Annals of Botany 97: 121–131, 2006
doi:10.1093/aob/mcj007, available online at www.aob.oxfordjournals.org
Changes in Growth and Nutrient Uptake in Brassica oleracea
Exposed to Atmospheric Ammonia
A N A C A S T R O , I N E K E S T U L E N , F R E E K S . P O S T H U M U S and L U I T J . D E K O K *
Laboratory of Plant Physiology, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands
Received: 24 June 2005 Returned for revision: 10 August 2005 Accepted: 27 September 2005 Published electronically: 16 November 2005
Background and Aims Plant shoots form a sink for NH3, and are able to utilize it as a source of N. NH3 was used as a
tool to investigate the interaction between foliar N uptake and root N uptake. To what extent NH3 can contribute to
the N budget of the plant or can be regarded as a toxin, was investigated in relation to its concentration and the
N supply in the root environment.
Methods Brassica oleracea was exposed to 0, 4 and 8 mL L 1 NH3, with and without nitrate in the nutrient solution.
Growth, N compounds, nitrate uptake rate, soluble sugars and cations were measured.
Key Results In nitrate-sufficient plants, biomass production was not affected at 4 mL L 1 NH3, but was reduced at
8 mL L 1 NH3. In nitrate-deprived plants, shoot biomass was increased at both concentrations, but root biomass
decreased at 8 mL L 1 NH3. The measured nitrate uptake rates agreed well with the plant’s N requirement for growth.
In nitrate-sufficient plants nitrate uptake at 4 and 8 mL L 1 NH3 was reduced by 50 and 66 %, respectively.
Conclusions The present data do not support the hypothesis that NH3 toxicity is caused by a shortage of sugars or a
lack of capacity to detoxify NH3. It is unlikely that amino acids, translocated from the shoot to root, are the signal
metabolites involved in the down-regulation of nitrate uptake, since no relationship was found between changes in
nitrate uptake and root soluble N content of NH3-exposed plants.
Key words: Atmospheric ammonia, Brassica oleracea, cations, nitrate requirement, nitrate uptake, nutrient, relative growth
rate, shoot : root ratio, soluble sugars, sulfate uptake, toxin.
INTRODUCTION
Roots of higher plants are able to take up inorganic N ions,
as nitrate and/or ammonium from the soil (Peuke et al.,
1998a, b; Britto et al., 2001; Glass et al., 2002). In addition,
plant shoots can also take up NH3 from the atmosphere. The
foliar uptake of NH3 is determined by the diffusive
conductance of the stomata, since the internal (mesophyll)
resistance to this gas is low due to its high solubility and
rapid dissociation into NH+4 in the water phase of the mesophyll cells (Hutchinson et al., 1972; Fangmeier et al., 1994).
The foliar uptake of NH3 shows diurnal variation and is
dependent on water status, temperature, light intensity,
internal CO2 concentration, nutrient availability, ontogeny
and developmental stage of the plant (Hutchinson et al.,
1972; Rogers and Aneja, 1980; Husted and Schjoerring,
1996; Schjoerring et al., 1998).
The foliarly absorbed NH3 may be metabolized by
the glutamine synthetase/glutamate synthase cycle (Lea
and Miflin, 1974; Pérez-Soba et al., 1994; Pearson and
Soares, 1998) and NH3 exposure may result in an increase
in total N content and N-containing metabolites (Van Dijk
and Roelofs, 1988; Pérez-Soba and Van der Eerden, 1993;
Clement et al., 1997; Gessler et al., 1998; Barker, 1999) and
changes in glutamine synthetase (GS; EC 6.3.1.2) and
nitrate reductase (NR; EC 1.6.6.1; Pérez-Soba et al.,
1994; Pearson and Soares, 1998). NH3 is potentially phytotoxic and high atmospheric levels may negatively affect
growth and alter the shoot : root (S/R) ratio (Van der
Eerden and Pérez-Soba, 1992; Pérez-Soba et al., 1994).
NH3 toxicity has been related to a shortage of soluble
* For correspondence. E-mail [email protected]
sugars, a lack of capacity to detoxify NH3, nutrient imbalances due to cation release (Fangmeier et al., 1994; Krupa,
2003) and effects on the acid–base balance (Yin and Raven,
1997). There is little information on interactions between
foliar uptake of atmospheric NH3 and N uptake by the roots.
Pérez-Soba and Van der Eerden (1993) concluded from
experiments with Pinus sylvestris exposed to 0075 mL L 1
NH3 that 90 % of the increase in needle N was derived from
the atmospheric N source, and that root N uptake was
reduced. Clement et al. (1997) exposed winter wheat
(Triticum aestivum) to 1 and 2 mL L 1 NH3 and found a
reduction in nitrate uptake rate of 80 and 138 %, respectively. Gessler et al. (1998), using concentrations of
005 mL L 1 NH3, on seedlings of beech (Fagus sylvatica),
described a reduction of 35 % in nitrate uptake rate by the
roots.
It needs to be established to what extent the foliar uptake
of atmospheric NH3 may contribute to the plant’s N requirement for growth and whether NH3 exposure affects N
uptake by the root. As far as is known, no experimental
studies are available in which the impact of atmospheric
NH3 concentrations on net nitrate root uptake was actually
measured in relation to changes in plant growth. The aim of
the present study was 3-fold. Firstly, the impact of atmospheric NH3 on root net nitrate uptake rate of Brassica
oleracea was assessed. Brassica oleracea was chosen as
a suitable species because of its high relative growth rate
(RGR), its preference for nitrate (Pearson and Stewart,
1993), as well as its tendency towards sensitivity to NH+4
as a member of the Brassicaceae (Britto and Kronzucker,
2002). The experiments were carried out at two atmospheric
NH3 concentrations (4 and 8 mL L 1), based on preliminary
The Author 2005. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: [email protected]
Castro et al. — Atmospheric NH3 and Brassica oleracea
122
experiments with a range of concentrations which showed
that growth of B. oleracea was not affected at 4 mL L 1 NH3
but was reduced at 8 mL L 1 NH3 (Castro et al., 2005). In
nitrate uptake experiments the effects of 4 and 8 mL L 1
NH3 on biomass production, RGR, dry matter partitioning,
nitrogenous compounds and nitrate reductase activity were
measured simultaneously. Since changes in nitrate uptake
rate can be caused by changes in RGR of the plant (Ter
Steege et al., 1998) the effect on sulfate uptake was also
measured, in order to be able to distinguish between direct
NH3- and growth-related effects. In order to establish
whether the measured nitrate uptake rates were of physiological significance, nitrate uptake rates were compared
with the plant’s N requirement for growth, calculated
from the RGR and the plant’s nitrogen content. The second
aim was to investigate to what extent atmospheric NH3 can
contribute to the N requirement of the plant, in the absence
of nitrate in the root environment. Thirdly, the effect of both
NH3 concentrations on soluble sugars, N compounds and
cations was measured, to assess whether changes in these
parameters could be related to NH3 toxicity.
MATERIALS AND METHODS
Plant material
Seeds of Brassica oleracea L. (curly kale ‘Arsis’; Royal
Sluis, The Netherlands) were germinated in vermiculite in a
climate-controlled room. Day and night temperatures were
20 and 16 C, respectively, with a relative humidity of
60–70 %. The photoperiod was 14 h, at a photon fluence
rate of 250–300 mmol m 2 s 1 (within the 400–700 nm
range), supplied by Philips HPI-T lamps (400 W).
Twelve-day-old seedlings were transferred to an aerated
25 % Hoagland nutrient solution (pH 6) for a 1-week period
(Castro et al., 2004).
Ammonia exposure
Nineteen-day-old seedlings (three plants per pot, five pots
per treatment, ten pots per cabinet) were transferred to
stainless-steel fumigation cabinets with a polycarbonate
top (Durenkamp and De Kok, 2002) and exposed to 0, 4
and 8 mL L 1 NH3 for 1 week. Day and night temperatures
were 20 and 16 C (61 C), respectively, relative humidity
was in a range of 40–50 % and the photoperiod was 14 h at a
photon fluence rate of 300–350 mmol m 2 s 1. Pressurized
NH3 diluted with N2 (1 mL L 1) was injected into the
incoming air stream and adjusted to the desired concentrations by AMS electronic mass flow controllers (Bilthoven,
The Netherlands). The air exchange rate was 40 L min 1
and a ventilator continuously mixed the air inside the cabinets. NH3 concentrations were verified during the experimental period by leading the air stream at a known flow rate
into a 1 mM EDTA solution for 2 h. Aliquots were taken
from the initial volume and NH+4 was measured colorimetrically at 410 nm (Starrcol SC-60-S, R&R Mechatronics, Hoorn, The Netherlands) with Nessler’s reagent A
(Merck, Germany) mixed 1 : 1 with 9 N NaOH (v/v). The
PVC pots were filled to 11 L with aereated 25 % Hoagland
nutrient solution. For the nitrate-deprived treatment, NO3
was replaced by Cl . In order to prevent formation of NH+4 ,
by dissolution of atmospheric NH3 into the nutrient solution, the PVC pots were sealed with soft adhesive rubber
(Terostat, Henkel Teroson GmbH, Heidelberg, Germany).
The effectiveness of the sealing procedure was checked by
exposing PVC pots with aerated nutrient solution (with and
without nitrate) and sealed with Terostat to 4 and 8 mL L 1
NH3 for 1 week. A test with Nessler’s reagent (as decribed
above) showed that under these conditions no detectable
level of atmospheric NH3 was absorbed in the nutrient
solutions.
Plant harvest and growth analysis
Plants were harvested 4 h after the start of the light period; the roots were rinsed in ice-cold demineralized water
and separated from the shoot. At this point, shoot and root
fresh weights were taken and used to calculate biomass
production and S/R ratio. The RGR of the total plant was
calculated from the ln-transformed fresh weight data
between days 19 and 26 (Hunt, 1982). Part of the plant
material was stored at 80 C, before it was freeze-dried
in a Heto LyoLab 3000 Freeze Dryer (Heto-Holten A/S,
Allerød, Denmark). Thereafter weights were used for dry
matter content (DMC) analysis, and shoot and root material
was pulverized by a Retsch Mixer-Mill (type MM2, Haan,
Germany) for further analyses. Fresh material was used for
all the analyses, except for cations, soluble sugar and total
sulfur contents, for which freeze-dried material was used.
Total nitrogen content
Soluble reduced nitrogen and insoluble reduced nitrogen
fractions of both fresh shoot and root material were measured using micro-Kjeldahl analysis according to Stulen et al.
(1981a). Reduced nitrogen was extracted in 3 % (v/v)
HCl : H2O. The soluble and insoluble fractions were separated by filtration and digested at 360 C in 96 % H2SO4 with
5 mg CuSO4 and K2SO4 (1 : 3 w/w) as catalyst. NH+4 was
measured colorimetrically at 410 nm as described above.
Nitrate and sulfate content
For nitrate and sulfate analysis, fresh shoot, and root
material was homogenized in demineralized water, with
an Ultra Turrax (T25 Basic Ika Labortechnik, Staufen,
Germany). The homogenate was incubated at 100 C for
10 min, filtered and centrifuged at 30 000 g for 15 min
(Beckman, Model J2-MC, Palo Alto, CA, USA). Nitrate
and sulfate were separated by HPLC on IonoSpher
A anion exchange column (Varian/Chrompack Benelux,
Bergen op Zoom, The Netherlands) and determined refractrometrically according to Buchner et al. (2004).
Free amino acids content
Fresh shoot and root material was extracted as described
for nitrate and sulfate content. The content of free amino
Castro et al. — Atmospheric NH3 and Brassica oleracea
123
acids in the supernatant was determined according to Rosen
(1957).
of the light period (Rubinigg et al., 2003), and expressed
as mmol g 1 f. wt plant day 1.
Nitrate reductase activity
Total sulfur content
Fresh material was used to determine the nitrate reductase
(EC 1.6.6.1) activity by both in vivo (Stulen et al., 1981a)
and in vitro assays (Kaiser et al., 1992). The latter assay was
carried out in the presence of EDTA so that the maximum
in vitro enzyme activity was measured. For determination of
in vivo nitrate reductase activity samples were taken in the
linear phase of the reaction, i.e. at 15 and 45 min for shoot
and at 5 and 10 min for root material, after the start of the
incubation. Nitrite in the samples was measured colorimetrically at 540 nm (Starrcol SC-60-S, R&R Mechatronics, Hoorn, The Netherlands). For in vitro nitrate reductase
activity, plant material was weighed in triplicate and ground
with a mortar and pestle with liquid N2. After an incubation
time of 10 min, samples were taken for nitrite measurements. Soluble protein content of the supernatants was
determined according to Bradford (1976).
Freeze-dried samples of both shoot and roots were used to
determine the total sulfur content by using a modification of
the method described by Jones (1995). The plant material
was weighed into porcelain ashing trays into which 50 %
Mg(NO3)2.6H2O was added slowly to saturate the material.
The samples were dried overnight at 100 C, transferred to a
650 C oven, for at least 12 h, in order to ash the material.
Aqua regia 20 % (50 ml conc. HNO3 and 150 ml conc. HCl
made up to 1 L with demineralized water) was added to
the ash residue, transferred quantitatively to a measuring
flask and made up to 100 ml with demineralized water.
SulfaVer4 Reagent Powder Pillow (HACH Permachem
reagents, Loveland, USA), which contains BaCl2, was
added and the turbidity was measured at 450 nm with a
spectrophotometer (HACH DR/400 V, Loveland, USA).
Soluble sugar content
Net nitrate and sulfate uptake rate
Long-term nitrate and sulfate uptake rate measurements
were performed as described by Westerman et al. (2000)
and calculated according to Stuiver et al. (1997). Plants
were placed in PVC pots, in triplicate, which were filled
exactly up to 11 L with aerated 25 % Hoagland nutrient
solution (375 mM nitrate and 05 mM sulfate as initial concentrations). Aliquots were taken at day 1 and at day 7 of the
NH3-exposure period, after re-filling the PVC pots with
demineralized water up to 11 L. Nitrate and sulfate contents
were analysed as described above. Net nitrate and sulfate
uptake rates were measured over a 1-week period and
expressed as mmol g 1 f. wt plant day 1.
Net nitrate uptake rate with
15
N nitrate
Short-term net nitrate uptake rate was measured with
NO3 after Ter Steege et al. (1998) and Rubinigg et al.
(2003). At day 7 of the NH3-exposure period, whole plants
were transferred from aerated 25 % Hoagland solution to
aerated labelled solution containing 99 % enriched 15KNO3.
Preliminary experiments showed that transfer of the plants
had no effect on nitrate fluxes in B. oleracea, in contrast to
the findings with Spinacia oleracea (Ter Steege et al.,
1998). Composition and pH of both solutions were kept
equal. The labelling period was 2 h according to Ter
Steege et al. (1998) and Rubinigg et al. (2003). At the
end of the labelling period, the roots were immersed in
demineralized water for 2· 45 s. Shoot and root fresh
weights of the plants were determined and the plants
dried at 70 C overnight. Total N and 15N concentrations
were detected with an isotope ratio mass spectrometer at the
Stable Isotope facility of the University of California,
Davis, USA (Europa Scientific Integra CN-Analyzer,
PDZ Europe Ltd, Norwich, UK) in both shoot and roots.
The measurements were performed 35–5 h after the starting
15
Soluble sugars were extracted from the freeze-dried
material with 80 % ethanol. The soluble and insoluble fractions were separated by centrifugation (Sorvall, GLC-2B,
Bergen op Zoom, The Netherlands). The soluble sugar fraction was determined in both shoot and roots according to
Fales (1951) and measured in a spectrophotometer at
620 nm (Starrcol SC-60-S, R&R Mechatronics, Hoorn,
The Netherlands) by using 0145 % (w/v) anthrone in
ethanol : H2O : H2SO4 4 : 15 : 50 (v/v/v).
Cation content
The cation content (Ca2+, Mg2+, K+, Na+) of freeze-dried
shoot and root samples was measured with an atomic
absorption spectrophotometer (Shimadzu AA-660, Kyoto,
Japan) after digestion with HNO3 : H2SO4 : HClO4
06 : 04 : 002 (v/v/v) in a high performance microwave
digestion unit (MLS 1200 Mega Microwave, Milestone,
Sorisole, Italy).
Statistical analysis
Statistical analysis was performed by using an unpaired
Student’s t-test, with level of significance P < 001. All
experiments were performed with six replicates [6standard
deviation (s.d.)], except for cation content and 15NO3 uptake
analyses with three replicates (6s.d.).
RESULTS
NH3 exposure and plant growth
The effect of NH3 on biomass production depended on the
atmospheric concentration, and differed between nitratesufficient and nitrate-deprived plants (Table 1). Exposure
of nitrate-sufficient plants to 4 mL L 1 NH3 had no effect
Castro et al. — Atmospheric NH3 and Brassica oleracea
124
T A B L E 1. The impact of NH3 exposure and nitrate nutrition on growth of Brassica oleracea
0 mL L
+ NO3
Biomass (S)
Biomass (R)
DMC (S)
DMC (R)
S/R
RGR (S + R)
1.51 6
0.49 6
9.6 6
6.3 6
3.4 6
0.21
0.15c
0.05b
0.9a
0.7a
0.4b
1
4 mL L
NH3
– NO3
0.55 6
0.41 6
17.2 6
6.3 6
1.4 6
0.11
0.09a
0.07b
0.4c
0.5a
0.3a
+ NO3
1.56 6
0.32 6
10.4 6
6.7 6
4.1 6
0.22
0.31c
0.09b
0.6a
0.9a
0.6b
1
8 mL L
NH3
– NO3
1.04 6
0.46 6
12.8 6
6.4 6
1.7 6
0.16
0.15b
0.14b
0.9b
0.8a
0.5a
1
NH3
+ NO3
1.07 6
0.18 6
12.6 6
6.0 6
6.3 6
0.15
0.34b
0.06a
0.7b
0.6a
0.9c
– NO3
0.95 6
0.20 6
13.9 6
6.5 6
5.4 6
0.14
0.14b
0.04a
1.6b
0.6a
0.5c
Plants were exposed for 1 week. Biomass S and Biomass R, biomass production in the shoot and root, respectively, calculated by subtracting the final fresh
weight from the initial fresh weight of the plant and expressed on a fresh weight basis (g f. wt); DMC S and DMC R, dry matter content as a percentage of the
fresh weight in the shoot and in the root, respectively (%); S/R, shoot : root ratio calculated on a fresh weight basis; RGR (S + R), relative growth rate calculated
over a 1-week period on a plant basis (g g 1 plant day 1). Data represent the mean of two experiments, with three measurements per experiment with three
plants in each (6s.d.). Means followed by different superscript letters are statistically different at P < 001.
on shoot and root biomass production (and RGR) of the
nitrate-sufficient plants. However, both shoot and root
biomass production (and RGR) of nitrate-sufficient plants
was significantly reduced at 8 mL L 1 NH3. Exposure of
nitrate-deprived plants to 4 and 8 mL L 1 NH3 resulted in a
significantly higher shoot biomass production (and RGR of
the plants); however, it was lower than that of nitratesufficient plants (at 0 mL L 1 NH3). The effects of NH3
on root biomass differed from those found for the shoot. A
concentration of 4 mL L 1 NH3 had no effect on root biomass, while exposure to 8 mL L 1 NH3 resulted in a significantly lower root biomass production of nitratesufficient as well as nitrate-deprived plants.
Shoot DMC increased with increasing NH3 concentration
in nitrate-sufficient plants, but decreased in nitrate-deprived
plants (Table 1). In the control plants shoot DMC of the
nitrate-deprived plants was much higher than that of the
nitrate-sufficient plants. Root DMC was overall significantly lower than the shoot DMC and not affected by
NH3 exposure and nitrate nutrition.
Increasing NH3 concentrations significantly increased the
S/R ratio of nitrate-sufficient plants (Table 1), due to a
relatively higher shoot biomass production and lower
root biomass production. The same effect on the S/R
ratio was found for nitrate-deprived plants, though the absolute values were lower in comparison to nitrate-sufficient
plants.
NH3 exposure and total N, nitrate and free
amino acid content
NH3 exposure resulted in a clear increase in shoot N
content, in particular the soluble N fraction, in both
nitrate-sufficient and nitrate-deprived plants, but did not
affect nitrate content in either of the NH3 treatments
(Fig. 1A). In all treatments, root N content was significantly
lower than that of the shoot (Fig. 1A and B). Root N content
of the nitrate-deprived plants exposed to 8 mL L 1 NH3 was
significantly lower than at 0 and 4 mL L 1 NH3. Compared
with the control plants, exposure to 4 mL L 1 NH3 resulted
in a 16-fold increase in free amino acids in the shoot
(Fig. 1C). At 8 mL L 1 NH3, an 185-fold increase was
found for the nitrate-sufficient plants, and a 17-fold increase
for the nitrate-deprived plants (Fig. 1C). Hence, the main
effect of NH3 exposure on N compounds is a considerable
increase in the free amino acid content.
NH3 exposure and nitrate reductase activity
Nitrate reductase activity (NRA) was measured by both
in vivo (Fig. 2A) and in vitro assays (Fig. 2B and C). NH3
exposure had no significant effect on in vivo NRA in the
shoot of nitrate-sufficient plants, but lead to a significant
increase at 8 mL L 1 in the nitrate-deprived plants, although
the values were very low (Fig. 2A). NH3 exposure significantly decreased in vitro NRA, expressed on a fresh weight
basis (Fig. 2B). When the in vitro NRA was expressed on a
soluble protein basis, however, the effect of NH3 was no
longer significant (Fig. 2C), showing that the effect of NH3
on in vitro NRA activity was mainly the result of changes in
protein content of the extracts. The value found with the
in vivo assay for the control, calculated on a 24-h basis
(96 mmol g 1 f. wt day1) was in the same range as the
calculated N requirement (70 mmol g 1 f. wt day1). A similar finding was observed for Plantago in vivo and it is a
better estimate of the actual nitrate reduction than in vitro
(Stulen et al., 1981b).
NH3 exposure and nitrate and sulfate uptake rate
Long-term net nitrate uptake rate, measured over a 7-d
interval, decreased upon NH3 exposure (Fig. 3A). The
uptake rates at 4 and 8 mL L 1 NH3 decreased by 50 %
and 66 %, respectively, of the control value (0 mL L 1 NH3).
Short-term net nitrate uptake rate, measured with
15
N-KNO3 over a 2-h period, showed a similar effect of
NH3 (Fig. 4), although the absolute values were lower than
those of the long-term measurements. The values at 4 and
8 mL L 1 NH3 decreased by 48 and 80 % of the control
value, respectively.
Net sulfate uptake rate at 0 and at 4 mL L 1 NH3 did not
differ. Exposure to 8 mL L 1 NH3 resulted in a significant
17-fold decrease (Fig. 3B).
In order to establish whether the measured uptake rates
were of physiological significance, the nitrate uptake rate
needed to cover the plant’s nitrogen requirement on a
125
Castro et al. — Atmospheric NH3 and Brassica oleracea
1400
Nitrate
Soluble N
Insoluble N
f
e
1050
d
700
c
b
a
350
5
A
Shoot
NR activity in vivo
(µmol g−1 f. wt h−1)
Total N (µmol g−1 f. wt)
1750
Nitrate
Soluble N
Insoluble N
250
200
150
Root
B
c
2
1
c
c
b
b
a
50
c
c
C
160
120
80
b
b
40
a
b
B
16
b
a
12
8
4
0
5
NR activity in vitro
(µmol mg−1 protein h−1)
Shoot
200
ab
a
20
0
Free amino acids (µmol g−1 f. wt)
3
c
d
100
c
4
0
NR activity in vitro
(µmol g−1 f. wt h−1)
Total N (µmol g−1 f. wt)
0
A
c
C
4
3
a
a
2
a
1
a
0
0
0
4
[NH3] µL
8
L–1
F I G . 1. The impact of NH3 exposure on nitrate, soluble and insoluble
nitrogen content of the shoot (A) and the root (B) and on the free amino
acid content of the shoot of Brassica oleracea (C). Plants were exposed to 0,
4, 8 mL L 1 NH3 for 1 week. Nitrate sufficient- and nitrate-deprived treatments are given in dark- and light-grey bars, respectively. The various N
compounds are given within each bar, from top to bottom. Data represent the
mean of two experiments, with three measurements per experiment with
three plants in each for total N, and measurements with three plants in each
for free amino acids content (6s.d.). Standard deviations and statistics
treatment refer to total N content (A and B). Different letters indicate
significant differences at P < 0.01.
0
4
[NH3] µL
8
L−1
F I G . 2. The impact of NH3 exposure on in vivo (A) and in vitro (B and C)
nitrate reductase activity (NRA) in the shoot of Brassica oleracea. Plants
were exposed to 0, 4, 8 mL L 1 NH3 for 1 week. In vivo NRA was determined
in the shoot (A). Nitrate-sufficient and nitrate-deprived treatments are given
in dark- and light-grey bars, respectively. In vitro NRA (B and C) was
expressed on a fresh weight (B) and a soluble protein (C) basis. For in vivo
NRA measurements, data represent the mean of two experiments, with three
measurements per experiment with three plants in each (6s.d.); for in vitro
NRA measurements data represent the mean of three measurements with
three plants in each (6s.d.). Different letters indicate significant differences
at P < 0.01.
NH3 exposure and soluble sugar content
whole-plant basis, and was estimated on the basis of RGR
times the plant’s N content, for the nitrate-sufficient plants.
The estimated nitrogen requirement was 70 mmol g 1 f. wt
plant day 1 which was within the range of measured uptake
rate over a 7-d interval (Fig. 3).
In nitrate-sufficient plants, shoot soluble sugar content
was unaffected by NH3 exposure (Fig. 6A). In nitratedeprived plants an accumulation of soluble sugars was
found at 0 mL L 1 NH3. In the roots of nitrate-deprived
plants a significant decrease at 8 mL L 1 NH3 was observed
(Fig. 6B).
NH3 exposure and total sulfur content
In nitrate-sufficient plants, shoot total S content
decreased at 4 and 8 mL L 1 NH3 (Fig. 5A). Root total S
content was unaffected upon NH3 exposure, for both nitrate
treatments (Fig. 5B).
NH3 exposure and cation content
In nitrate-sufficient plants a decrease in shoot K+ content
was only found at 4 mL L 1 NH3 (Fig. 7A). The effect of
Castro et al. — Atmospheric NH3 and Brassica oleracea
126
125
60
A
c
50
Total S
(µmol g−1 f. wt)
Net nitrate uptake rate
(µmol g−1 f. wt d−1)
150
100
75
b
50
a
c
A
abc
ab
b
40
30
a
20
10
25
0
60
0
Root
B
30
b
24
B
50
b
18
a
12
Total S
(µmol g−1 f. wt)
Net sulfate uptake rate
(µmol g−1 f. wt d−1)
Shoot
c
40
30
20
ab
ab
a
10
6
b
ab
a
0
0
0
4
[NH3] µL L−1
0
8
F I G . 3. The impact of NH3 exposure on net nitrate (A) and net sulfate (B)
uptake rate. Nitrate-sufficient plants were exposed to 0, 4, 8 mL L 1 NH3 for 1
week. (A) Data for long-term nitrate uptake rate measurement over a 1-week
period and expressed as mmol g 1 f. wt plant day 1; (B) data for long-term
sulfate uptake over a 1-week period expressed as as mmol g 1 f. wt day 1
(for experimental details see Materials and methods). Data represent the
mean of two experiments, with three measurements per experiment, with
three plants in each (6s.d.). Different letters indicate significant differences
at P < 0.01.
6
Soluble sugar
(µmol g−1 f. wt)
c
45
A
Shoot
b
4
3
2
a
a
a
a
a
1
b
30
0
a
15
0
0
4
[NH3] µL L−1
8
15
F I G . 4. The impact of NH3 exposure on the short-term NO3 net nitrate
uptake rate measurement, over a 2-h period and expressed as mmol g 1 f. wt
plant day 1. Nitrate-sufficient plants were exposed to 0, 4, 8 mL L 1 NH3 for
1 week. Data represent the mean of three measurements, with three
plants in each (6s.d.). Different letters indicate significant differences at
P < 0.01.
atmospheric NH3 on shoot K+ content was more pronounced
in nitrate-deprived plants, where a gradual decrease from
0 to 8 mL L 1 NH3 was found (Fig. 7A). In the roots the only
significant effect of NH3 on K+ content was found at
4 mL L 1 NH3 in nitrate-deprived plants (Fig. 7B). Neither
shoot nor root Na+ content was significantly affected by
Root
2·0
Soluble sugar
(µmol g−1 f. wt)
nitrate uptake rate
(µmol g−1 f. wt d−1)
15N
60
8
F I G . 5. The impact of NH3 exposure on total sulfur content in the shoot (A)
and in the roots (B). Plants were exposed to 0, 4, 8 mL L 1 NH3 for 1 week.
Nitrate-sufficient and nitrate-deprived treatments are given in dark- and
light-grey bars, respectively. Data represent the mean of two experiments,
with three measurements per experiment with three plants in each (6s.d.).
Different letters indicate significant differences at P < 0.01.
5
75
4
[NH3] µL L−1
B
1·5
abc
1·0
abc
c
c
0·5
b
a
0·0
0
4
8
[NH3] µL L−1
F I G . 6. The impact of NH3 exposure on the soluble sugar content in shoot
(A) and roots (B). Plants were exposed to 0, 4, 8 mL L 1 NH3 for 1 week.
Nitrate-sufficient and nitrate-deprived treatments are given in dark and lightgrey bars, respectively. Data represent the mean of two experiments, with
three measurements per experiment with three plants in each (6s.d.).
Different letters indicate significant differences at P < 0.01.
127
Castro et al. — Atmospheric NH3 and Brassica oleracea
Shoot
75
Root
A
d
[K+] content
(µmol g−1 f. wt)
60
c
c
b
B
c
a
45
b
ab
b
ab
a
30
ab
15
0
5
C
D
[Na+] content
(µmol g−1 f. wt)
4
b
3
a
a
a
a
ab
ab
ab
a
2
ab
a
a
1
0
105
d
d
E
[Ca2+] content
(µmol g−1 f. wt)
84
c
b
F
b
63
a
42
21
b
a
b
b
a
a
0
40
G
[Mg2+] content
(µmol g−1 f. wt)
32
24
H
e
d
c
c
a
16
b
abc
ab
a
a
bc
a
8
0
0·0
4·0
[NH3] µL
8·0
L−1
0·0
4·0
[NH3] µL
8·0
L−1
F I G . 7. The impact of NH3 exposure on cation content in the shoot (A, C, E and G) and in the roots (B, D, F and H). Plants were exposed to 0, 4, 8 mL L 1 NH3
for 1 week. Nitrate-sufficient and nitrate-deprived treatments are given in dark- and light-grey bars, respectively. Data represent the mean of three
measurements with three plants in each (6s.d.). Different letters indicate significant differences at P < 0.01.
exposure to NH3 (Fig. 7C and D) in either of the nitrate
treatments.
Exposure to NH3 had a more drastic effect on the content
of divalent cations, Ca2+ and Mg2+, especially in the shoot
(Fig. 7E and G). Exposure to 4 and 8 mL L 1 NH3 of the
nitrate-sufficient plants resulted in a significant decrease
compared with the control. In the shoot of nitrate-deprived
plants exposed to 4 mL L 1 NH3 a significant decrease of
22 and 40 % for Ca2+ and Mg2+ contents, respectively, compared with the control plants was found. At 8 mL L 1 NH3
the decrease in Ca2+ and Mg2+ content was 57 and 53 %,
respectively. Root Ca2+ content showed a significant
128
Castro et al. — Atmospheric NH3 and Brassica oleracea
decrease at 8 mL L 1 NH3 in both nitrate treatments, but the
decrease was less than that observed for the shoot (Fig. 7F).
For root Mg2+ content significant differences were found
between nitrate-sufficient and nitrate-deprived plants at
0 mL L 1 NH3 (Fig. 7H).
DISCUSSION
To what extent does NH3 exposure affect plant growth?
Previous experiments with B. oleracea exposed to 0, 2, 4, 6
and 8 mL L 1 NH3 (Castro et al., 2005) and the present
results (Table 1) revealed that B. oleracea can use atmospheric NH3 for growth up to a concentration of 4 mL L 1
NH3. Higher NH3 concentrations are toxic because biomass
production, especially of the roots, is reduced. Even though
the NH3 concentrations used in this study were much higher
than generally found in polluted areas (Fangmeier et al.,
1994; Stulen et al., 1998; Krupa, 2003), B. oleracea apparently can use an NH3 concentration of 4 mL L 1 as a nutrient
(Table 1). Since at 8 mL L 1 NH3 shoot as well as root
biomass production was severely impaired, both in the presence and absence of nitrate in the nutrient solution, the
present results with B. oleracea do not support the concept
of nitrate-ammonium synergism, in which the toxicity of
NH+4 may be alleviated by a simultaneous supply of nitrate
(Britto and Kronzucker, 2002). At 8 mL L 1 NH3 a significant increase in the S/R ratio was found, which is in agreement with other data (Table 1; Van der Eerden, 1982; Van
der Eerden and Pérez-Soba, 1992; Pérez-Soba et al., 1993).
Since the experimental set-up of the present experiments
prevented formation of NH+4 in the nutrient solution, the
negative effect on root biomass production must have
been mediated by a signal from the shoot as a result of
the foliar uptake of atmospheric NH3.
What is the impact of NH3 exposure at a metabolic level?
One of the best-documented effects of the foliar uptake of
atmospheric NH3 on metabolism is the increase in total N
and N-containing metabolites, such as amino acids (Van der
Eerden, 1982; Van Dijk and Roelofs, 1988; Fangmeier et al.,
1994; Pérez-Soba et al., 1994; Clement et al., 1997; Gessler
et al., 1998). The present results showed that the increase in
the total N content was mainly due to an increase in the
soluble N fraction (Fig. 1A). At 8 mL L 1 NH3, B. oleracea
was still able to detoxify atmospheric NH3 as shown by the
dramatic increase in the free amino acid content in the shoot
(Fig. 1C), which most likely is the effect of the reduction in
growth at this concentration.
In the experiments of Pearson and Soares (1998) with
bean (Phaseolus vulgaris), exposed to 47 mL L 1 NH3 for
1 h, a significant decrease in in vivo leaf NRA was found.
Amino acids, as end products of NH3 assimilation, have
been suggested as inhibiting the enzyme (Pearson and
Stewart, 1993). The present experiments, in which plants
were exposed for 1 week, showed that 4 mL L 1 NH3 had no
effect on in vivo NRA in the shoot. Only at 8 mL L 1 was a
reduction in in vivo NRA found (Fig. 2A). This effect cannot
be ascribed to a change in nitrate concentration, since the
nitrate pool was unaffected upon exposure to NH3 (Fig. 1A).
Changes in in vivo NRA may be caused by changes in the
amount of functional enzyme, and/or the endogenous supply of NADH to the enzyme (De Kok et al., 1986). There
was only a slight effect of 8 mL L 1 NH3 on in vitro NRA
(Fig. 2B and C), therefore it was unlikely that the effect on
in vivo NRA was primarily due to a change in amount of
enzyme. Apparently, at 8 mL L 1 NH3 the decrease in in vivo
NRA might be due to a change in endogenous NADH
supply (De Kok et al., 1986) and/or via modulation of
the catalytic activity of the existing NR protein (Kaiser
et al., 1992).
To what extent does NH3 exposure affect
nitrate uptake by the root?
In the present experiments, nitrate uptake rate on a plant
basis was measured in two ways: in a long-term experiment
in which nitrate depletion from the solution was measured
over a 7-d period; and in a short-term experiment, over a 2-h
period. Comparison of the measured uptake rates of the
nitrate-sufficient control plants (0 mL L 1 NH3; Fig. 3A)
with the N requirement for growth (N flux; RGR times the
plant’s nitrogen content; De Kok et al., 2000, 2002) showed
that the long-term uptake rate corresponded with the N
requirement for growth, and can therefore be considered
as physiologically relevant. The uptake rate in the shortterm experiment (Fig. 4) was lower than the N requirement
for growth. The difference may be caused by differences in
uptake rate during the day and/or night. Exposure to 4 and
8 mL L 1 NH3 resulted in a reduction of nitrate uptake
rate (long-term uptake experiment) of 50 and 66 %, respectively. A similar trend was found in the short-term uptake
experiment.
In nitrate-sufficient conditions, 4 mL L 1 NH3 had no
effect on the sulfate uptake rate, but decreased nitrate uptake
rate, so that the effect of NH3 can be considered direct, and
not mediated through changes in growth. A significant
decrease in the sulfate uptake rate (Fig. 4) and a concomitant
decrease in total S content (Fig. 5A and B) were only found
at the toxic concentration of 8 mL L 1 NH3, when RGR was
reduced. The effect of NH3 on nitrate and sulfate uptake rate
at this concentration, therefore, can be considered to be
indirect, and growth-related. This is in accordance with
the observation that changes in nitrate uptake rate are driven
by changes in RGR of the plant (Ter Steege et al., 1998).
An exact comparison between the present data and data in
the literature is difficult, because of differences in growth
conditions, which may have resulted in differences in RGR,
different atmospheric NH3 concentrations and exposure
times. Clement et al. (1997) exposed winter wheat (Triticum
aestivum) to 1 and 2 mL L 1 NH3, for 1 week, and found a
reduction of nitrate uptake rate of 8 and 138 %, respectively, at relatively low light conditions. These plants had a
rather low RGR (008 g g 1day 1). Gessler et al. (1998)
exposed seedlings of beech (Fagus sylvatica) to 005 mL
L 1 NH3, and found a reduction of 35 % in cumulative root
nitrate uptake over the last 24 h of the exposure. In these
experiments, no data were presented on RGR or physiological relevance of the measured uptake rates.
Castro et al. — Atmospheric NH3 and Brassica oleracea
The mechanisms underlying the regulation of nitrate
uptake by the roots as well as the signal metabolites
involved were dealt with in detail by Imsande and
Touraine (1994) and Touraine (2005). Phloem-translocated
amino acids acting as a regulatory signal between shoot and
root appear to be the explanation for a down-regulation of
nitrate uptake, under steady-state conditions, at a whole
plant level (Muller and Touraine, 1992; Imsande and
Touraine, 1994; Gessler et al., 1998). The present data,
however, showed no relationship between the effect of
NH3 on nitrate uptake rate and total root soluble N content
(amino acids and amides). Although shoot soluble N content
and free amino acid content were increased at both NH3
concentrations, in the root no significant changes in soluble
N content were found. However, a change in a specific
amino acid cannot be ruled out.
To what extent can atmospheric NH3 contribute to the
plant’s N requirement for growth?
From theoretical calculations with spinach, which contained an organic N content of 250 mmol g 1 plant fresh
weight and had an S/R ratio of 34, it was estimated that an
atmospheric NH3 concentration of 2 mL L 1 might be sufficient for the N requirement of a plant with an RGR of
005 g g 1 day, and might contribute to 50 % of the total N
requirement at an RGR of 02 g g 1 day (Stulen et al., 1998).
In a series of experiments in which sunflower plants were
grown in the absence of N in the root environment, plants
were able to grow on 2 mL L 1 NH3 as a sole N source
(Faller, 1972).
From estimations based on previous data (see Castro
et al., 2005) and the present data it is obvious that at
4 mL L 1 NH3 the foliar N uptake might contribute up to
65–70 % of the total N requirement of B. oleracea. From the
data on nitrate-deprived plants it was evident that at 4 and
8 mL L 1 NH3 indeed was utilized as a N source for growth;
however, plant biomass production was less than that of
plants in nitrate-sufficient conditions. Furthermore, it was
observed that in nitrate-sufficient plants the toxic effects of
NH4 started to prevail at >4 mL L 1. Apparently there was
no clear-cut transition in the level and rate of metabolism of
the foliar absorbed NH3 and the phytotoxicity upon exposure at 4 and 8 mL L 1.
Is the NH3 toxicity related to a lack of detoxification
capacity or a lack of carbohydrates?
A lack of detoxification capacity, occurring when uptake
exceeds the capacity to assimilate NH+4 via the GS/GOGAT
pathway, and a lack of carbon skeletons for the formation of
organic N compounds, are considered as primary causes of
NH3 toxicity (Van der Eerden, 1982; Fangmeier et al., 1994;
Krupa, 2003). The present data do not support these hypotheses. At 8 mL L 1 NH3, considered a toxic concentration,
the organic N content was higher than in plants exposed to
the non-toxic concentration of 4 mL L 1 NH3 (Fig. 1), which
shows that assimilation into N-compounds was still possible
at 8 mL L 1 NH3. It is therefore highly unlikely that the toxic
effect of 8 mL L 1 NH3 can be explained by a lack of
129
capacity to assimilate NH+4 formed in the mesophyll upon
exposure to atmospheric NH3.
Exposure to NH3 did not decrease shoot soluble sugar
content at any concentration (Fig. 6). The 2-fold higher
soluble sugar content in nitrate-deprived compared with
the nitrate-sufficient plants at 0 mL L 1 NH3 disappeared
upon exposure to 4 and 8 mL L 1 NH3. This can be
explained by the fact that the accumulated soluble sugars
in the nitrate-deficient plants were used as C-acceptors for
NH+4 and structural growth in the presence of an atmospheric
N source. This conclusion is corroborated by the changes in
DMC (Table 1). The present experiments, therefore, do not
support the hypothesis that NH3 toxicity is related to a lack
of availability of carbon skeletons. This conclusion is corroborated by findings from other authors. In a similar
experimental set-up to the present one, the impact of 1 and
2 mL L 1 NH3 on shoot soluble sugar and starch contents in
winter wheat was investigated by Clement et al. (1997).
Soluble sugar content was not significantly affected by
NH3, while plants exposed to 1 mL L 1 NH3 even had a
slightly higher starch content. In a different experimental
set-up in which NO3 - and NH+4 -containing solutions
(20 mM) were applied to the foliage of Ricinus communis,
grown without NO3 in the pedosphere, Peuke et al. (1998a)
found that the flow of C in the whole plant, as well as the
increment of C in different plant organs, were similar for
both foliar N-treatments. Similar results were found for
plants grown on NH+4 in the nutrient solution. Lang and
Kaiser (1994) showed that sugar levels in NH+4 -grown
barley plants were unchanged, or even increased, and
concluded that the growth impairment observed was not
related to impaired carbohydrate supply. Walch-Liu et al.
(2001) concluded from experiments in which the effects of
NO3 and NH+4 on tobacco were investigated that inhibitory
effects on shoot and root growth could not be related to
limitations in the N or C status of the plants, or to NH+4
toxicity.
Are changes in cations related to NH3 toxicity?
It is generally stated that atmospheric NH3 may lead to a
release of inorganic cations such as K+, Ca2+ or Mg2+,
leading to nutrient imbalances (Van der Eerden and
Pérez-Soba, 1992; Wollenweber and Raven, 1993;
Fangmeier et al., 1994; Pérez-Soba et al., 1994; Britto
and Kronzucker, 2002). Experimental data differ between
a change and no effect, and accordingly, caution should be
taken in making comparisons between results since NH3
concentrations and exposure times are rather different.
The present data showed hardly any changes in K+ content. Under nitrate-sufficient conditions, a decrease in shoot
K+ content was observed when NH3 acted as a nutrient
(4 mL L 1), but no change in K+ content when NH3 was
toxic (8mL L 1; Fig. 7). In contrast, Wollenweber and
Raven (1993) stated that the increase in K+ content was
the most striking effect in Lolium perenne, exposed to
04–18 mL L 1 NH3.
The effect of NH3 exposure on the divalent cations was
more pronounced. Ca2+ and Mg2+ contents of both shoot and
roots were decreased by NH3, independent of the nitrate
Castro et al. — Atmospheric NH3 and Brassica oleracea
130
supply (Fig. 7). Van Dijk and Roelofs (1988) and Van der
Eerden and Pérez-Soba (1992) observed shifts in the N : K,
N : Ca and N : Mg ratios but no decrease in K+, Ca2+ or Mg2+
content, in needles of P. sylvestris, after exposure to 008–
04 mL L 1 NH3 for 3 months. In the experiments of Peuke
et al. (1998b), in which Ricinus communis plants were
sprayed with NO3 and NH+4 solutions (20 mM), a strong
decrease in Mg2+ in NH+4 -sprayed plants compared with
the NO3-sprayed plants was observed. Lang and Kaiser
(1994) also found a drastic decrease (90 %) in Mg+ content
of roots of barley cultivated on NH+4 in the nutrient solution.
The decrease in Mg+ content, however, was not sufficient to
impair the energy status, as the ATP and carbohydrate concentrations. In the present experiments the effect of NH3 on
Mg2+ contents was much less, and did not differ much
between plants exposed to a non-toxic (4 mL L 1) or a
toxic (8 mL L 1) concentration. It is therefore highly
unlikely that in the present experiments cation imbalances
are the cause of the toxic effect of NH3.
Concluding remarks
From the results presented in this paper it is evident that
atmospheric NH3 can act as a nutrient, since it may contribute a considerable proportion of the total N requirement
of B. oleracea, at high levels, and in the absence of nitrogen
in the nutrient solution. The concentration at which NH3
changes from being a nutrient to a toxin is not clear-cut,
since NH3 can still be metabolized when growth is already
affected. The physiological basis of the phytotoxicity of
atmospheric NH3 is still largely unknown.
ACKNOWLEDGEMENTS
We thank M. Durenkamp for critical reading of the manuscript and B. Venema for technical assistance in the sample
preparation for cation and [15N]KNO3 determinations.
LITERATURE CITED
Barker AV. 1999. Foliar ammonium accumulation as an index of stress in
plants. Communication in Soil Science and Plant Analysis 30:
167–174.
Bradford MM. 1976. A rapid and sensitive method for the quantification of
microgram quantities of protein utilizing the principle of protein-dye
binding. Analytical Biochemistry 72: 248–254.
Britto DT, Kronzucker HJ. 2002. NH+4 toxicity in higher plants: a critical
review. Journal of Plant Physiology 159: 567–584.
Britto DT, Siddiqi MY, Glass ADM, Kronzucker HJ. 2001. Futile transmembrane NH+4 cycling: a cellular hypothesis to explain ammonium
toxicity in plants. Proceedings of the National Academy of Science of
the USA 7: 4255–4258.
Buchner P, Stuiver CEE, Westerman S, Wirtz M, Hell R,
Hawkesford MJ, et al. 2004. Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea L. as affected by
atmospheric H2S and pedospheric sulfate nutrition. Plant Physiology
136: 3396–3408.
Castro A, Aires A, Rosa E, Bloem E, Stulen I, De Kok LJ. 2004.
Distribution of glucosinolates in Brassica oleracea cultivars. Phyton
44: 133–143.
Castro A, Stulen I, De Kok LJ. 2005. Impact of atmospheric NH3 deposition
on plant growth and functioning: a case study in Brassica oleracea L.
In: Omasa K, Nouchi I, De Kok LJ eds. Plant responses to air pollution
and global changes. Tokyo: Springer-Verlag (in press).
Clement JMAM, Loorbach J, Meijer J, Van Hasselt PR. 1997. The
impact of atmospheric ammonia and temperature on growth and nitrogen metabolism in winter wheat. Plant Physiology and Biochemistry
34: 159–164.
De Kok LJ, Stulen I, Bosma W, Hibma J. 1986. The effect of short term
H2S fumigation on nitrate reductase activity in spinach leaves. Plant
and Cell Physiology 27: 1249–1254.
De Kok LJ, Westerman S, Stuiver CEE, Stulen I. 2000. Atmospheric H2S
as plant sulfur source: interaction with pedospheric sulfur nutrition – a
case study with Brassica oleracea L. In: Brunold C, Rennenberg H,
De Kok LJ, Stulen I, Davidian J-C, eds. Sulfur nutrition and sulfur
assimilation in higher plants: molecular, biochemical and physiological aspects. Bern, Switzerland: Paul Haupt, 41–56.
De Kok LJ, Castro A, Durenkamp M, Stuiver CEE, Westerman S,
Yang L, Stulen I. 2002. Sulphur in plant physiology. Proceedings
of the International Fertiliser Society, No. 500. York, UK: International Fertiliser Society, 1–26.
Durenkamp M, De Kok LJ. 2002. The impact of atmospheric H2S on
growth and sulfur metabolism of Allium cepa L. Phyton 42: 55–63.
Fales FW. 1951. The assimilation and degradation of carbohydrates by yeast
cells. Journal of Biological Chemistry 193: 113–124.
Faller N. 1972. Schwefeldioxid, Schwefelwasserstof, nitrose Gase und
Ammoniak als ausschliesliche S-bzw. N-Quellen der höheren Pflanze.
Zeitschrift für Pflanzenernahrung und Bodenkunde 131: 120–130.
Fangmeier A, Hadwiger-Fangmeier A, Van Der Eerden L, Jager H-J.
1994. Effects of atmospheric ammonia on vegetation – a review.
Environmental Pollution 86: 43–82.
Glass ADM, Britto DT, Kaiser BN, Kinghorn JR, Kronzucker HJ,
Kumar A, et al. 2002. The regulation of nitrate and ammonium transport systems in plants. Journal of Experimental Botany 370: 855–864.
Gessler A, Schultze M, Schrempp S, Rennenberg H. 1998. Interactions of
phloem–translocated amino compounds with nitrate net uptake by the
roots of beech (Fagus sylvatica) seedlings. Journal of Experimental
Botany 326: 1529–1537.
Hunt R. 1982. Plant growth curves. In: Functional approach to plant growth
analysis. London: Edward Arnold Publishers, 5–33.
Husted S, Schjoerring JK. 1996. Ammonia fluxes between oilseed rape
plants and the atmosphere in response to changes in leaf temperature,
light intensity and air humidity. Plant Physiology 112: 67–74.
Hutchinson GL, Millington RJ, Peters DB. 1972. Atmospheric ammonia:
absorption by plant leaves. Science 175: 771–772.
Imsande J, Touraine B. 1994. N demand and the regulation of nitrate
uptake. Plant Physiology 105: 3–7.
Jones Jr JB. 1995. Determining total sulphur in plant tissue using the HACH
kit spectrophotometer technique. Sulphur in Agriculture 19: 58–62.
Kaiser WM, Spill D, Brendle-Behnisch E. 1992. Adenine nucleotides are
apparently involved in the light-dark modulation of spinach-leaf nitrate
reductase. Planta 186: 236–240.
Krupa SV. 2003. Effects of atmospheric ammonia (NH3) on terrestrial
vegetation: a review. Environmental Pollution 124: 179–221.
Lang B, Kaiser WM. 1994. Solute content and energy status of roots of
barley plants cultivated at different pH on nitrate- or ammonium
nitrate. New Phytologist 128: 451–459.
Lea PJ, Miflin BJ. 1974. An alternative route for nitrogen assimilation in
higher plants. Nature 251: 614–616.
Muller B, Touraine B. 1992. Inhibition of NO3 uptake by various
phloem-translocated amino acids in soybean seedlings. Journal of
Experimental Botany 250: 617–623.
Pearson J, Soares S. 1998. Physiological responses of plant leaves to
atmospheric ammonia and ammonium. Atmospheric Environment
32: 533–598.
Pearson J, Stewart GR. 1993. The deposition of atmospheric ammonia
and its effects on plants. New Phytologist 125: 283–305.
Pérez-Soba M, Van der Eerden LJM. 1993. Nitrogen uptake in needles of
Scots pine (Pinus sylvestris L.) when exposed to gaseous ammonia and
ammonium fertilizer in the soil. Plant and Soil 153: 231–242.
Pérez-Soba M, Stulen I, Van der Eerden LJM. 1994. Effect of
atmospheric ammonia on the nitrogen metabolism of Scots pine
(Pinus sylvestris) needles. Physiologia Plantarum 90: 629–636.
Peuke AD, Jeschke WD, Dietz K-J, Schreiber L, Hartung W. 1998a.
Foliar application of nitrate and ammonium as sole nitrogen supply in
Castro et al. — Atmospheric NH3 and Brassica oleracea
Ricinus communis. I. Carbon and nitrogen uptake and inflows.
New Phytologist 138: 675–687.
Peuke AD, Jeschke WD, Hartung W. 1998b. Foliar application of nitrate
and ammonium as sole nitrogen supply in Ricinus communis. II. The
flows of cations, chloride and abcisic acid. New Phytologist 140:
625–636.
Rogers HH, Aneja PA. 1980. Uptake of atmospheric ammonia by selected
plant species. Environmental and Experimental Botany 20: 251–257.
Rosen H. 1957. A modified ninhydrin colorimetric analysis for amino acids.
Archives of Biochemistry and Biophysics. 67: 10–15.
Rubinigg M, Posthumus F, Ferschke M, Elzenga JTM, Stulen I. 2003.
Effects of NaCl salinity on 15N-nitrate fluxes and specific root length in
the halophyte Plantago maritima L. Plant and Soil 250: 201–213.
Schjoerring JK, Husted S, Mattson M. 1998. Physiological parameters
controlling plant-atmosphere ammonia exchange. Atmospheric
Environment 32: 491–498.
Stuiver CEE, De Kok LJ, Westerman S. 1997. Sulfur deficiency in
Brassica oleracea L.: development, biochemical, characterization
and sulfur/nitrogen interactions. Russian Journal of Plant Physiology
44: 505–513.
Stulen I, Lanting L, Lambers H, Posthumus F, Van de Dijk SJ, Hofstra R.
1981a. Nitrogen metabolism of Plantago lanceolata as dependent on
the supply of mineral nutrients. Physiologia Plantarum 51: 93–98.
Stulen I, Lanting L, Lambers H, Posthumus F, Van de Dijk SJ, Hofstra R.
1981b. Nitrogen metabolism of Plantago major ssp. major as dependent on the supply of mineral nutrients. Physiologia Plantarum 51:
108–114.
Stulen I, Pérez-Soba M, De Kok LJ, Van der Eerden L. 1998. Impact of
gaseous nitrogen deposition on plant functioning. New Phytologist
139: 61–70.
131
Ter Steege MW, Stulen I, Wiersma PK, Paans AJN, Vaalburg W,
Kuiper PJC, et al. 1998. Growth requirement for N as a criterion
to assess the effects of physical manipulation on nitrate uptake fluxes
in spinach. Physiologia Plantarum 103: 181–192.
Touraine B. 2005. Nitrate uptake by roots – transport and root
development. In: Amâncio S, Stulen I, eds. Nitrogen acquisition
and assimilation in higher plants. Dordrecht: Kluwer Academic
Publishers, 1–34.
Van der Eerden LJM. 1982. Toxicity of ammonia to plants. Agriculture
and Environment 7: 223–235.
Van der Eerden LJM, Pérez-Soba M. 1992. Physiological responses of
Pinus sylvestris to atmospheric ammonia. Trees 6: 48–53.
Van Dijk HFG, Roelofs JGM. 1988. Effects of excessive ammonium
deposition on the nutritional status and conditions of pine needles.
Physiologia Plantarum 73: 494–501.
Walch-Liu P, Neumann G, Engels K. 2001. Response of shoot and root
growth to supply of different nitrogen forms is not related to
carbohydrate and nitrogen status of tobacco plants. Journal of Plant
Nutrition and Soil Science 164: 97–103.
Westerman S, De Kok LJ, Stuiver CEE, Stulen I. 2000. Interaction
between metabolism of atmospheric H2S in the shoot and sulfate
uptake by the roots of curly kale (Brassica oleracea). Physiologia
Plantarum 109: 443–449.
Wollenweber B, Raven JA. 1993. Implications of N acquisition from atmospheric NH3 for acid-base and cation–anion balance of Lolium perenne.
Physiologia Plantarum 89: 519–523.
Yin Z-H, Raven JA. 1997. A comparison of the impacts of various
nitrogen sources on acid-base balance in C3 Triticum aestivum L.
and C4 Zea mays L. plants. Journal of Experimental Botany
48: 315–324.