 kg N ha^2?1 y^2?1 and depleted ¹³C‐CO₂ was used to increase the CO₂ concentration to 60 Pa pCO₂. The ¹³C–¹⁵N dual isotopic tracer enabled us to study the dynamics of newly sequestered C and N in the soil by aggregate size and fractions of particulate organic matter (POM), made up by intra‐aggregate POM (iPOM) and free light fraction (LF). Eight years of elevated CO₂ did not increase total C content in any of the aggregate classes or POM fractions at both rates of N application. The fraction of new C in the POM fractions also remained largely unaffected by N fertilization. Changes in the fractions of new C and new N (fertilizer‐N) under elevated CO₂ were more pronounced between POM classes than between aggregate size classes. Hence, changes in the dynamics of soil C and N cycling are easier to detect in the POM fractions than in the whole aggregates. Within N treatments, fractions of new C and N in POM classes were highly correlated with more new C and N in large POM fractions and less in the smaller POM fractions. Isotopic data show that the microaggregates were derived from the macro‐aggregates and that the C and N associated with the microaggregates turned over slower than the C and N associated with the macroaggregates. There was also isotopic evidence that N immobilized by soil microorganisms was an important source of N in the iPOM fractions. Under low N availability, 3.04 units of new C per unit of fertilizer N were sequestered in the POM fractions. Under high N availability, the ratio of new C sequestered per unit of fertilizer N was reduced to 1.47. Elevated and ambient CO₂ concentrations lead to similar ¹⁵N enrichments in the iPOM fractions under both low and high N additions, clearly showing that the SOM‐N dynamics were unaffected by prolonged elevated CO₂ concentrations.     

15N natural abundance during early and late succession in a middle-European dry acidic grassland

δ¹⁵N and total nitrogen content of above- and belowground tissues of 13 plant species from two successional stages (open pioneer community and ruderal grass stage) of a dry acidic grassland in Southern Germany were analysed, in order to evaluate whether resource use partitioning by niche separation and N input by N₂-fixing legumes are potential determinants for species coexistence and successional changes. Within each stage, plants from plots with different legume cover were compared. Soil inorganic N content, total plant biomass and δ¹⁵N values of bulk plant material were significantly lower in the pioneer stage than in the ruderal grass community. The observed δ¹⁵N differences were rather species- than site-specific. Within both stages, there were also species-specific differences in isotopic composition between above- and belowground plant dry matter. Species-specific δ¹⁵N signatures may theoretically be explained by (i) isotopic fractionation during microbial-mediated soil N transformations; (ii) isotopic fractionation during plant N uptake or fractionation during plant–mycorrhiza transfer processes; (iii) differences in metabolic pathways and isotopic fractionation within the plant; or (iv) partitioning of available N resources (or pools) among plant groups or differential use of the same resources by different species, which seems to be the most probable route in the present case. A significant influence of N₂-fixing legumes on the N balance of the surrounding plant community was not detectable. This was confirmed by the results of an independent in situ removal experiment, showing that after 3 years there were no measurable differences in the frequency distribution between plots with and without N₂-fixing legumes.     

Starvation and low feeding levels result in an enrichment of 13C in lipids and 15N in protein of Nile tilapia Oreochromis niloticus L.

The influence of different feeding levels below and slightly above maintenance on whole body δ¹³C and δ¹⁵N values of Nile tilapia Oreochromis niloticus was examined. The energy budget of each fish was determined by indirect calorimetry. The δ¹³C values of the lipid-free material of Nile tilapia fed below and slightly above maintenance level did not differ between the feeding groups, but the δ¹³C values in the lipids and the δ¹⁵N values of the lipid-free material showed small but significant differences. Those fish with a negative lipid retention had significantly higher δ¹³C values in the lipid fraction compared to fish that synthesized fatty acids. There was a significant negative correlation between the amount of energy metabolized by the fish and both the δ¹³C values in the lipids and the δ¹⁵N values of the lipid-free material. Fasting and feeding below the maintenance level may influence the isotopic composition of animals and should therefore be considered in ecological and nutritional studies.     

Pan-European δ13C values of air and organic matter from forest ecosystems

We present carbon stable isotope, δ¹³C, results from air and organic matter samples collected during 98 individual field campaigns across a network of Carboeuroflux forest sites in 2001 (14 sites) and 2002 (16 sites). Using these data, we tested the hypothesis that δ¹³C values derived from large‐scale atmospheric measurements and models, which are routinely used to partition carbon fluxes between land and ocean, and potentially between respiration and photosynthesis on land, are consistent with directly measured ecosystem‐scale δ¹³C values. In this framework, we also tested the potential of δ¹³C in canopy air and plant organic matter to record regional‐scale ecophysiological patterns. Our network estimates for the mean δ¹³C of ecosystem respired CO₂ and the related ‘discrimination’ of ecosystem respiration, δ_er and Δ_er, respectively, were ?25.6±1.9‰ and 17.8 ±2.0‰ in 2001 and ?26.6±1.5‰ and 19.0±1.6‰ in 2002. The results were in close agreement with δ¹³C values derived from regional‐scale atmospheric measurement programs for 2001, but less so in 2002, which had an unusual precipitation pattern. This suggests that regional‐scale atmospheric sampling programs generally capture ecosystem δ¹³C signals over Europe, but may be limited in capturing some of the interannual variations. In 2001, but less so in 2002, there were discernable longitudinal and seasonal trends in δ_er. From west to east, across the network, there was a general enrichment in ¹³C (～3‰ and ～1‰ for the 2 years, respectively) consistent with increasing Gorczynski continentality index for warmer and drier conditions. In 2001 only, seasonal ¹³C enrichment between July and September, followed by depletion in November (from about ?26.0‰ to ?24.5‰ to ?30.0‰), was also observed. In 2001, July and August δ_er values across the network were significantly related to average daytime vapor pressure deficit (VPD), relative humidity (RH), and, to a lesser degree, air temperature (T_a), but not significantly with monthly average precipitation (P_m). In contrast, in 2002 (a much wetter peak season), δ_er was significantly related with T_a, but not significantly with VPD and RH. The important role of plant physiological processes on δ_er in 2001 was emphasized by a relatively rapid turnover (between 1 and 6 days) of assimilated carbon inferred from time‐lag analyses of δ_er vs. meteorological parameters. However, this was not evident in 2002. These analyses also noted corresponding diurnal cycles of δ_er and meteorological parameters in 2001, indicating a rapid transmission of daytime meteorology, via physiological responses, to the δ_er signal during this season. Organic matter δ¹³C results showed progressive ¹³C enrichment from leaves, through stems and roots to soil organic matter, which may be explained by ¹³C fractionation during respiration. This enrichment was species dependent and was prominent in angiosperms but not in gymnosperms. δ¹³C values of organic matter of any of the plant components did not well represent short‐term δ_er values during the seasonal cycle, and could not be used to partition ecosystem respiration into autotrophic and heterotrophic components.     

Physiological and isotopic (δ13C and δ18O) responses of three tropical tree species to water and nutrient availability

Water-use efficiency and stable isotope composition were studied in three tropical tree species. Seedlings of Tectona grandis , Swietenia macrophylla and Platymiscium pinnatum were grown at either high or low water supply, and with or without added fertilizer. These three species previously exhibited low, intermediate and high whole-plant water-use efficiency ( TE ) when grown at high water supply in unfertilized soil. Responses of TE to water and nutrient availability varied among species. The TE was calculated as experiment-long dry matter production divided by cumulative water use. Species-specific offsets were observed in relationships between TE and whole-plant ¹³C discrimination (Δ¹³C_p). These offsets could be attributed to a breakdown in the relationship between Δ¹³C_p and the ratio of intercellular to ambient CO₂ partial pressures ( c _i/ c _a) in P. pinnatum , and to variation among species in the leaf-to-air vapour pressure difference ( v ). Thus, a plot of v · TE against c _i/ c _a showed a general relationship among species. Relationships between δ ¹⁸O of stem dry matter and stomatal conductance ranged from strongly negative for S. macrophylla to no relationship for T. grandis . Results suggest inter-specific variation among tropical tree species in relationships between stable isotope ratios ( δ ¹³C and δ ¹⁸O) and the gas exchange processes thought to affect them.