# Temporal variability in bioassays of the stomatal ammonia compensation point in relation to plant and soil nitrogen parameters in intensively managed grassland

M. Mattsson, B. Herrmann, Marie David, Benjamin Loubet, M. Riedo, M.R.

Theobald, M.A. Sutton, D. Bruhn, A. Neftel, J.K. Schjoerring

► To cite this version:

M. Mattsson, B. Herrmann, Marie David, Benjamin Loubet, M. Riedo, et al. Temporal variability in bioassays of the stomatal ammonia compensation point in relation to plant and soil nitrogen parameters in intensively managed grassland. Biogeosciences, 2009, 6 (2), pp.171-179. hal-01192090

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# Temporal variability in bioassays of the stomatal ammonia compensation point in relation to plant and soil nitrogen parameters in intensively managed grassland

M. Mattsson<fnref n="1" />,*, B. Herrmann<fnref n="2" />, M. David<fnref n="3" />, B. Loubet<fnref n="3" />, M. Riedo<fnref n="4" />, M. R. Theobald<fnref n="4" />, M. A. Sutton<fnref n="4" />, D. Bruhn<fnref n="1" />,
A. Neftel<fnref n="2" />, and J. K. Schjoerring<fnref n="1" />

Received: 5 May 2008 – Published in Biogeosciences Discuss.: 1 July 2008
Revised: 18 December 2008 – Accepted: 4 January 2009 – Published: 11 February 2009

**Abstract.** The exchange of ammonia between crop canopies and the atmosphere depends on a range of plant parameters and climatic conditions. However, little is known about effects of management factors. We have here investigated the stomatal ammonia compensation point in response to cutting and fertilization of a grass sward dominated by *Lolium perenne*. Tall grass had a very low NH₃ compensation point (around 1 nmol mol⁻<fnref n="1" />), reflecting the fact that leaf nitrogen (N) concentration was very low. During re-growth after cutting, leaf tissue concentrations of NO₃⁻, NH₄⁺, soluble N and total N increased along with apoplastic NH₄⁺ concentrations. In contrast, apoplastic pH decreased resulting in largely unaltered NH₃ compensation points. Nitrogen fertilization one week after cutting caused the apoplastic NH₄⁺ concentration of the newly emerging leaves to increase dramatically. The NH₃ compensation point peaked between 15 and 25 nmol mol⁻<fnref n="1" /> the day after the fertiliser was applied and thereafter decreased over the following 10 days until reaching the same level as before fertilisation. Ammonium concentrations in leaf apoplast, bulk tissue and litter were positively correlated (P=0.001) throughout the experimental period. Bulk tissue NH₄⁺ concentrations, total plant N and soil

NH₄⁺ concentrations also showed a positive correlation. A very high potential for NH₃ emission was shown by the plant litter.

## 1 Introduction

Ammonia is emitted from plants when the atmospheric NH₃ concentration is lower than the NH₃ compensation point, the latter being equal to the NH₃ concentration in the substomatal cavity (Farquhar et al., 1980; Husted et al., 1996). In the opposite situation, i.e. when the atmospheric NH₃ concentration exceeds the NH₃ compensation point, deposition of NH₃ occurs. The quantity of NH₃ exchanged between crop canopies and the atmosphere may vary between seasons, depending on climatic conditions (Schjoerring and Mattsson, 2001; Sommer et al., 2004). In particular, temperature is known to have a major effect on the NH₃ exchange under controlled environmental conditions (Husted and Schjoerring, 1996; Mattsson et al., 1997) as well as in the field (van Hove et al., 2002; Trebs et al., 2006).

The NH₃ emission potential of grasslands may vary with species composition (Horvath et al., 2005) because grass species differ in NH₃ compensation point (Hanstein et al., 1999; Herrmann et al., 2001; Mattsson and Schjoerring, 2002; Mattsson et al., 2008). In a non-fertilized managed

<figure><img src="image_3.png" /></figure>

Correspondence to: J. K. Schjoerring
(jks@life.ku.dk)

<footnote n="1">¹Plant and Soil Science Laboratory, University of Copenhagen, Faculty of Life Sciences, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark</footnote>

<footnote n="2">²Agroscope Reckenholz-Tänikon Rsearch Station ART, Reckenholzstrasse 191, 8046 Zürich, Switzerland</footnote>

<footnote n="3">³Institut National de la Recherche Agronomique (INRA), UMR Environnement et Grandes Cultures, Thiverval-Grignon, France</footnote>

<footnote n="4">⁴Natural Environmental Research Council, Centre for Ecology and Hydrology, Edinburgh Research Station, Penicuik EH260QB, Midlothian, Scotland</footnote>

<footnote>* now at: Section for Economy and Technology, Halmstad University, 30118 Halmstad, Sweden</footnote>
grassland in The Netherlands, NH₃ emission fluxes were fre-
quent, covering about 50% of the time in a warm, dry sum-
mer period (Wichink Kruit et al., 2007). In contrast, during a
wet, cool autumn period, deposition fluxes dominated (80%
of the time) due to small canopy compensation points caused
by low temperatures and a generally wet surface (Wichink
Kruit et al., 2007). Nitrogen fertilisation is one of the major
management factors of grasslands and NH₃ volatilisation can
be influenced by the form, timing and dosage of N fertiliser
(Riedo et al., 2002). Measurements of NH₃ volatilisation
under controlled laboratory conditions have shown that high
amounts of N supplied to the roots increase NH₃ emission
(Mattsson et al., 1998; Mattson and Schjoerring, 1996) and
NH₃ compensation points (Mattsson and Schjoerring, 2002).
Increasing the N availability to plant roots leads to elevated
steady state levels of different N pools within the plant tis-
sue. In a field experiment over two years, the NH₃ losses
from wheat, oilseed rape and barley increased under condi-
tions of high N concentration in the foliage (Schjoerring and
Mattsson, 2001). In a Scottish experiment, a higher NH₃
compensation point of the grass was seen after only one of
the two cuttings and fertilisations (Loubet et al., 2002). Lit-
tle is known about the NH₃ emission potential of grasslands
where repeated cuttings and N fertilisations are normal man-
agement practice. A better understanding of the component
parameters influencing the NH₃ emission potential is needed
in order to model NH₃ exchange between grasslands and the
atmosphere.

The aim of the present study was to estimate the NH₃
emission potential of grassland in relation to common man-
agement practice. In order to do this, the temporal variation
in the NH₃ compensation point and its underlying compo-
nents of grass leaves and soil were followed at a field site
(Sutton et al., 2008), starting with tall grass and spanning
subsequent events of cutting, lifting and N-fertilization.

**2 Materials and methods**

The investigation took place as part of the GRAMINAE
integrated experiment conducted on a field near Braun-
schweig from 22 May to 15 June 2000. The main field was
600×300 m in size and consisted of a mixed sward domi-
nated by *Lolium perenne* (around 60% abundance), *Phleum
pratense* (~15% abundance) and *Festuca pratensis* (~12%
abundance; Mattsson et al., 2008). The data presented for
leaves of tall grass plants are mean values of these 3 most
abundant species, weighted by their relative abundance in the
field. The grass was cut on 29 May and lifted for silage on
31 May. An area of 10×10 m was left uncut for additional
sampling of tall grass. Fertilizer (100 kg N ha⁻¹ in calcium
ammonium nitrate) was applied on the main field on the 5
June. A 10×10 m plot was left unfertilized and another plot
of the same size received 200 kg N ha⁻¹ in calcium ammo-

nium nitrate. Growth and development of the grass were as
described in Sutton et al. (2008).

2.1 Sampling of plant material

Throughout the entire experiment, plants were sampled al-
most every day between 12:00 and 03:00 p.m. (GMT). Cut
green leaves were immediately taken to the field laboratory
where the apoplastic solution was extracted using a vacuum
infiltration technique (Husted and Schjoerring, 1995). Whole
leaves were infiltrated in isotonic sorbitol solution (280 mM)
at a pressure of 16 bar under vacuum for 5 s. The procedure
was repeated 5 times in order to ensure full infiltration. In-
filtrated leaves were carefully blotted dry and kept in plastic
bags to equilibrate for 15 min in daylight. Leaf apoplastic so-
lution was extracted by centrifugation at 800 g for 10 min at
4°C. After extraction, pH of the apoplastic samples was mea-
sured with a micro-combination pH electrode (9810, Orion,
Beverly, USA) and samples were frozen at –18°C. Leaf sam-
ples for bulk tissue NH₄⁺ and NO₃⁻ analysis were also frozen
down at the same time for later extraction. Samples of litter
(senescent leaves) and stubble (cut stems) were frozen ev-
ery day after the grass was cut. For total N concentration,
samples of leaves, litter and stubbles were taken daily and
immediately dried in an oven (70°C) over night. Guttation
droplets were collected on the main field and the high fer-
tilized plot between 03:00 and 06:00 a.m. and immediately
frozen.

2.2 Plant analysis

Ammonium in apoplastic extracts was determined by fluo-
rometry on an HPLC system (Waters Corp. Milford, USA)
equipped with a pump, a column oven with a 3.3 m stain-
less steel reaction coil, an autosampler cooled to 2°C and
a scanning fluorescence detector. The reaction between
NH<sub>4</sub><sup>+</sup> and o-phthaldehyde (OPA) to form an alkylthioisoin-
dole fluorochrome was performed at neutral pH with 2-
mercaptoethanol as reducing agent. This fluorochrome was
detected at an excitation wavelength of 410 nm and an emis-
sion wavelength of 470 nm (Husted et al., 2000a).

The plant leaves, litter and stubble were homogenised in
10 mM formic acid in a cooled mortar with a little sand. The
homogenate was centrifuged at 25000 g (2°C) for 10 min and
the supernatant was transferred to polysulphone centrifuga-
tion filters (Size 500-µl, mesh 0.45 µm; Micro VectraSpin,
Whatman Ltd., Maidstone, UK) and spun at 5000 g (2°C)
for 5 min. The filtered solution was used for analysis of
NO₃⁻ and NH₄⁺ concentrations on a flow injection system
(Quick Chem instrument, Lachat Istruments INC, Milwau-
kee, USA). Tissue extracts were also analysed for total solu-
ble N concentration (so-called substrate N) using an ANCA-
SL Elemental Analyser coupled to a 20-20 Tracermass Mass
Spectrometer (SerCon Ltd., Crewe, UK). The same equip-
ment was used for analysis of total N and C concentrations
in oven dried plant material ground to a fine powder. For bulk tissue pH measurements, 0.2 g sample of leaf material was homogenized in 2 ml of deionized water in a cooled mortar with a little sand. The homogenate was centrifuged at 14 000 g (4°C) for 10 min and pH in the supernatant measured with a microelectrode (Metrohm, Herisau, Switzerland).

## 2.3 Soil sampling and analysis

Soil samples were taken at least every third to fourth day with a soil auger at random positions over the field. Soil cores were separated into two layers (0–10 cm and 10–30 cm) and frozen at –18°C. A sub-sample was analysed for moisture content by calculating % weight loss after drying the soil for 24 h at 108°C. Another sub-sample (10 g) was used for pH measurements after extraction for 1 h in 25 ml 0.01 M CaCl₂. Plant available NH₄⁺ and NO₃⁻ were analysed with flow injection after extraction of 25 g of soil in 50 ml 2M KCl.

## 2.4 Calculation of the NH₃ compensation point

The stomatal NH₃ compensation point ($\chi_{NH_3}$ mol NH₃ mol⁻¹ air) at 25°C was calculated by use of Eq. (1) derived from Husted and Schjoerring (1996) taking into account that $K_d \ll [H^+]_{apoplast}$ within the range of apoplastic pH values:

$$ ^{25}\chi_{NH_3} = K_{H.25} \times K_{d.25} \times \Gamma = 10^{-11.01} \times \Gamma \quad (1) $$

$\Gamma$ is the dimensionless ratio between the apoplastic NH₄⁺ and H⁺ concentrations, and $K_H$ and $K_d$ are thermodynamic constants of $10^{-1.76}$ atm·mol⁻¹ and $10^{-9.25}$ mol·l⁻¹ at 25°C, respectively. Equation (1) literally calculates the pressure of NH₃ (unit: atm), which according to Dalton's law of partial pressures is equal to the mol fraction (or volume fraction) at a given atmospheric pressure.

The calculated $\chi_{NH_3}$ at 25°C ($T_{ref}$) was adjusted to the actual canopy temperature $T_c$ by the following equation derived from Husted and Schjoerring (1996):

$$ \ln\left(\frac{T_c \chi_{NH_3}}{T_{ref} \chi_{NH_3}}\right) = \frac{(\Delta H_{dis}^0 + \Delta H_{vap}^0)}{R} \times \left(\frac{1}{T_{ref}} - \frac{1}{T_c}\right) = 34.868 - 10395.91/T_c \quad (2) $$

$T_c \chi_{NH_3}$ is the requested NH₃ compensation point at the actual canopy temperature $T_c$ (K), $\Delta H_{dis}^0$ the enthalpy of NH₄⁺ dissociation (52.21 kJ mol⁻¹), $\Delta H_{vap}^0$ the enthalpy of vaporization (34.18 kJ mol⁻¹), and R the gas constant (0.00831 kJ K⁻¹ mol⁻¹).

# 3 Results

## 3.1 Apoplastic parameters

The apoplastic NH₄⁺ concentration was below 50 µM in the tall grass both before and after the main field was cut

<figure><img src="image_4.png" /><figcaption>Fig. 1. Temporal variation in A) apoplastic [NH₄⁺], B) apoplastic pH, and C) the ratio of [NH₄⁺]_{apoplast} to [H⁺]_{apoplast} (Γ; dimensionless) in four treatments of a *Lolium perenne* dominated sward. The grass was cut on 29 May and lifted for silage on 31 May. An area of 100 m² was left uncut for additional sampling of tall grass. Fertilizer (100 kg N ha⁻¹ in calcium ammonium nitrate) was applied on the main field on 5 June. A 100 m² plot was left unfertilized and another plot of same size was applied 200 kg N ha⁻¹ in calcium ammonium nitrate. Vertical dotted lines indicate times of cutting and fertilisation, respectively. Values are means of three replicates ±S.E.</figcaption></figure>

(Fig. 1a). New grass leaves emerging after cutting showed slightly elevated apoplastic NH₄⁺ concentrations compared to leaves of the tall grass (Fig. 1a). Following application of 100 kg N ha⁻¹ to the main field 6 days after cutting of the grass, apoplastic NH₄⁺ concentrations rapidly
<table><caption>**Table 1.** Tissue extracts of green leaves, stems and senescent leaves (litter) of the main field analysed for pH, NH₄⁺ concentration and the ratio Γ between [NH₄⁺] and [H⁺]. Means of 3 replicates ±SE.</caption><thead><tr><th></th><th>pH</th><th>[NH<sub>4</sub><sup>+</sup>], mM</th><th>Γ</th></tr></thead><tbody><tr><td>Green leaves</td><td>6.33±0.02</td><td>1.79±0.01</td><td>3827±171</td></tr><tr><td>Stems</td><td>6.37±0.04</td><td>1.15±0.14</td><td>2696±282</td></tr><tr><td>Senescent leaves</td><td>7.03±0.05</td><td>16.2±1.2</td><td>173 586±12 917</td></tr></tbody></table>

peaked at around 500 µM, but thereafter decreased over the next 10 days until reaching almost the same level as before fertilisation (Fig. 1a). Plants on a plot receiving 200 kg N ha⁻¹ attained a maximum apoplastic NH₄⁺ concentration around 800 µM (Fig. 1a). When no nitrogen was applied (0 N plot) apoplastic NH₄⁺ concentrations remained below 100 µM throughout the experimental period.

Apoplastic pH was higher in the tall grass compared to the cut grass (Fig. 1b). Fertilisation caused a transient increase in apoplastic pH without showing any difference between plants receiving 100 or 200 kg N ha⁻¹ (Fig. 1b). The ratio between apoplastic NH₄⁺ and H⁺ concentrations (Γ<sub>apoplast</sub>) ranged from 10 to 150 before fertilisation (Fig. 1c). The slight increase in apoplastic NH₄⁺ following cutting was counteracted by decreasing pH (Fig. 1b). Accordingly, Γ<sub>apoplast</sub> hardly changed between cutting and fertilisation (Fig. 1c). During the first 2 days after fertilisation, Γ<sub>apoplast</sub> increased to above 1000, but thereafter started to decrease in parallel with the NH₄⁺ concentration (Fig. 1c). In the 0 N plot, Γ<sub>apoplast</sub> remained below 150 throughout the experiment.

The calculated stomatal NH₃ compensation point, χNH₃, corrected for temperature differences between different days (Eq. 2), was 1–2 nmol mol⁻¹ before cutting (data not shown). This level was maintained for unfertilised grass after cutting. After N fertilisation, χNH₃ peaked at 15–25 nmol mol⁻¹ but decreased to 3–4 nmol mol⁻¹ already 4 days after fertilisation (data not shown).

## 3.2 Bulk tissue nitrogen status

Bulk tissue NH₄⁺ concentrations of the tall grass as well as the cut grass prior to fertilisation were lower than 2 µmol g⁻¹ fresh weight (Fig. 2a). After fertilisation, bulk tissue NH₄⁺ increased rapidly and substantially, peaking around 14 µmol g⁻¹ FW with little difference between 100 and 200 kg N ha⁻¹ treatments (Fig. 2a). Plants not receiving N fertilizer (0 N treatment) maintained a bulk tissue NH₄⁺ level below 4 µmol g⁻¹ FW.

Bulk tissue NO₃⁻ concentrations were extremely low in the tall grass (Fig. 2b), while in the new leaves developing after cutting, NO₃⁻ increased considerably. Fertilisation caused a dramatic increase (4 to 5 fold) in bulk tissue NO₃⁻ and the

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Temporal variation in (A) leaf [NH₄⁺] and (B) leaf [NO₃⁻] on a fresh weight basis in four treatments of a *Lolium perenne* dominated sward. Details on experimental treatments are given in Fig. 1. Values represent means of three replicates ±S.E.</figcaption></figure>

high level remained until the end of the experiment (Fig. 2b). In unfertilised grass, NO₃⁻ concentrations decreased towards the end of the experiment to values similar to those of the tall grass.

Total N concentration in the tall grass leaves decreased from 3% (dry weight basis) before cutting to about 2% 9 days later (Fig. 3b). The remaining part of the cut stems (stubble) also had a total N concentration of ca. 2% throughout the rest of the experimental period. In the newly produced leaves of the 100 N treatment (main field), the total N concentration increased from around 3% just after cutting to around 5% at the end of the experiment (Fig. 3b). The final foliar N concentration in the 0 and 200 N treatments were 3.5 % and 5.5 %, respectively (data not shown).

Tissue extracts were also analysed for total soluble N concentration which can be interpreted as a dynamic N pool available for plant growth. This so-called “substrate N” was very high in leaves remaining or developing after cutting (Fig. 3a). Following fertilisation, plants in the 100 and 200 N treatments had significantly higher substrate N than unfertilised grass (0 N treatment). Substrate N constituted between 10 and 40% of total leaf N.
<table><caption>**Table 2.** Correlation coefficient table for all the different parameters measured and calculated in grass plants and soil from the 100 N treatment (main field). r² values with level of significance (*=0.05; **=0.01, ***=0.001). χNH₃ is the stomatal compensation point of green leaves.</caption><thead><tr><th></th><th>[NH<sub>4</sub><sup>+</sup>]<sub>lapo</sub></th><th>pH<sub>apo</sub></th><th>[NH<sub>4</sub><sup>+</sup>]<sub>tissue</sub></th><th>χ NH<sub>3</sub></th><th>[NH<sub>4</sub><sup>+</sup>]<sub>litter</sub></th><th>[NH<sub>4</sub><sup>+</sup>]<sub>guttation</sub></th><th>SubstN</th><th>TotN</th><th>[NH<sub>4</sub><sup>+</sup>]<sub>soil</sub></th></tr></thead><tbody><tr><td>[NH<sub>4</sub><sup>+</sup>]<sub>lapo</sub></td><td>0.08</td><td>—</td><td>0.60***</td><td>0.84***</td><td>0.50***</td><td>0.22</td><td>0.48***</td><td>0.27</td><td>0.23</td></tr><tr><td>pH<sub>apo</sub></td><td>0.08</td><td>—</td><td>0.02</td><td>0.21</td><td>0.07</td><td>0.02</td><td>0.16</td><td>0.15</td><td>0.004</td></tr><tr><td>[NH<sub>4</sub><sup>+</sup>]<sub>tissue</sub></td><td>0.60***</td><td>0.02</td><td>—</td><td>0.48***</td><td>0.73***</td><td>0</td><td>0.35**</td><td>0.42**</td><td>0.29</td></tr><tr><td>χ NH<sub>3</sub></td><td>0.84***</td><td>0.21</td><td>0.48***</td><td>—</td><td>0.46***</td><td>0.01</td><td>0.25*</td><td>0.15</td><td>0.21</td></tr><tr><td>[NH<sub>4</sub><sup>+</sup>]<sub>litter</sub></td><td>0.50***</td><td>0.07</td><td>0.73***</td><td>0.46***</td><td>—</td><td>0.34</td><td>0.40</td><td>0.21</td><td>0.47*</td></tr><tr><td>[NH<sub>4</sub><sup>+</sup>]<sub>guttation</sub></td><td>0.22</td><td>0.02</td><td>0</td><td>0.01</td><td>0.34</td><td>—</td><td>0.19</td><td>0.06</td><td>0.19</td></tr><tr><td>SubstN</td><td>0.48***</td><td>0.16</td><td>0.35**</td><td>0.25*</td><td>0.40</td><td>0.19</td><td>—</td><td>0.23</td><td>0.44</td></tr><tr><td>TotN</td><td>0.27</td><td>0.15</td><td>0.42**</td><td>0.15</td><td>0.21</td><td>0.06</td><td>0.23</td><td>—</td><td>0.68*</td></tr><tr><td>[NH<sub>4</sub><sup>+</sup>]<sub>soil</sub></td><td>0.23</td><td>0.004</td><td>0.29</td><td>0.21</td><td>0.47*</td><td>0.19</td><td>0.44</td><td>0.68*</td><td>—</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Temporal variation in (A) concentration of total soluble N ([N]substrate) in leaf tissue, and (B) total N in new leaves, stubble and hay of a *Lolium perenne* dominated sward. DW=dry weight. Details on experimental treatments are given in Fig. 1. Arrows indicate times of cutting and fertilisation, respectively. Values represent means of three replicates ±S.E.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Temporal variation in litter NH₄⁺ and NO₃⁻ concentrations in a *Lolium perenne* dominated sward. Details on experimental treatments are given in Fig. 1. Arrows indicate times of cutting and fertilisation, respectively. Values represent means of three replicates ±S.E.</figcaption></figure>

The litter component of the grassland consisting of senescent plant leaves either attached to the lower part of the stems or lying on the ground constituted about 20% of the total above-ground biomass before cutting (data not shown). Prior to cutting, litter concentrations of NH₄⁺ and NO₃⁻ were below 50 mM (Fig. 3), while 3 days after cutting the NO₃⁻ concentration in the litter had increased to about 350 mM, while litter NH₄⁺ remained below 50 mM (Fig. 4). After fertilization (100 kg N ha⁻¹), litter NH₄⁺ increased to around 500 mM but started to decrease again already after a few days (Fig. 4). Nitrate concentrations were slightly higher than NH₄⁺ concentrations after fertilisation but followed the same temporal pattern (Fig. 4).
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Temporal variation in [NH<sub>4</sub><sup>+</sup>] and [NO<sub>3</sub><sup>-</sup>] of the top layer of soil in three treatments of a *Lolium perenne* dominated sward. Details on experimental treatments are given in Fig. 1. Arrows indicate times of cutting and fertilisation, respectively.</figcaption></figure>

In parallel to apoplastic extracts, NH<sub>4</sub><sup>+</sup> and H<sup>+</sup> concentra-
tions in bulk tissue extracts can be used to derive Γ values for
different plant components of the sward in order to compare
their potential NH<sub>3</sub> exchange (Table 1). Bulk pH values of
green leaves and stems were similar to those in the apoplas-
tic solution ranging between 6.2 and 6.6 (Table 1; Fig. 1b).
Due to higher NH<sub>4</sub><sup>+</sup> concentrations in the tissue extracts (Ta-
ble 1) than in the apoplastic solution (Fig. 1a), the result-
ing Γ value in green leaves was around 40 times higher than
Γ<sub>apoplast</sub> (Fig. 1c). Extracts of plant litter showed both higher
pH and, particularly, NH<sub>4</sub><sup>+</sup> concentrations compared to leaves
and stems which resulted in a Γ<sub>litter</sub> value of around 173 500,
i.e. 45–60 times higher than Γ<sub>leaf</sub> and Γ<sub>stem</sub> (Table 1).

3.3 Soil parameters

Soil NH<sub>4</sub><sup>+</sup> and NO<sub>3</sub><sup>-</sup> concentrations (Fig. 5) were low be-
fore fertilisation with NH<sub>4</sub><sup>+</sup> concentrations being higher than
NO<sub>3</sub><sup>-</sup> concentrations in both the top soil fraction (0–10 cm)
and the deeper soil fraction (10–30 cm, not shown). The
top soil concentration of inorganic N increased slightly after
cutting of the grass (Fig. 5). Application of 100 kg N ha<sup>-1</sup>
caused a dramatic increase in both NH<sub>4</sub><sup>+</sup> and NO<sub>3</sub><sup>-</sup> (Fig. 5),
while soil inorganic N increased less in the 200 N treatment.
This difference may reflect the fact that the N fertiliser in the
200 N plot was applied by hand a few hours later and un-
der drier conditions compared to that in the 100 N treatment
of the main field where rainfall followed within a couple of
hours after the application.

3.4 Correlation analysis

The NH<sub>4</sub><sup>+</sup> concentrations in apoplastic solution, leaf tissue
and litter were mutually positively correlated (Table 2). The

NH₃ compensation point derived from the apoplastic mea-
surements was also positively correlated with leaf tissue
NH₄⁺, but not with total leaf N content. Apoplastic pH and
the NH₄⁺ concentration in guttation droplets were not signif-
icantly correlated with any of the other parameters (Table 2).

4 Discussion

Before cutting, the tall grass had low NH<sub>4</sub><sup>+</sup> concentrations in both leaf apoplast and bulk tissue (Fig. 1a, 2a). This resulted in NH<sub>3</sub> compensation points so low that the grass was not likely to emit NH<sub>3</sub> before cutting which is in agreement with atmospheric NH<sub>3</sub> concentration gradients above the canopy showing predominantly deposition fluxes (Milford et al., 2008). Extremely low tissue NO<sub>3</sub><sup>-</sup> concentrations (Fig. 2b) as well as low soil NH<sub>4</sub><sup>+</sup> and NO<sub>3</sub><sup>-</sup> levels (Fig. 5) also indicated that the small amounts of inorganic N available to the plants were efficiently taken up and utilised for growth and seed development at this stage. Before fertilisation, the soil content of NH<sub>4</sub><sup>+</sup> was 4 times higher than that of NO<sub>3</sub><sup>-</sup> (Fig. 5), which is not unusual for grassland soil (Whitehead, 1995).

Between cutting and fertilisation there was a re-growth pe-
riod of one week in which the grass leaves first showed in-
creased NO3- concentrations (Fig. 2b) and 5 days later also
increased NH4+ concentrations (Fig. 2a). Ryegrass has been
shown to rapidly accumulate NO3- in both leaves and stubble
after cutting as NO3- is involved in the osmotic adjustment
(Ourry et al., 1989). Also the soluble N and total N concen-
trations of the leaves showed higher values during re-growth
after cutting compared to the tall grass (Fig. 3a, b). The in-
crease in these N pools was paralleled by slightly increasing
NO3- and NH4+ concentrations in the soil. Several authors
have reported that during the first days after cutting, uptake
of N is inhibited (Bakken et al., 1998; Ourry et al., 1988).
In such case plants respond by allocating N from reserves in
root and stubble to the developing leaves. This inhibition of
uptake could explain the increasing levels of soil NO3- and
NH4+ after cutting. In *Lolium perenne* and *Bromus erectus*
grown in nutrient solution, tissue NH4+ concentrations of ex-
panding leaves did not start to increase until 6 days after cut-
ting (Sutton et al., 2001). In the same experiment, apoplastic
NH4+ concentrations increased in the new expanding leaves
3-6 days after cutting. Also in the present study, apoplas-
tic NH4+ concentrations showed slightly higher values in ex-
panding leaves during re-growth compared to the leaves of
the tall grass (Fig. 1a). Increasing tissue concentrations of
NH4+ and NO3- can also result from shortening of the leaf
growth zone and smaller dilution of the N transported to this
zone after defoliation compared to the fully expanded leaves
before cutting (Schäufele and Schnyder, 2001).
Grass cutting has in several cases been reported to lead to NH₃ volatilization from grassland (Milford et al., 1999, 2002; Loubet et al., 2001). The emitted NH₃ may originate from the plants as a consequence of increased N pools during the period of leaf expansion. However, the potential for NH₃ emission did not seem to increase after cutting in the present work since Γ<sub>apoplast</sub> (the ratio between apoplastic NH<sub>4</sub><sup>+</sup> and H<sup>+</sup>) were unaltered due to counteracting effects of decreased pH and increased NH<sub>4</sub><sup>+</sup> concentrations (Fig. 1c). Another source of NH₃ emission could be the litter, i.e. senescent leaves attached to the stems or lying on the ground surface. Ammonium concentrations were considerably higher in the litter material compared to green leaves (Fig. 4) due to the protein degradation processes going on in the litter (Mattsson and Schjoerring, 2003). Senescence-related processes were probably also enhanced after cutting when both the climatic conditions and the proportion of litter out of total biomass were changed at the bottom of the canopy (David et al., 2008). In a tall canopy, NH₃ emitted from the litter can be taken up by leaves positioned higher above the ground (Husted et al., 2000b; Nemitz et al., 2000) while in the absence of a tall canopy, the litter NH₃ may escape to the atmosphere. High NH<sub>4</sub><sup>+</sup> concentrations and relatively high pH values in the litter also resulted in an extremely high Γ<sub>litter</sub> value (Table 1) indicating a strong potential for NH₃ emission. In a non-fertilized grassland in the Netherlands, Wichink Kruit et al. (2007) observed an average canopy value of 2200, which is in line with that recorded for stems and young leaves in the present work (Table 1).

After fertilisation, all plant N pools increased with peak values already on the first day after fertilisation. Micrometeorological measurements also showed high NH₃ emissions after fertilisation with some contribution from the fertiliser itself during the first 2 days (Milford et al., 2008). The fertiliser was rapidly dissolved in the soil solution since it was raining the same afternoon as the main field was fertilised (Sutton et al., 2008). Consequently, the fertiliser contamination was restricted to a very short period. The fertilisation was also reflected in higher NH₄⁺ concentrations in guttation droplets collected at the leaf tips in the early mornings after fertilisation (not shown).

Ammonium concentrations in both leaf tissue and apoplast started to decrease again already a few days after fertilisation (Fig. 1a, 2a) while leaf tissue NO₃⁻ concentrations remained high for the rest of the experiment (Fig. 2b). This may partly reflect declining soil NH₄⁺ levels (Fig. 5) and partly rapid assimilation of NH₄⁺ in the plant cells, while NO₃⁻ was stored in the leaf cell vacuoles for later use. It has previously been shown that when NH₄NO₃ is supplied to plant roots, NH₄⁺ is absorbed more readily than NO₃⁻ (Bloom, 1981; Clarkson et al., 1986). Nitrate accumulates in grass herbage when the rate of uptake by the roots exceeds the rate of conversion to organic N (Whitehead, 1995). The NH₄⁺ concentrations in apoplast and bulk tissue were obviously sensitive parameters

responding rapidly to fluctuations in soil nitrogen availability. Also in a laboratory experiment with *Lolium perenne* and *Bromus erectus* both leaf tissue and apoplastic NH₄⁺ concentrations were shown to respond rapidly to changing NH₄⁺ concentrations in the nutrient solution (Mattsson and Schjoerring, 2002).

A correlation analysis revealed that the stomatal NH₃ compensation point calculated on the basis of apoplastic parameters was positively correlated with the bulk tissue NH₄⁺ concentration in leaves and litter (Table 2). Some previous investigations have likewise shown good correlation between apoplastic and leaf tissue NH₄⁺ concentrations (Mattsson et al., 1998; Mattsson and Schjoerring, 2002), suggesting that the tissue NH₄⁺ concentration may be used as an indicator of the NH₃ compensation point. Other studies have found the correlation to depend on growth conditions (Herrmann et al., 2008) or not to be present (Hill et al., 2002).

## 5 Conclusions

We conclude that the management practice has a major impact on the potential plant-atmosphere NH₃ exchange in grassland by influencing both plant and soil N parameters. The NH₃ compensation point derived from apoplastic measurements was positively correlated with bulk tissue NH₄⁺ concentrations in leaves and litter. This suggests that measurements of NH₄⁺ and pH in bulk extracts of plant material in grassland can be used as a simple indicator of the NH₃ exchange potential.

*Acknowledgements.* The authors gratefully acknowledge the support of many different funders and colleagues to this work. The measurements were conducted under the frame of the GRAMINAE project funded by the European Commission (ENV4-CT98-0722). Final synthesis of this paper was conducted as part of the NitroEurope Integrated Project.

Edited by: K. Pilegaard

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