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. 2002 Feb;128(2):742–750. doi: 10.1104/pp.010602

Dynamic and Steady-State Responses of Inorganic Nitrogen Pools and NH3 Exchange in Leaves of Lolium perenne and Bromus erectus to Changes in Root Nitrogen Supply1

Marie Mattsson 1, Jan K Schjoerring 1,*
PMCID: PMC148935  PMID: 11842177

Abstract

Short- and long-term responses of inorganic N pools and plant-atmosphere NH3 exchange to changes in external N supply were investigated in 11-week-old plants of two grass species, Lolium perenne and Bromus erectus, characteristic of N-rich and N-poor grassland ecosystems, respectively. A switch of root N source from NO3− to NH4+ caused within 3 h a 3- to 6-fold increase in leaf apoplastic NH4+ concentration and a simultaneous decrease in apoplastic pH of about 0.4 pH units in both species. The concentration of total extractable leaf tissue NH4+ also increased two to three times within 3 h after the switch. Removal of exogenous NH4+ caused the apoplastic NH4+ concentration to decline back to the original level within 24 h, whereas the leaf tissue NH4+concentration decreased more slowly and did not reach the original level in 48 h. After growing for 5 weeks with a steady-state supply of NO3− or NH4+, L. perenne were in all cases larger, contained more N, and utilized the absorbed N more efficiently for growth than B. erectus, whereas the two species behaved oppositely with respect to tissue concentrations of NO3−, NH4+, and total N. Ammonia compensation points were higher for B. erectus than for L. perenne and were in both species higher for NH4+- than for NO3−-grown plants. Steady-state levels of apoplastic NH4+, tissue NH4+, and NH3 emission were significantly correlated. It is concluded that leaf apoplastic NH4+ is a highly dynamic pool, closely reflecting changes in the external N supply. This rapid response may constitute a signaling system coordinating leaf N metabolism with the actual N uptake by the roots and the external N availability.

Grasses growing in terrestrial ecosystems receive the major part of their N as NH4+ derived from mineralization of soil organic matter (Whitehead, 1995). However, some nitrification occurs even in grassland soils and may lead to significant supply of nitrate to the roots (Hatch et al., 2000). Once in the root, nitrate can be stored, assimilated, or transported to the shoot. Ammonium absorbed by the roots has previously been assumed to be almost completely assimilated in the roots, but recent studies have shown that significant amounts of NH4+ can be transported in the xylem (Mattsson and Schjoerring, 1996; Finnemann and Schjoerring, 1999) and that plants can contain substantial concentrations of NH4+ in their tissues (Wang et al., 1993; Kronzucker et al., 1995). These NH4+ pools in leaf tissue and apoplast are important for regulating N utilization, including the exchange of gaseous NH3 between plant leaves and the atmosphere.

Because plants can act as both a source of and a sink for atmospheric NH3, it is important to know the physiological mechanisms that are involved in determining their NH3 compensation point, i.e. the NH3 concentration in the air within the sub-stomatal cavities at which no net exchange with the atmosphere takes place (Farquhar et al., 1980). The NH3 compensation point varies with the level of N nutrition (Mattsson et al., 1998), the developmental stage of the plant (Husted et al., 1996), and with the activity of Gln synthetase (Mattsson et al., 1997). Compensation points for NH3 are usually determined by gas exchange measurements in the field or in laboratory cuvettes. Alternatively, leaf apoplastic pH and NH4+ concentrations have been used as bio-indicators for the NH3 compensation point (Husted and Schjoerring, 1995; Mattsson et al., 1997; Hill et al., 2001). There exists, however, considerable discrepancies between results obtained by gas exchange measurements and apoplastic extracts (Hanstein et al., 1999; Sutton et al., 2001).

This investigation was conducted to study dynamic and steady-state effects of N supply to the roots (NO3− or NH4+) on plant NH4+ pools and NH3 exchange with the atmosphere. Two different grass species were compared: Lolium perenne, which is characteristic of N-fertilized grasslands, and Bromus erectus, characteristic of N-poor grasslands. A key question asked was: What are the dynamic responses of apoplastic pH and NH4+ concentrations after changing the source of N supplied to the roots?

RESULTS

Dynamic Response to Switches in N Source

When 3 mm NH4+ was introduced to plants that had been growing in nutrient solution with 3 mm NO3−, the NH4+ concentration in the apoplastic solution rapidly increased and became 3 to 6 times greater than the initial value within 3 h (Fig. 1A). After 3 to 9 h, the apoplastic NH4+ concentration reached a maximum and after 24 h of NH4+ supply it started to decrease again. Tissue NH4+ concentrations did not increase as dramatically, but still showed a 2- to 3-fold increase after 3 h of exposure to NH4+ and thereafter stayed at about the same level throughout the remaining 45 h of the experimental period (Fig. 1B). Concomitant with the change in NH4+ concentration, apoplastic pH showed a transient decrease of about 0.4 pH units (Fig. 2). Switching back from NH4+ to NO3− nutrition after 24 h of NH4+ treatment, i.e. when the NH4+ concentration was still at the maximum level, resulted in a decrease in both apoplastic and bulk tissue NH4+ concentrations (Fig. 1, C and D). After 24 h, the apoplastic NH4+ concentration was down at the original level again, whereas the tissue NH4+ concentration decreased more slowly and did not reach the initial level in 48 h. The two species showed similar responses to the changes in N source, although tissue NH4+ concentrations and apoplastic pH values were generally higher for B. erectus than for L. perenne.

Figure 1.

Figure 1

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Apoplastic NH4+ concentrations (A and C) and leaf tissue NH4+ concentrations (B and D) of 11-week-old L. perenne and B. erectus over a 48-h period after a change in N source from 3 mm NO3− to 3 mm NH4+ (A and B) or from 3 mm NH4+ to 3 mm NO3− (C and D). Values are means ± se of four replicate samples.

Figure 2.

Figure 2

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Apoplastic pH of 11-week-old L. perenne and B. erectus during 48 h after a change in N source from 3 mm NO3− to 3 mm NH4+. Values are means ± se of four replicate samples.

Growth at Different Steady-State N Supplies

Both species remained in the vegetative stage of growth throughout the experimental period. After 6 weeks of growth at three different steady-state N treatments (3 mm KNO3, 3 mm NH4HCO3, or 6 mm NH4HCO3), L. perenne generally had higher root and shoot fresh weights than B. erectus (Fig. 3). L. perenne plants growing at 3 mm NO3− were the largest, followed by those growing on the same concentration of NH4+. A doubling of the NH4+ concentration retarded both shoot and root growth although no stress symptoms were visible. Fresh weights of B. erectus shoots and roots were similar across all the 3 n treatments. Leaf areas were 3 to 4 times higher for L. perenne (2,400–2,800 cm2) than for B. erectus (680–780 cm2).

Figure 3.

Figure 3

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Shoot and root fresh weights of 11-week-old L. perenne and B. erectus plants grown for 6 weeks with different N treatments (3 mm KNO3, 3 mm NH4HCO3, or 6 mm NH4HCO3) in the nutrient solution. Values are means ± se of three replicate samples.

Shoot dry matter N concentration of L. perenne was lowest (2.2%) in NO3−-grown plants and highest (4.9%) in the 6 mm NH4+ treatment (Table I). B. erectus had higher shoot N concentration (4.4%–6%) and much lower shoot C to N ratio than L. perenne at all the N treatments. The total amount of N contained in the shoots at the end of the experimental period was twice as high in L. perenne as in B. erectus, whereas the N content did not differ between 3 mm NO3−- and NH4+-fed plants in either species, amounting to 0.34 g N plant−1 for L. perenne and 0.13 g N plant−1 for B. erectus. Shoots of plants growing at double-strength NH4+ contained 0.39 and 0.21 g N plant−1 for L. perenne and B. erectus, respectively.

Table I.

Total shoot N concentration, shoot C to N ratio, photosynthesis, transpiration, and stomatal conductance of 11-week-old L. perenne and B. erectus

Grass Species N Treatment Total N C to N Ratio Photosynthesis Transpiration Stomatal Conductance
% in dry matter μmol m−2 s−1 mmol m−2 s−1
L. perenne KNO3 2.2 ± 0.1 18.8 ± 1.1 2.39 ± 0.10 0.48 ± 0.01 21.1 ± 0.5
NH4HCO3 3.1 ± 0.2 14.4 ± 1.2 2.90 ± 0.20 0.55 ± 0.01 24.0 ± 0.4
NH4HCO3 × 2 4.9 ± 0.2 8.9 ± 0.4 2.58 ± 0.35 0.59 ± 0.10 25.8 ± 4.4
B. erectus KNO3 4.9 ± 0.3 8.1 ± 0.5 6.18 ± 1.04 1.59 ± 0.04 69.6 ± 1.9
NH4HCO3 4.4 ± 0.3 10.2 ± 1.3 4.68 ± 1.27 1.22 ± 0.07 56.4 ± 3.4
NH4HCO3 × 2 6.0 ± 0.2 7.4 ± 0.2 6.36 ± 0.75 1.53 ± 0.11 66.9 ± 4.7
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Plants were grown for 6 weeks with different N supplies (3 mm KNO3, 3 mm NH4HCO3, or 6 mm NH4HCO3) in the nutrient solution. Values are means ± se of three replicate samples.

Tissue Concentrations of Inorganic N at Different Steady-State N Supplies

Both roots and leaves of NO3−-grown plants had low bulk tissue NH4+ concentrations (Fig. 4, B and C). When NH4+ was the N source, roots had 4- to 7-fold higher NH4+ concentration than leaves. Nitrate-grown B. erectus leaves contained considerable amounts of NO3− (45 ± 4 and 68 ± 12 μmol g−1 fresh weight) in root and leaf tissue, respectively, whereas L. perenne had very low tissue NO3− concentrations (5 ± 2 and 1 ± 0.4 μmol g−1 fresh weight). B. erectus showed higher tissue NH4+ concentrations compared with L. perenne in all cases except in roots of plants grown with double-strength NH4+ (Fig. 4, B and C). Leaf concentrations of free Asn and Gln increased 5- to 10-fold upon provision of NH4+ instead of NO3− (data not shown). Also Ser, Arg, and the nonvolatile amine γ-amino buturic acid were highest (P < 0.05) in NH4+-grown plants, the latter only in B. erectus though (data not shown).

Figure 4.

Figure 4

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Average daytime shoot NH3 emission (A), leaf (B), and root (C) tissue NH4+ concentrations of 11-week-old L. perenne and B. erectus grown for 6 weeks at different N treatments (3 mm KNO3, 3 mm NH4HCO3, or 6 mm NH4HCO3). Values are means ± se of three replicate samples.

Leaf Apoplastic NO3− and NH4+ Concentrations at Different Steady-State N Supplies

The variation in apoplastic NH4+ concentration between species and N treatments closely resembled that of bulk tissue NH4+ (Table II). NO3−-grown plants had lower apoplastic NH4+ concentrations than leaves from 3 mm NH4+- grown plants and the 6 mm NH4+ treatment resulted in further increased apoplastic NH4+ concentrations. The apoplast of NO3−-grown B. erectus also had a high concentration of NO3− (2.4 ± 0.7 mm), which was not the case for L. perenne (0.1 ± 0.05 mm).

Table II.

Leaf apoplastic pH and NH4+ concentration and calculated NH3 compensation points of 11-week-old L. perenne and B. erectus

Grass Species N Treatment NH4+ Concentration pH Calculated Compensation Point
μm nmol mol−1
L. perenne KNO3 21 ± 5 6.7 ± 0.1 0.6 ± 0.1
NH4HCO3 49 ± 12 6.2 ± 0.1 0.8 ± 0.2
NH4HCO3 × 2 170 ± 38 6.3 ± 0.1 2.5 ± 0.7
B. erectus KNO3 33 ± 16 6.4 ± 0.1 1.0 ± 0.8
NH4HCO3 45 ± 19 6.6 ± 0.1 1.8 ± 1.0
NH4HCO3 × 2 369 ± 81 6.5 ± 0.1 10.4 ± 2.6
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Plants were grown for 6 weeks with different N treatments (3 mm KNO3, 3 mm NH4HCO3, or 6 mm NH4HCO3) in the nutrient solution. Values are means ± se of four replicate samples.

Apoplastic pH values were lower in NH4+-grown L. perenne plants (6.2–6.3 ± 0.1) compared with NO3−-grown plants (6.7 ± 0.1), whereas for B. erectus the pH values of the apoplastic solution were similar (6.4–6.6 ± 0.1) in all treatments (Table II).

Measured and Calculated Compensation Points in Plants Growing at Different Steady-State N Supply

The exchange of ammonia between shoots and the atmosphere responded linearly to changes in NH3 concentrations between 0 to 20 nmol mol−1 in both species (Fig. 5). Compensation points were 5.0 and 6.8 nmol mol−1 for NO3−-grown plants of L. perenne and B. erectus, respectively. The corresponding values for NH4+-grown plants were 5.8 and 9.0 nmol mol−1. The 6 mm NH4+ treatment resulted in very high NH3 compensation points of 11.9 and 18.3 nmol mol−1 for L. perenne and B. erectus, respectively. B. erectus showed greater response to the NH3 supply than L. perenne as evidenced by 2 to 3 times higher leaf NH3 conductance (slope of the curve). This difference was mainly related to differences in the stomatal conductance, which also was 2 to 3 times higher in B. erectus (Table I).

Figure 5.

Figure 5

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Ammonia fluxes at different external concentrations above the leaves of 11-week-old L. perenne and B. erectus grown for 6 weeks with 3 mm KNO3 (A), 3 mm NH4HCO3 (B), or 6 mm NH4HCO3 (C) in the nutrient solution. Positive fluxes denote NH3 emission and negative fluxes represent NH3 absorption. Curves are typical examples of two replicate measurements. Correlation coefficients were >0.99 for all curves.

Compensation points estimated on the basis of apoplastic pH and NH4+ concentrations (Table II) showed the same pattern in relation to N supply as those measured in the fumigation experiments, but the calculated values were from 2- to 9-fold lower than the measured.

Gas-Exchange Measurements in Plants Growing at Different Steady-State N Supply

Ammonia emission, photosynthesis, and transpiration were measured after 1.5 h of acclimatization in the cuvette with no external NH3 added. All plants emitted NH3 in the light period. Emissions were lowest for NO3−-grown plants, tended to increase in 3 mm NH4+-grown plants, and were particularly high in B. erectus growing at 6 mm NH4+ (Fig. 4A).

Net photosynthesis and transpiration rates did not change with the N treatment but were on a leaf area basis more than two times higher for B. erectus compared with L. perenne (Table I).

DISCUSSION

Ammonium concentrations in leaf tissue and apoplast increased rapidly in both L. perenne and B. erectus as a response to the switch from NO3− to NH4+ nutrition (Fig. 1). This demonstrates that NH4+ is rapidly taken up from the nutrient solution, translocated in the xylem from root to shoot (Mattsson and Schjoerring, 1996; Finnemann and Schjoerring, 1999), further transferred to the leaf apoplast, and absorbed into the leaf cells. A transient decrease in apoplastic pH of 0.4 pH unit (Fig. 2) coincided with the increase in apoplastic NH4+ concentration that followed upon the switch to NH4+ nutrition. Thus, uptake of NH4+ from the apoplast into the symplast of the cell was associated with a net release of H+ (see also Hoffmann et al., 1992; Mattsson et al., 1998). The oppositely directed responses of apoplastic NH4+ and pH resulted in roughly unchanged NH3 compensation points (not shown). Ammonium concentrations in the apoplast decreased again and pH values were back to normal after 48 h of NH4+ treatment (Fig. 1), suggesting repression of NH4+ uptake in the roots due to feedback regulation elicited by amino acids, especially Gln, or by NH4+ itself (Kronzucker et al., 1998). Removal of the NH4+ after 24 h of NH4+ treatment resulted in a rapid decrease of NH4+ concentrations in both leaf apoplast and tissue. Both the increase and decrease of NH4+ concentration were more pronounced in the apoplast than in the leaf tissue, indicating that the apoplastic NH4+ pool is a very dynamic N pool rapidly responding to changes in external N supply. This rapid response may constitute a signaling system coordinating leaf N metabolism with the actual N uptake by the roots and the external N availability.

L. perenne plants have been shown to prefer NH4+ over NO3−, both when supplied individually (Griffith and Streeter, 1994) or in combination (Clarkson et al., 1992). With the N treatments used in the present study it was, however, clear that, although NO3− and NH4+ were taken up at similar rates, NO3− produced the largest L. perenne plants (Fig. 3). The fact that only small amounts of NO3− were present in the leaves of L. perenne (see “Results”) indicates that all the NO3− taken up was utilized for growth. B. erectus, on the other hand, utilized N similarly regardless of the form taken up. Part of the higher leaf N concentration in NH4+- compared with NO3−-grown plants could be attributed to 10 to 20 times higher Gln and Asn concentrations, which are well known responses to NH4+ nutrition (Goodchild and Givan, 1990; Clarkson et al., 1992). The reduced growth of L. perenne under NH4+ nutrition compared with NO3− nutrition may be due to factors such as intracellular pH disturbance, increased contents of polyamines, disturbance in osmoregulation, or lack of C skeletons in the root (Gerendás et al., 1997).

At the high NH4+ treatment (6 mm), growth was retarded in L. perenne and total N concentration was further increased. Growth inhibition by high external NH4+ concentrations has often been demonstrated (Shelp, 1987; Magalhaes and Huber, 1989; Cramer and Lewis, 1993; Raab and Terry, 1995). Ammonium supplied at a high concentration (6 mm) to B. erectus produced plants of the same size as the 3 mm treatments but with increased total N concentration (Fig. 3, Table I), indicating that B. erectus was not able to utilize the extra NH4+ taken up for increasing growth.

The 6-week NH4+ treatment increased leaf tissue and apoplastic NH4+ concentrations only slightly compared with NO3−-grown plants (Fig. 4, Table II). Seen together with the short-term responses to NH4+ nutrition (Fig. 1), this indicates that at the 3 mm level of supply, NH4+ was efficiently removed from the apoplast into the symplast. An increase in NH4+ supply from 3 to 6 mm resulted, however, in further increased tissue NH4+ concentrations, particularly in the roots, and in dramatically increased NH4+ concentrations of the apoplast, indicating intracellular overloading.

It has been suggested (Sutton et al., 2001) that the tissue NH4+ concentration can be used to estimate the NH3 compensation point because it often shows a proportional increase to apoplastic NH4+. Linear relationships between the external NH4+ concentration and that in leaf tissue, xylem sap, or leaf apoplastic solution have been shown for barley (Hordeum vulgare) and oilseed rape (Brassica napus; Mattsson et al., 1998; Finnemann and Schjoerring, 1998, 1999). The correlation between leaf tissue NH4+ concentrations and leaf apoplastic NH4+ concentrations in the present experiment was not close (Fig. 6A, r2 = 0.5), but still significant (P = 0.02). Emission of NH3 for both L. perenne and B. erectus followed the same pattern as the apoplastic NH4+ concentration and the leaf tissue NH4+ concentration (Fig. 4); therefore, NH3 emission was closely correlated (r2 = 0.94, P = 0.001) with the NH4+ concentration in the leaf tissue (Fig. 6B).

Figure 6.

Figure 6

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Correlation between leaf tissue NH4+ concentrations and apoplastic NH4+ (A) and NH3 emission (B) of 11-week-old L. perenne and B. erectus grown for 6 weeks at different N treatments (3 mm KNO3, 3 mm NH4HCO3, or 6 mm NH4HCO3).

The plants supplied with 6 mm NH4+ experienced pH fluctuations from 6 to 8.5 in the nutrient solution. The very high concentrations of NH4+ in the root tissue and in the leaf apoplast of these plants (Fig. 4, Table II) may at least partly be due to NH3 influx (Gerendás et al., 1997) because at high external pH the NH3/NH4+ ratio increases and NH3 may diffuse uncontrolled over the plasma membrane (Walch-Liu et al., 2000).

The compensation points measured by gas exchange (Fig. 5) were higher for B. erectus than for L. perenne in all the treatments. B. erectus also showed a 3-fold higher leaf NH3 conductance (slope of the curves in Fig. 5) and higher stomatal conductances (Table I) than L. perenne. A relatively high NH3 conductance for B. erectus was also observed by Hanstein et al. (1999), who in addition found a high correlation between stomatal conductance and total leaf NH3 conductance. The NH3 compensation points reported by Hanstein et al. (1999) were, however, very low compared with the NH3 compensation points obtained in the present study. The differences between the two experiments likely reflect the fact that in the experiment of Hanstein et al. (1999), the leaf N concentrations were considerably lower (3.3% on a dry matter basis) than in the present work (4.4% for 3 mm NH4+-grown plants; Table I).

The NH3 compensation points determined on the basis of NH3 flux measurements in the present experiment were severalfold higher than the NH3 compensation points derived from apoplastic NH4+ concentrations and pH (Fig. 5, Table I). The general pattern showing the lowest compensation points for NO3−-grown plants, somewhat higher values for NH4+-grown plants, and much higher NH3 compensation points for plants growing at double-strength NH4+ was, however, the same for both measured and calculated values (Fig. 5, Table I). In vegetative growth stages of oilseed rape, a good agreement was found between measured and calculated NH3 compensation points, whereas during later stages several cases of discrepancy were observed (Husted and Schjoerring, 1996). In Luzula sylvatica, the NH3 compensation points determined by NH3 fumigation in cuvettes were 2 to 30 times higher than those calculated on the basis of apoplast extracts (Hill et al., 2001). Also, compared with NH3 compensation points derived from micrometeorological measurements in the field, the apoplastic bio-assay has in several cases resulted in lower NH3 compensation points (Sutton et al., 2001). One reason for the discrepancy may be temporal and spatial variability in apoplastic pH (Hanstein and Felle, 1999; Yu et al., 2000) and NH4+ concentration. The latter may be due to convection of NH4+ in the transpiration stream of water, which would lead to an enrichment of NH4+ at the sites of evaporation and cause NH3 compensation points to be greater in practice (gas exchange experiments). Despite uncertainties about the actual level of the NH3 compensation point, the apoplastic bio-assay was still capable of predicting relative effects of N treatment and species differences. In general, NH3 compensation points increased with NH4+ nutrition as compared with NO3− nutrition and also increased with increasing levels of N fertilization.

CONCLUSIONS

The apoplast is a highly dynamic NH4+ pool that rapidly responds to changes in external N. Both the N source (NO3− or NH4+) and amount of N supplied to the plants largely influence tissue and apoplastic NH4+ concentrations and NH3 emission to the atmosphere.

Increasing N availability leads to higher steady-state levels of inorganic N compounds and higher NH3 compensations points in leaves of a grass species adapted to growth under N-poor conditions as compared with a species naturally occurring in N-rich ecosystems.

MATERIALS AND METHODS

Plant Culturing

Seeds of Lolium perenne L. cv Bastion and Bromus erectus Huds. were germinated on wet filter paper in the dark for 7 d at 20°C. Three seedlings were transferred to hydroponics in 4-L high-density polyethylene containers and grown in a greenhouse with a day/night period of 16/8 h. To keep the day light intensity above 400 μmol m−2 s−1, supplementary light was given by HQI lamps (Power Star 400W, Osram, Munich). Day/night temperatures were 20°C ± 3°C/15°C ± 2°C. The nutrient solution consisted of: HxPO4(3 − x)− (0.2 mm), K+ (1.2 mm), Mg2+ (0.6 mm), Ca2+ (0.3–0.9 mm), SO42− (0.5 mm), Na+ (0.1 mm), Cl− (0.1 mm), Fe-EDTA (50 μm), Mn (7 μm), B (2 μm), Zn (0.7 μm), Cu (0.8 μm), and Mo (0.8 μm). All plants were supplied with 3 mm KNO3 during the first 4 weeks of the experiment. After this, the plants were grown for 6 weeks at three different N regimes: 3 mm KNO3, 3 mm NH4HCO3, and 6 mm NH4HCO3. The solutions were renewed once a week. N was supplied and pH was adjusted three times a week.

Gas-Exchange Measurements

Gas-exchange measurements were performed during the 5th week of growth with different N treatments at a total plant age of 10 to 11 weeks. Uptake and emission of gaseous NH3 in the shoots was monitored in NH3 concentrations ranging from 0 to 20 nmol mol−1 air. A computerized cuvette system designed for simultaneous measurements of NH3 exchange, photosynthesis, and transpiration was used (Mattsson and Schjoerring, 1996). Into this cuvette, 50 L min−1 of pressurized filtered air with the stated NH3 concentration was led. The plant cuvette was installed in a growth chamber in which air temperature, light, and relative humidity of the air were controlled. The temperature was 20°C, the relative humidity was 65%, and the photon flux density was 300 μmol m−2 s−1. Ammonia was measured continuously by sampling of the air stream leaving the cuvette in a rotating denuder and analysis of the collected NH3 by conductometry as described by Wyers et al. (1993). CO2 and water were measured using a combined infrared gas analyser (Ciras-1, PP-Systems, Herts, UK). All data were logged once a minute and NH3 flux, photosynthesis, and transpiration were calculated using the air flow rates through the cuvette and leaf areas of the plants. Estimates of stomatal conductances were based on transpiration data and a leaf temperature of 20°C.

Plant Harvest

Some containers with NO3−-grown plants were utilized for studying effects over time of switching the N source from NO3− to NH4+ and back again. Plant material from these containers was harvested for apoplastic extractions and tissue NH4+ determinations at 0, 3, 6, 9, 24, and 48 h after start of the NH4+ treatments, or following a 24-h NH4+ treatment at 0, 3, 8, 24, and 48 h after switching back to NO3− treatment. The other containers were all harvested after 11 weeks of growth and the fresh weights and leaf areas of the plants were monitored and roots were rinsed in deionized water. Some of the leaf material was used for apoplastic extractions, whereas other tissue material was immediately frozen in liquid N2 and then stored at −80°C until extraction. The rest of the plant material was dried at 70°C, ground to a fine powder, and used for total C/N analysis.

Extraction of Leaf and Root Tissue

The plant tissue was homogenized in 10 mm formic acid in a cooled mortar with a little sand. The homogenate was centrifuged at 25,000g (2°C) for 10 min and the supernatant was transferred to 500-μL 0.45-μm polysulphone centrifugation filters (Micro VectraSpin, Whatman Ltd, Maidstone, UK) and spun at 5,000g (2°C) for 5 min. The filtered solution was used for analysis of NO3− and NH4+ concentrations.

Extraction of Apoplastic Fluid

Apoplastic solution was extracted with a vacuum infiltration technique slightly modified from the method described by Husted and Schjoerring (1995). The technique is based on vacuum infiltration of leaves with isotonic sorbitol solutions (490 mOsm for L. perenne and 530 mOsm for B. erectus corresponding to 392 and 424 mm sorbitol, respectively). Fifty-milliliter plastic syringes were mounted on a hydraulic arm that automatically moved the plunger up and down to infiltrate the leaf. The infiltrator exposed the leaves to a pressure of 4 atm and vacuum for 10 s, and repeated the procedure five times, thereby ensuring full infiltration. Infiltrated leaves were blotted dry and left in sealed plastic bags for 15 min at 18°C to allow full equilibration between apoplast and symplasm. After this time, the leaf apoplast was extracted by centrifugation at 2,000g for 10 min at 4°C and the apoplastic solution (20–100 μL) was collected in small vials (Eppendorf Scientific, Westbury, NY). pH of the apoplast was determined with a microelectrode (Metrohm, Herisau, Switzerland) and then the apoplast extracts were stabilized with ice-cold 20 mm HCOOH in a 1:1 (v/v) ratio. Apoplastic extracts were used for determination of NH4+ concentrations.

Analysis of N Compounds

Ammonium was determined by fluorimetry on an HPLC system (Waters Corp., Milford, MA) equipped with a pump, a column oven with a 3.3-m stainless steel reaction coil, an autosampler cooled to 2°C, and a scanning fluorescence detector. The reaction between NH4+ and o-phthaldehyde to form an alkylthioisoindole fluorocrome was performed at neutral pH with 2-mercaptoethanol as reducing agent. This fluorochrome was detected at an excitation wavelength of 410 nm and an emission wavelength of 470 nm (Husted et al., 2000).

Nitrate concentration in tissue and apoplast was determined according to Cataldo et al. (1975), where nitration of salicylic acid is recorded at 410 nm.

Amino acids were analyzed by the AccQ-Tag method developed by Cohen and Michaud (1993), following derivatization with 6-N-aminoquinolyl-N-hydroxysuccinimidyl carbamate and quantification in a HPLC system (method described in detail by Husted et al., 2000).

Total C and N concentrations were determined on an elemental analyzer (20-20, Europa Scientific, Crewe, UK) according to the Dumas method.

Compensation Point Determination

Ammonia compensation points were determined by two different procedures: (a) on the basis of NH3 fumigation experiments in which the NH3 exchange fluxes with the atmosphere were measured, and (b) by using the apoplastic NH4+ and H+ concentrations (Husted and Schjoerring, 1995) according to the equation:

graphic file with name M1.gif

where χNH3 is the NH3 compensation point, Ctot is the NH4+/NH3 concentration of the apoplast, and (H+) is the H+ activity of the apoplast. Kd and KH are thermodynamic constants at 20°C.

Footnotes

1

This work was supported by the European Commission (GRAMINAE project; contract no. ENV4–CT98–0722).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.010602.

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