Journal of Experimental Botany, Vol. 53, No. 366, pp. 139-146,
January 1, 2002
© 2002 Oxford University Press
Original Papers |
Leaf–atmosphere NH3 exchange of white clover (Trifolium repens L.) in relation to mineral N nutrition and symbiotic N2 fixation
1 Swiss Federal Research Station for Agroecology and Agriculture, Zuerich-Reckenholz and Liebefeld-Bern, Schwarzenburgstrasse 155, Liebefeld, CH-3003 Bern, Switzerland
2 Plant Nutrition Laboratory, Department of Agricultural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark
Received 21 May 2001; Accepted 3 September 2001
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Abstract |
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Plant–atmosphere NH3 exchange was studied in white clover (Trifolium repens L. cv. Seminole) growing in nutrient solution containing 0 (N2 based), 0.5 (low N) or 4.5 (high N) mM
The aim was to show whether the NH3 exchange potential is influenced by the proportion of N2 fixation relative to 
supply. During the treatment, inhibition of N2 fixation by was followed by in situ determination of total nitrogenase activity (TNA), and stomatal NH3 compensation points (
NH3) were calculated on the basis of apoplastic 
concentration ([]) and pH. Whole-plant NH3 exchange, transpiration and net CO2 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 [] as well as NH3 were equal in the three treatments. In NH3-free air, net NH3 emission rates of <1 nmol m-2 s-1 were observed in both high-N and N2-based plants. When plants were supplied with air containing 40 nmol mol-1 NH3, the resulting net NH3 uptake was higher in plants which acquired N exclusively from symbiotic N2 fixation, compared to grown plants. The results indicate that symbiotic N2 fixation and mineral N acquisition in white clover are balanced with respect to the pool leading to equal NH3 in plants growing with or without At atmospheric NH3 concentrations exceeding NH3, the NH3 uptake rate is controlled by the N demand of the plants. Key words: Ammonia, apoplast, clover, compensation point, nitrogen fixation.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ammonia (NH3) is recognized as a major atmospheric pollutant affecting soil chemistry, biodiversity and stability of ecosystems (Gundersen and Rasmussen, 1990
; Bobbink et al., 1992). Considerable losses of NH3 from shoots of agricultural plants have been measured in field investigations (Harper et al., 1987; Sutton et al., 1993; Yamulki et al., 1996; Schjoerring and Mattsson, 2001). However, chamber studies revealed that plants may either emit or absorb NH3, depending on the NH3 concentration gradient between the atmosphere and the substomatal cavity of the leaves (Farquhar et al., 1980). A higher NH3 concentration in the substomatal cavity compared to the atmosphere results in NH3 emission while NH3 uptake occurs in the opposite case. The NH3 concentration at which NH3 emission and uptake are balanced is called the stomatal NH3 compensation point (NH3; Farquhar et al., 1980). The influence of N nutrition on NH3 has been demonstrated in several studies. In Hordeum vulgare NH3 emission increased with increasing addition rates of mineral N, and NH3 emission was generally higher in plants receiving 
compared to plants supplied with 
(Mattsson and Schjoerring, 1996; Mattsson et al., 1998). In Brassica napus, NH3 was highly correlated with apoplastic and total leaf tissue [] (Mattsson et al., 1998).
The dominating N source for legumes is in many cases N2. In a grass/clover field without N fertilization annual N inputs via symbiosis of up to 300 kg N ha-1 a-1 have been reported (Boller and Nösberger, 1987). The reduction of atmospheric N2 to is mediated by the bacterial enzyme nitrogenase, which is negatively influenced by the availability of mineral N (Minchin et al., 1986; Streeter, 1988; Kaiser et al., 1997). Symbiotic N2 fixation may, therefore, be tuned to the N demand of the plant, depending on the external concentration of mineral N. However, it is not known whether the two pathways of primary N acquisition influence the [] and thus NH3 of the leaves to the same extent. produced by symbiotic N2 fixation is assimilated in the root nodules and transported to the shoot in the form of amides or ureides (Raven and Smith, 1976). In contrast, N supplied as is partly translocated to the shoot in unaltered form. In white clover the ratio between assimilated in the shoot and in the root increases with the applied concentration (Pate, 1971).
The objective of the present study was to elucidate the role of the N source, i.e. nutrition versus N2 derived from fixation, on NH3 and NH3 exchange of white clover. It could be expected that with application, NH3 and thus NH3 exchange would be influenced by the N supply rate, whereas with only N2 fixation, a more balanced N budget, which is tuned to the N demand, would result in lower NH3. Changes in in situ nitrogenase activity and NH3, the latter derived from measurements of [] and pH in leaf apoplast solution, were followed over a period of 10 d after application of either 0.5 or 4.5 mM to plants that previously had acquired all their N from symbiotic N2 fixation. The NH3 could thus be followed during the transition from N2 fixation to predominantly nutrition. The contribution of symbiotic N2 fixation to plant N accumulation was quantified using the 15N dilution method. Whole-plant NH3 exchange was related to net CO2 exchange and transpiration.
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Materials and methods |
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Plant cultivation
Seeds of white clover (Trifolium repens L. cv. Seminole) were germinated on moist vermiculite, inoculated with Hup- Rhizobium leguminosarum biovar trifolii strain WPBS5 (IGER, Aberystwyth, Wales) and kept in the greenhouse. At 20 d the seedlings were transferred to 4.0 l containers (five plants per container) with aerated N-free nutrient solution of the following composition (in mM): P 0.2, K 0.6, S 0.5, Ca 1.0, Cl 2.5, Mg 0.5, Fe 0.05, Na 0.1, and (in µM) Mn 7.0, B 2.0, Cu 0.8, Mo 0.8, Zn 0.7. The plants were mounted on the lid with a silicon rubber sealant (Qubit Systems Inc., Kingston, Ontario, Canada) in order to set up an air-tight seal between shoot and root. They were grown in a climate chamber at a day/night temperature of 20/15 °C (70% RH) with a daily 16 h light period (400 µmol m-2 s-1) followed by 8 h of darkness. The nutrient solution was changed once a week. From 49 d and onwards, the plants received either 0.5 mM N (low N) or 4.5 mM N (high N) applied as 10% enriched Ca(15NO3)2, or N-free nutrient solution (N2-based). Plants from three containers of each treatment were used for nitrogenase measurements as well as apoplastic infiltration. From the high-N and the N2-based treatment another set of three containers each were used for gas exchange measurements. Each container was considered as one replicate. During the measuring period of 10 d the nutrient solution was changed daily.
Determination of nitrogenase activity
Nitrogenase (EC 1.7.99.2) activity was measured in situ as H2 evolution. The sealed nodulated root systems were provided with a continuous flow of H2-free gas (1.0 l min-1) and H2 concentration in the effluent gas stream was measured by a hydrogen sensor (Qubit Systems Inc., Kingston, Ontario, Canada). For the determination of apparent nitrogenase activity (ANA) the gas consisted of a 80:20 (v/v) mixture of N2:O2 until stabilization was reached. The peak value observed after switching to a 80:20 (v/v) mixture of Ar:O2 was used to determine total nitrogenase activity (TNA). Electron allocation coefficients (EAC) were calculated by the following equation:
 | (001) |
Gas exchange measurements
Ammonia exchange, net CO2 exchange, and transpiration of the clover shoots were monitored simultaneously in a computer-controlled cuvette system. The system was placed in a growth chamber (Mattsson and Schjoerring, 1996) and supplied with a continuous flow of 50 l min-1 of pressurized N2-free air. The growth chamber was operated with a photon flux density of 400 µmol m-2 s-1 during the 16 h photoperiod at 20 °C and 15 °C during the dark period and a relative air humidity of 60%. The air stream leaving the cuvette was sampled in a rotating denuder to collect NH3 which was analysed by conductometry as described previously (Wyers et al., 1993). NH3 concentration was logged once every minute and NH3 fluxes were calculated using the difference in the NH3 concentration between the empty cuvette and the cuvette containing the plant, the flow rate through the cuvette, and total leaf area of the plant. For an estimation of NH3 plants were exposed to air containing five NH3 concentrations ranging from 0 to 40 nmol mol-1. Measured NH3 fluxes were plotted against the NH3 concentration and the intercept of the regression line with the abscissa yielded NH3 whereas the slope of the regression line yielded leaf NH3 conductance (g(NH3)). Simultaneously with NH3 exchange, net CO2 exchange and transpiration were monitored using a combined infrared gas analysis system (CIRAS-1, PP-systems, Herts, UK). The stomatal conductance for water vapour (gs(H2O)) was obtained from the transpiration rate, E (mol m-2 s-1), the mole fraction of water vapour in the cuvette, 0 (mol mol-1) and the mole fraction of water vapour in the intercellular air space of the leaves, i (mol mol-1):
 | (002) |
i was derived from leaf temperature assuming that the intercellular air space was close to saturation.
The stomatal conductance for NH3 (gs(NH3)) was derived from gs(H2O) using the ratio between the diffusion coefficients of NH3 and H2O:
 | (003) |
Plant analysis
On selected days during the exposure to inorganic nitrate in the root medium eight fully developed green leaves from each container were collected for extraction of apoplastic solution by vacuum infiltration (Husted and Schjoerring, 1995). The pH of the extracts were measured directly after infiltration with a microelectrode (9802 BN Orion, Boston, USA). The leaves were infiltrated with a 280 mM sorbitol solution at a pressure of 16 bar and under vacuum for 5 s. This procedure was repeated five times. After infiltration, leaves were carefully blotted dry, kept in plastic bags to equilibrate for 20 min in the climate chamber, and centrifuged for 10 min at 4 °C and 1800 g. Concentrations of in the extracted solution were determined by fluorimetry on a HPLC system (Waters Corp., Milford, USA) (Husted et al., 2000). Apoplastic pH was measured directly after infiltration with a microelectrode (9802 BN Orion, Boston, USA). Cytoplasmic contamination of the apoplast was below 1%, as assessed by measuring the activity of malate dehydrogenase (EC 1.1.1.37) in the apoplast relative to the activity in bulk leaf extracts (Husted and Schjoerring, 1995).
After centrifugation the infiltrated leaves were dried at 60 °C for 24 h, ground to a fine powder and used for analysis of 15N abundance and total N concentration as a percentage of shoot dry weight (Ntot) using the Dumas dry combustion method in a system consisting of an ANCA-SL Elemental Analyser coupled to a 20–20 Tracermass Mass Spectrometer (Europa Scientific Ltd., Crewe, UK).
N derived from the nutrient solution (%N) was calculated (Vose and Victoria, 1986):
 | (004) |
and Ntot yielded total N which was taken up from the nutrient solution (
N) and Nfix was the difference between Ntot and N.
For the analysis of bulk tissue [], four leaves of each container were collected, immediately frozen in liquid N2 and stored at -80 °C. The leaves were ground to a fine powder and 0.2 g of the powder were extracted in 2 ml ice-cold 10 mM formic acid in a cooled mortar. The extract was centrifuged at 25000 g and 4 °C for 10 min and the supernatant transferred to 500 µl 0.45 µm polysulphone centrifugation filters (Micro VectraSpin, Whatman Ltd., Maidstone, UK) and centrifuged at 5000 g and 4 °C for 5 min. and concentration of the supernatant was analysed using a flow injection system (Husted et al., 2000).
After gas exchange and nitrogenase activity measurements the plants were separated into shoots and roots and the fresh weight was determined separately. The plant material was dried at 60 °C for 24 h, weighed, and ground to a fine powder which was used for analysis of total N and 15N abundance as described above.
Stomatal NH3 compensation points (NH3)
Estimates of NH3 were derived from measured [] and pH of the extracted apoplastic solution (according to Husted and Schjoerring, 1995). The calculation of NH3 was based on the equilibrium of and NH3 (Kd=10-9.25, 25 °C) in the aqueous phase of the apoplast and the equilibrium between NH3 in the aqueous phase and NH3 in the apoplastic gas phase (KH=10-1.76, 25 °C). NH3 was corrected for ionic strength and temperature using Debye–Hückel and Clausius-Clapeyron equations, respectively. Apoplastic [] was not corrected for dilution since the leaves were equilibrated for 20 min after infiltration. Similar to measurements in oilseed rape (Nielsen and Schjoerring, 1998) homeostasis was completely reached after 20 min (JK Schjoerring, unpublished data).
Statistical analysis
Treatment effects on apoplastic parameters, N concentration and plant weight were evaluated by analysis of variance (ANOVA) for every single measuring time point using NCSS 2000 software (NCSS, Kaysville, Utah, USA). All experiments were carried out twice.
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Results |
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Plant weight and N concentration
After 10 d of N treatment, total plant dry weight was equal in plants receiving nitrate and plants depending exclusively on N2 fixation (P>0.05; Table 1
). The increase in root biomass was smaller in high-N plants compared to N2-based plants. Nodule dry weight was highest in N2-based plants and decreased with increasing N application. Total N concentration in the shoot increased with the amount of the applied (Table 2). The increase in total N during the 10 d treatment was around 20% in N2-based plants, 33% in low-N plants and 54% in high-N plants. In low-N plants, 55% of the total N increase was derived from symbiotic N2 fixation, whereas in high-N plants this fraction only accounted for 20% of the total N increase.
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). Data are means of 7 replicates ±SE.
Apoplastic and bulk tissue 
concentration, apoplastic pH and stomatal NH3 compensation points
An increase in concentration of plant tissue was seen from the first day after application in both low-N and high-N plants (Fig. 1A). After 7 d of N treatment, concentrations were 7 and 40 µmol g FW-1 in low-N and high-N plants, respectively (Fig. 1A).
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Bulk tissue [
] did not differ among the N treatments (Fig. 1B
). In all plant tissues [] was between 0.5 and 2 µmol g-1 FW and decreased throughout the 10 d of N treatment. In low-N and high-N plants, bulk tissue [] was significantly lower after 10 d of N treatment than before the treatment.
No consistent differences between treatments or changes with time were observed for apoplastic [] during the 10 days of N application (Fig. 2A). In all treatments, apoplastic [] ranged between 0.1 and 0.3 mM. Apoplastic pH ranged between 5.3 and 5.6 with no consistent pattern of differences among treatments (Fig. 2B). For all N treatments, NH3 did not change significantly over the experimental period (P>0.05).
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Nitrogenase activity
Total nitrogenase activity (TNA) in high-N plants started to decrease from day 2 after the beginning of application to 60% of the initial activity after 10 d (Fig. 3). In contrast, TNA increased over the same period in N2-based plants and reached 160% of the initial activity after 10 d. TNA of low-N plants increased although these plants received in the root medium. Yet the increase was smaller compared to that of N2-based plants. Mean electron allocation coefficient (EAC) was 0.7±0.01, 0.66±0.01 and 0.60±0.02 for N2-based, low-N and high-N plants, respectively.
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Gas exchange measurements
In NH3-free air NH3 was emitted from both N2-based and high-N plants, with peak emission shortly after the light was switched on as seen for an N2-based plant in Fig. 4. A small NH3 emission was still measured during the dark period. Mean NH3 emission was higher in N2-based plants, yet the variability among replicates was high in both treatments (Table 3). Net CO2-exchange and transpiration on a leaf area basis were slightly higher in N2-based plants compared to high-N plants.
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Exposing plants to air containing 40 nmol mol-1 NH3 for 12 h resulted in net NH3 uptake. The rate of uptake per unit leaf area during daytime was twice as high in N2-based plants compared to high-N plants (Table 3
; Fig. 5). During the night, NH3 uptake decreased much less in high-N plants than in N2-based plants and the difference between day and night-time NH3 uptake was less pronounced in high-N plants (Fig. 5). Exposing plants to 40 nmol mol-1 NH3 had no influence on net CO2 exchange and transpiration in both treatments (data not shown).
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NH3 uptake by shoots in both N2-based plants and high-N plants increased linearly with air NH3 concentration in the cuvette in the range 0–40 nmol mol-1 (Fig. 6
). gs(NH3) as derived from the slopes of the regression line in Fig. 6 were 175 mmol m-2 s-1 and 108 mmol m-2 s-1 in N2-based and high-N plants, respectively. The intercept of the regression line with the abscissa representing NH3 was around 4 nmol mol-1 in both N treatments.
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Discussion |
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Initial exposure of clover plants to nitrate enabled the NH3 exchange potential during the change from N2 fixation to
nutrition to be followed. High tissue [] in both low-N and high-N plants showed that a high proportion of the 
which was taken up by the plants, was transported to the shoot and partly stored in an unaltered form (Fig. 1A). A relatively immediate induction and high rates of uptake in plants upon first exposure to nitrate has been observed before (Michaelson-Yeates et al., 1998; Kronzucker et al., 1995). Simultaneously, TNA decreased rapidly after N application in both low and high-N plants compared to N2-based plants (Fig. 3; see also Silsbury et al., 1986; Macduff et al., 1996; Svenning et al. 1996). Although in the present investigation high amounts of were stored in the shoot tissue, the content did not fully account for the difference in the total N concentration among the treatments. Tissue only accounted for 19% and 44% of the difference in total N concentration between N2-based plants and low- and high-N plants, respectively. Thus, the N assimilation capacity was not saturated in N2-based plants.
Despite the higher total N content and the higher concentration in plants supplied with tissue and apoplastic [] were similar among the treatments, as also reflected in NH3 (Figs 1B, 2). Furthermore, the change from N2 fixation to predominantly nutrition in N-treated plants, as seen in the gradual increase in [] and concomitant decrease in TNA, caused no change in [] over the whole period of the N treatment. Thus, in shoots of white clover a stable [] was maintained, independent of the ratio between N2 fixation and acquisition and independent of the total N status of the shoot. This confirms measurements in the field which, on a seasonal scale, revealed similar apoplastic [] in clover plants of a grass/clover crop treated with either 80 or 160 kg N ha-1 a-1 (Herrmann et al., 2001).
When plants were kept in a NH3-free atmosphere daytime, NH3 emission was below 1 nmol m-2 s-1 in N2-based and high-N clover plants and, therefore, in the same range as NH3 emission measured in pre-senescing non-leguminous plants grown on nitrate (Olsen et al., 1995; Schjoerring, 1991). Mean NH3 emission per unit leaf area was on average higher in N2-based plants compared to plants receiving nitrate. In contrast to measurements in non-leguminous plants (Parton et al., 1988; Schjoerring, 1991), NH3 volatilization on a total shoot basis did not increase with the level of N application in the clover plants (data not shown). This suggests that the contribution from clover to NH3 volatilization over grassland ecosystems is of minor importance.
Exposing plants to an atmosphere containing 40 nmol mol-1 NH3 for 12 h resulted in an NH3 uptake rate which was twice as high in N2-based plants compared to high-N plants. Consistent with the NH3 uptake measurements, g(NH3) was higher in N2-based plants than in high-N plants. Higher NH3 uptake rates were also found in low-N plants of Bromus erectus, Arrhenatherum elatius (Hanstein et al., 1999) and in Lolium multiflorum (Whitehead and Lockyer, 1987) compared to high-N plants. In the present study this difference was partly resulting from a higher gs(NH3) in N2-based plants. However, the difference in gs(NH3) between the treatments was lower than the difference in NH3 uptake, indicating that the NH3 exchange rate is not exclusively controlled by stomatal conductance. Cuticular uptake of NH3 was shown to be negligible in several chamber studies and can therefore not explain the discrepancy between gs(NH3) and g(NH3) (van Hove et al., 1991). Similar to the present study g(NH3) in Arrhenatherum elatius was reported to decrease upon suppression of photorespiration while gs(NH3) was not reduced, showing that the two parameters may be regulated independently (Hanstein et al., 1999). This was attributed to a lower mesophyll NH3 conductance resulting from a reduced assimilation rate, which could not keep pace with NH3 supply. A difference in the mesophyll NH3 conductance, which represents the NH3 uptake capacity into the leaf mesophyll, could be responsible for the difference in NH3 uptake between N2-based and high-N clover plants. Accordingly, mesophyll NH3 conductance would be higher in N2-based plants, which showed a lower total N concentration compared to high-N plants. The fact, that a higher NH3 uptake rate in N2-based plants coincided with a lower total N concentration of the shoot compared to high-N plants indicates that mesophyll NH3 conductance may be linked to the N demand of the plant. Whether the rate of assimilation in the leaf cells may be responsible for the NH3 uptake capacity into the leaf remains to be elucidated.
NH3 uptake by clover shoots increased linearly with atmospheric NH3 concentration from 0 to 40 nmol mol-1 (Fig. 6). Thus, the capacity to absorb NH3 was not exceeded in these plants even at the highest level of NH3 used. Consistent with NH3 calculated from apoplastic [] and [H+], NH3 derived from the fumigation experiment was similar for the two treatments. However, NH3 derived from fumigation was on average seven times higher than calculated NH3. The discrepancy between the two methods is in agreement with previous studies (Mattsson et al., 1997; Hill et al., 2001), but the reason for the deviation is still unsolved, since none of the possible errors which could occur during extraction of apoplastic solution could account for the underestimation of NH3 (Hill et al., 2001). Spatial variability of pH and [] within the foliar apoplast might be responsible for the discrepancy. Alternatively, the relatively low pH values measured in the present investigation indicate that an underestimation of pH might be responsible for the low NH3.
From the present study it is concluded that NH3 of white clover is not influenced by the proportion of N2 fixation relative to supply. Although total N concentration is higher in plants supplied with NH3 is equal in plants grown with or without . However, at elevated atmospheric NH3 concentrations NH3 uptake rate is higher in plants which receive nitrogen exclusively from symbiotic N2 fixation, showing that mesophyll conductance might be linked to the N demand of the plants.
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Acknowledgments |
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This work was part of the EU project GRAMINAE (ENV4-CT98-0722) and was funded by the Swiss Federal Office of Education and Science (contract 97.0031) and by the Danish Research Agency (contract no. 643-00-0050/11).
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Notes |
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3 To whom correspondence should be addressed. Fax: +45 352 83460. E-mail: jks{at}kvl.dk
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References |
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