Elevated atmospheric CO2 decreases the ammonia compensation point of barley plants.

The ammonia compensation point ( ) controls the direction and magnitude of NH3 exchange between plant leaves and the atmosphere. Very limited information is currently available on how responds to anticipated climate changes. Young barley plants were grown for 2 weeks at ambient (400 μmol mol(-1)) or elevated (800 μmol mol(-1)) CO2 concentration with or NH4NO3 as the nitrogen source. The concentrations of and H(+) in the leaf apoplastic solution were measured along with different foliar N pools and enzymes involved in N metabolism. Elevated CO2 caused a threefold decrease in the concentration in the apoplastic solution and slightly acidified it. This resulted in a decline of the from 2.25 and 2.95 nmol mol(-1) under ambient CO2 to 0.37 and 0.89 nmol mol(-1) at elevated CO2 in the and NH4NO3 treatments, respectively. The decrease in at elevated CO2 reflected a lower N concentration (-25%) in the shoot dry matter. The activity of nitrate reductase also declined (-45 to -60%), while that of glutamine synthetase was unaffected by elevated CO2. It is concluded that elevated CO2 increases the likelihood of plants being a sink for atmospheric NH3 and reduces episodes of NH3 emission from plants.

Introduction

Ammonia (NH3) plays a major role in atmospheric chemistry and radiative forcing (Hertel ). When deposited in sensitive terrestrial ecosystems, NH3/ammonium () contributes to acidification and loss of biodiversity (Dise ). Exchange of NH3 between the atmosphere and vegetation constitutes an important process in the global NH3 budget, but very little information is available on how anticipated climate changes will affect this exchange. The magnitude and direction of NH3 fluxes between vegetation and the atmosphere are controlled by plant physiological processes in interaction with environmental conditions (Schjoerring ; Massad ). The fundamental parameters characterizing plant–atmosphere NH3 exchange are the stomatal NH3 compensation point () together with the conductance to diffusion of NH3 through the stomata and boundary layers surrounding the leaves (Kruit ). Both of these parameters can be expected to be modified by future climate changes, including elevated atmospheric carbon dioxide (CO2), rising air temperature, and more frequent drought periods (Fowler ).

The NH3 compensation point in plants is defined as the NH3 concentration in the air within the substomatal cavity at which no net NH3 exchange with the ambient atmosphere takes place (Farquhar ). NH3 may be emitted when the atmospheric NH3 concentration falls below and absorbed by plants when the atmospheric NH3 concentration is higher than . The value reflects the concentration of and H+ in the leaf apoplastic solution as these two parameters control the concentration of dissolved NH3 in equilibrium with gaseous NH3 within the substomatal cavities (Husted ; 2000).

There is currently very limited data on how atmospheric CO2 affects . Theoretically, elevated CO2 would be expected to decrease due to repression of photorespiration, which is a major -generating process implicated in foliar NH3 exchange (Mattsson ; Husted ; Kumagai ). The more efficient photosynthetic carbon acquisition under elevated CO2 will in many cases stimulate plant growth without increasing root nitrogen (N) uptake, leading to an overall decline in plant N status (Taub and Wang, 2008), which would be expected to decrease . In addition, more carbon skeletons, energy and reducing power would be available for assimilation via the glutamine synthetase/glutamate synthase cycle (Forde and Lea, 2007). An uncertain factor in the budget will be how nitrate () reduction is affected. Elevated CO2 has in some cases been reported to inhibit shoot reduction in C3 species, including barley (Bloom ), while in other cases the foliar nitrate reductase activity of barley was only slightly suppressed (Sicher, 2001) or was even significantly enhanced (Robredo ).

Plant–atmosphere NH3 exchange is strongly affected by apoplastic pH (Husted and Schjoerring, 1995). When CO2 dissolves in water, it forms carbonic acid, which is a weak acid. In addition, photosynthetic CO2 fixation leads to intracellular generation of stoichiometric amounts of H+. Elevated CO2 would therefore theoretically be expected to decrease the apoplastic pH, assuming a low buffer capacity of the apoplastic solution (Nielsen and Schjoerring, 1998). Very high atmospheric CO2 concentrations (1–16%) have been shown to rapidly decrease the apoplastic pH (Oja ; Savchenko ). However, transient additions of CO2 in the physiologically relevant CO2 range (0–800 μmol mol–1) alkalinized the apoplastic solution (Hedrich ; Felle and Hanstein, 2002). All these studies of elevated CO2 exposure were conducted over a short period (minutes to hours). Information is still lacking on whether or not the buffering system of the apoplast can compensate for the pH changes after prolonged exposure to elevated CO2.

The aim of the present study was to investigate the effect of elevated CO2 on and relate this effect to N metabolic responses. Our hypothesis was that elevated atmospheric CO2 would enhance the capacity for assimilation and therefore decrease . Barley plants were grown in 2mM or 1mM NH4NO3 solution at ambient (400 μmol mol–1) or elevated (800 μmol mol–1) CO2 for 2 weeks. The apoplastic solution was extracted for determination of and related to key N pools and N-assimilatory enzymes in shoots and roots.

Materials and methods

Plant cultivation

Barley seeds (Hordeum vulgare L., cv. Golden Promise) were germinated at 22 °C in vermiculite. After 5 d, uniform seedlings were selected and transferred to opaque 4.5 l cultivation units, each holding four plants. The units were filled with aerated nutrient solution containing 0.9mM Ca(NO3)2.4H2O, 0.6mM KNO3, 0.3mM Mg(NO3)2.6H2O, 0.2mM KH2PO4, 0.2mM K2SO4, 0.3mM MgSO4.7H2O, 0.1mM NaCl, 0.8 μM Na2MoO4.2H2O, 0.7 μM ZnCl2, 0.8 μM CuSO4.5H2O, 1 μM MnCl2.4H2O, 2 μM H3BO3, and 50 μM Fe(III)-EDTA-Na. The pH of the nutrient solution was kept at 6.5–7.5 by addition of CaCO3. The cultivation units were placed in two growth chambers at 380–400 μmol CO2 mol–1 air, 70–75% relative air humidity, and 20 and 18 °C during a 16h day/8h night light cycle with a photosynthetic photon flux density of 500–600 μmol photons m–2 s–1 during the day.

At the three-leaf stage 1 week later (d 7), all the cultivation units were redistributed randomly between the two growth chambers. CO2 was maintained at 400 μmol mol–1 in one chamber and 800 μmol mol–1 in the other. Half of the cultivation units in each chamber received 2mM [0.2mM Mg(NO3)2.6H2O, 0.6mM Ca(NO3)2.4H2O, and 0.4mM KNO3] and the other half received 1mM [0.1mM Mg(NO3)2.6H2O, 0.3mM Ca(NO3)2.4H2O, and 0.2mM KNO3] and 1mM [0.5mM (NH4)2SO4]. Two units with different N forms without plants were placed in each chamber as blank controls (nutrient solution without plants) in order to correct for possible N volatilization and water loss through evaporation from the nutrient solutions. The other conditions and nutrient concentrations were the same as before the CO2 treatment. The CO2 treatment lasted 15 d. The N concentration in the nutrient solution was checked daily and, based on these measurements, N was topped up or the nutrient solution renewed every 3 d. During every change of the solution, each cultivation unit was weighed and 12ml solution was sampled for and measurement. Water consumption was calculated on the basis of the difference between water losses in cultivation units with and without plants. All the plants were harvested and divided into shoots and roots at d 22. Two plants from each cultivation unit were freeze dried to determine dry weight and water content. The other two plants were quickly rinsed with deionized water and thereupon immediately immersed in liquid N2. All samples were stored at –80 °C and ground to a fine powder in liquid N2 before biochemical analyses.

Leaf gas exchange

Photosynthesis and transpiration were measured at d 21. The youngest fully expanded leaf was placed in the cuvette of a CIRAS-2 portable photosynthesis and transpiration monitor (CIRAS-2 Portable Photosynthesis System; PP Systems, Amesbury, MS, USA). The concentration of CO2 in the cuvette was controlled at the level corresponding to that in growth chamber, i.e. 400 or 800 μmol mol–1 for the ambient and elevated CO2 treatments, respectively, and the other conditions were similar to the growth chamber conditions. In order to assess photosynthetic acclimation, the photosynthesis of plants that were growing at 400 μmol mol–1 CO2 was also measured at 800 μmol mol–1 CO2.

N pools and assimilation

The extraction of apoplastic solution was carried out at d 22 after a 6h light period. After rinsing in deionized water, the fully expanded leaves were cut into 2–3cm segments and infiltrated with a 350 mOsm isotonic sorbitol solution (280mM), after which the apoplastic extracts were collected in microtubes by centrifuging at 2000g for 15min (Mattsson ). Xylem sap was collected from cut stems for a maximum of 30min.

, , and amino acids were extracted at 4°C from roots and shoots in 10mM formic acid with 0.25mM α-aminobutyric acid as an internal standard for amino acid measurement. After centrifugation at 20 000g at 4 °C for 10min, the supernatant was filtered (Qmax syringe filter, 0.45 μm pore size; Frisenette Aps, Knebel, DK) and stored at –80 °C until analysis. was derivatized with o-pthaldialdehyde and measured by fluorometry (Wang ). was determined by spectrophotometric detection (Häusler ). Amino acids were derivatized by AccQ-Taq Ultra reagent (Waters, Milford, MA, USA) and afterwards measured on an Acquity UPLC System (BEH C18 1.7 μM column; Waters). For analysis of bulk tissue pH, plant tissues were ground in Milli-Q water and the pH recorded by use of a microelectrode (Metrohm, Herisau, Switzerland). Lyophilized samples were used for analysis of total C and N by mass spectrometry in a system consisting of an ANCA-SL Elemental Analyzer (Wang ).

Frozen tissues were ground with a mortar and pestle for determination of nitrate reductase (NR), glutamine synthetase (GS), and glutamate dehydrogenase (GDH) activities. For NR, the shoot and root material was extracted in a buffer containing 100mM HEPES/KOH (pH 7.6), 20mM MgCl2, 0.2mM phenylmethylsulfonyl fluoride, 5mM dithiothreitol (DTT), 10 μM leupeptin, 1mM Pefabloc, 10 μm flavin adenine dinucleotide, 0.6% polyvinylpolypyrrolidone (PVPP), and 0.05% casein. The assay was performed in the presence of Mg2+ or EDTA in order to obtain the actual and maximum activity, respectively, and the NR activation state (ratio between actual and maximum activity).

Shoot and root tissues for assays of GS and GDH activity were extracted in the same buffer solution containing 50mM HEPES/KOH (pH 7.8), 5mM MgCl2, 0.5mM EDTA-Na2, 5mM DTT, 20% glycerol, and 0.6% PVPP. GS activity was measured by incubation of extracts in a reaction buffer containing 100mM HEPES/KOH (pH 7.8), 150mM glutamate, 10mM MgCl2, 15mM ATP, 10mM hydroxylamine, and 2mM EDTA. After 20min at 30 °C, stop solution consisting of 8% (w/v) trichloroacetic acid, 3.3% (w/v) FeCl3, and 2M HCl was added. NADH-GDH activity was determined in a reaction buffer containing 100mM Tris/HCl (pH 8), 1mM MgCl2, 50mM hydroxylamine, 13mM 2-ketoglutarate, and 0.25mM NADH. NAD-GDH activity was determined in a reaction buffer consisting of 100mM Tris/HCl (pH 9), 1mM CaCl2, 35mM sodium glutamate, and 0.25mM NAD. Total soluble protein in the supernatant of GS and GDH extraction was determined according to the method of Bradford (1976) using bovine serum albumin as the standard.

Calculations and statistical analysis

The at 25 °C was calculated on the basis of the apoplastic concentrations of and H+ according to the following equation (Mattsson ):

where K H and K d are thermodynamic constants of 10−1.76 atm l mol−1 and 10−9.25 mol l−1 at 25 °C, respectively, Γ is the /H+ ratio, and and H+ are the concentration and the proton concentration (H+=10−pH) in the apoplastic extracts. Bulk tissue Γ values were calculated as the ratio between and H+ in bulk tissue extracts.

Two-way analysis of variance (ANOVA) was carried out to test CO2, N form, and their interactions. Duncan’s test was conducted to compare the mean values.

Results

Plant growth

Elevated CO2 increased total plant biomass by 36 and 21% in the and NH4NO3 treatments, respectively (P <0.01; Fig. 1). The corresponding root/shoot ratios remained unchanged in the treatment but decreased in the NH4NO3 treatment (P <0.01; data not shown). The total leaf area was increased by elevated CO2 (P <0.05; Fig. 1), while the water consumption decreased by 8–9% (P <0.01; Fig. 1). More water was consumed when plants were supplied with NH4NO3 compared with those supplied with (P <0.01; Fig. 1).

Fig. 1.

Effect of elevated atmospheric CO2 on plant weight (dry matter basis), leaf area (LA), and water consumption (WC). The experimental treatments were: ambient CO2 and 1mM (A-, open bars), elevated CO2 and 1mM (E-, grey shaded bars), 1mM NH4NO3 (A-NH4NO3, open hatched bars), and elevated CO2 and 1mM NH4NO3 (E-NH4NO3, grey hatched bars). Values are means ± standard error (SE) (n=8). Different letters above columns indicate significant differences (P ≤0.05) between mean values inside same plant organ (shoot, root, or total). In the ANOVA table, * and ** denote significant differences at P ≤0.05 and P ≤0.01, respectively; n.s., no significant difference.

Elevated CO2 promoted a clear increase in net photosynthesis, while stomatal conductance and transpiration decreased (P <0.01; Fig. 2). Measurements of net photosynthesis at elevated CO2 showed similar rates in plants previously grown at ambient or elevated CO2, thus demonstrating that photosynthetic acclimation had not occurred (Fig. 2a). Plants receiving in addition to responded more to elevated CO2 and obtained higher net photosynthetic rates than plants supplied exclusively with (Fig. 2a).

Fig. 2.

Effect of elevated atmospheric CO2 on (a) net photosynthesis (P n), (b) stomatal conductance (g s), and (c) transpiration (E). Values are means ± SE (n=6) measured under similar CO2 levels as in the preceding growth period, i.e. ambient and elevated. Net photosynthesis of plants previously grown at ambient CO2 was also recorded at elevated CO2. Plant growth conditions and symbols are as in Fig. 1.

Nitrogen and carbon levels

The N concentration in the dry matter of both shoot and root tissues exhibited a remarkable decrease at elevated CO2 (P <0.01; Fig. 3a). This decrease was similar whether N was supplied as (–19% in shoot and –18% in roots) or NH4NO3 (–15% in shoot and –22% in roots). The decline in N concentration resulted in a marked increase in the C/N ratio at elevated CO2 (P <0.01; Fig. 3b). However, due to the increased biomass at elevated CO2, the total N content per plant was not changed (Fig. 3c).

Fig. 3.

Effect of elevated CO2 on (a) N concentration per unit dry weight, (b) C/N ratio, and (c) N content. Plant growth conditions and symbols are as in Fig. 1. Values are means ± SE (n=8).

Apoplastic and pH

Elevated CO2 caused about a threefold decrease in the apoplastic concentration (P <0.01; Fig. 4a). At the same time, a small (P <0.05) acidification of the apoplastic solution occurred (Fig. 4b). These changes decreased the stomatal from 2.25 and 2.95 nmol mol–1 under ambient CO2 to 0.37 and 0.89 nmol mol–1 at elevated CO2 in the and NH4NO3 treatments, respectively (P <0.01; Fig. 4c). The corresponding apoplastic Γ values, i.e. the ratio between the concentration of and H+ in the apoplastic solution, were 230 and 302 under ambient CO2 and 38 and 91 at elevated CO2 in the and NH4NO3 treatments, respectively.

Fig. 4.

Effect of elevated CO2 on (a) apoplastic concentration ([]), (b) apoplastic pH, and (c) NH3 compensation point (). Plant growth conditions and symbols are as in Fig. 1. Values are means ± SE (n=6).

Tissue and xylem and pH

Elevated CO2 significantly reduced the concentration in shoots of plants in the treatment but not in the NH4NO3 treatment (Fig. 5a). In the roots, the concentration increased in response to elevated CO2 in the NH4NO3 treatment (Fig. 5a). Elevated CO2 also led to acidification of the leaf tissues (Fig. 5b). The bulk tissue Γ value, i.e. the ratio between bulk tissue and H+ concentration, decreased significantly (P <0.01) from 646 at ambient CO2 to 280 at elevated CO2 in plants supplied with , while in plants receiving the corresponding decrease (P=0.30) was from 667 to 563 (Fig. 5c).

Fig. 5.

Effect of elevated CO2 on (a) bulk tissue concentration, (b) pH, and (c) the ratio between and H+ (Γ). Plant growth conditions and symbols are as in Fig. 1. Values are means ± SE (n=8).

The concentration in the xylem sap was around 1mM and showed a small, statistically non-significant decrease in response to elevated CO2 (data not shown). Elevated CO2 reduced the xylem concentration from around 30 to 22mM in plants receiving only and from around 20 to 13mM in plants receiving NH4NO3 (P <0.01). The pH in the xylem sap was around 6.0 in all treatments and showed a small (about 0.2 pH units) increase under elevated CO2 in the treatment (P <0.05; data not shown).

Tissue , amino acids, and soluble protein

The concentration in shoot and root tissues was significantly reduced by elevated CO2 (P <0.01; Fig. 6a). In addition, the level of soluble (free) amino acids in shoot tissue decreased under elevated CO2, while no significant change was observed in the roots (Fig. 6b). There was no significant effect of elevated CO2 on the soluble protein concentration (Fig. 6c).

Fig. 6.

Effect of elevated CO2 on (a) bulk tissue concentration ([]), (b) total concentration of free amino acids, and (c) total soluble protein. Plant growth conditions are as in Fig. 1. Values are means ± SE (n=6).

NR, GS, and GDH activity

The actual activity of NR in the shoots was about 45% lower under elevated CO2 (P <0.01; Fig. 7). Concurrently, the maximum NR activity in the shoots decreased by 55–60%, reducing the activation state from around 45% under ambient CO2 to 40% under elevated CO2 (Fig. 7). In roots, no significant changes in NR activity were observed (Fig. 7). GS activity was not affected by elevated CO2 (Fig. 8), as was also the case for the aminating activity of GDH (NADH-GDH), while the deaminating GDH activity (NAD-GDH) increased by around 15 and 16–34% in shoots and roots, respectively (data not shown).

Fig. 7.

Effect of elevated CO2 on (a) NR activity expressed on the basis of fresh weight or (b) per unit soluble protein. Full-length columns represent maximum NR activities (Max.), while the horizontal line within columns shows actual NR activities (Act.). Filled circles above columns in (a) show the NR activation state. Plant growth conditions and symbols are as in Fig. 1. Values are means ± SE (n=5–8).

Fig. 8.

Effect of elevated CO2 on GS activity expressed on the basis of (a) fresh weight and (b) soluble protein. Plant growth conditions and symbols are as in Fig. 1. Values are means ± SE (n=8).

Discussion

Plant growth and photosynthesis

Plants exposed to elevated CO2 are likely to become N limited as they achieve a faster growth rate (Conroy and Hocking, 1993). The N limitation will cause a decrease in the N concentration or in the levels of N metabolites (Stitt and Krapp, 1999). In order to avoid N limitation, previous studies have explored a very high N supply (more than 10mM N) to grow barley plants (Sicher and Bunce, 2008; Robredo ), which is far above physiologically relevant N concentrations in soil solution. In the present study, plants received 2mM or 1mM NH4NO3 and the N was topped up or renewed every 3 d. The N concentrations in shoot and root tissues were above 3.8% (Fig. 3) and total soluble protein remained unchanged for all treatments (Fig. 6), showing that the N status of the plants was optimum.

Elevated CO2 dramatically increased leaf photosynthesis as well as shoot and root biomass (Figs 1 and 2a). Increased biomass is a general feature of CO2 responses in C3 crops (Kimball ). However, the initial stimulation of photosynthesis often gradually declines with prolonged exposure to elevated CO2, a phenomenon known as photosynthetic acclimation (Stitt and Krapp, 1999; Ainsworth and Long, 2005; Seneweera ). This acclimation can mechanistically and quantitatively be attributed to a decline in apparent in vivo Rubisco activity (V c,max) and reduced investment in Rubisco, accompanied by a decrease in foliar soluble protein (Rogers and Humphries, 2000). However, in the present study, the amounts of total soluble protein in shoots were similar at ambient and elevated CO2 in both the and NH4NO3 treatments (Fig. 6c). Moreover, plants grown at elevated CO2 did not show any decline in photosynthesis, as evidenced by the fact that their photosynthesis was similar to that of plants grown at ambient CO2 and only exposed to elevated CO2 during the short measurement period (Fig. 2a).

Increased CO2 assimilation at elevated CO2 would be expected to stimulate N acquisition in order to match the faster growth rates (Kruse ). However, declines in N concentration of shoot and root tissues at elevated CO2, accompanied by increasing C/N ratios, are often observed in C3 plants (Cotrufo ; Kimball ). This decrease may derive from a dilution effect accompanying the faster biomass growth (Taub and Wang, 2008). That dilution was a main factor is corroborated by the fact that the quantity of N per plant, i.e. the product of biomass and N concentration, was not different between plants grown at elevated and ambient CO2 (Fig. 3c). It is not clear why a dilution of internal N does not give rise to enhanced uptake capacity by the roots. and uptake by roots is controlled by a number of specific transporters, which are regulated at both transcriptional and post-transcriptional levels (Lanquar ; Laugier ). These transporters are known to respond to the size of the pool of free amino acids, particularly that of glutamine, which provides a signal that can upregulate uptake when plant N status is insufficient or vice versa (Miller ). Plants growing at elevated CO2 actually had 30–50% lower concentrations of glutamine in both roots and shoots (data not shown), but this was apparently not sufficient to stimulate N uptake. uptake also depends on energy from mitochondrial respiration. The potential alterations in root respiration by elevated CO2 are still poorly documented (Gonzelez-Meller ; Leakey ). In the present work, root biomass was increased by elevated CO2 (Fig. 1), which may have increased growth respiration at the expense of energy for ion uptake. The signalling pathways involved in this regulation and their interactions with plant growth and C and N metabolism under elevated CO2 is an area where further knowledge is required (Foyer ). Bloom ) ascribed the lack of increased N uptake under elevated CO2 to decreased capacity for shoot assimilation rather than to a direct effect on N absorption. However, provision of N in the form of did not in the present work result in higher total N uptake (Fig. 3c), suggesting that the lack of stimulation of N acquisition under elevated CO2 was not specifically associated with assimilation.

Plant content and assimilation

The decline in concentration in shoot and root tissues was pronounced for all plants at elevated CO2 (Fig. 6a). Approximately 30% lower concentration in the xylem sap at elevated CO2 in parallel with lower transpiration rates (Figs 1 and 2c) indicated that less was transported to the shoot. In terms of assimilation, elevated CO2 resulted in a marked decrease (45–60%) in shoot maximum and actual NR activities compared with ambient CO2 (Fig. 7). This decline may reflect the lower tissue levels promoting degradation of NR transcripts and protein (Galangau ) and leading to de-induction of transporter and NR activities (Stitt and Krapp, 1999). Elevated CO2 inhibited assimilation in leaves of wheat, tomato, and Arabidopsis (Bloom , 2010; Searles and Bloom, 2003). In a recent study, Bloom ) demonstrated that elevated CO2 inhibited assimilation in eight different C3 species including barley, whereas it remained unchanged in three different C4 species. This indicates that the mode of C fixation is a main factor determining the response in assimilation to elevated CO2. In the work of Bloom and co-workers, plants were grown at a relatively low N supply (<1mM N). However, at higher N supplies (>10mM N), the foliar NR activity of barley was only slightly suppressed (Sicher, 2001) or even significantly enhanced (Robredo ) in response to elevated CO2. In the latter case, plants were grown at very high supply (20mM) to ensure that any possible downregulation of photosynthesis resulting from elevated CO2 was unrelated to nitrogen limitation.

In many plant species, including barley, assimilation predominantly occurs in root tissue when the supply is low (<1mM), and the importance of shoot assimilation increases with increasing concentrations (Andrews ). Root NR activity only accounted for a small proportion of the assimilation in the present work (Fig. 7). Elevated CO2 did not increase the activity of NR in roots, but, due to the relatively large decrease in shoot NR activity, a larger proportion of total plant NR was present in roots (Fig. 7; see also Kruse , 2003). This change in NR distribution may partly alleviate inhibition of shoot assimilation under elevated CO2 due to reduced availability of NADH and ferredoxin (Bloom ).

NH3 compensation point, tissue , and NH3 exchange potential

Elevated CO2 showed a clear negative effect on , which declined from 2.25 and 2.95 nmol mol–1 under ambient CO2 to 0.37 and 0.89 nmol mol–1 at elevated CO2 in the and NH4NO3 treatments, respectively (Fig. 4c). The decrease was derived mainly from lower apoplastic concentrations, while apoplastic H+ concentrations only became marginally lower (Fig. 4a, b).

The apoplastic concentration is controlled by the balance between processes generating and assimilating (Schjoerring ). Root uptake is an important process leading to increased shoot levels (Mattsson and Schjoerring, 2002) and NH3 emission from young barley plants (Mattsson and Schjoerring, 1996). translocated from roots to shoots via the xylem sap enters the leaf apoplastic solution and ultimately the leaf cells (Finnemann and Schjoerring, 1999). Elevated CO2 caused only a non-significant decrease in the xylem concentration but markedly decreased the transpiration rate (Figs 1 and 2c), thereby suppressing the root-to-shoot transport and thus lowering the apoplastic concentration.

Photorespiration is quantitatively one of the most important processes generating in young leaves of C3 plants (Carvalho ) and repression of photorespiration is widely accepted to explain the decreased foliar concentration at elevated CO2 (Geiger ; Sicher, 2001). Suppression of photorespiration has been shown to lead to reduced tissue and decreased NH3 emission (Mattsson ; Husted ; Kumagai ). However, the decrease in tissue was only observed in shoots receiving and not in the NH4NO3 treatment (Fig. 5a), showing the importance of derived from root uptake for bulk tissue levels (Finnemann and Schjoerring, 1999; Mattsson and Schjoerring, 2002).

The activity of the main assimilating enzyme, GS, has been shown to be essential for tissue and apoplastic concentrations in barley (Mattsson ). The GS activity did not show a significant response to elevated CO2 in either shoots or roots (Fig. 8). This suggests that GS may be not the key factor regulating apoplastic under elevated CO2. The enzyme GDH was for many years assumed to be involved in assimilation, but seems on the contrary to play a role in the liberation of by catalysing the oxidative deamination of glutamate, thereby providing carbon skeletons for respiration and oxidative phosphorylation (Labboun ). In agreement, elevated CO2 did not significantly alter the aminating activity of GDH (NADH-GDH), but significantly increased its deaminating activity (NAD-GDH) in shoots and roots (data not shown). Increasing NAD-GDH activity could be relevant to meet energy requirements for assimilation in competition with CO2 assimilation at elevated CO2 (Bloom ).

The stomatal NH3 compensation point, , is a central driver in the exchange of NH3 between plant leaves and the atmosphere (Personne ; Massad ; Zhang ). Lower in response to elevated CO2 will increase the sink strength of plant leaves for atmospheric NH3 and reduce the likelihood of foliar NH3 emissions. Besides , the actual stomatal NH3 flux at a given atmospheric NH3 concentration will also depend on the stomatal conductance. This parameter decreases markedly under elevated CO2 (Fig. 2b; Leakey ), implying that the reduction in under elevated CO2 will not result in a proportional increase in NH3 flux.

Conclusions

It is concluded that elevated atmospheric CO2 decreased in barley leaves. This reflected a pronounced decrease in the concentration in the apoplastic solution, while the pH only changed marginally. The primary reasons for the lower apoplastic concentration were decreased input via the transpiration stream and decreased production via reduction and photorespiration. The activity of GS, the first step in the assimilation of , did not increase in response to elevated CO2.