Natural abundance of 13C and 15N in C3 and C4 vegetation of southern Africa: patterns and implications

The mean annual rainfall in southern Africa is found to explain over half of the observed variance in the stable nitrogen (N) isotopic signatures of C₃ vegetation in southern Africa (r²=0.54, P<0.01). The inverse relationship between the stable N isotopic signatures of foliar samples from C₃ vegetation and long‐term southern African rainfall is found on a scale larger than previously observed. A modest relationship is found between stable carbon (C) isotopic signatures of C₃ vegetation and rainfall across the region (r²=0.20, P<0.01). No such relationship is found between stable C and N isotopic signatures of C₄ vegetation and rainfall. The explanation of the relationship between ¹⁵N in C₃ vegetation and the mean annual rainfall presented here is that nutrient availability varies inversely with water availability. This suggests that water‐limited systems in southern Africa are more open in terms of nutrient cycling and therefore the resulting natural abundance of foliar ¹⁵N in these systems is enriched. The use of this relationship may be of value to those researchers modeling both the dynamics of vegetation and biogeochemistry across this region. The use of the isotopic enrichment in C₃ vegetation as a function of rainfall may provide an insight into nutrient cycling across the semi‐arid and arid regions of southern Africa. This finding has implications for the study of global change, especially as it relates to vegetation responses to changing regional rainfall regimes over time.     

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.     

δ 13C of CO2 respired in the dark in relation to δ13C of leaf carbohydrates in Phaseolus vulgaris L. under progressive drought

The variations in δ ¹³C in both leaf carbohydrates (starch and sucrose) and CO₂ respired in the dark from the cotyledonary leaves of Phaseolus vulgaris L. were investigated during a progressive drought. As expected, sucrose and starch became heavier (enriched in ¹³C) with decreasing stomatal conductance and decreasing p _i/ p _a during the first half (15 d) of the dehydration cycle. Thereafter, when stomata remained closed and leaf net photosynthesis was near zero, the tendency was reversed: the carbohydrates became lighter (depleted in ¹³C). This may be explained by increased p _i/ p _a but other possible explanations are also discussed. Interestingly, the variations in δ ¹³C of CO₂ respired in the dark were correlated with those of sucrose for both well-watered and dehydrated plants. A linear relationship was obtained between δ ¹³C of CO₂ respired in the dark and sucrose, respired CO₂ always being enriched in ¹³C compared with sucrose by ≈ 6‰. The whole leaf organic matter was depleted in ¹³C compared with leaf carbohydrates by at least 1‰. These results suggest that: (i) a discrimination by ≈ 6‰ occurs during dark respiration processes releasing ¹³C-enriched CO₂; and that (ii) this leads to ¹³C depletion in the remaining leaf material.     

Canopy-scale δ13C of photosynthetic and respiratory CO2 fluxes: observations in forest biomes across the United States

The δ¹³C values of atmospheric carbon dioxide (CO₂) can be used to partition global patterns of CO₂ source/sink relationships among terrestrial and oceanic ecosystems using the inversion technique. This approach is very sensitive to estimates of photosynthetic ¹³C discrimination by terrestrial vegetation (Δ_A), and depends on δ¹³C values of respired CO₂ fluxes (δ¹³C_R). Here we show that by combining two independent data streams – the stable isotope ratios of atmospheric CO₂ and eddy‐covariance CO₂ flux measurements – canopy scale estimates of Δ_A can be successfully derived in terrestrial ecosystems. We also present the first weekly dataset of seasonal variations in δ¹³C_R from dominant forest ecosystems in the United States between 2001 and 2003. Our observations indicate considerable summer‐time variation in the weekly value of δ¹³C_R within coniferous forests (4.0‰ and 5.4‰ at Wind River Canopy Crane Research Facility and Howland Forest, respectively, between May and September). The monthly mean values of δ¹³C_R showed a smaller range (2–3‰), which appeared to significantly correlate with soil water availability. Values of δ¹³C_R were less variable during the growing season at the deciduous forest (Harvard Forest). We suggest that the negative correlation between δ¹³C_R and soil moisture content observed in the two coniferous forests should represent a general ecosystem response to the changes in the distribution of water resources because of climate change. Shifts in δ¹³C_R and Δ_A could be of sufficient magnitude globally to impact partitioning calculations of CO₂ sinks between oceanic and terrestrial compartments.     

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.     

A new method for measuring 13carbon abundance in tracer experiments

Abstract. A new technique for the precise measurement of ¹³C-abundance and concentration is described. It is based on the differences in infra-red spectra between ¹²CO₂ and ¹³CO₂ and can be applied to gas mixtures or organic materials which have been oxidized to CO₂. The gas mixture is first dried and then passed through two infra-red gas analysers (IRGAs) connected in parallel. The two IRGAs are fitted with different optical filters so they differ in their relative sensitivities to ¹²CO₂ and ¹³CO₂. Once these sensitivities are known then simple algebra allows the concentrations of ¹²CO₂ and ¹³CO₂ to be calculated from the two readings. Two variants of this basic system have been tested. In both, one IRGA was a normal commercial instrument with a narrow band pass interference filter making it highly specific for ¹²CO₂; the second instrument was fitted with either a wide-band pass filter covering both the ¹²CO₂ and ¹³CO₂ absorption bands, or a narrow band pass filter specific for ¹³CO₂. These variations convey different advantages in operation. The wide-band system can be easily calibrated using a single natural abundance ¹²CO₂ standard but is only moderately precise at low abundances. It is particularly valuable for continuous monitoring of the relatively high abundance sources used in plant photosynthesis experiments. The narrow-band system gives high precision but requires a more complex standardization procedure. It is recommended for measurements on low-abundance samples resulting from tracer experiments. Here, its high sensitivity permits measurements on samples as small as 3 μmole C, thus enabling plant fractions and individual metabolites to be investigated. While the wide-band system can be manually operated under field conditions, it is necessary for highest precision to use computerized data collection and linearization. These processes are described, as are novel techniques for standardization, the preparation of small quantities of CO₂ of known abundance, and the transfer of gas samples from oxidizer to analyser. Determinations by the wide band system of % abundance in standard gas mixtures gave a standard error of ±0.03% but this increased to over ±0.1% for abundances below 20%. Corresponding values for the narrow-band system were ±0.01% over the whole abundance range an accuracy almost identical to that observed with an organic mass spectrometer. Two pulse-chase experiment with ¹³CO₂ are described in which the technique was used for studies on growth and metabolism of Lemna minor. The first demonstrated that ¹³C-accumulation within the plants matched closely the predictions from the net assimilation rate and measurements of ¹³C-abundance in the gas phase. The second revealed the rapid changes in the ¹³C-labelling of some plant components during pulse and chase phases. These examples demonstrate the potential of the method for studies in plant physiology and biochemistry. In view of its relative cheapness, ease of maintenance and operation, accuracy, and sensitivity, it is suggested that this new method may encourage a wider use of the safe stable ¹³C for biological and medical applications.     

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].     

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.     

Beyond CO2-fixation by Rubisco – an interpretation of 13C/12C variations in tree rings from novel intra-seasonal studies on broad-leaf trees

Evidence is presented for a very specific, seasonally recurring tri‐phase carbon isotope pattern in tree rings of broad‐leaf deciduous tree species. It is derived from highly resolved intra‐annual measurements of ¹³C/¹²C ratios of wood and cellulose from tree rings of Fagus sylvatica, Populus nigra, Quercus petraea and Morus alba. Investigations on δ¹³C from buds and leaves of Fagus sylvatica revealed a similar tri‐phase δ¹³C pattern. At the very beginning of a growing season, the δ¹³C trend of tree rings and foliage shows a marked increase of up to 5‰. The maximum δ¹³C‐value of each vegetation period always occurs in young heterotrophic leaves shortly after bud burst and persistently in the early wood of each tree ring, when growth depends on carbon reserves. Thereafter, δ¹³C profiles represent the autotrophic stage of the leaves, which show different patterns of variation, by and large characterized by a decline. The minimum δ¹³C‐value always shows up in the late wood of each tree ring. At the very end of each tree ring δ¹³C‐values start rising again. This increase in δ¹³C marks the gradual switch‐over to storage‐dependent growth and can also be observed in senescent leaves. Seasonal changes of more than 4‰ were measured, whereas contiguous δ¹³C values rarely differed from each other by more than 0.3‰. This tri‐phase pattern cannot be explained by the common model of carbon isotope fractionation during photosynthesis. It appears to be primarily an indication of seasonal changes in down‐stream processes of the carbohydrate metabolism. Environmental influences on the carbon isotope fractionation during photosynthesis are presumably of secondary importance and expressed by certain peculiarities showing up during the autotrophic phase, i.e. the mid‐section of the seasonal δ¹³C pattern.     

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.     

Soil carbon inventories and δ13C along a moisture gradient in Botswana

We present a study of soil organic carbon (SOC) inventories and δ¹³C values for 625 soil cores collected from well‐drained, coarse‐textured soils in eight areas along a 1000 km moisture gradient from Southern Botswana, north into southern Zambia. The spatial distribution of trees and grass in the desert, savannah and woodland ecosystems along the transect control large systematic local variations in both SOC inventories and δ¹³C values. A stratified sampling approach was used to smooth this variability and obtain robust weighted‐mean estimates for both parameters. Weighted SOC inventories in the 0–5 cm interval of the soils range from 7 mg cm^?2 in the driest area (mean annual precipitation, MAP=225 mm) to 41±12 mg cm^?2 in the wettest area (MAP=910 mm). For the 0–30 cm interval, the inventories are 37.8 mg cm^?2 for the driest region and 157±33 mg cm^?2 for the wettest region. SOC inventories at intermediate sites increase as MAP increases to approximately 400–500 mm, but remain approximately constant thereafter. This plateau may be the result of feedbacks between MAP, fuel load and fire frequency. Weighted δ ¹³C values decrease linearly in both the 0–5 and 0–30 cm depth intervals as MAP increases. A value of –17.5±1.0‰ characterizes the driest areas, while a value of ?25±0.7‰ characterizes the wettest area. The decrease in δ ¹³C value with increasing MAP reflects an increasing dominance of C₃ vegetation as MAP increases. SOC in the deeper soil (5–30 cm depth) is, on average, 0.4±0.3‰ enriched in ¹³C relative to SOC in the 0–5 cm interval.     

Photosynthesis and the influence of CO2-enrichment on δ13C values in a C3 halophyte

Abstract Shifts in ?¹³C of the graminaceous C₃ halophyte Puccinellia nuttalliana (Schultes) Hitch. can be induced by salinization. To investigate this phenomenon, three approaches were taken: assay of carboxylases, CO₂-enrichment studies, and gas exchange analysis. Although ribulose-1,5-bisphosphate carboxylase activity decreased with salinity, phosphoenolpyruvate carboxylase activity did not increase and its levels were not atypical of C₃ plants. When plants were grown at four NaCl concentrations under atmospheres of 310 and 1300 cm³ m^?3 CO₂, the CO₂-enrichment enhanced the effects of salinity on ?¹³C. This is consistent with a biophysical explanation for salt-induced shifts in ?¹³C, whereby there is a steepening of the CO₂ diffusion gradient into the leaf. Gas exchange analysis indicated that intercellular CO₂ concentrations were depressed in the leaves of salt-affected plants. This resulted from a greatly decreased stomatal conductance coupled with only small effects on intrinsic photosynthetic capacity. Water-use efficiency was enhanced.     

Changes in leaf Δ13C of herbarium plant species during the last 3 centuries of CO2 increase

Δ¹³C were determined for herbarium specimens of 12 C₃ plants (trees, shrubs and herbs) collected during the last 240 years in Catalonia, an area with a Mediterranean climate. Values were 19.91 (S.E. = 0.32, n= 21) for 1750–1760, 19–86 (S.E. = 0.21, n= 49) for 1850–1890 and 19.95 (S.E. = 0.29, n= 25) for 1925–1950, and decreased significantly to 18.87 (S.E. = 0.31, n= 29) for 1982–1988. More irregular temporal changes were found in Δ¹³C of two C₄ species, but they also suggest a decrease in discrimination in recent decades. These results suggest that either carbon assimilation rates have increased or stomatal conductance has decreased, and therefore, that there has been an increase in water use efficiency over the last few decades.