Combined effects of chronic ozone and elevated CO2 on Rubisco activity and leaf components in soybean Glycine max

Content and activity of Rubisco and concentrations of leaf nitrogen, chlorophyll and total non-structural carbohydrates (TNC) were determined at regular intervals during the 1993 and 1994 growing seasons to understand the effects and interactions of [O3] and elevated [CO2] on biochemical limitations to photosynthesis during ontogeny. Soybean (Glycine max var. Essex) was grown in open-top field chambers in either charcoal-filtered air (CF, 20 nmol mol^-1) or non-filtered air supplemented with 1.5 x ambient [O3] (c. 80 nmol mol^-1) at ambient (AA, 360 mol mol^-1) or elevated [CO2] (700 mol mol^-1). Sampling period significantly affected all the variables examined. Changes included a decrease in the activity and content of Rubisco during seed maturation, and increased nitrogen (N), leaf mass per unit area (LMA) and total non-structural carbohydrates (TNC, including starch and sucrose) through the reproductive phases. Ontogenetic changes were most rapid in O2-treated plants. At ambient [CO2], O3 decreased initial activity (14-64% per unit leaf area and 14-29% per unit Rubisco) and content of Rubisco (9-53%), and N content per unit leaf area. Ozone decreased LMA by 17-28% of plants in CF-AA at the end of the growing season because of a 24-41% decrease in starch and a 59-80% decrease in sucrose. In general, elevated CO2], in CF or O3-fumigated air, reduced the initial activity of Rubisco and activation state while having little effect on Rubisco content, N and the chlorophyll content, per unit leaf area. Elevated CO2 decreased Rubisco activity by 14-34% per unit leaf area and 15-25% per unit Rubisco content of plants in grown CF-AA, nd increases LMA by 27-74% of the leaf mass per unit area in CF-AA because of a 23-148% increase in starch. However, the data suggest that, at elevated [CO2], increases in starch and sucrose are not directly responsible for the deactivation of Rubisco. Also, there was little evidence of an adjustment of Rubisco activity in response to starch and sucrose metabolism. Significant interactions between elevated [CO2] and [O3] on all variables examined generally resulted in alleviation or amelioration of the O3 effects at elevated CO2. These data provide further support to the idea that elevated atmospheric CO2 will reduce or prevent damage from pollutant O3.     

Leaf and canopy responses to elevated CO2 in a pine forest under free-air CO2 enrichment

Physiological responses to elevated CO₂ at the leaf and canopy-level were studied in an intact pine (Pinus taeda) forest ecosystem exposed to elevated CO₂ using a free-air CO₂ enrichment (FACE) technique. Normalized canopy water-use of trees exposed to elevated CO₂ over an 8-day exposure period was similar to that of trees exposed to current ambient CO₂ under sunny conditions. During a portion of the exposure period when sky conditions were cloudy, CO₂-exposed trees showed minor (7%) but significant reductions in relative sap flux density compared to trees under ambient CO₂ conditions. Short-term (minutes) direct stomatal responses to elevated CO₂ were also relatively weak (5% reduction in stomatal aperture in response to high CO₂ concentrations). We observed no evidence of adjustment in stomatal conductance in foliage grown under elevated CO₂ for nearly 80 days compared to foliage grown under current ambient CO₂, so intrinsic leaf water-use efficiency at elevated CO₂ was enhanced primarily by direct responses of photosynthesis to CO₂. We did not detect statistical differences in parameters from photosynthetic responses to intercellular CO₂ (A _net-C _i curves) for Pinus taeda foliage grown under elevated CO₂ (550 mol mol^–1) for 50–80 days compared to those for foliage grown under current ambient CO₂ from similar-sized reference trees nearby. In both cases, leaf net photosynthetic rate at 550 mol mol^–1 CO₂ was enhanced by approximately 65% compared to the rate at ambient CO₂ (350 mol mol^–1). A similar level of enhancement under elevated CO₂ was observed for daily photosynthesis under field conditions on a sunny day. While enhancement of photosynthesis by elevated CO₂ during the study period appears to be primarily attributable to direct photosynthetic responses to CO₂ in the pine forest, longer-term CO₂ responses and feedbacks remain to be evaluated.     

Mechanical properties of spruce and beech wood grown in elevated CO2

Biomechanical responses of stems of 6- to 7-year-old spruce [Picea abies (L.) Karst.] and beech (Fagus sylvatica L) trees were studied after 4 years of growth in elevated atmospheric CO₂ in combination with a nitrogen treatment and on two different soil types. At the end of the treatment, stems were harvested and tested in fresh and air-dried status. Bending characteristics of juvenile wood (modulus of elasticity, termed rigidity) were determined by bending tests. Fracture characteristics (termed toughness) were determined by stroke-pendulum tests. From the base disk of each stem densitometric data were obtained. In spruce, wood produced under elevated CO₂ was tougher on both soil types; enhanced N deposition made wood less rigid only on acidic soils. In contrast, beech wood samples showed no significant reaction to CO₂ but were significantly tougher under high nitrogen depositions on acidic soil. Effects on wood density of both CO₂ and N treatments were not significant, but wood density was higher on acidic soil and so were rigidity and toughness (soil effect). Different genotypes of spruce and beech reacted significantly differently to the treatments. Some genotypes reacted strongly to CO₂ or N, whereas others did not react or showed interactions between CO₂ and N. This underlines the importance of genetic diversity in tree communities.     

氮素对高大气CO2浓度下小麦叶片光合作用的影响

通过测定小麦拔节期叶片的光合气体交换参数和光强-光合速率（P_n）响应曲线,研究了氮素对长期高大气CO₂浓度（760 μmol·mol^-1）下小麦叶片光合作用的影响.结果表明：在长期高大气CO₂浓度下,增施氮肥能提高小麦叶片P_n、蒸腾速率（T_r）和瞬时水分利用效率（WUE_i）;与正常大气CO₂浓度相比,高大气CO₂浓度下小麦叶片的P_n和WUE_i增加,气孔导度（G_s）和胞间CO₂浓度(C_i）降低.随光合有效辐射的增强,高大气CO₂浓度下小麦叶片的P_n和WUE_i均高于正常大气CO₂浓度处理,G_s则较低,而C_i和T_r无显著变化.高氮水平下小麦叶片G_s与P_n、T_r、WUE_i呈线性正相关,G_s与C_i在正常大气CO₂浓度下呈线性负相关,但高大气CO₂浓度下二者无相关性;低氮水平下小麦叶片的G_s与P_n、WUE_i无相关性,而与C_i和T_r呈线性正相关,表明高大气CO₂浓度下低氮水平的小麦叶片P_n由非气孔因素限制.     

Increase of photosynthesis and starch in potato under elevated CO2 is dependent on leaf age

Potato plants (Solanum tuberosum cv. Bintje) were grown in open top chambers under ambient (400 microL L(-1)) and elevated CO2 (720 microL L(-1)). After 50 days one half of each group was transferred to the other CO2 concentration and the effects were studied in relation to leaf age (old, middle-aged and young leaves) in each of the four groups. Under long-term exposure to elevated CO2, photosynthesis increased between 10% and 40% compared to ambient CO2. A subsequent shift of the same plants to ambient CO2 caused a 20-40% decline in photosynthetic rate, which was most pronounced in young leaves. After shifting from long-term ambient to elevated CO2, photosynthesis also increased most strongly in young leaves (90%); these experiments show that photosynthesis was downregulated in the upper young fully expanded leaves of potato growing long-term under elevated CO2. Soluble sugar content in all leaf classes under long-term exposure was stable irrespective of the CO2 treatment, however under elevated CO2 young leaves showed a strongly increased starch accumulation (up to 400%). In all leaf classes starch levels dropped in response to the shift from 720 to 400 microL L(-1) approaching ambient CO2 levels. After the shift to 720 microL L(-1), sucrose and starch levels increased, principally in young Leaves. There is clear evidence that leaves of different age vary in their responses to changes in atmospheric CO2 concentration.     

Mechanism for increased leaf growth in elevated CO2

The effect of exposure to elevated CO₂ on the processes of leafcell production and leaf cell expansion was studied using primaryleaves of Phaseolus vulgaris L. Cell division and expansionwere separated temporally by exposing seedlings to dim red lightfor 10 d (when leaf cell division was completed) followed byexposure to bright white light for 14 d (when leaf growth wasentirely dependent on cell expansion). When plants were exposedto elevated CO₂ during the phase of cell expansion, epidermalcell size and leaf area development were stimulated. Three piecesof evidence suggest that this occurred as a result of increasedcell wall loosening and extensibility, (i) cell wall extensibility(WEx, measured as tensiometric extension using an Instron) wassignificantly increased, (ii) cell wall yield turgor (V, MPa)was reduced and (iii) xyloglucan endotransglycosylase (XET)enzyme activity was significantly increased. When plants wereexposed to elevated CO₂ during the phase of cell division, thenumber of epidermal cells was increased whilst final cell sizewas significantly reduced and this was associated with reducedfinal leaf area, WEx and XET activity. When plants were exposedto elevated CO₂ during both phases of cell division and expansion,leaf area development was not affected. For this treatment,however, the number of epidermal cells was increased, but cellexpansion was inhibited, despite exposure to elevated CO₂ duringthe expansion phase. Assessments were also made of the spatialpatterns of WEx across the expanding leaf lamina and the datasuggest that exposure to elevated CO₂ during the phase of leafexpansion may lead to enhanced extensibility particularly atbasal leaf margins which may result in altered leaf shape. The data show that both cell production and expansion were stimulatedby elevated CO₂, but that leaf growth was only enhanced by exposureto elevated CO₂ in the cell expansion phase of leaf development.Increased leaf cell expansion is, therefore, an important mechanismfor enhanced leaf growth in elevated CO₂, whilst the importanceof increased leaf cell production in elevated CO₂ remains tobe elucidated. Key words: Phaseolus vulgaris L., dwarf beans, elevated CO², biophysics of cell expansion, xyloglucan endotransglycosylase, XET, water relations     

Higher daytime leaf temperatures contribute to lower freeze tolerance under elevated CO2

Elevated atmospheric CO2 adversely affects freezing tolerance in many evergreens, but the underlying mechanism(s) have been elusive. We compared effects of elevated CO2 with those of daytime warming on acclimation of snow gum (Eucalyptus pauciflora) to freezing temperatures under field conditions. Reduction in stomatal conductance g(c) under elevated CO2 was shown to cause leaf temperature to increase by up to 3 degrees C. In this study, this increase in leaf temperature was simulated under ambient CO2 conditions by using a free air temperature increase (FATI) system to warm snow gum leaves during daytime, thereby increasing the diurnal range in temperature without affecting temperature minima. Acclimation to freezing temperatures was assessed using measures of electrolyte leakage and photosynthetic efficiency of leaf discs exposed to different nadir temperatures. Here, we show that both elevated CO2 and daytime warming delayed acclimation to freezing temperatures for 2-3 weeks after which time freeze tolerance of the treated plants in both the FATI and open top chamber (OTC) experiments did not differ from control plants. Our results support the hypothesis that delayed development of freezing tolerance under elevated CO2 is because of higher daytime leaf temperatures under elevated CO2. Thus, potential gains in productivity in response to increasing atmospheric CO2 and prolonging the growing season may be reduced by an increase in freezing stress in frost-prone area.     

Interactive effects of elevated CO2 and drought stress on leaf water potential and growth in Caragana intermedia

We studied the responses of leaf water potential (Ψ_w), morphology, biomass accumulation and allocation, and canopy productivity index (CPI) to the combined effects of elevated CO₂ and drought stress in Caragana intermedia seedlings. Seedlings were grown at two CO₂ concentrations (350 and 700 μmol mol⁻¹) interacted with three water regimes (60–70%, 45–55%, and 30–40% of field capacity of soil). Elevated CO₂ significantly increased Ψ_w, decreased specific leaf area (SLA) and leaf area ratio (LAR) of drought-stressed seedlings, and increased tree height, basal diameter, shoot biomass, root biomass as well as total biomass under the all the three water regimes. Growth responses to elevated CO₂ were greater in well-watered seedlings than in drought-stressed seedlings. CPI was significantly increased by elevated CO₂, and the increase in CPI became stronger as the level of drought stress increased. There were significant interactions between elevated CO₂ and drought stress on leaf water potential, basal diameter, leaf area, and biomass accumulation. Our results suggest that elevated CO₂ may enhance drought avoidance and improved water relations, thus weakening the effect of drought stress on growth of C. intermedia seedings.     

Polyphenoloxidase activity in coffee leaves and its role in resistance against the coffee leaf miner and coffee leaf rust

In plants, PPO has been related to defense mechanism against pathogens and insects and this role was investigated in coffee trees regarding resistance against a leaf miner and coffee leaf rust disease. PPO activity was evaluated in different genotypes and in relation to methyl-jasmonate (Meja) treatment and mechanical damage. Evaluations were also performed using compatible and incompatible interactions of coffee with the fungus Hemileia vastatrix (causal agent of the leaf orange rust disease) and the insect Leucoptera coffeella (coffee leaf miner). The constitutive level of PPO activity observed for the 15 genotypes ranged from 3.8 to 88 units of activity/mg protein. However, no direct relationship was found with resistance of coffee to the fungus or insect. Chlorogenic acid (5-caffeoylquinic acid), the best substrate for coffee leaf PPO, was not related to resistance, suggesting that oxidation of other phenolics by PPO might play a role, as indicated by HPLC profiles. Mechanical damage, Meja treatment, H. vastatrix fungus inoculation and L. coffeella infestation caused different responses in PPO activity. These results suggest that coffee resistance may be related to the oxidative potential of the tissue regarding the phenolic composition rather than simply to a higher PPO activity.     

The effect of plant material grown under elevated CO2 on soil respiratory activity

The biodegradability of aerial material from a C4 plant, sorghum grown under ambient (345 µmol mol^–1) and elevated (700 µmol mol^–1) atmospheric CO₂ concentrations were compared by measuring soil respiratory activity. Initial daily respiratory activity (measured over 10 h per day) increased four fold from 110 to 440 cm³ CO₂ 100g dry weight soil^–1 in soils amended with sorghum grown under either elevated or ambient CO₂. Although soil respiratory activity decreased over the following 30 days, respiration remained significantly higher (t-test;p>0.05) in soils amended with sorghum grown under elevated CO₂ concentrations. Analysis of the plant material revealed no significant differences in C:N ratios between sorghum grown under elevated or ambient CO₂. The reason for the differences in soil respiratory activity have yet to be elucidated. However if this trend is repeated in natural ecosystems, this may have important implications for C and N cycling.     

The effects of chamber pressurization on soil-surface CO2 flux and the implications for NEE measurements under elevated CO2

Soil and ecosystem trace gas fluxes are commonly measured using the dynamic chamber technique. Although the chamber pressure anomalies associated with this method are known to be a source of error, their effects have not been fully characterized. In this study, we use results from soil gas-exchange experiments and a soil CO₂ transport model to characterize the effects of chamber pressure on soil CO₂ efflux in an annual California grassland. For greater than ambient chamber pressures, experimental data show that soil-surface CO₂ flux decreases as a nonlinear function of increasing chamber pressure; this decrease is larger for drier soils. In dry soil, a gauge pressure of 0.5 Pa reduced the measured soil CO₂ efflux by roughly 70% relative to the control measurement at ambient pressure. Results from the soil CO₂ transport model show that pressurizing the flux chamber above ambient pressure effectively flushes CO₂ from the soil by generating a downward flow of air through the soil air-filled pore space. This advective flow of air reduces the CO₂ concentration gradient across the soil–atmosphere interface, resulting in a smaller diffusive flux into the chamber head space. Simulations also show that the reduction in diffusive flux is a function of chamber pressure, soil moisture, soil texture, the depth distribution of soil CO₂ generation, and chamber diameter. These results highlight the need for caution in the interpretation of dynamic chamber trace gas flux measurements. A portion of the frequently observed increase in net ecosystem carbon uptake under elevated CO₂ may be an artifact resulting from the impact of chamber pressurization on soil CO₂ efflux.     

Decomposition of tree leaf litters grown under elevated CO2: Effect of litter quality

Ash (Fraxinus excelsior L.), birch (Betula pubescens Ehrh.), sycamore (Acer pseudoplatanus L.) and Sitka spruce (Picea sitchensis (Bong.) Carr.) leaf litters were monitored for decomposition rates and nutrient release in a laboratory microcosm experiment. Litters were derived from solar domes where plants had been exposed to two different CO₂ regimes: ambient (350 L L^-1 CO₂) and enriched (600 L L^-1 CO₂).Elevated CO₂ significantly affected some of the major litter quality parameters, with lower N, higher lignin concentrations and higher ratios of C/N and lignin/N for litters derived from enriched CO₂. Respiration rates of the deciduous species were significantly decreased for litters grown under elevated CO₂, and reductions in mass loss at the end of the experiment were generally observed in litters derived from the 600 ppm CO₂ treatment. Nutrient mineralization, dissolved organic carbon, and pH in microcosm leachates did not differ significantly between the two CO₂ treatments for any of the species studied. Litter quality parameters were examined for correlations with cumulative respiration and decomposition rates: N concentration, C/N and lignin/N ratios showed the highest correlations, with differences between litter types. The results indicate that higher C storage will occur in soil as a consequence of litter quality changes resulting from higher atmospheric concentrations of CO₂.Abbreviations CHO soluble carbohydrates - DOC dissolved organic carbon - HCel holocellulose - WTREM weight remaining     

Rhizodeposition under ambient and elevated CO2 levels

As global CO₂ levels rise, can soils store more carbon and so buffer atmospheric CO₂ levels? Answering this question requires a knowledge of the rates of C inputs to soil and of CO₂ outputs via decomposition. Below-ground inputs from roots are a major component of the C flow into soils but are still poorly understood. In this article, new techniques for measuring rhizodeposition are reviewed and discussed and the need for cross-comparisons between methods is identified. One component of rhizodeposition, root exudation, is examined in more detail and evidence is presented which suggests that current estimates of exudate flow into soils are incorrect. A mechanistic mathematical model is used to explore how exudate flows might change under elevated CO₂.     

Interactive effects of elevated CO2 and precipitation change on leaf nitrogen of dominant Stipa L. species

Nitrogen (N) serves as an important mineral element affecting plant productivity and nutritional quality. However, few studies have addressed the interactive effects of elevated CO₂ and precipitation change on leaf N of dominant grassland genera such as Stipa L. This has restricted our understanding of the responses of grassland to climate change. We simulated the interactive effects of elevated CO₂ concentration and varied precipitation on leaf N concentration (N_mass) of four Stipa species (Stipa baicalensis, Stipa bungeana, Stipa grandis, and Stipa breviflora; the most dominant species in arid and semiarid grassland) using open-top chambers (OTCs). The relationship between the N_mass of these four Stipa species and precipitation well fits a logarithmic function. The sensitivity of these four species to precipitation change was ranked as follows: S. bungeana > S. breviflora > S. baicalensis > S. grandis. The N_mass of S. bungeana was the most sensitive to precipitation change, while S. grandis was the least sensitive among these Stipa species. Elevated CO₂ exacerbated the effect of precipitation on N_mass. N_mass decreased under elevated CO₂ due to growth dilution and a direct negative effect on N assimilation. Elevated CO₂ reduced N_mass only in a certain precipitation range for S. baicalensis (163–343 mm), S. bungeana (164–355 mm), S. grandis (148–286 mm), and S. breviflora (130–316 mm); severe drought or excessive rainfall would be expected to result in a reduced impact of elevated CO₂. Elevated CO₂ affected the N_mass of S. grandis only in a narrow precipitation range. The effect of elevated CO₂ reached a maximum when the amount of precipitation was 253, 260, 217, and 222 mm for S. baicalensis, S. bungeana, S. grandis, and S. breviflora, respectively. The N_mass of S. grandis was the least sensitive to elevated CO₂. The N_mass of S. breviflora was more sensitive to elevated CO₂ under a drought condition compared with the other Stipa species.     

Forest carbon balance under elevated CO2

Free-air CO₂ enrichment (FACE) technology was used to expose a loblolly pine (Pinus taeda L.) forest to elevated atmospheric CO₂ (ambient + 200 µl l^-1). After 4 years, basal area of pine trees was 9.2% larger in elevated than in ambient CO₂ plots. During the first 3 years the growth rate of pine was stimulated by ~26%. In the fourth year this stimulation declined to 23%. The average net ecosystem production (NEP) in the ambient plots was 428 gC m^-2 year^-1, indicating that the forest was a net sink for atmospheric CO₂. Elevated atmospheric CO₂ stimulated NEP by 41%. This increase was primarily an increase in plant biomass increment (57%), and secondarily increased accumulation of carbon in the forest floor (35%) and fine root increment (8%). Net primary production (NPP) was stimulated by 27%, driven primarily by increases in the growth rate of the pines. Total heterotrophic respiration (R_h) increased by 165%, but total autotrophic respiration (R_a) was unaffected. Gross primary production was increased by 18%. The largest uncertainties in the carbon budget remain in separating belowground heterotrophic (soil microbes) and autotrophic (root) respiration. If applied to temperate forests globally, the increase in NEP that we measured would fix less than 10% of the anthropogenic CO₂ projected to be released into the atmosphere in the year 2050. This may represent an upper limit because rising global temperatures, land disturbance, and heterotrophic decomposition of woody tissues will ultimately cause an increased flux of carbon back to the atmosphere.     

Effects of elevated CO2 on leaf area dynamics in nodulating and non-nodulating soybean stands

Aims

The effects of elevated CO₂ on leaf area index (LAI) vary among studies. We hypothesized that the interactive effects of CO₂ and nitrogen on leaf area loss have important roles in LAI regulation.

Methods

We studied the leaf area production and loss using nodulating soybean and its non-nodulating isogenic line in CO₂-controlled greenhouse systems.

Results

Leaf area production increased with elevated CO₂ levels in the nodulating soybean stand and to a lesser extent in the non-nodulating line. Elevated CO₂ levels accelerated leaf area loss only in nodulating plants. Consequently, both plants exhibited a similar stimulation of peak LAI with CO₂ elevation. The accelerated leaf loss in nodulating plants may have been caused by newly produced leaves shading the lower leaves. The nodulating plants acquired N throughout the growth phase, whereas non-nodulating plants did not acquire N after flowering due to the depletion of soil N. N retranslocation to new organs and subsequent leaf loss were faster in non-nodulating plants compared with nodulating plants, irrespective of the CO₂ levels.

Conclusion

LAI regulation in soybean involved various factors, such as light availability within the canopy, N acquisition and N demands in new organs. These effects varied among the growth stages and CO₂ levels.     

Throughfall chemistry in a loblolly pine plantationunder elevated atmospheric CO2 concentrations

Accelerated tree growth under elevatedatmospheric CO₂ concentrations may influencenutrient cycling in forests by (i) increasingthe total leaf area, (ii) increasing the supplyof soluble carbohydrate in leaf tissue, and (iii) increasing nutrient-use efficiency. Here wereport the results of intensive sampling andlaboratory analyses of NH ₄ ⁺ , NO ₃ ^– , PO ₄ ^3– , H⁺, K⁺, Na⁺,Ca²⁺, Mg²⁺, Cl^–, SO ₄ ^2– , and dissolved organic carbon (DOC) in throughfallprecipitation during the first 2.5+ years of the DukeUniversity Free-Air CO₂ Enrichment (FACE)experiment. After two growing seasons, a largeincrease (i.e., 48%) in throughfall deposition of DOCand significant trends in throughfall volume and inthe deposition of NH ₄ ⁺ , NO ₃ ^– , H⁺, and K⁺ can be attributed to the elevatedCO₂ treatment. The substantial increase indeposition of DOC is most likely associated withincreased availability of soluble C in plant foliage,whereas accelerated canopy growth may account forsignificant trends toward decreasing throughfallvolume, decreasing deposition of NH ₄ ⁺ ,NO ₃ ^– , and H⁺, and increasing deposition of K⁺ under elevated CO₂. Despiteconsiderable year-to-year variability, there wereseasonal trends in net deposition of NO ₃ ^– ,H⁺, cations, and DOC associated with plant growthand leaf senescence. The altered chemical fluxes inthroughfall suggest that soil solution chemistry mayalso be substantially altered with continued increasesin atmospheric CO₂ concentrations in the future.     

Nutrient relations in calcareous grassland under elevated CO2

Plant nutrient responses to 4 years of CO₂ enrichment were investigated in situ in calcareous grassland. Beginning in year 2, plant aboveground C:N ratios were increased by 9% to 22% at elevated CO₂ (P < 0.01), depending on year. Total amounts of N removed in biomass harvests during the first 4 years were not affected by elevated CO₂ (19.9 ± 1.3 and 21.1 ± 1.3 g N m⁻² at ambient and elevated CO₂), indicating that the observed plant biomass increases were solely attained by dilution of nutrients. Total aboveground P and tissue N:P ratios also were not altered by CO₂ enrichment (12.5 ± 2 g N g⁻¹ P in both treatments). In contrast to non-legumes (>98% of community aboveground biomass), legume C/N was not reduced at elevated CO₂ and legume N:P was slightly increased. We attribute the less reduced N concentration in legumes at elevated CO₂ to the fact that virtually all legume N originated from symbiotic N₂ fixation (%N_dfa ≈ 90%), and thus legume growth was not limited by soil N. While total plant N was not affected by elevated CO₂, microbial N pools increased by +18% under CO₂ enrichment (P = 0.04) and plant available soil N decreased. Hence, there was a net increase in the overall biotic N pool, largely due increases in the microbial N pool. In order to assess the effects of legumes for ecosystem CO₂ responses and to estimate the degree to which plant growth was P-limited, two greenhouse experiments were conducted, using firstly undisturbed grassland monoliths from the field site, and secondly designed `microcosm' communities on natural soil. Half the microcosms were planted with legumes and half were planted without. Both monoliths and microcosms were exposed to elevated CO₂ and P fertilization in a factored design. After two seasons, plant N pools in both unfertilized monoliths and microcosm communities were unaffected by CO₂ enrichment, similar to what was found in the field. However, when P was added total plant N pools increased at elevated CO₂. This community-level effect originated almost solely from legume stimulation. The results suggest a complex interaction between atmospheric CO₂ concentrations, N and P supply. Overall ecosystem productivity is N-limited, whereas CO₂ effects on legume growth and their N₂ fixation are limited by P. Received: 12 July 1997 / Accepted: 15 April 1998