118
DOI: 10.1002/jpln.200420418
J. Plant Nutr. Soil Sci. 2006, 169, 118–126
Nitrogen metabolism in flag leaf and grain of wheat in response to
irrigation regimes
Zhen-Zhu Xu1, 2 and Zhen-Wen Yu1*
1
Key Laboratory of Wheat Physiology and Genetic Improvement, Ministry of Agriculture, Shandong Agricultural University,
Taian, Shandong 271018, P. R. China
2 Laboratory of Quantitative Vegetation Ecology, Institute of Botany, CAS, 20 Nanxincun, Xiangshan, Beijing 100093,
P. R. China
Accepted November 5, 2005
PNSS P04/18P
Summary
Although scarcity of irrigation water is one of the key limiting
factors for wheat production in many regions of the world, by
using partial irrigation at strategic times during the growing
season, it might be possible to enhance productivity. We
measured the changes in various parameters related to nitrogen (N) metabolism in flag leaf and grain of wheat (Triticum
aestivum L.) plants (cv. Jinan 17 and Lumai 21), which were
subjected to five irrigation regimes until physiological maturity. Severely deficient or excessive irrigation during grain
filling decreased the photosynthetic rate (A), the concentrations of N, free amino acid, and soluble protein, as well as the
activities of nitrate reductase (NR) and glutamine synthetase
(GS) and increased malondialdehyde (MDA) accumulation
1 Introduction
Environmental constraints are considerable in crop production in many regions of the world (Boyer, 1982; Ghahraman
and Sepaskhah, 1997). Although water deficit significantly
reduces the final grain weight per ear and yield (Boyer, 1982;
Ewert et al., 2002), water stress imposed during the grain
filling of wheat can enhance remobilization of prestored carbon and N reserves to grains and grain-filling rate (Bidinger
et al., 1977; Yang et al., 2001). In addition, transfer of N from
leaves to the grain can be accelerated by limited N uptake
from dry soil (Sinclair et al., 2000), suggesting that mild water
stress might increase grain yield and improve quality. Thus,
knowledge of current plant N and water status is needed to
optimize cropping practices in the field, such as irrigation and
nitrogen application (Pande and Becker, 2003; Behrens et
al., 2004).
Nitrogen concentration of plants varies with soil N status
(Blankenau et al., 2000) and soil moisture (Pande and
Becker, 2003) in which drought results in a decrease in leaf N
concentration (Sinclair et al., 2000; Xu and Zhou, 2005), and
drought stress and N limitation significantly reduce net photosynthetic rate and Rubisco activity, although drought alone
does not affect Rubisco activity (Heitholt et al., 1991). However, the decreases in Rubisco activity and A of plants as a
response to drought are in agreement with the lower foliar N
concentrations (Llorens et al., 2003). Nitrate reductase (NR)
and glutamine synthetase (GS) are the key enzymes of N
metabolism and are also involved in carbohydrate metabolism (Solomonson and Barber, 1990; Lam et al. 1996; Hirel
et al., 2005). Endopeptidase (EP) is another key enzyme
* Correspondence: Prof. Z.-W. Yu; e-mail: [email protected]
and endopeptidase (EP) activity, though grain protein concentration might mainly depend on genotype. The activities of
NR and GS were significantly positively correlated with A, but
those of EP were significantly negatively correlated with A.
The results indicate that while severe water stress aggravates the adverse effect on nitrogen metabolism, excessive
soil moisture is also not useful during the grain-filling stage,
resulting in lower grain yield and quality. Our results suggest
that applying an optimal irrigation regime in wheat fields still
plays an important role in the improvement of grain yield and
quality.
Key words: irrigation / water stress / nitrogen / key enzymes / grain
filling / wheat
related to N catabolism (Maki and Morohashi, 2002) and
might also play an important role in plant senescence caused
by drought. Nitrate reductase and GS activities rapidly
decrease under water stress (Somers et al., 1983; Sibout
and Guerrier, 1998). An increase in the content of molecules,
such as proline (Pro), which act as osmotic regulators, is a
major cause responsible for the typical changes undergone
by these key enzymes in the assimilatory ammonia pathway
when plants are water-stressed (Bourgeais-Chaillou et al.,
1992; Sibout and Guerrier, 1998). Unfortunately, the effects
of soil drought on the activities of the key enzymes of nitrogen
metabolism are not well understood.
Lipid peroxidation is often used as a marker of oxidative
damage under environmental stress (Foyer and Noctor,
2002; Hernández and Almansa, 2002). Environmental stresses lead to cellular damage through the direct action of reactive oxygen species and indirect action of lipid peroxidation
products (Foyer and Noctor, 2002; Taylor et al., 2004). As
malondialdehyde (MDA) is a marker for lipid peroxidation and
shows greater accumulation under environmental stresses
(Cakmak and Horst, 1991; Benhassaine-Kesri et al., 2002;
Hernández and Almansa, 2002), in the present study, MDA
concentration was used as an indicator of cellular-membrane
damage to assess the effect of water stress.
Therefore, to help fill the gap that still exists between physiological and agronomic studies, we determined N and MDA
concentration, the activities of NR, GS, and EP, as well as A,
in flag leaves of wheat. The main aims were to study (1) how
the parameters in relation to nitrogen metabolism change
with grain filling under different irrigation regimes, (2) the relationships between nitrogen parameters and photosynthesis,
and (3) how irrigation regimes affect plant and grain growth.
 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1436-8730/06/0102-118
J. Plant Nutr. Soil Sci. 2006, 169, 118–126
2 Materials and methods
N metabolism in wheat in response to irrigation regimes
119
face to a depth of 1.40 m. The average soil moisture content
(0–1.40 m depth) was expressed as a volume percentage.
2.1 Plant material and growing conditions
The experiment was conducted from fall of 1999 to summer
of 2000 at the agricultural experimental station of Shandong
Agricultural University, Shandong, China, which is located in
the central zone in the Huang (Yellow River)-Huai (Huai
River)-Hai (Hai River) plain of China, one of China’s most
productive winter wheat–growing areas. In the area, winter
wheat is commonly sown in October, and harvested in early
June of the following year. The winter wheat crops were cultivated in rain-sheltered troughs (2.5 m length, 2.5 m width,
and 1.6 m depth), constructed of bricked wall mortared by
concrete, but with no wall mortar on the bottom. The water
table was below 13 m, and the mean rainfall during the growth
period of the wheat was 195 mm over the past 50 years.
The soil was a fine loam. From the soil surface to a depth
0.60 m, bulk density, available water, and wilting moisture
were 1.50 g cm–3, 108 mm, and 7.7% (v/v), respectively. In
the top 0.25 m layer, the organic-carbon and total nitrogen
concentrations were 13.1 (s.e. 1.47) g kg–1 and 0.80 (s.e.
0.05) g kg–1, respectively.
Two winter wheat varieties (Jinan 17 and Lumai 21) were
chosen with two apparently contrasting characteristics, based
on experience over several years: cultivar Jinan 17 has a
higher grain protein concentration, earlier maturity, and is
less drought-resistant than Lumai 21 (Fang and Liu, 1996;
Xu et al., 2003). Seeds were sown in twelve rows spaced
0.31 m apart on Oct . 1, 1999, giving a density of 135 plants
m–2. Fertilizers were applied to each trough at 3 d prior to
sowing (105 kg N ha–1, 173 kg P2O5 ha–1, and 108 kg
K2O ha–1) and at the jointing stage (105 kg N ha–1). The crops
were irrigated using drip tubes positioned at a depth of 0.20 m
in the soil and between alternate crop rows.
The experiment employed a 5 × 2 (five irrigation regimes and
two varieties) factorial design with ten treatment combinations
and three replicates, in a completely randomized block design.
A rain shelter consisting of a double-rail steel frame covered
with a plastic sheet was used to protect the plots when it rained.
The irrigation treatments were applied at the appropriate stages:
treatment A represented irrigation at sowing; B at sowing, jointing, and booting; C at sowing, winter stage, jointing, and booting;
D at sowing, winter stage, jointing, booting, and flowering; and E
at sowing, winter stage, jointing, booting, flowering, filling, and
yellowing. The irrigation amount each time was 60 mm, which
resulted in irrigation amounts of 60, 180, 240, 300, and 420 mm
for treatments A, B, C, D, and E, respectively. Each drip tube
was installed with a water meter in order to control the amount of
irrigation to be applied.
2.2 Measurements and analyses
Soil moisture was measured using a neutron probe (Troxler
Electronics Laboratory, NC, USA) at the key growth stages,
which had been calibrated using soil from the experimental site.
Measurements were made at 0.20 m intervals from the soil sur 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Dates of anthesis and maturity were noted. Anthesis was
recorded when anthers in the central spikelets of 50% of ears
in a plot had extruded, and maturity when most of the ears in
a plot were no longer green.
The net photosynthetic rate per unit leaf area was measured
on attached flag leaves using a 0.25 L chamber connected to
a portable photosynthesis system (LI-6200, Li-Cor, Inc.,
Lincoln, Nebraska, USA) at 9:00–11:00 a.m. at 7 d intervals.
Readings were terminated after 30 s.
Thirty plants were cut at ground level and separated into flag
leaves and grains. For N determinations, all plant samples were
oven-dried at 80°C, weighed, and ground in a Wiley Mill through
a 1 mm opening screen. Nitrogen concentrations in the plant tissue and grain were determined by the standard macro-Kjeldahl
procedure (Nitrogen analysis system, Büchi, Switzerland) and
expressed as a percentage of the dry weight. Nitrogen-translocation efficiencies (NTE) were calculated according to a
previous formula (Papakosta and Gagianas, 1991):
N-translocation amount of a vegetative organ = N amount of
a vegetative organ at anthesis – N amount of the organ at
maturity.
N-translocation efficiency = (N-translocation amount of a
vegetative organ / N amount of the organ at anthesis) × 100.
The grain protein concentration was calculated by multiplying
values of the grain N concentration by 5.7. Detached flag
leaves (about 0.5 g fresh weight) were cut and weighed
immediately to obtain fresh weight (FW). They were then
placed in a beaker (25 mL) filled with water overnight in the
dark and reweighed to obtain turgid fresh weight (TW) the
next morning and dry weight (DW) after drying at 80°C for
24 h in a drying oven. The relative water content (RWC) of
the leaves was calculated thus:
RWC = [(FW – DW) / (TW – DW)] × 100.
Fresh leaf samples destined for measurement of soluble-protein, free–amino acid concentrations, and activities of key
enzymes were frozen in liquid N2 for 1 min and then stored at
–80°C. Subsequently, about 1 g of leaves was homogenized
with 10 mL of 50 mM sodium phosphate, pH 7.8, containing
2 mM EDTA and 80 mM L-ascorbic acid. After centrifugation
at 15,000 g for 20 min, the supernatants obtained were used
for determination of soluble-protein, free–amino acid concentrations, and the activities of the key enzymes (Cruz et al.,
1970; Alvim et al., 2001).
Soluble-protein and free–amino acid (FAA) concentrations in
the leaves were determined according to Bradford (1976)
and Moore (1968), respectively. Nitrate reductase (EC
1.6.6.1), glutamine synthetase (EC 6.3.1.2), and endopeptidase (EC 3.4.24.11) activities were determined according to
the methods reported by Baki et al. (2000), Elliot (1953), and
Wittenbach (1979), respectively.
www.plant-soil.com
Xu, Yu
All statistical ANOVA analyses were performed using SPSS
10.0 (SPSS for Windows, Chicago, Illinois, USA). Data obtained from the measurements and biochemical analyses
were evaluated statistically at the factorial level by means of
variance analyses (ANOVA) and their significance levels
(p < 0.05 or p < 0.01) were determined. A correlation analysis was also conducted to examine the relationship between
the activities of key enzymes of N metabolism and net photosynthetic rates. Duncan’s multiple-range tests were used to
compare treatment means.
3 Results
3.1 Changes in soil moisture
Figure 1 illustrates the progression of soil-moisture changes
from booting to maturity. The soil moisture (0–140 cm soil
layer) resulting from irrigation only at sowing (treatment A)
decreased gradually during grain filling. Irrigation during the
winter season maintained the soil moisture at a higher level
until anthesis (treatments C, D, and E), while irrigation after
anthesis significantly increased soil moisture during grain filling (treatments D and E). The excessive-irrigation treatment
(E) caused the highest water concentrations (17.6%) at
maturity, while irrigation treatment (A) produced the lowest
level (8.7%). According to the two-way ANOVA, there were
significant effects of irrigation and time on soil moisture
(p < 0.01). Their interaction was significant (p < 0.01),
because the effect of irrigation on soil moisture also depends
on irrigation time.
3.2 Leaf relative water content
As shown in Tab. 1, the leaf relative water content (RWC)
ranged from 82.0% to 86.6% for Jinan 17 and from 82.2% to
91.7% for Lumai 21 at 14 d after anthesis (DAA) and from
50.0% to 64.7% for Jinan 17, and from 54.6% to 65.9% for
Lumai 21 at 21 DAA. Increasing the irrigation quantity
obviously increased leaf RWC in the two varieties at the two
grain-filling stages. Relative water content at the late stage
was lower than at the early stage for both varieties, but Lumai
21 had a higher RWC compared to Jinan 17.
 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
25
20
Soil moisture (V%)
For measurement of malondialdehyde (MDA), the flag leaves
were cut, and the material (300 mg) was homogenized in
2 mL 0.1% trichloroacetic acid (TCA) solution. The homogenate was centrifuged at 15,000 g for 10 min, and 0.5 mL of
the supernatant obtained was added to 1.5 mL thiobarbituric
acid (TBA) in 20% TCA. The mixture was incubated at 90°C
in a shaking water bath for 20 min, and the reaction stopped
by placing the reaction tubes in an ice-water bath. The
samples were then centrifuged at 10,000 g for 5 min and the
absorbance of the supernatant measured at 532 nm (Hernández and Almansa, 2002). The value for nonspecific absorption at 600 nm was subtracted. The amount of MDA present
was calculated according to the methods reported by
Cakmak and Horst (1991). Absorbance was determined
using a Beckman DU®-640 spectrophotometer (DU 640,
Beckman Instruments Inc., USA).
J. Plant Nutr. Soil Sci. 2006, 169, 118–126
15
10
I: p < 0.01
T: p < 0.01
I×T: p < 0.01
5
0
-7
0
7
14
21
28
35
Days after anthesis
A
B
C
D
E
Figure 1: Changes of soil moisture in the 0–140 cm soil layer under
different irrigation regimes. Treatment A represents the irrigation
treatment at sowing stage; B at sowing, jointing, and booting; C at
sowing, winter, jointing, and booting; D at sowing, winter, jointing,
booting, and flowering; and E at sowing, winter, jointing, booting,
flowering, filling, and yellowing, respectively. Each irrigation quantity
was 60 mm. I and T represent irrigation and time, respectively. Bars
are SEM, n = 3.
Table 1: Effects of irrigation regimes on the relative water content
(%) in flag leaves of wheat. Means within the same time with same
letters are not significantly different at the 0.05 level using Duncan’s
multiple-range test.
Varieties Days after
anthesis
A
B
Jinan 17
14
82.0b
84.2a
85.6a
86.1a
86.1a
21
50.0d
52.4d
56.9c
60.2b
64.7a
Lumai 21 14
82.2c
88.3b
90.6a
91.7a
91.7a
21
54.6d
56.3d
59.6c
63.4b
65.9a
Irrigation regimes
C
D
E
30
Jinan 17
Lumai 21
25
Leaf photosynthetic rate
(µmol CO2 m–2 s –1)
120
20
15
10
I: p < 0.01
V: p < 0.01
I×V: p < 0.01
5
0
-7
0
A
7
14 21
-7
Days after anthesis
B
C
0
D
7
14
21
E
Figure 2: Changes of net photosynthetic rate (A) of flag leaves of two
wheat varieties in response to irrigation regimes (for an explanation of
irrigation treatments see legend to Fig. 1). I and V represent irrigation
and variety, respectively, n = 6.
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J. Plant Nutr. Soil Sci. 2006, 169, 118–126
N metabolism in wheat in response to irrigation regimes
3.3 Leaf net photosynthetic rate
121
(mg (g DW)–1)
3
2
1
Jinan 17
Lumai 21
–1
(mg (g DW) )
0
45
40
35
30
25
20
15
10
5
0
Jinan 17
I: p < 0.01
V: p < 0.01
I×V: p < 0.05
7
(mg (g DW) )
The grain protein concentration ranged from 11.8% to 15.7%.
Until the end of grain filling, the grain protein concentration of
treatment A was lowest for Jinan 17, but highest for Lumai 21.
For Jinan 17, treatment E produced a lower protein concentration compared to treatment C, indicating that excessive irrigation might limit grain protein accumulation. Generally, Jinan 17
had a higher grain protein concentration than Lumai 21.
Total free amino acid content
As shown in Tab. 2, averaged over all treatments, the leaf N
concentration gradually decreased from 3.8% at anthesis to
0.9% at maturity for Jinan 17 and from 4.2% to 1.1% for
Lumai 21, respectively. Increasing the irrigation quantity
obviously increased leaf N in the two varieties at anthesis
and 21 DAA. Lumai 21 had a higher leaf N concentration than
Jinan 17, especially at the late stage, indicating that Lumai 21
retained more N in the leaves. Both the severe irrigation
shortage and excessive irrigation decreased the N-translocation efficiency (NTE), and Lumai 21 had a lower NTE, except
for irrigation treatment C.
I: p < 0.01
V: p < 0.01
I×V: p < 0.05
4
–1
3.3 Nitrogen, protein, and free–amino acid
concentrations
Soluble protein content
Averaged over all irrigation treatments, the net photosynthetic
rate (A) of flag leaves gradually decreased from 21.1 at
–7 DAA to 7.4 lmol CO2 m–2 s–1 at 21 DAA for Jinan 17 and
from 20.4 to 8.2 lmol CO2 m–2 s–1 for Lumai 21, respectively.
That of Jinan 17 decreased more sharply during the late grain
filling (Fig. 2). Soil-water deficits (irrigation treatments A
and B) significantly decreased, but irrigations applied after
anthesis remarkably increased A. There were significant
effects of irrigation and variety and their interaction on flag
leaf A, because of the different responses of the two varieties
to irrigation treatment (p < 0.01).
Total free amino acid content
5
14
180
160
140
120
100
80
60
40
20
0
21
28
7
14
Jinan 17
21
28
Lumai 21
I: p < 0.01
V: p < 0.01
I×V: p < 0.05
0
7
14
21 28
0
7
14
21
28
Days after anthesis
A
As shown in Fig. 3 (upper panel), in flag leaves, there was a
minor difference in the total free–amino acid (FAA) concentration between different irrigation regimes at 7 DAA, but the
FAA significantly decreased with intensifying water deficit at
14 DAA. For treatments A and B, the FAA was maintained at
a constant level between anthesis and 21 DAA, but markedly
Lumai 21
B
C
D
E
Figure 3: Changes of total free–amino acid (FAA) concentration of
flag leaves (upper panel) and grains (middle panel) and solubleprotein concentration in leaves (low panel) of two wheat varieties in
response to irrigation regimes (for an explanation of irrigation
treatments see legend to Fig. 1). I and V represent irrigation and
variety, respectively, n = 6.
Table 2: Effects of different soil-moisture treatments (A–E; explanation see legend to Fig. 1) on N concentrations and N-translocation efficiencies (NTE) in the flag leaves and grain protein concentrations of two wheat varieties (Jinan 17, Lumai 21) at maturity. Means within columns
with same letters are not significantly different at the 0.05 level using Duncan's multiple-range test.
0
A
B
C
D
E
3.47b
3.74ab
3.88a
3.88a
3.88a
Jinan 17
Leaf N (%)
Days after anthesis
21
31
1.86b
1.75b
2.01ab
2.37a
2.47a
0.93a
0.96a
0.88a
0.88a
0.98a
NTE (%)
73.2b
74.3b
77.3a
77.3a
74.7b
Grain
protein
concentration
(%)
0
13.9b
14.9ab
15.7a
15.0ab
14.1b
3.51b
3.81b
4.59a
4.59a
4.59a
Lumai 21
Leaf N (%)
Days after anthesis
21
31
1.95c
2.62b
2.98ab
3.10a
3.11a
1.09b
1.08b
0.91b
1.12b
1.33a
NTE (%)
Grain
protein
concentration
(%)
69.0c
13.9a
12.8b
12.4b
12.3b
11.8b
71.7c
80.2a
75.6b
71.0c
NTE denotes N-translocation efficiencies, which could be calculated as N-translocation efficiency = (N-translocation amount of a vegetative
organ / N amount of the organ at anthesis) × 100.
 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Xu, Yu
declined at 28 DAA. Irrigations at anthesis and filling stages
also caused an increase in FAA; grain FAA gradually
decreased with filling. The FAA for treatment A remained at
the lowest level during whole grain-filling stage, and irrigation
caused an increase in grain FAA (Fig. 3, middle panel). The
main effects and their interaction on FAA were significant (p <
0.05). The significant interaction between irrigation and variety might result from the different changes of FAA between
the two varieties when the plants were subjected to different
irrigation treatments.
J. Plant Nutr. Soil Sci. 2006, 169, 118–126
NR activity
(µg NO 2 (g DW) –1 min –1 )
122
14
I: p < 0.01
V: p < 0.01
I×V: p < 0.01
12
10
8
6
4
Jinan 17
0
-7 0
-
(∆ A 540 (g DW) min )
As shown in Fig. 4 (upper panel), averaged over all treatments, NR activity increased from 7.5 at –7 DAA to a peak
value of 9.5 at 7 DAA, followed by a decrease to 2.1 lg NO2
(g DW)–1 min–1 at the end of grain filling. Jinan 17 had a higher NR activity than Lumai 21 prior to 21 DAA, but the latter
maintained a higher level at 28 DAA. Treatment A stimulated
NR activity at –7 and 0 d after anthesis, but thereafter significantly decreased it. The effects of irrigation, variety, and their
interaction were significant (p < 0.01). The significant interaction could mainly result from the fact that Jinan 17 had higher
range of NR activity than Lumai 21. Nitrate reductase activities of flag leaves were positively and significantly correlated
with A (r = 0.634**, p < 0.01, n = 25 for Jinan 17; r = 0.671**,
p < 0.01, n = 25 for Lumai 21). In the grains, the NR activity
was very low, and the effect of irrigation regime was not significant (data not shown).
As shown in Fig. 4 (middle panel), the GS activities in flag
leaves peaked at anthesis and thereafter declined during
grain filling. The effects of irrigation and variety on GS activity
were significant (p < 0.01), and their interaction was also significant (p < 0.01), because the irrigation effects depended
on variety. Severe water deficit (treatment A) significantly
decreased the GS activity during grain filling for the two varieties, while moderate irrigation (treatment C and D) caused a
higher level of GS activity after anthesis until maturity. The
GS activity in the grains peaked for the two varieties at
14 DAA, except for treatment A at 7 DAA (Fig. 4, low panel).
The GS activities of the flag leaves were positively correlated
with A (r = 0.458*, p < 0.05, n = 15 for Jinan 17; r = 0.535*,
p < 0.05, n = 15 for Lumai 21). Treatment A produced the
lowest GS activity during grain filling, although the excessive 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
–1
GS activity
3.5 Nitrate reductase and glutamine synthetase
activities
-7 0
7 14 21 28
I: p < 0.01
V: p < 0.01
I×V: p < 0.01
35
30
25
20
15
10
5
0
Jinan 17
0
–1
Figure 3 (low panel) demonstrates that the soluble-protein
concentration of flag leaves gradually decreased during grain
filling, the decrease being particularly steep from 14 to
21 DAA. Treatment A (irrigation at sowing) produced a lower
soluble-protein concentration during grain filling for both
Jinan 17 and Lumai 21, whereas excessive irrigation (treatment E) produced a higher level for the two varieties until
21 DAA. The main effects and interaction were also significant (p < 0.05). The interaction was significant, because
Jinan 17 had a higher range of change for soluble-protein
concentration compared to Lumai 21.
7 14 21 28
40
GS activity
(∆ A 540 (g DW)–1 min–1)
3.4 Soluble-protein concentration
Lumai 21
2
9
8
7
6
5
4
3
2
1
0
7
Lumai 21
14 21 28
0
7
14 21 28
I: p < 0.01
V: p < 0.01
I×V: p < 0.01
Jinan
17
7
Lumai 21
14 21 28 35
7
14 21
28 35
Days after anthesis
A
B
C
D
E
Figure 4: Changes of nitrate reductase (NR) activity (upper panel)
and glutamine synthetase (GS) activity of flag leaves (middle panel)
and grains (low panel) of two wheat varieties in response to irrigation
regimes (for an explanation of irrigation treatments see legend to
Fig. 1). I and V represent irrigation and variety, respectively, n = 6.
irrigation regime (E) also led to a decrease compared to the
moderate irrigations. There were significant effects of irrigation and variety on GS activities in grains (p < 0.01), and their
interaction was significant (p < 0.01), because of the different
response of the two varieties to irrigation treatment.
3.6 Endopeptidase activity and malondialdehyde
concentrations
As shown in Fig. 5 (upper panel), averaged over all treatments, the EP activity in the flag leaves increased by 273%
and 289% at the end of grain filling for Jinan 17 and Lumai
21, respectively. Severe water deficit (treatments A and B)
markedly increased EP activity, but increasing the irrigation
amount significantly decreased it. Jinan 17 had a higher level
of EP activity than Lumai 21 after 7 DAA, suggesting that
Jinan 17 might be more sensitive to water stress. Thus, there
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was a different response to irrigation treatment for the two
varieties. The irrigation and variety effects and the interactions between irrigation and variety were significant for EP
activities in the flag leaves (p < 0.01). The EP activities of the
flag leaves were negatively and significantly correlated with A
(r = –0.882**, p < 0.01, n = 20 for Jinan 17; r = –0.890**, p <
0.01, n = 20 for Lumai 21). We also measured the activity of
EP in the grains: there was very low activity, and the effects
of irrigation regimes were not significant (data not shown).
We also examined the effect of different irrigation regimes on
the malondialdehyde (MDA) concentration of flag leaves
(Fig. 5, low panel). Averaged over all the irrigation treatments,
the MDA concentration increased by 281% and 270% from
anthesis to the end of grain filling for Jinan 17 and Lumai 21,
respectively. Severely deficient irrigation obviously increased
the MDA concentration, but moderate irrigation decreased it.
More increase with grain filling was observed for Jinan 17
compared to Lumai 21, particularly at late grain filling, indicating that the Lumai 21 plants might possess a higher drought
resistance. The effects of irrigation and variety and their interaction on leaf MDA concentration were all significant
(p < 0.01). The significant interaction was due to the different
response of the two varieties to irrigation treatment.
3.7 Plant growth and grain yield
Figure 6 (upper panel) demonstrates that the different irrigation regimes markedly affected plant growth during grain filling. Severe soil-water deficit significantly limited, but irrigation
( ∆A 280 (g DW) –1 h –1 )
Endopeptidase activity
8
7
I: p < 0.01
V: p < 0.01
I×V: p < 0.01
6
5
4
3
2
Jinan 17
1
0
Lumai 21
400
123
I: p < 0.05
V: p < 0.01
I×V: p < 0.05
350
300
250
200
150
100
Jinan 17
Lumai 21
50
160
140
120
100
80
60
40
20
0
I: p < 0.05
V: p < 0.01
I×V: p < 0.05
Jinan 17
0
7
14 21 28 35
A
Lumai 21
0
7 14 21 28 35
Days after anthesis
B
C
D
E
Figure 6: Changes of plant dry weight (upper panel) and grain yield
(low panel) of two wheat varieties in response to irrigation regimes
(for an explanation of irrigation treatments see legend to Fig. 1). I and
V represent irrigation and variety, respectively, n = 3.
at anthesis and during grain filling enhanced plant growth,
especially in the later grain-filling stage. The grain dry weight
increased with grain filling (Fig. 6, low panel). The severely
deficient irrigation led to a decline in grain yield after 28 DAA,
while moderate irrigation (treatment D) enhanced grain
growth at the late grain-filling stages. Excessive irrigation
enhanced the plant growth of Lumai 21 compared to Jinan
17, but moderate irrigation promoted the grain filling of Lumai
21 more highly compared to Jinan 17. For both the plant and
grain dry matter, there were significant effects of irrigation
(p < 0.05) and variety (p < 0.01). The interaction between irrigation and variety was significant (p < 0.05).
4 Discussion
140
120
MDA content
(µmol (g DW)–1)
Plant dry weight (g plant–1)
N metabolism in wheat in response to irrigation regimes
Grain dry weight (g plant–1 )
J. Plant Nutr. Soil Sci. 2006, 169, 118–126
I: p < 0.01
V: p < 0.01
I×V: p < 0.01
100
80
60
40
20
Jinan 17
Lumai 21
0
0
7
14
A
21 28
0
7 14
Days after anthesis
B
C
D
21
28
E
Figure 5: Changes of endopeptidase (EP) activity (upper panel) and
malondialdehyde (MDA) concentration (low panel) in flag leaves of
two wheat varieties in response to irrigation regimes (for an
explanation of irrigation treatments see legend to Fig. 1). I and V
represent irrigation and variety, respectively, n = 6.
 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Shortage of water is the most important factor limiting crop
production in many parts of the world, especially in arid and
semiarid regions (Boyer, 1982; Ewert et al. 2002; Shah and
Paulsen, 2003), yet more land can become productive by
using partial irrigation at strategic times during the growing
season (Ghahraman and Sepaskhah, 1997). In many experiments, it has been found that wheat-leaf N concentration
decreases with increasing soil drought, but modest soil
drought is considered likely to enhance N transfer from the
leaf to the grain (Sinclair et al., 2000). However, the total
N-translocation amount decreases under water stress,
because early leaf senescence enhanced by drought can
lead to a decrease in leaf N levels (Seligman and Sinclair,
1995). The present results demonstrate that severely deficient irrigation can decrease grain yield and leaf N concentration through adversely affecting N metabolism with early flagleaf senescence.
www.plant-soil.com
124
Xu, Yu
Leaf is an important N source for the grain after flowering
(Guitman et al., 1991; Barneix et al., 1992), and photosynthetic rate and Rubisco activity increase with increasing leaf
N concentration (Evans, 1983; Llorens et al., 2003; Xu and
Zhou, 2005). Metabolic imbalance caused by water stress
results in amino acid accumulation, decreased ATP and
RuBP synthesis, and altered protein synthesis (Lawlor and
Cornic, 2002). Drought can also have adverse effects on both
nitrate reductase (NR) expression and activity (Hsiao, 1973;
Huber and Kaiser, 1996; Baki et al., 2000; El-Komy et al.,
2003; Burman et al., 2004), which is involved in carbohydrate
metabolism (Solomonson and Barber, 1990). For example,
high carbohydrate assimilation can increase nitrate uptake
and nitrate reductase activity when the plant is growing on
nitrate (Matt et al. 2001). Nitrate assimilation requires eight
electrons to reduce NO3 to NH‡
4 , which is mainly catalyzed
by NR, and is thus the highest energy-consuming reaction of
metabolism after CO2 assimilation. Consequently, NR might
play an important role in maintaining the balance between C
and N (Champigny, 1995). A decrease in NR activity was
positively correlated with a decrease in soluble-protein concentration and photosynthetic capacity under water stress
(El-Komy et al., 2003; Burman et al., 2004). Nitrate reductase
also plays a key role in regulating the drought response of
plants (Singh and Usha, 2003). In addition, glutamate is the
main precursor of proline, which is considered to be involved
in osmoregulation of plants subjected to drought (Venekamp,
1989). Enhanced plant growth has been closely correlated
with higher nitrate levels and the higher activities of NR and
glutamine synthetase (GS) (Lam et al., 1996; Chen et al.,
2003). Moreover, in detached rice leaves, water stress
decreases chlorophyll and protein concentrations, and increases NH‡
4 concentration due to the decreased GS activity,
which in turn results in an enhancement of chlorophyll and
protein loss (Hsu et al., 2003). Glutamine synthetase activity
is closely associated with protein hydrolysis (Tsai et al., 2003)
and the ability of adapting to drought (Bianchi et al., 2002). In
the present study, severely deficient irrigation reduced the
activities of the two enzymes (NR and GS) involved in N
assimilation and increased the activity of endopeptidase
(EP). Furthermore, the activities of NR and GS were positively and significantly correlated with the net photosynthetic
rates (A), although that of EP was negatively and significantly
correlated with A. Therefore, it was suggested that the Nmetabolism regulation could play an important role in the
photosynthetic adaptation of wheat plants to water stress.
In wheat seedlings, moderate soil-water stress can enhance
antioxidant defense, but severe water stress or water logging
causes loss of catalase activity (an antioxidant enzyme for
scavenging toxic oxygen species; Keles and Öncel, 2002).
The present results demonstrate that severe water deficit and
over-irrigating provoke MDA accumulation in flag leaves. In
conclusion, the reduction in the activities of the two enzymes
(NR and GS) involved in N assimilation and enhancement in
the activities of endopeptidase are involved in the decline of
photosynthetic capacity, plant growth, and grain yield caused
by severe drought.
Nitrogen-concentration change in wheat is associated with
genotype and cultural practices (Papakosta and Gagianas,
 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Plant Nutr. Soil Sci. 2006, 169, 118–126
1991). In the present study, grain protein concentration ranged from 11.8% to 15.7%, and was higher for Jinan 17 than
Lumai 21 (Tab. 2), supporting that concept that grain protein
concentration mainly depends on genotype. However, changes in leaf N concentration and key enzyme activities for the
two varieties were complex. This could have resulted from
different responses of the different genotypes to drought.
Limited irrigation and a higher level of N can induce better utilization of soil moisture (Khan et al., 1990) and improve grain
quality (Barber and Jessop, 1987; Xu et al., 2003). The study
of Cheng et al. (1996) indicated that in the whole growth period of winter wheat, if total evapotranspiration amounts (total
consumption in soil can attain about 440 mm), water-use efficiency might be higher. In the study area, the average precipitation during the total wheat plant–growing period is 195 mm.
If moderate irrigation is applied, a higher grain yield could be
obtained, in view of saving fresh-water resources. However,
when we consider the grain nitrogen concentrations related
to quality, the effects of genotype are also important. The
experiment highlights that N-translocation efficiency will be
enhanced by a moderate irrigation quantity (treatment C and
D) and weakened by severely deficient irrigation (treatment
A) or excessive irrigation (E). Based on N-status changes in
flag leaves and grains during grain filling, the selection of an
optimum-irrigation regime will still be an important task in the
future.
Acknowledgments
We are grateful to the support of the National Natural Science
Foundation (Grant No. 30471026), Agricultural Foundation of
Ministry of Science-Technology (2002BA516A12), and the
National Key Basic Research Special Foundation Project
(2006CB400502) of China.
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