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.     

Carbon dioxide fixation in orchid aerial roots

Acidity fluctuation, CO₂ gas exchange, δ¹³C value, PEP carboxylase and RuBP carboxylase activities in aerial roots of selected thick-leaved orchid hybrids ( Arachnis and Aranthera ) were studied. Both aerial roots and leaves showed acidity fluctuation over a 24 h period. Dark acidification in aerial roots was enhanced at low temperature (15°C). Aerial roots had δ¹³C values close to those of leaves which have been previously demonstrated to possess crassulacean acid metabolism. Variation in δ¹³C values along the length of the roots was observed; the root tip having a less negative δ¹³C value (—13.34%‰) than the older portions of the roots (—14.55%‰). There was no net CO₂ fixation by aerial root, although ¹⁴³²CO₂ fixation was observed in light and in darkness. The pattern of fluctuation in activities of PEP carboxylase and RuBP carboxylase in aerial roots was similar to that obtained for the leaves. In both aerial roots and leaves, PEP carboxylase activity was several times higher than that of RuBP carboxylase.     

Photosynthesis and carbon metabolism in photoautotrophic cell suspensions of Chenopodium rubrum from different phases of batch growth

Rapidly dividing photoautotrophic cell suspensions from Chenopodium rubrum L. assimilated about 85 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. During the late stationary phase of culture growth, CO₂ fixation rate was reduced to about 60 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. Actively dividing cells characteristically incorporated a smaller proportion of ¹⁴C into starch than cells from non-dividing stationary phases. In rapidly dividing cells, [¹⁴C]-turnover from free sugars, sugar-phosphates, organic and amino acids was substantially higher compared to non-dividing cells from stationary growth phase. Higher proportions of photosynthetically fixed carbon were channelled into proteins, lipids and structural components in actively dividing cells than in non-dividing cells. In the latter. ¹⁴C was preferentially channeled into starch, and a striking increase in starch accumulation was observed. The transfer of non-dividing, stationary growth-phase cells into fresh culture medium resulted in an increase in the maximum extractable activities of some enzymes involved in the glycolytic and dark respiratory pathways and in the citric acid cycle. In contrast, the maximum extractable activities of the chloroplastic enzymes, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.38) and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were highest after the cells had reached the stationary growth phase.     

Comparative studies of sucrose and mannitol utilization in celery (Apium graveolens)

Utilization of sucrose and mannitol, the major forms of translocatable assimilate in celery ( Apium graveolens L. cv. Giant Pascal), was investigated in intact plants, excised leaves and leaf discs by estimating the soluble carbohydrate pools, starch levels and oxidation of [¹⁴C]-sucrose or mannitol in the light and after extended dark treatments. In detached mature fully-expanded leaves, mannitol pools remained constant, while sucrose decreased during a 48 h dark treatment. In attached leaves on plants trimmed to a single compound leaf, however, mannitol levels decreased after a dark treatment. In leaf discs floated on bathing solutions containing [¹⁴C]-sucrose or [¹⁴C]-mannitol, oxidation of mannitol was restricted to young leaf tissues, whereas sucrose was metabolized to CO₂ regardless of leaf age. Uptake of labelled mannitol, however, was greater than that of sucrose in the light in leaves of every age. Although both mannitol and sucrose are translocated out of leaf tissues, leaf age differences indicate that, unlike sucrose, mannitol utilization is restricted to active sink tissues. The results suggest different roles for mannitol and sucrose with mannitol representing a more rigorously sequestered transport carbohydrate.     

13C-isotope analyses reveal that chemolithoautotrophic Gamma- and Epsilonproteobacteria feed a microbial food web in a pelagic redoxcline of the central Baltic Sea

Marine pelagic redoxclines are zones of high dark CO₂ fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO₂ fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO₂ incorporation in ¹³C-bicarbonate pulse experiments. The incorporation of ¹³C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in ¹³C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO₂-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the ¹³C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO₂-fixing Proteobacteria within this habitat.     

PYRUVATE METABOLISM IN THE LOBSTER NERVE AS AFFECTED BY THE PARTIAL PRESSURE OF CARBON DIOXIDE: OBSERVATIONS ON THE SYNTHESIS OF ACETYLCHOLINE AND ON METABOLIC COMPARTMENTATION

Abstract— In the lobster nerve the fixation of CO, at various levels of _pCO2 was studied by the incorporation of [l-¹⁴C]pyruvate. Incorporation of ¹⁴C was solely dependent on CO₂ fixation since the C-1 was decarboxylated in the formation of acetyl-CoA. Paired-nerve studies with [2-¹⁴C]pyruvate afforded a study of pyruvate metabolism in the lobster nerve. [I¹⁴C]Pyruvate was incorporated to nearly the same extent at all levels of pCO₂ including zero pCO₂, a finding that suggested metabolic recycling of CO₂. The magnitude of the metabolic recycling of C-1 of pyruvate or pyruvate dismutation was estimated to be nearly 20 per cent of total CO₂ fixation. Re-evaluation of the relative contributions of the CO₂ fixation. and acetyl-CoA pathways on the basis of more extensive data gave a ratio of 2:3.
The pCO₂ affected synthesis of ACh and the level of citrate. With increasing pCO₂, the specific radioactivity of ACh decreased much more than the content of ACh. The decrease in the specific radioactivity of ACh but not that of citrate further suggested metabolic compartmentation. The implication of these findings is discussed.
Alanine functioned as a metabolic sink for the incorporated pyruvate. Pyruvate levels were estimated to be approximately 0.1 nmol/mg of protein.     

Diurnal and seasonal variation in the carbon isotope composition of leaf dark-respired CO2 in velvet mesquite (Prosopis velutina)

We evaluated diurnal and seasonal patterns of carbon isotope composition of leaf dark-respired CO₂ ( δ ¹³C_l) in the C₃ perennial shrub velvet mesquite ( Prosopis velutina ) across flood plain and upland savanna ecosystems in the south-western USA. δ ¹³C_l of darkened leaves increased to maximum values late during daytime periods and declined gradually over night-time periods to minimum values at pre-dawn. The magnitude of the diurnal shift in δ ¹³C_l was strongly influenced by seasonal and habitat-related differences in soil water availability and leaf surface vapour pressure deficit. δ ¹³C_l and the cumulative flux-weighted δ ¹³C value of photosynthates were positively correlated, suggesting that progressive ¹³C enrichment of the CO₂ evolved by darkened leaves during the daytime mainly resulted from short-term changes in photosynthetic ¹³C discrimination and associated shifts in the δ ¹³C signature of primary respiratory substrates. The ¹³C enrichment of dark-respired CO₂ relative to photosynthates across habitats and seasons was 4 to 6‰ at the end of the daytime period (1800 h), but progressively declined to 0‰ by pre-dawn (0300 h). The origin of night-time and daytime variations in δ ¹³C_l is discussed in terms of the carbon source(s) feeding respiration and the drought-induced changes in carbon metabolism.     

Modelling environmental controls on ecosystem photosynthesis and the carbon isotope composition of ecosystem-respired CO2 in a coastal Douglas-fir forest

We developed and applied an ecosystem-scale model that calculated leaf CO₂ assimilation, stomatal conductance, chloroplast CO₂ concentration and the carbon isotope composition of carbohydrate formed during photosynthesis separately for sunlit and shaded leaves within multiple canopy layers. The ecosystem photosynthesis model was validated by comparison to leaf-level gas exchange measurements and estimates of ecosystem-scale photosynthesis from eddy covariance measurements made in a coastal Douglas-fir forest on Vancouver Island. A good agreement was also observed between modelled and measured δ ¹³C values of ecosystem-respired CO₂ ( δ _R). The modelled δ _R values showed strong responses to variation in photosynthetic photon flux density (PPFD), air temperature, vapour pressure deficit (VPD) and available soil moisture in a manner consistent with leaf-level studies of photosynthetic ¹³C discrimination. Sensitivity tests were conducted to evaluate the effect of (1) changes in the lag between the time of CO₂ fixation and the conversion of organic matter back to CO₂; (2) shifts in the proportion of autotrophic and heterotrophic respiration; (3) isotope fractionation during respiration; and (4) environmentally induced changes in mesophyll conductance, on modelled δ _R values. Our results indicated that δ _R is a good proxy for canopy-level C _c/ C _a and ¹³C discrimination during photosynthetic gas exchange, and therefore has several applications in ecosystem physiology.     

Light period of Crassulacean Acid Metabolism: Control of carbon transfer from malic acid to carbohydrates by CO2 concentration

In the CAM plants, Kalanchoë tubiflora (Harvey) Hasset, Sedum morganianum E. Walth and Sedum rubrotinctum R. T. Clausen, the effects of CO₂ concentrations on the light-dependent ¹⁴C transfer from the nocturnally synthetized [14C]-malic acid to starch have been studied. CO₂ concentrations up to 5 × 103 μ1 1–1 did not inhibit this carbon transfer. Higher CO₂ concentrations, however, were increasingly inhibitory. At 104 μl 1–1 CO₂, the carbon transfer was practically prevented.
The malic acid consumption in the light showed the same response to CO₂ concentrations as the [^l4C]-transfer. Photosynthesis itself was not inhibited by the CO₂ concentrations applied. It is assumed that, during phase III of CAM, light controls the internal CO₂ concentration via photosynthesis; and that the internal CO2 concentration then controls the rate of malate decarboxylation.     

Photoinhibition of respiratory CO2 release in the green alga Scenedesmus

¹⁴CO₂ evolution of prelabeled Scenedesmus obliquus Kütz, has been followed in the dark and in the light. In the light, no carbon dioxide is evolved. Addition of unlabeled NaHCO, leads to ¹⁴CO₂ release attaining 20 to 30% of the dark rate. Double-reciprocal plots of NaHCO₃ concentrations vs ¹⁴CO₂ release results in a straight line, indicative of competition between exogenously supplied bicarbonate and endogenously evolved carbon dioxide. With this method, it is possible to measure CO₂ evolved by respiration in the light and to show that true photoinhibition of respiration occurs in Scenedesmus . In the light. DCMU substantially increases ¹⁴CO₂ evolution; in the presence of the uncoupler carbonyl cyanide- m -chlorophenylhydrazone. ¹⁴CO₂ evolution is comparable to that in the dark. ¹⁴CO₂ release and oxygen uptake in the dark are only slightly affected by cyanide, indicative of a cyanide-resistant respiration and/or fermentation as the essential CO₂-yielding processes in the presence of cyanide. These results, compared with concurrent ATP levels, lead us to assume that energy charge is not the only factor responsible for photoinhibition of respiration.     

Elevated atmospheric CO2 does not affect per se the preference for symbiotic nitrogen as opposed to mineral nitrogen of Trifolium repens L.

The objective of this investigation was to examine the effect of an elevated atmospheric CO₂ partial pressure ( p CO₂) on the N-sink strength and performance of symbiotic N₂ fixation in Trifolium repens L. cv. Milkanova. After initial growth under ambient p CO₂ in a nitrogen-free nutrient solution, T. repens in the exponential growth stage was exposed to ambient and elevated p CO₂ (35 and 60 Pa) and two levels of mineral N (N-free and 7·5 mol m^–3 N) for 36 d in single pots filled with silica sand in growth chambers. Elevated p CO₂ evoked a significant increase in biomass production from day 12 after the start of CO₂ enrichment. For plants supplied with 7·5 mol m^–3 N, the relative contribution of symbiotically fixed N (%N_sym) as opposed to N assimilated from mineral sources (¹⁵N-isotope-dilution method), dropped to 40%. However, in the presence of this high level of mineral N, %N_sym was unaffected by atmospheric p CO₂ over the entire experimental period. In plants fully dependent on N₂ fixation, the increase in N yield reflects a stimulation of symbiotic N₂ fixation that was the result of the formation of more nodules rather than of higher specific N₂ fixation. These results are discussed with regard to physiological processes governing symbiotic N₂ fixation and to the response of symbiotic N₂ fixation to elevated p CO₂ in field-grown T. repens .     

Effect of elevated CO2 on carbon partitioning and exudate release from Plantago lanceolata seedlings

Plantago lanceolata L. seedlings were grown in sand microcosm units over a 43‐day experimental period under two CO₂ regimes (800 or 400 µmol mol⁻¹) to investigate the effect of elevated atmospheric CO₂ concentration on carbon partitioning and exudate release. Total organic carbon (TOC) content of the collected exudate material was measured throughout the experimental period. After 42 days growth the seedlings were labelled with [¹⁴C]‐CO₂ and the fate of the label within the plant and its release by the roots monitored. Elevated CO₂ significantly (P ≤ 0.001) enhanced shoot, root and total dry matter production although the R:S ratio was unaltered, suggesting no alteration in gross carbon partitioning. The cumulative release of TOC (in mg C) over 0‐42 days was unaltered by CO₂ treatment however, when expressed as a percentage of net assimilated C, ambient‐grown plants released a significantly (P≤ 0.001) higher percentage from their roots compared to elevated CO₂‐grown plants (i.e. 8 vs 3%). The distribution of ¹⁴C‐label was markedly altered by CO₂ treatment with significantly (P≤ 0.001) greater per cent label partitioned to the roots under elevated CO₂. This indicates increased partitioning of recent assimilate below‐ground under elevated CO₂ treatment although there was no significant difference in the percentage of ¹⁴C‐label released by the roots. Comparison of plant C budgets based on ¹⁴C‐pulse‐chase methodology and TOC measurements is discussed.     

Effects of mycorrhizal colonization on biomass production and nitrogen fixation of black locust (Robinia pseudoacacia) seedlings grown under elevated atmospheric carbon dioxide

Interactive effects of elevated atmospheric CO₂ and arbuscular mycorrhizal (AM) fungi on biomass production and N₂ fixation were investigated using black locust ( Robinia pseudoacacia ). Seedlings were grown in growth chambers maintained at either 350 μmol mol⁻¹ or 710 μmol mol⁻¹ CO₂. Seedlings were inoculated with Rhizobium spp. and were grown with or without AM fungi. The ¹⁵N isotope dilution method was used to determine N source partitioning between N₂ fixation and inorganic fertilizer uptake. Elevated atmospheric CO₂ significantly increased the percentage of fine roots that were colonized by AM fungi. Mycorrhizal seedlings grown under elevated CO₂ had the greatest overall plant biomass production, nodulation, N and P content, and root N absorption. Additionally, elevated CO₂ levels enhanced nodule and root mass production, as well as N₂ fixation rates, of non- mycorrhizal seedlings. However, the relative response of biomass production to CO₂ enrichment was greater in non-mycorrhizal seedlings than in mycorrhizal seedlings. This study provides strong evidence that arbuscular mycorrhizal fungi play an important role in the extent to which plant nutrition of symbiotic N₂-fixing tree species is affected by enriched atmospheric CO₂.     

Regulation of Rubisco activity in vivo

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is not able to achieve and maintain adequate CO₂ and Mg²⁺ activation under physiological conditions. Higher plants and green algae contain Rubisco activase, a soluble protein which not only facilitates Rubisco activation in situ but also regulates enzyme activity in response to irradiance and other factors. Regulation of Rubisco activity by modulation of activation state coordinates the rate of CO₂ fixation with the rate of substrate regeneration. This regulation may be required to ensure that the levels of photosynthetic metabolites in the chloroplast are optimal for photosynthesis under a variety of environrmental conditions. Some plant species also appear to regulate Rubisco activity by synthesizing 2-carboxyarabinitol 1-phosphate, an inhibitor of Rubisco in the dark. This inhibitor may function primarily as a regulator of metabolite binding in the dark rather than as a modulator of Rubisco activity in the light.     

The inhibition of photosynthesis and photorespiration in isolated mesophyll cells of Phaseolus and Lycopersicon by reduced osmotic potentials

Mesophyll cells isolated from Phaseolus vulgaris and Lycopersicon esculentum show decreasing photosynthetic rates when suspended in media containing increasing concentrations of osmoticum. The photosynthetic activity was sensitive to small changes in osmotic potential over a range of sorbitol concentrations from 0.44 M (−1.08 MPa) to 0.77 M (−1.88 MPa). Photorespiration assayed by ¹⁴CO₂ release in CO₂-free air and by ¹⁴CO₂ release from the oxidation of [1–¹⁴C] glycolate also decreased as the osmotic potential of the incubation medium was reduced. The CO₂ compensation points of the cells increased with increasing concentration of osmoticum from approximately 60 μ I⁻¹1 at −1.08 MPa to 130 μl 1⁻¹ for cells stressed at −1.88 MPa. Changes in photosynthetic and photorespiratory activities occurred at moderate osmotic potentials in these cells suggesting that in whole leaves during a reduction in water potential, non- stomatal inhibition of CO₂ assimilation and glycolate pathway metabolism occurs simultaneously with stomatal closure.     

Re-evaluation of proposed C4 photosynthetic characteristics in the genus Larix

It has been suggested previously that Japanese larch ( Larix kaempferi ) exhibits characteristics of C₄ photosynthesis. To further evaluate this suggestion, stable carbon isotope ratios were determined for leaf and bark tissue of Larix gmelini, L. kaempferi, L. laricina, L. Iyallii, L. occidentalis , and L. sibirica. All δ¹³C values were more negative than –22‰. Short-term labeling with ¹⁴CO₂ showed that phosphoglyceric acid and other phosphorylated compounds were the first products of photosynthesis in L. sibirica. Both of these results strongly suggest that the initial fixation of atmospheric CO₂ in these six Larix species is accomplished solely via the C₃ photosynthetic pathway.     

Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain

Abstract: Chains of lumbar sympathetic ganglia from 15-day-old chicken embryos were incubated for 4 h at 36°C in a bicarbonate-buffered salt solution equilibrated with 5% CO₂-95% O₂. Glucose (1–10 m M ), lactate (1–10 m M ), [U-¹⁴C]glucose, [1-¹⁴C]glucose, [6-¹⁴C]glucose, and [U-¹⁴C]lactate were added as needed. ¹⁴CO₂ output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO₂ from [U-¹⁴C]glucose, and glucose reduced that from [U-¹⁴C]lactate. When using uniformly labeled substrates in the presence of 5.5 m M glucose, the output of CO₂ from lactate exceeded that from glucose when the lactate concentration was >2 m M . The combined outputs at each concentration tested were greater than those from either substrate alone. The ¹⁴CO₂ output from [1-¹⁴C]glucose always exceeded that from [6-¹⁴C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood-brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.     

Using stable isotopes to determine soil carbon input differences under ambient and elevated atmospheric CO2 conditions

Quantitative estimates of soil C input under ambient (35 Pa) and elevated (60 Pa) CO₂-partial pressure (pCO₂) were determined in a Free-Air Carbon dioxide Enrichment (FACE) experiment. To facilitate ¹³C-tracing, Trifolium repens L. was grown in a soil with an initial δ¹³C distinct by at least 5‰ from the δ¹³C of T. repens grown under ambient or elevated pCO₂. A shift in δ¹³C of the soil organic C was detected after one growing season. Calculated new soil C inputs in soil under ambient and elevated pCO₂ were 2 and 3 t ha^–1, respectively. Our findings suggest that under elevated CO₂ conditions, soil C sequestration may be altered by changes in plant biomass production and quality.     

Time-course of uptake of dissolved inorganic carbon through willow roots in light and in darkness

Uptake of dissolved inorganic carbon (DIC) from a nutrient solution by willow roots was measured in light and darkness and the distribution in the plant of DIC taken up by the roots was determined. It was also studied whether the transport system could be activated by preincubation with dissolved inorganic carbon.
Willow plants ( Salix cv. Aquatica gigantea) grown in hydroponic culture media were preincubated for 2 days with or without 0.74 mM NaHCO₃. After preincubation, either unlabelled or [¹⁴C]-labelled NaHCO₃ was injected into the media and after 1, 5, 10 and 24 h either in light or in darkness the plants were harvested in pieces into liquid nitrogen, lyophilized and burned in a combustion chamber.
¹⁴C was transported through the roots to the shoots and leaves both in light and in darkness, although incorporation of ¹⁴C in darkness was only half of that in light at the end of the 24-h feeding period. Both in light and in darkness the amount of ¹⁴C increased in all parts of willow plants with time. In light the rate of labelling was highest into cuttings and shoots. In darkness more than half of the total label was detected in cuttings of both the non-activated and the activated treatments.
In the shoots the middle part was most strongly labelled after 5 and 10 h, but after 24 h ¹⁴C moved towards the base of the shoot. In the leaves at all feeding times most radioactivity was incorporated into the young, fully open leaves on the upper part of the shoots. Preincubation of plants with unlabelled NaHCO³ in growth media had no clear effect on the rate of DIC uptake either in light or in darkness.     

Symbiotic N2 fixation in a high Alpine grassland: effects of four growing seasons of elevated CO2

1. Increasing carbon dioxide concentration (E: 680 μl CO₂ litre^–1 vs ambient, A: 355 μl CO₂ litre^–1) around late-successional Alpine sedge communities of the Swiss Central Alps (2450 m) for four growing seasons (1992–1995) had no detectable effect on symbiotic N₂ fixation in Trifolium alpinum —the sole N₂-fixing plant species in these communities (74 ± 30 mg N m^–2 year^–1, A and E plots pooled).
2. This result is based on data collected in the fourth growing season showing that elevated CO₂ had no effect on Trifolium above-ground biomass (4·4 ± 1·7 g m^–2, A and E plots pooled, n = 24) or N content per unit land area (124 ± 51 mg N m^–2, A and E pooled), or on the percentage of N Trifolium derived from the atmosphere through symbiotic N₂ fixation (%Ndfa: 61·0 ± 4·1 across A and E plots) estimated using the ¹⁵N dilution method.
3. Thus, it appears that N inputs to this ecosystem via symbiotic N₂ fixation will not be dramatically affected in the foreseeable future even as atmospheric CO₂ continues to rise.