Evidence that P deficiency induces N feedback regulation of symbiotic N2 fixation in white clover (Trifolium repens L.)

Trifolium repens L. was grown to test the following hypotheses: when P is deficient (i) N2 fixation decreases as a result of the plant's adaptation to the low N demand, regulated by an N feedback mechanism, and (ii) the decrease in the photosynthetic capacity of the leaves does not limit N2 fixation. Severe P deficiency prevented nodulation or stopped nodule growth when the P deficiency occurred after the plants had formed nodules. At low P, the proportion of whole-plant-N derived from symbiotic N2 fixation decreased, whereas specific N2 fixation increased and compensated partially for poor nodulation. Leaf photosynthesis was reduced under P deficiency due to low Vc,max and Jmax. Poor growth or poor performance of the nodules was not due to C limitation, because (i) the improved photosynthetic performance at elevated pCO2 had no effect on the growth and functioning of the nodules, (ii) starch accumulated in the leaves, particularly under elevated pCO2, and (iii) the concentration of WSC in the nodules was highest under P deficiency. Under severe P deficiency, the concentrations of whole-plant-N and leaf-N were the highest, indicating that the assimilation of N exceeded the amount of N required by the plant for growth. This was clearly demonstrated by a strong increase in asparagine concentrations in the roots and nodules under low P supply. This indicates that nodulation and the proportion of N derived from symbiotic N2 fixation are down-regulated by an N feedback mechanism.     

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 .     

Mycorrhizas improve nitrogen nutrition of Trifolium repens after 8 yr of selection under elevated atmospheric CO2 partial pressure

Altered environmental conditions may change populations of arbuscular mycorrhizal fungi and thereby affect mycorrhizal functioning. We investigated whether 8 yr of free-air CO2 enrichment has selected fungi that differently influence the nutrition and growth of host plants. In a controlled pot experiment, two sets of seven randomly picked single spore isolates, originating from field plots of elevated (60 Pa) or ambient CO2 partial pressure (pCO2), were inoculated on nodulated Trifolium repens (white clover) plants. Fungal isolates belonged to the Glomus claroideum or Glomus intraradices species complex, and host plants were clonal micropropagates derived from nine genets. Total nitrogen (N) concentration was increased in leaves of plants inoculated with fungal isolates from elevated-pCO2 plots. These isolates took up nearly twice as much N from the soil as isolates from ambient-pCO2 plots and showed much greater stimulation of biological N2 fixation. The morpho-species identity of isolates had a more pronounced effect on N2 fixation and on root length colonized than isolate identity. We conclude that rising atmospheric pCO2 may select for fungal strains that will help their host plants to meet increased N demands.     

Phosphorus availability and elevated CO2 affect biological nitrogen fixation and nutrient fluxes in a clover-dominated sward

The response of biological nitrogen fixation (BNF) to elevated CO(2) was examined in white clover (Trifolium repens)-dominated swards under both high and low phosphorus availability. Mixed swards of clover and buffalo grass (Stenotaphrum secundatum) were grown for 15 months in 0.2 m2 sand-filled mesocosms under two CO2 treatments (ambient and twice ambient) and three nutrient treatments [no N, and either low or high P (5 or 134 kg P ha(-1)); the third nutrient treatment was supplied with high P and N (240 kg N ha(-1))]. Under ambient CO2, high P increased BNF from 410 to 900 kg ha(-1). Elevated CO2 further increased BNF to 1180 kg ha(-1) with high P, but there was no effect of CO2 on BNF with low P. Allocation of N belowground increased by approx. 50% under elevated CO2 irrespective of supplied P. The results suggest that where soil P availability is low, elevated CO2 will not increase BNF, and pasture quality could decrease because of a reduction in aboveground N.     

Nitrogen‐15 budget in model ecosystems of white clover and perennial ryegrass exposed for four years at elevated atmospheric pCO2

Although there are many indications that N cycling in grassland ecosystems changes under elevated atmospheric CO₂ partial pressure (pCO₂), most information has been obtained in short‐term studies. Thus, N budgets were established for four years under ambient and 60 Pa pCO₂ at two levels of N fertilization in two contrasting model ecosystems: Trifolium repens L. (white clover) and Lolium perenne L. (perennial ryegrass) were planted in soil in boxes in the Swiss FACE experiment. While T. repens showed an 80% increase in harvested biomass with no change in biomass allocation under elevated atmospheric pCO₂ compared to ambient conditions, L. perenne showed an increase only in the biomass of the roots. During the four years of the experiment, the systems gained N both from N retained in the soil and from stubble/stolon and roots left after the final harvest; in total between 11 and 86 gN m⁻². Nitrogen retention in the soil was between 4 and 64 g m². The L. perenne system gained the most N and retained the most N in the soil at high N fertilization and elevated atmospheric pCO₂. The input of new C and N into the soil correlated well in the L. perenne systems but not in the T. repens systems. Elevated atmospheric pCO₂ led neither to an increase in N retention in the soil nor did it reduce the loss of N from the soil. In the L. perenne systems, N fertilization played the main role in both the retention of N and the sequestration of C, while in the T. repens systems symbiotic N₂ fixation may have controlled N retention in the soil.     

Variation in acclimation of photosynthesis in Trifolium repens after eight years of exposure to Free Air CO2 Enrichment (FACE)

The initial stimulation of photosynthesis observed on elevation of [CO2] in grasslands has been predicted to be a transient phenomenon constrained by the loss of photosynthetic capacity due to other limitations, notably nutrients and sinks for carbohydrates. Legumes might be expected partially to escape these feedbacks through symbiotic N2 fixation. The Free-Air Carbon dioxide Enrichment (FACE) experiment at Eschikon, Switzerland, has been the longest running investigation of the effects of open-air elevation of [CO2] on vegetation. The prediction of a long-term loss of photosynthetic capacity was tested by analysing photosynthesis in Trifolium repens L. (cv. Milkanova) in the spring and autumn of the eighth, ninth and tenth years of treatment. A high and low N treatment also allowed a test of the significance of exogenous N-supply in maintaining a stimulation of photosynthetic capacity in the long-term. Prior work in this Free Air CO2 Enrichment (FACE) experiment has revealed that elevated [CO2] increased both vegetative and reproductive growth of T. repens independent of N treatment. It is shown here that the photosynthetic response of T. repens was also independent of N fertilization under both current ambient and elevated (600 micro mol mol-1) [CO2]. There was a strong effect of season on photosynthesis, with light-saturated rates (Asat) 37% higher in spring than in autumn. Higher Asat in the spring was supported by higher maximum Rubisco carboxylation rates (Vc,max) and maximum rates of electron transport (Jmax) contributing to RuBP regeneration. Elevated [CO2] increased Asat by 37% when averaged across all measurement periods and both N fertilization levels, and decreased stomatal conductance by 25%. In spring, there was no effect of elevated [CO2] on photosynthetic capacity of leaves, but in autumn both Vc,max and Jmax were reduced by approximately 20% in elevated [CO2]. The results show that acclimation of photosynthetic capacity can occur in a nitrogen-fixing species, in the field where there are no artificial restrictions on sink capacity. However, even with acclimation there was a highly significant increase in photosynthesis at elevated [CO2].     

Direct evidence that symbiotic N2 fixation in fertile grassland is an important trait for a strong response of plants to elevated atmospheric CO2

Although legumes showed a clearly superior yield response to elevated atmospheric pCO₂ compared to nonlegumes in a variety of field experiments, the extent to which this is due to symbiotic N₂ fixation per se has yet to be determined. Thus, effectively and ineffectively nodulating lucerne (Medicago sativa L.) plants with a very similar genetic background were grown in competition with each other on fertile soil in the Swiss FACE experiment in order to monitor their CO₂ response. Under elevated atmospheric pCO₂, effectively nodulating lucerne, thus capable of symbiotically fixing N₂, strongly increased the harvestable biomass and the N yield, independent of N fertilization. In contrast, the harvestable biomass and N yield of ineffectively nodulating plants were affected negatively by elevated atmospheric pCO₂ when N fertilization was low. Large amounts of N fertilizer enabled the plants to respond more favourably to elevated atmospheric pCO₂, although not as strongly as effectively nodulating plants. The CO₂‐induced increase in N yield of the effectively nodulating plants was attributed solely to an increase in symbiotic N₂ fixation of 50–175%, depending on the N fertilization treatment. N yield derived from the uptake of mineral N from the soil was, however, not affected by elevated pCO₂. This result demonstrates that, in fertile soil and under temperate climatic conditions, symbiotic N₂ fixation per se is responsible for the considerably greater amount of above‐ground biomass and the higher N yield under elevated atmospheric pCO₂. This supports the assumption that symbiotic N₂ fixation plays a key role in maintaining the C/N balance in terrestrial ecosystems in a CO₂‐rich world.     

The effects of elevated CO2 on symbiotic N2 fixation: a link between the carbon and nitrogen cycles in grassland ecosystems

The response of plants to elevated CO₂ is dependent on the availability of nutrients, especially nitrogen. It is generally accepted that an increase in the atmospheric CO₂ concentration increases the C:N ratio of plant residues and exudates. This promotes temporary N-immobilization which might, in turn, reduce the availability of soil nitrogen. In addition, both a CO₂ stimulated increase in plant growth (thus requiring more nitrogen) and an increased N demand for the decomposition of soil residues with a large C:N will result under elevated CO₂ in a larger N-sink of the whole grassland ecosystem. One way to maintain the balance between the C and N cycles in elevated CO₂ would be to increase N-import to the grassland ecosystem through symbiotic N₂ fixation. Whether this might happen in the context of temperate ecosystems is discussed, by assessing the following hypothesis: i) symbiotic N₂ fixation in legumes will be enhanced under elevated CO₂, ii) this enhancement of N₂ fixation will result in a larger N-input to the grassland ecosystem, and iii) a larger N-input will allow the sequestration of additional carbon, either above or below-ground, into the ecosystem. Data from long-term experiments with model grassland ecosystems, consisting of monocultures or mixtures of perennial ryegrass and white clover, grown under elevated CO₂ under free-air or field-like conditions, supports the first two hypothesis, since: i) both the percentage and the amount of fixed N increases in white clover grown under elevated CO₂, ii) the contribution of fixed N to the nitrogen nutrition of the mixed grass also increases in elevated CO₂. Concerning the third hypothesis, an increased nitrogen input to the grassland ecosystem from N₂ fixation usually promotes shoot growth (above-ground C storage) in elevated CO₂. However, the consequences of this larger N input under elevated CO₂ on the below-ground carbon fluxes are not fully understood. On one hand, the positive effect of elevated CO₂ on the quantity of plant residues might be overwhelming and lead to an increased long-term below-ground C storage; on the other hand, the enhancement of the decomposition process by the N-rich legume material might favour carbon turn-over and, hence, limit the storage of below-ground carbon.     

Due to symbiotic N2 fixation,five years of elevated atmospheric pCO2 had no effect on the N concentration of plant litter in fertile,mixed grassland

Experimental findings indicate that, in terrestrial ecosystems, nitrogen cycling changes under elevated partial pressure of atmospheric CO₂ (pCO₂). It was suggested that the concentration of N in plant litter as well as the amount of litter are responsible for these changes. However, for grassland ecosystems, there have been no relevant data available to support this hypothesis. Data from five years of the Swiss FACE experiment show that, under fertile soil conditions in a binary plant community consisting of Lolium perenne L. and Trifolium repens L., the concentration of litter N does not change under elevated atmospheric pCO₂; this applies to harvest losses, stubble, stolons and roots as the sources of litter. This is in strong contrast to the CO₂ response of L. perenne swards without associated legumes; in this case the above-ground concentration of biomass N decreased substantially. Increased symbiotic N₂ fixation in T. repens nodules and a greater proportion of the N-rich T. repens in the community are regarded as the main mechanisms that buffer the increased C introduction into the ecosystem under elevated atmospheric pCO₂. Our data also suggest that elevated atmospheric pCO₂ results in greater amounts of litter, mainly due to increased root biomass production. This study indicates that, in a fertile grassland ecosystem with legumes, the concentration of N in plant litter is not affected by elevated atmospheric pCO₂ and, thus, cannot explain CO₂-induced changes in the cycling of N. This revised version was published online in June 2006 with corrections to the Cover Date.     

Arbuscular mycorrhiza infection enhances the growth response of Lolium perenne to elevated atmospheric pCO(2)

Elevated atmospheric pCO(2) increases the C-availability for plants and thus leads to a comparable increase in plant biomass production and nutrient demand. Arbuscular mycorrhizal fungi (AMF) are considered to play an important role in the nutrient uptake of plants as well as to be a significant C-sink. Therefore, an increased colonization of plant roots by AMF is expected under elevated atmospheric pCO(2). To test these hypotheses, Lolium perenne L. plants were grown from seeds in a growth chamber in pots containing a silica sand/soil mixture for 9 weeks with and without inoculation with Glomus intraradices (Schenck and Smith). The growth response of plants at two different levels of N fertilization (1.5 or 4.5 mM) combined with ambient (35 Pa) and elevated atmospheric pCO(2) (60 Pa) was compared. The inoculation with G. intraradices, the elevated atmospheric pCO(2) and the high N fertilization treatment all led to an increased plant biomass production of 16%, 20% and 49%, respectively. AMF colonization and high N fertilization increased the plant growth response to elevated atmospheric pCO(2); the plant growth response to high N fertilization was also increased by AMF colonization. The root/shoot ratio was reduced by high N fertilization or elevated atmospheric pCO(2), but was not affected by AMF colonization. The unchanged specific leaf area indicated that if AMF colonization represented an increased C-sink, this was fully covered by the plant. Elevated atmospheric pCO(2) strongly increased AMF colonization (60%) while the high N fertilization had a slightly negative effect. AMF colonization neither improved the N nor P nutrition status, but led to an improved total P uptake. The results underline the importance of AMF for the response of grassland ecosystems to elevated atmospheric pCO(2).     

Legume species identity and soil nitrogen supply determine symbiotic nitrogen-fixation responses to elevated atmospheric [CO2

In nitrogen (N)-limited systems, the response of symbiotic N fixation to elevated atmospheric [CO2] may be an important determinant of ecosystem responses to this global change. Experimental tests of the effects of elevated [CO2] have not been consistent. Although rarely tested, differences among legume species and N supply may be important. In a field free-air CO2 enrichment (FACE) experiment, we determined, for four legume species, whether the effects of elevated atmospheric [CO2] on symbiotic N fixation depended on soil N availability or species identity. Natural abundance and pool-dilution 15N methods were used to estimate N fixation. Although N addition did, in general, decrease N fixation, contrary to theoretical predictions, elevated [CO2] did not universally increase N fixation. Rather, the effect of elevated [CO2] on N fixation was positive, neutral or negative, depending on the species and N addition. Our results suggest that legume species identity and N supply are critical factors in determining symbiotic N-fixation responses to increased atmospheric [CO2].     

N2 fixation and cycling in Alnus glutinosa, Betula pendula and Fagus sylvatica woodland exposed to free air CO2 enrichment

We measured the effect of elevated atmospheric CO(2) on atmospheric nitrogen (N(2)) fixation in the tree species Alnus glutinosa growing in monoculture or in mixture with the non-N(2)-fixing tree species Betula pendula and Fagus sylvatica. We addressed the hypotheses that (1) N(2) fixation in A. glutinosa will increase in response to increased atmospheric CO(2) concentrations, when growing in monoculture, (2) the impact of elevated CO(2) on N(2) fixation in A. glutinosa is the same in mixture and in monoculture and (3) the impacts of elevated CO(2) on N cycling will be evident by a decrease in leaf δ(15)N and by the soil-leaf enrichment factor (EF), and that these impacts will not differ between mixed and single species stands. Trees were grown in a forest plantation on former agricultural fields for four growing seasons, after which the trees were on average 3.8 m tall and canopy closure had occurred. Atmospheric CO(2) concentrations were maintained at either ambient or elevated (by 200 ppm) concentrations using a free-air CO(2) enrichment (FACE) system. Leaf δ(15)N was measured and used to estimate the amount (N(dfa)) and proportion (%N(dfa)) of N derived from atmospheric fixation. On average, 62% of the N in A. glutinosa leaves was from fixation. The %N(dfa) and N(dfa) for A. glutinosa trees in monoculture did not increase under elevated CO(2), despite higher growth rates. However, N(2) fixation did increase for trees growing in mixture, despite the absence of significant growth stimulation. There was evidence that fixed N(2) was transferred from A. glutinosa to F. sylvatica and B. pendula, but no evidence that this affected their CO(2) response. The results of this study show that N(2) fixation in A. glutinosa may be higher in a future elevated CO(2) world, but that this effect will only occur where the trees are growing in mixed species stands.     

Contrasting growth response of an N2-fixing and non-fixing forb to elevated CO2: dependence on soil N supply

With the ability to symbiotically fix atmospheric N₂, legumes may lack the N-limitations thought to constrain plant response to elevated concentrations of atmospheric CO₂. The growth and photosynthetic responses of two perennial grassland species were compared to test the hypotheses that (1) the CO₂ response of wild species is limited at low N availability, (2) legumes respond to a greater extent than non-fixing forbs to elevated CO₂, and (3) elevated CO₂ stimulates symbiotic N₂ fixation, resulting in an increased amount of N derived from the atmosphere. This study investigated the effects of atmospheric CO₂ concentration (365 and 700 mol mol^–1) and N addition on whole plant growth and C and N acquisition in an N₂-fixing legume (Lupinus perennis) and a non-fixing forb (Achillea millefolium) in controlled-chamber environments. To evaluate the effects of a wide range of N availability on the CO₂ response, we incorporated six levels of soil N addition starting with native field soil inherently low in N (field soil + 0, 4, 8, 12, 16, or 20 g N m^–2 yr^–1). Whole plant growth, leaf net photosynthetic rates (A), and the proportion of N derived from N₂ fixation were determined in plants grown from seed over one growing season. Both species increased growth with CO₂enrichment, but this response was mediated by N supply only for the non-fixer, Achillea. Its response depended on mineral N supply as growth enhancements under elevated CO₂ increased from 0% in low N soil to +25% at the higher levels of N addition. In contrast, Lupinus plants had 80% greater biomass under elevated CO₂ regardless of N treatment. Although partial photosynthetic acclimation to CO₂ enrichment occurred, both species maintained comparably higher A in elevated compared to ambient CO₂ (+38%). N addition facilitated increased A in Achillea, however, in neither species did additional N availability affect the acclimation response of A to CO₂. Elevated CO₂ increased plant total N yield by 57% in Lupinus but had no effect on Achillea. The increased N in Lupinus came from symbiotic N₂ fixation, which resulted in a 47% greater proportion of N derived from fixation relative to other sources of N. These results suggest that compared to non-fixing forbs, N₂-fixers exhibit positive photosynthetic and growth responses to increased atmospheric CO₂ that are independent of soil N supply. The enhanced amount of N derived from N₂ fixation under elevated CO₂ presumably helps meet the increased N demand in N₂-fixing species. This response may lead to modified roles of N₂-fixers and N₂-fixer/non-fixer species interactions in grassland communities, especially those that are inherently N-poor, under projected rising atmospheric CO₂.     

Elevated pCO(2 )favours nitrate reduction in the roots of wild-type tobacco (Nicotiana tabacum cv. Gat.) and significantly alters N-metabolism in transformants lacking functional nitrate reductase in the roots

The impact of elevated pCO(2 )on N-metabolism of hydroponically grown wild-type and transformed tobacco plants lacking root nitrate reduction was studied in order to elucidate the effects on (i) nitrate uptake, (ii) long-distance transport of N, (iii) nitrate reduction with emphasis on root-NR, and (iv) the allocation of N between the root and shoot. The findings were related to alterations of growth rates. At elevated pCO(2 )the wild type exhibited higher growth rates, which were accompanied by an increase of NO(3)(-)-uptake per plant, due to a higher root:shoot ratio. Furthermore, elevated pCO(2 )enhanced nitrate reduction in the roots of the wild type, resulting in enhanced xylem-loading of organic N (amino-N) to supply the shoot with sufficient nitrogen, and decreased phloem-transport of organic N in a basipetal direction. Transformed tobacco plants lacking root nitrate reduction were smaller than the wild type and exhibited lower growth rates. Nitrate uptake per plant was decreased in transformed plants as a consequence of an impeded root growth and, thus, a significantly decreased root:shoot ratio. Surprisingly, transformed plants showed an altered allocation of amino-N between the root and the shoot, with an increase of amino-N in the root and a substantial decrease of amino-N in the shoot. In transformed plants, xylem-loading of nitrate was increased and the roots were supplied with organic N via phloem transport. Elevated pCO(2 )increased shoot-NR, but only slightly affected the growth rates of transformed plants, whereas carbohydrates accumulated at elevated pCO(2 )as indicated by a significant increase of the C/N ratio in the leaves of transformed plants. Unexpectedly, the C/N balance and the functional equilibrium between root and shoot growth was disturbed dramatically by the loss of nitrate reduction in the root.     

The relative contribution of symbiotic N2 fixation and other nitrogen sources to grassland ecosystems along an altitudinal gradient in the Alps

The significance of symbiotic N₂ fixation in legumes (Trifolium alpinum L., T. nivale Sieber, T. pratense L., T. badium Schreber, T. thalii Vill., T. repens L., Lotus alpinus [DC.] Schleicher, L. corniculatus L., Vicia sativa L.) and other N sources for the N budget of grassland ecosystems was studied along an altitudinal gradient in the Swiss Alps. The total annual symbiotic N₂ fixation was compared with other sources of N for plant growth of the total plant community (mineralisation and wet deposition). The contribution of symbiotically fixed N to total above-ground N yield of the swards decreased from at least 16% to 9% with increasing altitude where legumes were present. This decrease was due to a decrease in the yield proportion of legumes from 15% at 900 and 1380 m a.s.l. to 5% at 2100 and 2300 m a.s.l. (no legumes were found above 2750 m a.s.l.) and not to a decline in the activity of symbiotic N₂ fixation. With increasing altitude legumes are more patchily distributed. The high symbiotic N₂ fixation of individual plants up to their altitudinal limit is not primarily the result of low mineral N availability since an addition of NH₄ ⁺ or NO₃ ⁻ fertiliser at 2300 m a.s.l. led either to no decrease or only to a minor decrease in symbiotic N₂ fixation. At 1380 m a.s.l., N mineralisation (13.45 g N m⁻² yr⁻¹) appeared to be the main source of N for growth of the sward; N from symbiosis (at least 1.0 g to 2.6 g N m⁻² yr⁻¹) and wet deposition (0.4 g to 0.6 g m⁻² yr⁻¹) was not a significant N source for plant growth at this altitude. At 2100 m a.s.l., the combined amounts of N from symbiotic N₂ fixation (at least 0.1 g N m⁻² yr⁻¹) and wet deposition (0.3 g N m⁻² yr⁻¹) appeared to be similarly important for plant growth as soil N mineralisation (0.47 g N m⁻² yr⁻¹). At high altitudes, wet N deposition and symbiotic N₂ fixation together represent a significant source of N for the grassland ecosystem while at low altitudes these N inputs appear to be much less important. This revised version was published online in June 2006 with corrections to the Cover Date.     

The fate of photosynthetically-fixed carbon in Lolium perenne grassland as modified by elevated CO2 and sward management

Prediction of the impact of climate change requires the response of carbon (C) flow in plant-soil systems to increased CO(2) to be understood. A mechanism by which grassland C sequestration might be altered was investigated by pulse-labelling Lolium perenne swards, which had been subject to CO(2) enrichment and two levels of nitrogen (N) fertilization for 10 yr, with (14)CO(2). Over a 6-d period 40-80% of the (14)C pulse was exported from mature leaves, 1-2% remained in roots, 2-7% was lost as below-ground respiration, 0.1% was recovered in soil solution, and 0.2-1.5% in soil. Swards under elevated CO(2) with the lower N supply fixed more (14)C than swards grown in ambient CO(2), exported more fixed (14)C below ground and respired less than their high-N counterparts. Sward cutting reduced root (14)C, but plants in elevated CO(2) still retained 80% more (14)C below ground than those in ambient CO(2). The potential for below-ground C sequestration in grasslands is enhanced under elevated CO(2), but any increase is likely to be small and dependent upon grassland management.     

Effects of elevated CO2 and N addition on growth and N2 fixation of a legume subshrub (Caragana microphylla Lam.) in temperate grassland in China

It is well demonstrated that the responses of plants to elevated atmospheric CO(2) concentration are species-specific and dependent on environmental conditions. We investigated the responses of a subshrub legume species, Caragana microphylla Lam., to elevated CO(2) and nitrogen (N) addition using open-top chambers in a semiarid temperate grassland in northern China for three years. Measured variables include leaf photosynthetic rate, shoot biomass, root biomass, symbiotic nitrogenase activity, and leaf N content. Symbiotic nitrogenase activity was determined by the C(2)H(2) reduction method. Elevated CO(2) enhanced photosynthesis and shoot biomass by 83% and 25%, respectively, and the enhancement of shoot biomass was significant only at a high N concentration. In addition, the photosynthetic capacity of C. microphylla did not show down-regulation under elevated CO(2). Elevated CO(2) had no significant effect on root biomass, symbiotic nitrogenase activity and leaf N content. Under elevated CO(2), N addition stimulated photosynthesis and shoot biomass. By contrast, N addition strongly inhibited symbiotic nitrogenase activity and slightly increased leaf N content of C. microphylla under both CO(2) levels, and had no significant effect on root biomass. The effect of elevated CO(2) and N addition on C. microphylla did not show interannual variation, except for the effect of N addition on leaf N content. These results indicate that shoot growth of C. microphylla is more sensitive to elevated CO(2) than is root growth. The stimulation of shoot growth of C. microphylla under elevated CO(2) or N addition is not associated with changes in N(2)-fixation. Additionally, elevated CO(2) and N addition interacted to affect shoot growth of C. microphylla with a stimulatory effect occurring only under combination of these two factors.     

Leaf-atmosphere NH(3) exchange of white clover (Trifolium repens L.) in relation to mineral N nutrition and symbiotic N(2) fixation.

Plant-atmosphere NH(3) exchange was studied in white clover (Trifolium repens L. cv. Seminole) growing in nutrient solution containing 0 (N(2) based), 0.5 (low N) or 4.5 (high N) mM NO(3)(-). The aim was to show whether the NH(3) exchange potential is influenced by the proportion of N(2) fixation relative to NO(3)(-) supply. During the treatment, inhibition of N(2) fixation by NO(3)(-) was followed by in situ determination of total nitrogenase activity (TNA), and stomatal NH(3) compensation points (chi(NH(3))) were calculated on the basis of apoplastic NH4(+) concentration ([NH4(+)]) and pH. Whole-plant NH(3) exchange, transpiration and net CO(2) exchange were continuously recorded with a controlled cuvette system. Although shoot total N concentration increased with the level of mineral N application, tissue and apoplastic [NH4(+)] as well as chi(NH(3)) were equal in the three treatments. In NH(3)-free air, net NH(3) emission rates of <1 nmol m(-2) s(-1) were observed in both high-N and N(2)-based plants. When plants were supplied with air containing 40 nmol mol(-1) NH(3), the resulting net NH(3) uptake was higher in plants which acquired N exclusively from symbiotic N(2) fixation, compared to NO(3)(-) grown plants. The results indicate that symbiotic N(2) fixation and mineral N acquisition in white clover are balanced with respect to the NH4(+) pool leading to equal chi(NH(3)) in plants growing with or without NO(3)(-). At atmospheric NH(3) concentrations exceeding chi(NH(3)), the NH(3) uptake rate is controlled by the N demand of the plants.