Environmental and Experimental Botany 44 (2000) 141 – 149

www.elsevier.com/locate/envexpbot

Nitrogen nutrition and water stress effects on leaf

photosynthetic gas exchange and water use efficiency in
winter wheat
Z.P. Shangguan a,*, M.A. Shao a, J. Dyckmans b
a
State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conser6ation,
Chinese Academy of Sciences, Northwest Sci-Tech Uni6ersity of Agriculture and Forestry, Yangling, Shannxi 712100, P.R. China
b
Institute of Soil Science and Forest Nutrition, Uni6ersity of Göttingen, Büsgenweg 2, 37077 Göttingen, Germany

Received 14 December 1999; received in revised form 21 June 2000; accepted 21 June 2000

Abstract

The responses of gas exchange and water use efficiency to nitrogen nutrition for winter wheat were investigated
under well-watered and drought conditions. The photosynthetic gas exchange parameters of winter wheat are
remarkably improved by water and nitrogen nutrition and the regulative capability of nitrogen nutrition is influenced
by water status. The effects of nitrogen nutrition on photosynthetic characteristics and on the limited factors to
photosynthesis are not identical under different water status. Intrinsic water use efficiency (WUEi) of the plants at the
high-N nutrition was decreased by a larger value than that of the plants in the low-N treatment due to a larger
decrease in photosynthetic rate than in transpiration rate. Carbon isotope composition of plant material (dp) is
increased by the increase of drought intensity. The dp at a given level of Ci/Ca is reduced by nitrogen deficiency. Leaf
carbon isotope discrimination (D) is increased by the increase of nitrogen nutrition and decreased by the increase of
drought intensity. Transpirational water use efficiency (WUEt) is negatively correlated with D in both nitrogen supply
treatments and increased with the nitrogen supply. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Carbon isotope discrimination; Drought; Nitrogen supply; Photosynthesis; Triticum aesti6um; Wheat

1. Introduction Similar problems are encountered worldwide

Drought stress and nitrogen deficiency are ma-
jor constraints to winter wheat production and
yield stability in most rainfed regions in China.
* Corresponding author. Tel.: +86-29-7019107; fax: + 86-
29-7012210.
E-mail addresses: zhouping@public.xa.sn.cn
Shangguan), jdyckma@gwdg.de (J. Dyckmans).
(Fischer and Turner, 1978; Lawlor, 1995). An
efficient use of limited water resources and better
growth under both limited water and nitrogen
supply are desirable traits for crops in drought
environments.
Physiological responses of plants to either
drought or nitrogen deficiency have been well
(Z.P. documented. However the interactions between
these two factors on plant morphological and

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PII: S 0 0 9 8 - 8 4 7 2 ( 0 0 ) 0 0 0 6 4 - 2
142 Z.P. Shangguan et al. / En6ironmental and Experimental Botany 44 (2000) 141–149
physiological responses have received relatively
little attention (Mcdonald and Davies, 1996). In
beans (Shimshi, 1970), coffee (Tesha and Kumar,
1978), and winter wheat (Shangguan, 1997), the
stomatal conductance (gs) increased with nitrogen
nutrition under well-watered conditions and be-
came more sensitive to leaf water potential. It
decreased as soil water became less available.
Other works on tea (Nagarajah, 1981) and cotton
(Radin and Ackerson, 1989) have indicated an
opposite response, i.e. the stomatal sensitivity to
leaf water potential was decreased by high nitro-
gen nutrition. A strong correlation was found
between leaf conductance and leaf nitrogen con-
tent; however, this relationship was weaker than
that between stomatal conductance and water po-
tential (Radin and Parker, 1979; Bolton and
Brown, 1980; Morgan, 1986; Shangguan and
Chen, 1990; Ciompi et al., 1996). Sugiharto et al.
(1990) found a significiant positive correlation
between the photosynthetic capacity of leaves and
their leaf nitrogen concentration suggesting that
most of the nitrogen used for synthesis of compo-
nents of the photosynthetic apparatus. In particu-
lar, Rubisco, the leaf protein playing the major
role in carbon assimilation, was strongly affected
by nitrogen deficiency (Seemann et al., 1987).
Although CO2 and NO− 3 assimilation are linked,
it is not completely clear to what extent they are
coupled (Lawlor, 1995). Therefore, further eluci-
dation of the relation of leaf nitrogen content to
the gas exchange and water use is needed.
Water use efficiency indicates the performance
of a crop growing under any environmental con-
straint. At the leaf level, intrinsic water use effi-
ciency (WUEi) is defined as the ratio of
photosynthetic rate (Pn) to transpiration rate. To
achieve the same Pn at a lower gs, a higher
Rubisco activity and capacity for electron trans-
port is required and thus a higher concentration
of nitrogen in the leaf does not necessarily mean a
proportional increase in the rate of photosynthesis
(Shangguan et al., 1999). In crop plants with C3
photosynthetic pathway, carbon isotope discrimi-
nation (D) has been used to provide time-inte-
grated estimates of plant intrinsic water use
efficiency (Farquhar and Richards, 1984; Far-
quhar et al., 1989). Foliar D values of C3 plants
have also been used as an integrated measure of
the response of photosynthetic gas exchange to
environmental variables such as humidity (Winter
et al., 1982), salinity (Guy et al., 1986), light
intensity (Zimmerman and Ehleringer, 1990), soil
water availability (Meinzer et al., 1992), and ele-
vated CO2 (Picon et al., 1996). A high but nega-
tive correlation was found between carbon isotope
discrimination (D) and WUEt (Ehdaie and Hall,
1991; Ismail and Hall, 1992) and D with WUEi
(Wright et al., 1988, 1994). Meinzer et al. (1990)
reported simultaneous reductions in stomatal con-
ductance and D with increased WUE for water
stressed cowpea. Rao and Wright (1994) showed
significant effects of location and water regime on
D for cowpea. Ehleringer et al. (1991) reported a
high correlation between D and Ci/Ca. To our
knowledge, there is little information on the ef-
fects of the two major environmental constraints
on photosynthetic gas exchange and D in winter
wheat.
Since different stress histories could signifi-
cantly have effects on a number of physiological
mechanisms in wheat (Morgan, 1984), the present
study was designed to eliminate the uncertain
effect of nitrogen nutrition stress with growing
plants in solution culture and imposing water
stress with polyethylene glycol (PEG 6000). In this
study, it was hypothesized that: (1) there would be
interaction between drought and nitrogen nutri-
tion on photosynthetic gas exchange and water
use efficiency, and the effects of nitrogen nutrition
on plants depend on solution water status; (2)
WUEt and WUEi would be increased by the
nitrogen nutrition supply. For verifying the hy-
potheses winter wheat was grown under different
combinations of nitrogen nutrition and water sup-
ply levels and the study was focused on the effects
of nitrogen deficiency and water stress on leaf
water status, photosynthesis, nitrogen content,
and water use.
2. Materials and methods
2.1. Plant material and growth conditions
Seeds of winter wheat (Triticum aesti6um L. cv
 Z.P. Shangguan et al. / En6ironmental and Experimental Botany 44 (2000) 141–149
Xiaoyuan 6) were initially germinated in dark-
ness at a constant temperature of 25°C on moist
filter paper in dishes for 5 days. Upon emer-
gence, the uniform seedlings were transferred to
plastic pots, 20 cm in height and 10 cm in di-
ameter, containing a mixture of vermiculite and
perlite (1:1, v/v) (four seedlings per pot). All
plants were irrigated weekly with a nutrient so-
lution and placed in a growth chamber. The nu-
trient solutions were modified half-strength
Hoagland solutions containing either 15 mM
NO− 3 (high-N) or 1.5 mM NO− 3 (low-N). All
other ions in the solutions were constant, except
for SO24 − and Cl−, which were used in equal
portions to maintain charge balance. Between
nutrient additions, deionized water was applied
as needed. Pots were covered with plastic beads
to reduce evaporation from the soil surface. The
conditions in the growth chamber were: a photo-
synthetic photon flux density of 700 mmol m − 2
s − 1 over plants; day/night temperature: 25/189
2°C; midday relative humidity: 60 95%; and a
12-h light period.
The first measurement was performed 45 days
after planting. In the following measurements,
PEG (6000) was added to the solutions in both
nitrogen treatments and the seedlings had been
kept in the solutions for 9 days. Drought stress
was achieved by adding different amount of
PEG, the osmotic potential of the solutions were
−0.24 MPa (well watered) and − 1.25 MPa
(droughted), respectively.
2.2. Leaf water potential and gas exchange mea-
surements
Leaf water potential (8w) was measured with a
pressure chamber (LI− COR 3005, Inc., Lincoln,
NE) on the first fully expanded leaves. A port-
able infrared CO2 analyzer (LCA− 3, ADC,
Hoddesdon, UK) was used to measure Pn, gs, E
and intercellular CO2 concentration (Ci) during
the 9 days. The leaf was then removed for carbon
isotope analysis. The youngest fully expanded leaf
of three plants of each treatment were enclosed
into the gas exchange chamber between 10:00 and
11:30 h for gas exchange measurements. Before
143
the gas exchange measurements, the print of the
leaf was taken for leaf area measurement by an
area meter. Plant WUEi was determined as the
ratio of Pn to E.
2.3. Carbon isotope composition analysis
In order to calculate WUEt, all leaves of the
plants were used for d 13C and nitrogen determi-
nations within each water and nitrogen treatment.
The leaves were oven-dried at 70°C for at least 48
h and then finely ground. Relative abundancies of
13
C and 12C were analyzed by mass spectrometry
(Delta S, Finnigan Mat). Carbon isotope compo-
sition (d) was expressed as the 13C/12C ratio rela-
tive to that of the Pee Dee Belemnite standard
(PDB). The resulting d 13C values were used to
calculate D (Farquhar and Richards, 1984):
da − dp
D (‰) = × 1000 (1)
1000+ dp
where da and dp refer to the isotopic compositions
of air (−8.0‰) and the plant material,
respectively.
D is related to the time-integrated value of the
ratio of the Ci to ambient CO2 concentration (Ca)
and thus to plant WUEi (Farquhar et al., 1989):
Ci 1.6× 10 − 36 Pn
D= a+ (b− a) = 1− (2)
Ca Ca E
where a and b are the discrimination coefficients
against 13CO2 during diffusion into the leaf
and carboxylation. The values of a and b are
estimated to be 4.4 and 27.0‰, respectively
(Farquhar et al., 1989). 6 (mmol mol − 1) is the
mean value of leaf-to-air water vapor pressure
difference during the growing period.
Plant transpirational water use efficiency
(WUEt) is related to D by (Farquhar and
Richards, 1984):
Ca b− D 2
WUEt = (1− F) (3)
1.6×10 6 b− a 3k
−3
where Ca (mmol mol − 1) is the mean ambient CO2
concentration, k is the ratio of plant carbon mass
144 Z.P. Shangguan et al. / En6ironmental and Experimental Botany 44 (2000) 141–149

to plant mass, and F is the proportion of net
daytime fixation of carbon lost by mitochondrial
respiration, fine root mortality or exudates by the
whole plant.
2.4. Nitrogen measurements
For determining leaf nitrogen concentration,
200 mg of powdered material was analyzed with a
modified Kjeldahl analysis using concentrated sul-
phuric and salicylic acid and Na2SO4, K2SO4 and
Se as a catalyst in a ratio of 62:1:1 (w/w). The
N-concentration of the digests was determined on
a continuous flow analyzer (Skalar, Breda,
Netherlands).
2.5. Statistical analysis
S.E., variance, regression and correlation coeffi-
cients, and significant differences among regres-
sion coefficients were calculated by standard
methods with the DAPS statistical package (Feng
and Tang, 1997).
3. Results and discussion
3.1. Water relations and nitrogen concentration of
lea6es
Leaf water potential decreased significantly
with water stress development in all treatments.
On the 9th day of drought stress, 8w was 0.25
MPa lower in the drought and high-N supply
Table 1
Water potential (8w) and nitrogen concentration of winter wheat leavesa
than that in the drought and low-N supply treat-
ment (Table 1). At the same soil water conditions,
drought that plants experienced was more severe
under high-N supply than under low-N supply.
These results are in agreement with Morgan
(1984, 1986) who reported that 8w of N-deficient
leaves were less affected by decrease in leaf water
content compared to leaves grown under a high
nitrogen regime. Some of the xeromorphic charac-
teristics are modified, such as cell wall thickness,
volumetric modulus of elasticity and cell volume,
which have been changed by plants growing in
nitrogen deficiency media. These structural
changes of the plant tissue could have profound
effects on plant internal water relations. There-
fore, the different 8w values for each treatment
support the hypothesis that the effect of water
stress is related to the level of nitrogen supply.
Under well-watered conditions, leaf nitrogen con-
tent at high-N supply was significantly (PB 0.01)
higher than that at low-N supply while the de-
creased water availability led to a lower leaf nitro-
gen content (Table 1). Leaf nitrogen
concentration on the 9th day of the drought pe-
riod was reduced by 40.3% for the low-N supply
and by 25.4% for the high-N supply treatments,
respectively. These results are in agreement with
Morgan (1984) who reported that drought in
spring wheat reduced leaf nitrogen content. How-
ever, opposite result have been reported in two
wheat cultivars (Van den Boogaard et al., 1995),
i.e. that drought stress led to higher leaf nitrogen
content. The mechanism that leads to these con-
tradictory results are not clear and further study is
needed.

Treatment Well-watered Droughted ANOVA

Low-N High-N Low-N High-N N Drought Interaction

8w (MPa) −0.27c −0.32c −1.42b −1.67a * ** *

Leaf nitrogen concentration (10−2 g g−1 dry 4.74b 6.25a 2.83c 4.66b ** ** *
wt)

a
Each value is the mean of three measurements taken on the 9th day after imposition of drought stress. Results of Fisher’s test
are shown where means followed by the same letter are not significantly different at the 5% level.
* PB0.05;
** PB0.01; two-way ANOVA where the effects of nitrogen, water and nitrogen/water interaction are significantly different.
 Z.P. Shangguan et al. / En6ironmental and Experimental Botany 44 (2000) 141–149 145

Table 2
Rate of photosynthesis (Pn), leaf conductance for water vapour (gs ), transpiration rate (E) and intrinsic water-use efficiency (WUEi)
of winter wheat leavesa

Treatment Well-watered Droughted ANOVA

Low-N High-N Low-N High-N N Drought Interaction

Pn (mmol m−2 s−1), 12.46b 15.24a 6.35c 5.63c * * **

gs (mmol m−2 s−1) 156.5b 180.2a 82.0c 85.7c ** * **
E (mmol m−2 s−1) 2.93a 3.01a 2.53b 2.41b ns ** ns
Ci (mmol mol−1) 217.6b 209.7c 221.1b 239.8a ** * **
WUEI (mmol mol−1) 4.25b 5.06a 2.51c 2.34c * * **

a
Measurements were carried out at ambient CO2 and O2 concentrations (345 mmol mol−1 and 21%, respectively) and at 700 mmol
−2
m s−1 photosynthetic photon flux density on the 9th day after imposition of drought stress. Each value is the mean of three
replications. Results of Fisher’s test are shown where means followed by the same letter are not significantly different at the 5% level.
* P B 0.01;
** P B0.05; two-way ANOVA where the effects of nitrogen, water and nitrogen/water interaction are significantly different.

3.2. Leaf photosynthetic gas exchange
Under well-watered conditions, Pn and gs were
increased in the high-N treatment as compared to
the low-N treatment. WUEi of the plants was
significantly higher in the high-N treatment than
that in the low-N treatment (Table 2). Under
drought conditions, Pn of the plants in the high-N
treatment was significantly inhibited while no sig-
nificant nitrogen effect was noticed for gs. At low
N supply WUEi was higher than that at high N
supply. The observation that gs of plants at high-
N supply appeared to be greater than that of
plants at low N supply under well-watered condi-
tions is in agreement with previous studies (Wong
et al., 1979; Bolton and Brown, 1980). Wong et al.
(1979) attributed this relation to the determina-
tion of stomatal aperture by the mesophyll capac-
ity to fix CO2 because high plant nitrogen content
results in great photosynthetic capacity and fairly
stable Ci between nitrogen treatments. Lower Ci
of wheat plants in high-N solution indicated
greater photosynthetic capacity of the mesophyll.
However, in this study, stomatal control was not
sufficient to maintain Ci constant between nitro-
gen treatments. Treatment differences in non-
stomatal sensitivity to reduction in 8w caused
WUEi to either increase or decrease from 8w
induced changes in gs in the high-N and low-N
treatment, respectively. While Pn and WUEi were
initially greater for the high-N treatment because
at the start of the stress cycle the apparent greater
sensitivity of mesophyll photosynthetic capacity
made decrease in 8w. Then gs resulted in similar
values of Pn and WUEi at low gs. For the drought
stress treatments gs and Pn both decreased. The
reaction to drought is different between high- and
low-N treatments. WUEi of the plants at high-N
solution was decreased more than that of the
plants in the low-N treatment due to stronger
decrease in Pn than in E.
3.3. dp and the gas exchange-deri6ed 6alues of
Ci /Ca
Because of the lag in time-integration scales
between the A/E and isotope data, no quantita-
tive interpretation can be derived from the dP
versus Ci/Ca relationships obtained in the present
study, as can be done when isotope discrimination
is assessed simultaneously with gas exchange
(Lloyd et al., 1992). Nitrogen deficiency strongly
increased dp at a given Ci/Ca derived from gas
exchange measurements. Averagely speaking, dp
was difference between the high-N treatment and
the low-N treatment was 6.9‰. dp were weakly
negatively correlated with the Ci/Ca values derived
from leaf photosynthetic gas exchange in both
nitrogen supply treatment (Fig. 1). This is consis-
tent with the two-step carbon isotope discrimina-
tion theory (stomatal diffusion followed by
carboxylation) (Farquhar et al., 1989). The phe-
146 Z.P. Shangguan et al. / En6ironmental and Experimental Botany 44 (2000) 141–149

patchy leaf function and to gm, conductance for

CO2 diffusion in liquid phase from the intercellu-
lar air spaces to the sites of carboxylation in the
chloroplasts was negligible in this case. gm was
decreased during drought (Shangguan et al.,
1999). Such an effect would also tend to decrease
leaf carbon isotope discrimination — and thus to
increase dP — while decreasing the A/E ratio (or
increasing the gas exchange-derived Ci/Ca) (Lloyd
et al., 1992; Parkhurst, 1994).

3.4. Transpirational water use efficiency and

carbon isotope discrimination

D increased with nitrogen supply. Nine days

Fig. 1. Relationship between leaf carbon isotope composition
and gas exchange-derived values of Ci/Ca of winter wheat.
Points are the mean of three measurements taken daily after
imposition of water stress. Symbols: ( ) high-N and well-wa-
tered; (
) high-N and droughted; () low-N and well-wa- respectively. In the drought treatments, D was
tered; (
) low-N and droughted. 3.96 and 3.29‰ lower as compared to the well-
nomenon was also observed in Pinus pinaster
subject to drought and elevated CO2 (Picon et al.,
1996). In contrast, the results of Meinzer et al.
(1992), Picon et al. (1996) on Coffea arabica and
Quercus petraea, respectively, are not in accor-
dance with the simple two-step discrimination
model.
However, Vivin et al. (1995) using Q. robur, a
species with a leaf structure identical to that of
Q. petraea, they found a good agreement between
season-long WUE determined gravimetrically and
dP, using the simple two-step discrimination
model. This result suggests that the effects due to
after imposition of drought, D differences between
the high-N and the low-N treatment were 1.5 and
2.3‰ in well-watered and drought conditions,
watered treatments for low-N and high-N supply,
respectively (Table 3). WUEt of the plants in the
high-N treatment was increased by 62.2% in the
case of the well-watered plants but only by 42.0%
in the drought treatment. Drought increased
WUEt by 67.7% for low-N supply and by 46.8%
for high-N supply. WUEt was increased by N
supply both for the leaf gas exchange and at the
whole plant and time integrated level. dp was
increased with the increase of drought intensity
(Fig. 2). This reflects the effect of drought on
isotope discrimination of the carbon photoassimi-
lated during the drying cycle. This interpretation
is also supported by a public garden study in
which water-stressed western larch seedlings

Table 3
Transpiration efficiency (WUEt) and isotope discrimination (D) of winter wheat plantsa

Treatment Well-watered Droughted ANOVA

Low-N High-N Low-N High-N N Drought Interaction

D (‰) 16.53ab 18.20a 12.57c 14.91b * * *

WUEt (mg g−1) 5.61c 9.10b 9.41b 13.36a ** ** **

a
Each value is the mean of three measurements taken on the 9th day after imposition of drought stress. Results of Fisher’s test
are shown where means followed by the same lower case letter are not significantly different at the 5% level.
* PB0.05;
** PB0.01; two-way ANOVA where the effects of nitrogen, water and nitrogen/water interaction are significantly different.
 Z.P. Shangguan et al. / En6ironmental and Experimental Botany 44 (2000) 141–149
Fig. 2. Relationship between leaf carbon isotope composition
and leaf water potential of winter wheat. Points are the mean
of three measurements taken daily after imposition of water
stress. Symbols: ( ) high-N and well-watered; (
) high-N
and droughted; () low-N and well-watered; (
) low-N and
droughted.
Fig. 3. Relationship between transpiration efficiency (WUEt)
and leaf carbon isotope discrimination () of winter wheat.
Points are the mean of three measurements taken daily after
imposition of water stress. Symbols: ( ) high-N and well-wa-
tered; (
) high-N and droughted; () low-N and well-wa-
tered; (
) low-N and droughted. This research was funded by the major state
showed 2‰ less discrimination of 13C than that of
well-watered seedlings (Zhang et al., 1994). WUEt
was negatively correlated with D in both nitrogen
supply treatments, but there was a noticeable
147
difference in the values of WUEt between the two
nitrogen treatments (Fig. 3). In the high-N treat-
ment, WUEt was not increased by drought despite
of the decreased D values. According to Farquhar
et al. (1989), D is mainly caused in C3 species by
(i) fractionation due to CO2 diffusion; (ii) changes
in stomatal resistance or assimilation rate, which
affect the ratio of internal to ambient concentra-
tion of CO2 (Ci/Ca); and (iii) fractionation by the
enzyme ribulose-1,5-bisphosphate carboxylase-
oxygenase (Rubisco).
This discrepancy between WUEt and D may be
explained based on Eq. (3). Ca and 6 were the
same in all treatments and for a given species, k is
contant among the treatments. Because it is also
likely that the term (1−F) was increased by
nitrogen supply and the parameter F was de-
creased by drought (Bowman et al., 1989;
Von Caemmerer et al., 1997), it can be con-
culuded that (b− D) is decreased and that D,
which is also positively linear with Ci/Ca, is in-
creased by nitrogen supply, no matter of what
water availability. The linear relationship between
D and WUE was in agreement with the theory of
the isotope effect (Farquhar and Richards, 1984)
and other experiments (Wright et al., 1988, 1994).
The relationship between D and nitrogen was not
significant although as reported earlier by Eh-
leringer (1990). D values in plant tissues (leaves,
stubble and roots) were higher (less negative) in
NH+ 4 -grown plants of T. aesti6um and Zea mays
than those in those supplied with NH4NO3 or
NO− 3 (Yin and Raven, 1998). It can be concluded
that nitrogen form and water conditions can in-
teract to influence WUEt, but the underlying
physiological mechanisms need further elucida-
tion.
Acknowledgements
basic research development projects of China
(Project No: G19 990 117) and the key project of
resources, ecological and environmental research
of the Chinese Academy of Sciences (Project No:
KZ 951-B1-211).
148 Z.P. Shangguan et al. / En6ironmental and Experimental Botany 44 (2000) 141–149
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