Journal of Experimental Botany, Vol. 47, No. 297, pp. 477-484, April 1996
Journal of
Experimental
Botany
Ammonia emission from young barley plants: influence
of N source, light/dark cycles and inhibition of
glutamine synthetase
Marie Mattsson1 and Jan K. Schjoerring
Plant Nutrition Laboratory, Department of Agricultural Sciences, The Royal Veterinary and Agricultural
University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark
Received 10 August 1995; Accepted 1 December 1995
Abstract
Introduction
Barley (Hordeum vulgare L. cv. Golf) plants were
grown at two different relative addition rates; 0.1 and
0.2 d 1 of nitrate. Three to five days before measurements started the plants were transferred to a nutrient
solution with 2 mM nitrate or ammonium. The ammonium-grown plants showed increased ammonium
levels in both shoots and roots and also increased
ammonium concentrations in xylem sap.
Ammonia emission measured in cuvettes connected
to an automatic NH3 monitor was close to zero for
nitrate-grown plants but increased to 0.59 and
0.88 nmol NH3 m" 2 s~1 for plants transferred to ammonium after growing at RA = 0.2 and 0.1 d ~ \ respectively. In darkness, NH3 emission decreased together
with photosynthesis and transpiration, but increased
rapidly when the light was turned on again.
Addition of 0.5 mM methionine sulphoximine (MSO)
to the plants caused an almost complete inhibition of
both root and shoot glutamine synthetase (GS) activity
after 24 h. Ammonia emission increased dramatically
and photosynthesis and transpiration decreased in
both nitrate- and ammonium-grown plants as a result
of the GS inhibition. At the same time plant tissue and
xylem sap ammonium concentrations increased, indicating the importance of GS in controlling plant ammonium levels and thereby NH3 emission from the leaves.
Substantial losses of applied nitrogen from shoots of
agricultural plants have been observed in many investigations (Stutte and Weiland, 1978; Farquhar et al., 1980;
Abrol et al., 1986; Mattsson et al., 1993). The most
accepted pathway of this loss is through ammonia volatilization from the plant leaves. Flux measurements of NH3
exchange over agricultural crops have usually indicated
emission in the range 0-5 kg ha" 1 year"1 (Sutton et al,
1995). Chamber measurements with different NH3 air
concentrations have, however, shown that both emission
and deposition can occur (Farquhar et al., 1980; Morgan
and Parton, 1989; Husted and Schjoerring, 1995).
Farquhar et al. (1980) established that plants have a
compensation point for NH3. At ambient NH 3 concentrations above the compensation point, NH 3 is absorbed by
the leaves, while at concentrations below the compensation point NH3 is lost to the atmosphere. The ammonia
compensation point for young plants of several species
was found to be about 1-4 ^gm~ 3 , which is near the
normal concentration of ammonia in air over agricultural
areas (Farquhar et al., 1980).
The magnitude and direction of the NH 3 fluxes may
change on hourly, diurnal and seasonal scales, depending
on environmental conditions, crop growth characteristics
and timing of fertilizer application (Sutton et al., 1994).
The highest rates of ammonia emission is usually shown
during senescence and grain filling (Morgan and Parton,
1989; Schjoerring et al., 1993). A positive correlation
between temperature and ammonia losses (Farquhar
et al., 1980; Stutte and da Silva, 1981) as well as between
transpiration and NH3 losses (Stutte et al., 1979) has also
been shown.
Key words: Hordeum vulgare, ammonia emission, ammonium, glutamine synthetase, nitrogen nutrition, photosynthesis, transpiration.
1
To whom correspondence should be addressed.
Abbreviations: MSO = methionine sulphoximine; GS = glutamine synthetase; RA = relative addition rate.
© Oxford University Press 1996
478 Mattsson and Schjoerring
Increased plant N status may influence ammonia volatilization and, particularly during senescence, high N seems
to result in higher NH 3 emission (Parton et al., 1988;
Schjoerring, 1991). It may be expected that high plant N
content would result in high NH 3 emission, since the
potential for plants to emit ammonia depends on the
NH 3 concentration gradient between the substomatal
cavity and the ambient atmosphere (Parton et al., 1988).
This gradient will reflect the concentration of NH^ in
plant tissues. The most important ammonium/ammoniaproducing processes in plants are nitrate reduction,
ammonium uptake through roots, photorespiration,
deamination, and protein degradation during senescence
(Joy, 1988).
The usual pathway of ammonium assimilation is the
glutamine synthetase/glutamate synthase cycle. Since
ammonium is released and reassimilated in large amounts
in different processes of nitrogen metabolism, the enzymes
must be operating with high efficiency (Miflin and Lea,
1977). Inhibition of GS by methionine sulphoximine
(MSO) causes a large increase in ammonium concentrations in plant tissues (Martin et al., 1983; Fentem et al.,
1983) and in the inhibition of photosynthesis (Platt and
Anthon, 1981).
No studies on ammonia emission in relation to nitrogen
nutrition have been carried out on young plants grown
under controlled conditions and so far the large amount
of studies on GS inhibition have only been concerned
with ammonia accumulation and photosynthesis inhibition and not with ammonia emission. Measurements of
NH 3 fluxes are generally limited by the capabilities of the
NH 3 detection system. This investigation was performed
using a continuous NH 3 wet rotating annular denuder
system, with online detection of NH^ by conductivity
(Wyers et al., 1993). This system is suitable for precise
determinations of NH 3 fluxes over short time-scales.
The aim of this study was to monitor ammonia emission
from young barley plants grown under controlled conditions at two different nitrogen levels and with nitrate or
ammonium as nitrogen sources. The responses of these
plants to MSO, an inhibitor of GS, was examined for 24 h.
Materials and methods
8h darkness. The temperature was 17°C throughout and the
relative humidity was 70%.
Nitrogen nutrition
Nitrogen additions started at day 8 and were calculated from
the equation:
where yV, and No are the nitrogen contents of the plants at
times t and zero, respectively, and RA is the relative addition
rate of nitrogen. The daily additions, given by Nt-N0, was
added continuously to the plants, by means of an infusion
pump (Harvard 22 pump, Harvard apparatus, USA). The
addition solution containing nitrate-N was the same as described
previously (Mattsson et al., 1991).
The plants were grown at two different RA, namely 0.1 and
0.2 d~' until day 23 for the high RA plants and day 31 for the
low RA plants. At this time the plants were transferred to
nutrient solutions containing either 2 mM NH^ or NO3" for
3-5 d before the measurements of ammonia emission started.
During this time pH was adjusted daily to 6.0 in the ammonium
cultures. Methionine sulphoximine (MSO) was added to the
culture media at a concentration of 0.5 mM.
Ammonia emission measurements
Ammonia emission was monitored in a computerized cuvette
system (Fig. 1) in which plexiglas cuvettes with a volume of 8 1
were clamped airtight on to the plant holders. Pressurized,
filtered air with a temperature of 24 °C and a relative humidity
of 40% was let into the cuvette at a rate of 30 1 min "' through
a distributing nozzle at the bottom of the cuvette. Light was
supplied by Osram PowerStar high pressure sodium lamps to a
photon flux density of c. 300 /
m~2 s~~'' and the temperature
in the cuvettes was 24 °C. The air inside the cuvette had a slight
overpressure in order to avoid the intrusion of outside
atmospheric air. Through an opening at the top of the cuvette
the air was sent to the ammonia monitor. Ammonia emission
was monitored by switching between two parallel cuvettes every
2 h during 24 h.
Emitted ammonia was sent to the NH3 monitor (AMANDA,
Anasys B.V., Albergen, NL) and collected in an absorption
solution (NaHSO4) in a rotating denuder and detected by
conductometry (Wyers et al., 1993). At the same time changes
in CO2- and H2O-air concentrations due to plant photosynthesis
and transpiration were monitored on a combined infrared gas
analysis system (CIRAS-1, PP-systems, Herts, UK). All data
were logged once a minute on a computer and using flow rates
through the cuvettes and leaf areas of the plants, ammonia
emission, photosynthesis and transpiration could be calculated.
Estimates of leaf conductances were based on transpiration
data, and a leaf temperature of 25 °C.
Plant material
Plant analysis
Barley seeds (Hordeum vulgare L. cv. Golf) were germinated
for 4 d on filter paper moistened with de-ionized water. The
seedlings were then transplanted to plexiglas holders with
neoprene plugs around the stems in order to create an airtight
seal between shoot and root. Each holder with 9 plants was
mounted at the surface of 41 of aerated N-free nutrient solution
(Mattsson et al., 1991). Nutrient solutions were changed once
a week and the pH was maintained between 5.5 and 6.5. The
plants were kept in a growth chamber with 16 h light (photon
flux density in the range 400-700 nm, 550 ^mol m~2 s"1) and
After measurements for 8 or 24 h, shoots were excised 2 cm
above the root and xylem sap was collected for 30 min. After
leaf area and fresh weight measurements, shoot and root
samples were dried at 70 °C for 24 h.
Dry material was weighed and ground before the analysis of
total nitrogen was performed by the Dumas method on an
automatic nitrogen analyser (Elemental Analyser, Carlo Erba
1108, Milano, Italy). Nitrate and ammonium were extracted
from root and shoot material in 0.05 M H2SO4 for 1 h. After
filtration, concentrations in extracts and xylem sap of nitrate
Ammonia emission from barley leaves
Computer
NHg monitor
II
CO2 /H2O monitor
—
Filtered
air ~
Flow meters
Plant cuvettes
Reference
Fig. 1. Plant cuvette system for continuous measurement of NH3 by
annular denuder sampling and on-line analysis (AMANDA) and of
CO2 and H2O by infrared gas analysis (CIRAS).
and ammonium were determined according to Cataldo et al.
(1975) and Krom (1980), respectively.
Ammonium transport from root to shoot was calculated by
multiplying transpiration rate by xylem N H ^ concentration.
This calculation was made with the assumption that the
ammonium concentration was not affected by the water flux
(see Mattsson et al., 1988).
Total extractable glutamine synthetase activity was determined on fresh shoot and root material extracted in a buffer
solution containing 100 mM TRIS-HC1, 1 mM EDTA, 1 mM
DTT, 10 mM MgSO4, 100 mM KC1, 0.5% Triton X-100, and
20% glycerol with a pH of 7.8. Activity was measured according
to Lea et al. (1990) by incubation of extracts in an assay
medium containing 100 mM TRIS-HC1, 50 mM glutamate,
5 mM hydroxylamine hydrochloride, 50 mM MgSO4, and
20 mM ATP for 20 min at 30 °C.
Replicates
Five identical plant cultures were performed. In each experiment
data were obtained from four parallel groups of nine plants for
each treatment.
479
Results
The two relative addition rates of nitrogen (0.1 and
0.2 d" 1 ) resulted at day 23 in relative growth rates of 0.1
and 0.15 d" 1 (not shown). Thus, the plants grown at RA
0.1 d ~' were nitrogen limited and utilized all the nitrogen
added. In contrast, plants grown at RA 0.2 d" 1 were
nitrogen sufficient and did not utilize all the nitrogen
added. After the 3-5 d treatment with 2 mM NO^ or
NH^, dry weights and total nitrogen contents were still
higher for the plants grown at RA 0.2 d~\ while tissue
nitrate contents and xylem sap nitrate concentrations
were approximately equal for both RA (Table 1). Nitrategrown plants showed somewhat higher dry weights and
root total nitrogen contents than ammonium-grown
plants of both RA (Table 1). The tissue nitrate content
was equally high in roots and shoots of nitrate-grown
plants and was low in ammonium-grown plants (Table 1).
Ammonium contents of shoot and root were twice as
high in the ammonium-grown plants compared to the
nitrate-grown ones (Table 2). Plants grown at RA 0.1 d"1
showed higher NH/ contents of both shoot and root
compared to plants growth at RA 0.2 d" 1 (Table 2).
Ammonia emission was low (0.05 nmol m~2 s"1) for
the nitrate-grown plants of both RA (Table 2). In fact,
in many cases no emission at all could be detected. For
ammonium-grown plants NH3 emissions were significantly higher, 0.59 and 0.88 nmol m" 2 s" 1 for RA = 0.2
and 0.1 d"1, respectively (Table 2), which corresponded to cuvette NH 3 concentrations of 1.2-1.7 /xgm"3.
Photosynthesis, transpiration and leaf conductance
showed higher values for plants grown at RA = 0.\ d~'
than at RA 0.2 d"1, but showed no response to ammonium or nitrate nutrition (Table 3). Ammonia emission
showed a diurnal pattern together with photosynthesis
and transpiration (Fig. 2). Emission rates were usually
constant during the day, but decreased to about half
during the night. Transpiration also decreased to about
half during the night while photosynthesis stopped
completely (Fig. 2). Xylem sap NH^ concentrations
and xylem NH4+ transport were 2-fold higher in ammonium-grown plants compared to nitrate-grown ones
(Table 5).
Table 1. Dry weights, total N content, nitrate content and xylem sap nitrate concentration of barley plants grown at two levels of
nitrate-N nutrition (RA=0.I and 0.2 d~l) and with a subsequent treatment of 2 mM nitrate or ammonium for 3-5 d
RA
(d"1)
Plant part
Treatment
Dry weight
(mg plant" 1 )
Total N
(mgg-'DW)
Tissue nitrate
(fimol g"1 tissue water)
0.1
Shoot
Root
Shoot
Root
Shoot
Root
Shoot
Root
NH4+
218+15
156 + 9
370 + 32
198+17
503 ± 7
194±13
647 ±37
277+ 11
34.9+1.8
36.4 + 3.1
34.8 + 5.5
46.9 + 4.6
46.7 + 2.9
38.9 + 3.4
46.9 + 3.5
49.2 + 2.6
13.1 ±2.1
3.5 + 1.1
76.0+12.9
89.9 + 14.1
12.9 + 3.2
5.1+0.6
75.1+3.9
75.5 + 25
0.1
0.2
0.2
NO3"
NH 4 +
NOi"
Xylem nitrate
(mM)
0.16±0.1
14.7+4.1
1.56 + 0.2
14.3 ±1.4
480 Mattsson and Schjoerring
Table 2. Tissue ammonium contents and ammonia emission of barley plants grown at two levels of nitrate-N nutrition
( RA = 0.1 and 0.2 d~1) and with a subsequent treatment of 2 mM nitrate or ammonium for 3-5 d and inhibition of GS for
8 or 24 h with 0.5 mM MSO
RA
(d" 1 )
0.1
0.1
0.2
0.2
Treatment
NH4+
+ MSO 8 h
+ MSO 24 h
NO3"
+ MSO 8 h
+ MSO24h
NH4+
+ MSO 8 h
+ MSO24h
NO 3 + MSO 8 h
+ MSO24h
Tissue ammonium
(ixmo) g" 1 tissue water)
Ammonia emission
(nmol m" 2 s' 1 )
Shoot
Root
18.9+1 3
17.2±1.2
73.6±15
11.8 + 2.2
14.9+1.9
37.4 + 8.2
16.4+1.9
12.6 + 2.9
49.0±4.5
6.8 ±0.4
6.7 + 1.4
22.8+1.6
83 + 1.6
12.1 + 1.2
13.9 + 2.1
4 3+0.3
9.3 + 0.5
12.2 + 2.8
5.8+1.1
73 + 1.0
11.1 + 1.0
3.3±0.2
4.3 ±0.8
9.4+1.3
0.88 + 0.1
5.25 + 2.9
52.8+19
0.06 + 0.02
7.75 + 2.9
35.0+15
0.59 + 0.1
9.15 + 3.2
29.6 ±5.0
0.05 + 0.02
6.42 + 2.2
40.7 + 2.6
Table 3. Photosynthesis, transpiration and leaf conductance of barley plants grown at two levels of nitrate-N nutrition
(RA = 0.1 and 0.2 d~l) and with a subsequent treatment of 2 mM nitrate or ammonium for 3-5 d and inhibition of GS for
8 or 24 h with 0.5 mM MSO
RA
(d" 1 )
Treatment
Photosynthesis
(ftmol m~ 2 s" 1 )
Transpiration
(mmol m " 2 s " ' )
Leaf conductance
(mmol m~ 2 s" 1 )
0.1
NH4+
+ MSO 8 h
+ MSO24h
NO3+ MSO 8 h
+ MSO24h
NH 4 +
+ MSO 8 h
+ MSO 24 h
NO3"
+ MSO 8 h
+ MSO 24 h
7.54 + 0.82
3.04 + 0.47
0.35 + 0 08
6.37+1.33
4 57+0.85
1.12 + 0.26
4.59 + 0.38
2.10 + 0.58
093+0.12
5.04 + 0.39
2.60 + 0.76
0.81+0.26
3 91+0.29
2.42 + 0 33
0.36 + 0.18
3.39 + 0.45
2.28 + 0.83
0.51+0.13
2.50 + 0.77
1.45 + 0.06
0.74 + 0.30
2.58 + 0.21
1.59 + 0.17
0.84 ±0.08
208.0
128.7
19.1
180.3
121.3
27.1
133.0
77.1
39.4
137.2
84.6
44.7
0.1
0.2
02
Addition of 0.5 mM MSO to the root medium resulted
after 4-5 h in increasing ammonia emission for both
nitrate- and ammonium-grown plants (Fig. 3). At the
same time (4-5 h after MSO addition) photosynthesis
and transpiration started to decrease (Fig. 3). After 8 h,
ammonia emission had increased to between 5 and
10 nmol m~2 s" 1 in both nitrate- and ammonium-grown
plants of both RA (Fig. 3; Table 2). Photosynthesis had,
after 8 h of MSO treatment, decreased to about half the
original level and transpiration had decreased slightly less
(Table 3). Ammonia emission increased with a slower
rate during the following hours and then decreased
during the night together with photosynthesis and transpiration (Fig. 3). When the light was turned on again
ammonia emission showed a dramatic increase up to
30-50 nmol m~ 2 s~' (24 h after MSO addition), while
photosynthesis and transpiration seemed to level off at a
low, but still detectable, level (Fig. 3; Table 3).
After MSO addition the total extractable activity of
glutamine synthetase decreased rapidly in shoots and
roots of both nitrate- and ammonium-grown plants
(Table 4). After 8 h of MSO treatment root GS activity
had decreased to a lower level than shoot GS activity
and after 24 h activities in both shoots and roots were
very low (Table 4).
Root ammonium content showed a marked increase
8 h after the MSO addition and was approximately
doubled after 24 h (Table 2). Shoot ammonium contents
were not changed after 8 h but were, after 24 h, three
times higher than before MSO was added (Table 2).
Xylem sap ammonium concentrations started to increase
already after 8 h and after 24 h concentrations were three
times higher than before the MSO addition (Table 5).
Calculated xylem transport of ammonium from root to
shoot showed a marked increase after 8 h of MSO treatment in nitrate-grown plants while in ammonium-grown
plants, ammonium transport was unaffected after 8 h
(Table 5). After 24 h, however, xylem transport of
Ammonia emission from barley leaves
Table 4. Total extractable GS activity of barley plants grown at
RA 0.2 d'1 with 2 mM nitrate or ammonium for 3-5 d and
subsequent inhibition of GS for 8 or 24 h with 0.5 mM MSO
Treatment
NH4+
+ MSO 8h
+ MSO24h
NO3+ MSO 8h
+ MSO24h
481
sap concentrations of nitrate were not affected by the
MSO treatment (not shown).
Glutamine synthetase activity
(/xmolg-'FWrT 1 )
Discussion
Shoot
Root
Effects of nitrogen nutrition on ammonia emission
36.0 + 4.5
5.2 + 2.2
1.7+0.7
34.1 ±6.7
10.1+2.2
2.9 + 0.01
30.9±7.5
1.4 + 0.4
0.9±0.2
22.4 ±2.8
2.2 ±2.0
0.3±0.1
The barley plants were, in this experiment, grown with
two relative addition rates of nitrate in order to establish
two sets of plants with different nitrogen status (Mattsson
et al., 1991). Treatments with 2 mM nitrate or ammonium
were short (3-5 d) in order to minimize differences in
growth and total nitrogen contents while still achieving
large differences in tissue ammonium and nitrate contents.
Small differences in dry weight and total N between
nitrate- and ammonium-grown plants could, however,
not be completely avoided which was also seen by Raab
and Terry (1995) and Cramer and Lewis (1993). Plants
grown on ammonium showed increased ammonium contents in both shoot and root tissues compared to nitrategrown plants (Table 2). This, together with increased
amide-N and lower organic acid contents, are well-known
effects of ammonium nutrition (Goyal and Huffaker,
1984). Also, increased xylem sap ammonium concentration as a result of growth on ammonium has been shown
before (Lee and Ratcliffe, 1991; Cramer and Lewis, 1993).
The increase in tissue ammonium and xylem sap ammonium concentrations was probably the reason for the
higher ammonia emission rates seen from ammoniumgrown plants (Table 2). The higher ammonia emission
from plants grown at RA 0.1 d" 1 compared to plants
grown at RA 0.2 d" 1 could be explained by the somewhat
higher tissue ammonium content, which was probably the
result of a higher uptake capacity in the previously
N-limited, RA 0.1 d" 1 plants (Mattsson et al., 1991).
Also, photosynthesis and transpiration were higher in
plants grown at RA 0.1 d~'. This indicates that the leaves
of plants grown at RA =0.2 d" 1 were, already at this
stage, subject to self-shading and thus had a slower rate
of photosynthesis on a leaf area basis. The higher total
N content of the high RA plants thus had no effect on
ammonia emission. Other experiments have indicated that
NH 3 emission on a total shoot basis may increase with
increasing N concentration in senescing plants (Parton
et al., 1988; Schjoerring, 1991; Schjoerring et al., 1993).
Expressed on a leaf area basis there was, however, only
small differences between low- and high N plants (Parton
et al., 1988; Morgan and Parton, 1989; Schjoerring, 1991).
The potential for plants to emit ammonia seems to
depend on the concentration of ammonium in plant
leaves, or rather the leaf apoplast, and also on the pH of
the apoplast. The apoplastic N H / concentration corresponds to an equilibrium NH3 concentration within the
substomatal cavities of leaves (Sutton et al., 1995). Few
determinations of apoplastic ammonium concentrations
14
Fig. 2. Ammonia emission, photosynthesis and transpiration during
the light and dark (21.00 h to 03.00 h) period of young barley plants
growth with 2 mM NH^ for 4 d after previous growth on nitrate at a
relative addition rate of 0.1 d"1.
had decreased to about the same level in both nitrateand ammonium-grown plants. This decrease in ammonium transport was more pronounced in plants grown at
RA 0.1 d" 1 (Table 5). Tissue nitrate contents and xylem
482 Mattsson and Schjoerring
6O
a
50
7
emissi
0
a
"o
4O
30
X
20
z
IO
0
IO
M
8
•H
<A
0
"fi
6
a
d
4
0
0
A
0,
d"
0
"S
ran
aa
u
2
a
-2
0
0
4
CO
3
'rt
0
"o
2
1
h
i i i
10
15
2O
01
Time, h
O6
10
15
i i i
20
01
Time, h
O6
11
Fig. 3. Ammonia emission, photosynthesis and transpiration during the light and dark (21.30 h to 03.30 h) period of young barley plants grown
with either 2 mM NO3~ (A) or N H ^ (B) for 4d after previous growth on nitrate at a relative addition rate of 0.1 d" 1 . At 11.00 h 0.5 mM
methionine sulphoximine (MSO) was added to both cultures.
Table 5. Xylem sap NH^ concentrations and calculated xylem
NH4 transport of barley plants grown at two levels of nitrate-N
nutrition (RA =0.1 and 0.2 d~x) and with a subsequent treatment
of 2 mM nitrate or ammonium for 3-5 d and inhibition of GS for
8 or 24 h with 0 05 mM MSO
RA
(d"1)
Treatment
Xylem NH4+
concentration
(mM)
Xylem NH4+
transport
(nmol m~ 2 s" 1 )
O.I
NH;
+ MSO 8 h
+ MSO24h
NO3"
+ MSO 8 h
+ MSO 24 h
NH4+
+ MSO 8 h
+ MSO24h
NO3+ MSO 8 h
+ MSO24h
2.2 + 0.4
3.7 + 0.1
6.4 + 0.6
0.9 ±0.1
3.8+0.1
4.5 + 0.7
2.2 + 0.4
3.9 + 0.4
6 5 + 0.3
1.3 + 0.1
3.6 + 0.4
4.4 + 0.8
159.1
160.4
41.2
57.3
155.8
41.8
99.0
102.3
85.5
60.3
101.8
66.7
0.1
0.2
0.2
seem to exist, but will most likely depend on the transpiration stream ammonium concentration in the xylem
which, in these experiments, was 2.2 mM (Table 2).
The calculated xylem ammonium transport was
159 nmol m" 2 s""1 for ammonium grown plants at RA
0.1 d" 1 . Since, in this case, the ammonia emission was
only 0.88 nmol m~2 s~' this indicates a very high capacity
for ammonium influx at the plasmalemma of leaf meso-
phyll cells (Raven and Farquhar, 1981) and a high
assimilation rate of ammonium in the leaf cells. The GS
activity was shown to increase in both roots and leaves
after transfer of sugar beet seedlings from nitrate to
ammonium nutrition (Raab and Terry, 1995). In this
experiment no significant difference in GS activity
between NO^~- and NH^-grown plants could be seen,
probably because the ammonium treatment was too short.
The concentration of NH^ in plant tissues and xylem sap
is often reported to be low (Lewis et al., 1982; Lee and
Ratcliffe, 1991) due to toxic effects of ammonia and the
fast tissue NH^ assimilation. High ammonium concentrations in xylem sap (Cramer and Lewis, 1993) and plant
leaves (Walker et al., 1984; Maheswari et al., 1988) have,
however, also been reported, which may throw some
doubt upon the toxic effects of ammonium in plant
tissues. The explanation for the high measured concentration of ammonium in plants with no visible damage,
might be that the ammonium is located in organelles with
low pH and/or high tolerance for ammonium. Studies
using nuclear magnetic resonance have shown that high
concentrations of ammonium can be present in the vacuoles of maize root cells (Lee and Ratcliffe, 1991; Roberts
and Pang, 1992). Besides the ammonium concentration
the pH of the apoplast is maybe the most important
factor determining leaf ammonium exchange and there is
evidence that ammonium-fed plants may have reduced
Ammonia emission from barley leaves
apoplastic pH compared to nitrate-fed plants (Hoffman
et al, 1992).
Effects of MSO on ammonia emission
The addition of 0.5 mM MSO to the barley plants caused,
after a few hours, an accumulation of ammonium in plant
tissues of both nitrate- and ammonium-grown plants
(Table 3). This is in agreement with other studies (Fentem
et al., 1983; Platt and Anthon, 1981; Walker et al, 1984),
although the effect was slower in this experiment due to
the rather low concentration of MSO (0.5 mM).
Increasing concentrations up to 2.5 mM MSO, resulted
in increasing accumulation of ammonium in wheat
(Martin et al, 1983). Also the length of the lag period
was dependent on the concentration of MSO (Platt and
Anthon, 1981). Most studies on MSO effects on leaf
ammonium accumulation have also been carried out on
excised leaves or leaf discs which probably makes uptake
of MSO faster. Ammonium concentrations increased
faster in roots (Table 3) probably due to a rapid deactivation of root GS (Fentem et al, 1983). Total extractable
GS also showed lower values in the roots compared to
shoots after 8 h of MSO treatment. In xylem sap the
ammonium concentration also increased as a result of the
MSO treatment, but since transpiration decreased at the
same time, xylem transport of ammonium to the shoot
after 8 h had only increased in nitrate-grown plants due
to the low xylem ammonium concentrations in these
plants prior to MSO treatments (Table 5). After 24 h
xylem ammonium transport had decreased in both nitrateand ammonium-grown plants. This decrease was entirely
due to decreased transpiration since xylem concentrations
of NH^ actually increased. In the experiments of Fentem
et al. (1983) xylem transport appeared to stop shortly
after MSO feeding to the roots. Inhibition of GS with
MSO also led to the appearance of ammonium in the
xylem sap of both nitrate- and ammonium-grown maize
plants (Lee and Ratcliffe, 1991).
The MSO treatment also decreased photosynthesis and
transpiration (Fig. 3; Table 3). This effect has been attributed to the toxic effect of the accumulation of ammonium
in plant tissue (Givan, 1979). Treatments with high
concentrations of NH^ could, however, increase the
tissue NH^ content more than MSO treatment, but
without decreasing photosynthesis to the same level as
MSO treatment (Walker et al, 1984; Ikeda, 1985). This
suggests that some other factor like depletion of photosynthetic intermediates might be responsible for the inhibition
of photosynthesis (Walker et al, 1984). Photosynthesis
was, however, not completely inhibited, but could also
be detected after 24 h of MSO treatment when plant
metabolism was poisoned and ammonia emission had
risen to 80-100 nmol m" 2 s"1 (Table 3), probably due to
protein breakdown and amino acid catabolism (Platt and
483
15
N-NH4+
Anthon, 1981; Lea, 1991). Using
Rhodes et al.
(1986) were able to show that protein turnover was the
major source of the amino acids that accumulated in
response to MSO. It is notable that some ammonia was
emitted and that the plants also showed some transpiration in the dark (Fig. 3). This could be a consequence
of the large NH 3 concentrations in plant tissue which
may limit stomatal closure during the night (Sutton
et al, 1994).
Conclusions
Young healthy barley plants grown under favourable
climatic conditions can emit NH3 from the leaves to the
atmosphere. The NH 3 emission rates from 0.05 to
0.9 nmol m~2 s"1 were very much in accordance with
other measurements under field conditions (Schjoerring
et al, 1993). These amounts were, in all cases, smaller
than 1% of the simultaneous N transport in barley plants
not subjected to inhibition of GS. Thus, seen in relation
to total plant N economy, the measured NH 3 emission
was of minor significance. However, seen in the context
of atmospheric NH 3 pollution, fluxes of the observed
magnitude (Table 2) still merit attention (see Sutton
et al, 1995).
Growth with 2 mM NH4 in the root medium and
inhibition of glutamine synthetase activity with 0.5 mM
MSO resulted in increased tissue NH^" concentrations
and increased NH 3 emission. The high levels of ammonia
emission caused by the MSO treatment, indicates the
importance of a high GS activity for controlling N H |
levels in plant tissues.
Acknowledgements
The authors gratefully acknowledge financial support from the
DANVIS programme under the Danish Research Academy
and from the Hofmangave Foundation.
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