372 S. Husted et al. Agricultural and Forest Meteorology 105 2000 371–383
Sutton et al., 1995, which is a composite parame- ter expressing the emission and deposition potential
of NH
3
fromto the canopy as influenced by the dif- ferent pathways, i.e. stomatal exchange, NH
3
sorption on leaf surfaces and liberation from degrading litter
material. Ammonia emission from plant leaves oc- curs if the mole fraction of NH
3
in the atmosphere is smaller than the mole fraction of gaseous NH
3
above the leaf apoplastic water phase, while in the oppo-
site case NH
3
absorption takes place Farquhar et al., 1980; Husted and Schjoerring, 1996. Plants grow-
ing in low N input ecosystems like moorlands and forests generally appear to have NH
3
compensation points close to zero and tend to absorb NH
3
from the atmosphere Sutton et al., 1992; Langford and Fehsen-
feld, 1992; Kesselmeier et al., 1993. However, agri- cultural crop species often have compensation points
from 1 to 6 nmol NH
3
mol
− 1
air 0.7–4.2 mg m
− 3
under field conditions Dabney and Bouldin, 1990; Schjoerring et al., 1993; Sutton et al., 1995; Yamulki
et al., 1996 and significant losses of NH
3
may occur. The NH
3
flux is linked to plant ontogeny and N supply Husted et al., 1996 and substantial NH
3
losses have been observed in several agricultural crops, especially
following flowering, where nitrogen compounds are remobilised from source organs leaves and stems to
seed capsules and seeds Schjoerring, 1997.
Controlled environment studies have clearly shown that NH
3
emission from plants are controlled by the stomatal NH
3
compensation point Husted and Schjoerring, 1996, but there is very little information
available on how stomatal NH
3
compensation points influence NH
3
fluxes from plants under field condi- tions. Thus, the aim of the present study was to apply a
recently developed vacuum infiltration technique that enables analysis of the apoplastic H
+
and NH
4 +
con- centrations with very little cytoplasmic contamination
2 Husted and Schjoerring, 1995. On the basis of apoplastic H
+
and NH
4 +
concentrations measured at different leaf heights above the ground, the stom-
atal NH
3
compensation points were calculated and compared with micrometeorological NH
3
flux mea- surements and within-canopy NH
3
concentrations. The field experiments were made in an oilseed
rape B. napus field in central Scotland, UK, dur- ing June 1995 as part of the EU EXAMINE project
North Berwick Experiment Sutton et al., 2000a. The measurements here represent the first occasion
that micrometeorological data on NH
3
exchange have been directly related to stomatal NH
3
compensation points calculated on the basis of apoplastic H
+
and NH
4 +
concentrations.
2. Materials and methods
2.1. Plant material and growth conditions The oilseed rape field near North Berwick was lo-
cated about 35 km east of Edinburgh, with experi- ments made in the period 7–17 June 1995. Fertilizer
was applied on 29 August 1994, 23 February 1995, 15 March 1995 and 31 March 1995 with 60, 85, 85
and 55 kg N ha
− 1
, respectively. The oilseed rape plants were in the maturation stage where seeds had devel-
oped but were still green and much leaf abscission had taken place leaves 1–12 in acropetal direction were
lost. This ontogenetic stage is defined as growth stage 5.3 on the FAO scale.
2.2. Plant sampling and preparation Table 1 summarizes the plant sampling procedures
and steps in analytical methods for determination of stomatal NH
3
compensation points for the North Berwick experiment.
Bulk tissue NH
4 +
and apoplastic H
+
and NH
4 +
concentrations were analysed in the period from 8 to 15 June 1995. Leaves were sampled at 9 a.m., 1 p.m.
and 5 p.m. GMT from four different leaf positions within the canopy 0, 0.50, 0.75 and 1.25 m above
ground with four replicates. In two periods from 9 to 10 June and 14 to 15 June 1995, respectively, the
analysis programme was extended in order to analyse diurnal variations in apoplastic NH
4 +
and H
+
concen- trations in relation to micrometeorological conditions.
In both diurnal campaigns, leaves were sampled ev- ery 2 h from 9 a.m. and the following 24 h. It should
be noted that this was a major task, which including subsequent extraction employed two full time staff in
the field.
After sampling, leaves were immediately brought to a field laboratory where the apoplast was extracted
with a vacuum infiltration technique described in de- tail by Husted and Schjoerring 1995 and Schjoerring
and Husted 1997. The technique is based on vacuum
S. Husted et al. Agricultural and Forest Meteorology 105 2000 371–383 373
Table 1 Summary of steps in the methods used for determination of NH
3
compensation points in oilseed rape leaves in the North Berwick field experiment. The table notes tasks performed in the field laboratory immediately after sampling, and tasks performed on frozen samples in
the institute laboratory Component task
Field laboratory immediately after
sampling Institute laboratory
frozen samples All determinations
A or selected samples S
Harvesting, cutting leaf sections, weighing and freezing of bulk leaf material
+ A
Apoplastic air volume vacuum infiltration with silicon fluid and weighing
+ S — daily on all leaf levels
Apoplastic water volume vacuum infiltration with indigo carmine, cooled centrifugation
and freezing of sample +
S — daily on all leaf levels Spectrophotometric measurement of dye dilution
+ S — daily on all leaf levels
Apoplast extraction vacuum infiltration with sorbitol solution and freezing of samples
+ A
Drying of bulk leaf material, homogenization and extraction
+ A
pH determination of apoplastic extracts +
A pH determination in senescent plant tissue
suspension +
S NH
4 +
determination in apoplast and bulk leaf extracts
+ A
Total cation and anion analysis of apoplast +
S Malate dehydrogenase contamination assay
+ S
infiltration of small leaf segments 25 mm × 50 mm with a 350 mOsm sorbitol solution 280 mM, which
is isotonic with the leaf protoplast. The infiltration takes place in a 50 ml plastic syringe mounted on a hy-
draulic arm that automatically moves the plunger up and down in order to infiltrate the leaf alternating un-
der pressure and vacuum. The infiltrator exposed the leaf discs to 4 atm pressure and vacuum for 10 s, and
repeated the procedure eight times, ensuring full infil- tration. Immediately after infiltration the leaf apoplast
was extracted by centrifugation at 2000g for 10 min at 8
◦
C and the apoplastic solution 20–100 ml was col- lected in small Eppendorf vials. The apoplast extracts
and plant tissue samples were frozen at −18
◦
C for a period of 14 days maximum in the field laboratory be-
fore they were transferred to the institute laboratory and frozen down to −80
◦
C until final analysis. 2.3. Plant analysis
Bulk tissue NH
4 +
was determined by drying plant material at 80
◦
C for 12 h, followed by extraction of NH
4 +
with 0.025 M H
2
SO
4
. Comparison of extractable NH
4 +
in oven-dried leaves with that of lyophilized leaves showed no significant difference
between the two methods. The concentration of NH
4 +
and NH
3
in the bulk tis- sue extracts and in apoplast fluid was measured by flow
injection analysis FIA in combination with online dialysis Tecator FIA Star 5020, Höganäs, Sweden.
The system had a detection limit of 1 mM NH
4 +
in 50 ml 50 pmol NH
3
. If less than 50 ml apoplastic fluid was obtained, samples were diluted with 18.2 M cm
water Milli-Q plus, Ultra pure water system, Milli- pore, MA.
Apoplastic pH was measured with a micro-com- bination-electrode enabling precise measurement in
volumes down to 5 ml model 9802B, Orion, Boston, MA. Calibrations of pH were performed in low ionic
strength buffers Cat. No. 700001, Orion, Boston, MA in order to obtain precise H
+
activity measurements in the dilute samples of apoplastic fluid.
In order to estimate the ionic strength in the apoplast, cations in the apoplast were analysed on an
ion chromatograph Waters, Milford, MA equipped with a 3.9 mm × 150 mm cation MD column. The
374 S. Husted et al. Agricultural and Forest Meteorology 105 2000 371–383
mobile phase was 4.0 mM HNO
3
, 0.1 mM EDTA and 2.5 mM 18-crown-6-ether Sigma, St. Louis, MO
which enables detection of small amounts of NH
4 +
in the presence of high concentrations of Na
+
and K
+
by isocratic elution at 1.0 ml min
− 1
. Inorganic anions Cl
−
, NO
3 −
, NO
2 −
, H
2
PO
4 −
, SO
4 2−
, CO
3 2−
were analysed by a traditional isocratic procedure on the
ion chromatograph Technical Bulletin 091064TP-O, Waters, MA.
The apoplastic extracts were screened for cyto- plasmic contamination by measuring their activity of
malate dehydrogenase MDH relative to bulk leaf extracts Husted and Schjoerring, 1995. The apoplast
could not be isolated from the senescing leaves on the soil surface as the cytoplasmic marker enzyme
activity of MDH was too high data not shown. Thus, only bulk tissue NH
4 +
concentrations were analysed in senescent leaves. In order to estimate the partial
pressure of NH
3
in the senescent leaves, pH was de- termined in a 1:1 tissue:water suspension made on
the basis of frozen samples and corrected for dilution. 2.4. Determination of apoplastic air and water
volumes Analysis of apoplastic air and water volumes are
prerequisites for determination of the dilution factor, which is used for correcting NH
4 +
and H
+
concen- trations in extracted solution to their actual concen-
trations in the leaf apoplastic solution for details see Schjoerring and Husted, 1997.
Apoplastic air was determined by weighing and in- filtrating leaf segments with high-viscosity silicon oil
viscosity: 5 cs; density: 0.904 g cm
− 3
; Dow Corning, Poole, UK which is impermeable to the plasma mem-
brane. The leaf segments were thereafter blotted dry with thin paper tissues, reweighed and the weight in-
crease, corrected for the density of the silicone oil, used for calculation of the apoplastic air volume V
air
, cm
3
air cm
− 3
tissue. The amount of silicone oil ad- sorbed to the cuticle resulted in an insignificant weight
increase. The volume of apoplastic water was assessed on
the basis of the dilution of the dye indigo carmine indigo-5,5
′
-disulphonic acid,
Merck Chemicals
which does not adsorb to the cell walls Husted and Schjoerring, 1995. Leaf segments were weighed and
infiltrated with 50 mM indigo carmine dissolved in 50 mM phosphate buffer at pH 6.2. Immediately after
infiltration, leaves were reweighed and the weight increase, which corresponds to the infiltration volume
V
i
, recorded. The leaf segment was then put into a small zippered polyethylene bag to eliminate water
evaporation and stored for a maximum of 15 min at 4
◦
C before extraction by centrifugation. After extrac- tion the decrease in absorbance at 610 nm 1A
610
was measured spectrophotometrically. The apoplastic water volume V
apo
and dilution factor f
dil
could then be calculated using the following equations:
V
apo
= 1A
610
V
i
1 − 1A
610
, where 0 ≤ 1A
610
≤ 1
1 f
dil
= V
apo
+ V
air
V
apo
2 On the basis of Eq. 2, the true apoplastic ion
X
±
concentration was calculated as [X
±
]
apo
= [X
±
]
fluid
f
dil
. 2.5. Calculation of ammonia compensation points
Knowing the apoplastic NH
4 +
and H
+
concentra- tions, the following equations can be used to calculate
the stomatal NH
3
compensation point χ
s
: χ
s
= K
d
K
H
[NH
4 +
] [H
+
] 3
or since C
tot
= [NH
4 +
] + [NH
3
]
aq
is measured by most analytical procedures:
[NH
3
]
aq
= C
tot
K
d
K
d
+ [H
+
] 4
χ
s
= [NH
3
]
aq
K
H
5 Combining Eqs. 4 and 5 gives
χ
s
= K
H
K
d
C
tot
K
d
+ [H
+
] 6
Since the physiological ionic strength I in the apoplast deviates considerably from zero, the dissoci-
ation constant for NH
4 +
K
d
must be adjusted. This was done by measuring the total ionic composition in
the apoplast for leaves at 0.75 m above ground level. An ionic strength of 31.4 mM was found giving a cor-
rected dissociation constant K
d ′
of 10
− 9.32
using the extended Debye–Hückel equation Atkins, 1990. The
S. Husted et al. Agricultural and Forest Meteorology 105 2000 371–383 375
Henry’s law constant K
H
of 10
− 1.76
is not affected by the ionic strength. All equilibrium constants listed
above are given at 25
◦
C 298.13 K and at a pressure of 1 atm and valid only at I = 0 Wagman, 1968.
The temperature dependence of the stomatal NH
3
compensation point can be described by the Clausius–Clapeyron equation Husted and Schjoer-
ring, 1996:
ln χ
s2
χ
s1
= 1H
dis
+ 1H
vap
R 1
T
1
− 1
T
2
7 where χ
s1
is the actual stomatal NH
3
compensation point at the temperature T
1
and χ
s2
is the requested NH
3
compensation point at a new temperature T
2
. R denotes the gas constant 8.31 J K
− 1
mol
− 1
and the enthalpies of dissociation 1H
dis
and vaporisation 1H
vap
are 52.21 and 34.18 kJ mol
− 1
, respectively. The stomatal NH
3
compensation point for the whole plant canopy was calculated by weighting data from
the leaf fractions in 0.50, 0.75 and 1.25 m height ac- cording to the leaf area for each level. Moreover,
stomatal NH
3
compensation points were corrected to the mean diurnal temperature Eq. 7 for each plant
height and compared with the net NH
3
flux obtained by micrometeorological techniques see below.
2.6. Determination of NH
3
flux and NH
3
concentration profiles in canopy air The NH
3
concentrations at different heights above the canopy were continuously measured with two dif-
ferent techniques viz. replicated continuous wet de- nuders of the type described by Wyers et al. 1993
and filter packs see Sutton et al., 2000b in order to obtain the net NH
3
flux. However, during the last di- urnal campaign 14–15 June 1995 denuders and fil-
ter packs were placed at three different heights above the soil surface in the canopy h = 1.38 m, whereas
two additional denuders were used to measure the net NH
3
flux above the canopy. 2.7. Statistical analysis
The ratio between apoplastic concentrations of NH
4 +
and H
+
was used to calculate the stomatal compensation point for NH
3
see Eq. 3. The com- bined standard error for this ratio see Figs. 1 and 2
was derived from the following equation:
S [NH
4 +
] H
+
= s
1 H
+ 2
S
2 [NH
4 +
]
+ −[NH
4 +
] H
+ 2
S
2 [H
+
]
8
3. Results
