agricultural water management 95 (2008) 1261–1270

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Effects of N fertilization on root development and activity of

water-stressed cotton (Gossypium hirsutum L.) plants

Rui-Xian Liu a, Zhi-Guo Zhou a,b,*, Wen-Qi Guo a, Bing-Lin Chen a, Derrick M. Oosterhuis b
a
Key Laboratory of Crop Growth Regulation, Ministry of Agriculture, Nanjing Agricultural University,
Nanjing 210095, Jiangsu Province, PR China
b
Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, United States

article info abstract

Article history: The objective of this investigation was to study effects of nitrogen on drought resistance in
Received 15 January 2008 terms of changes in cotton (Gossypium hirsutum L.) root dry matter accumulation, N con-
Accepted 8 May 2008 centration, antioxidant enzyme activities and root vigor during short-duration water stress
Published on line 24 June 2008 (withholding water for 8 days and then permitting to 10 days recover by re-watering). Cotton
plants were grown in pots with three N levels (0, 240, and 480 kg N ha1). Soil-relative water
Keywords: content decreased with increasing N supply during the soil water stress period, while leaf
Nitrogen area, dry matter production and N accumulation were enhanced. The root/shoot ratio and
Water stress root-N/shoot-N ratio increased with water stress, and were smallest at 240 kg N ha1.
Cotton Application of N increased the activities of peroxidase (POD) and catalase (CAT) of cotton
Root development root, but decreased superoxide dismutase (SOD) activity during water stress as well as
Antioxidant enzyme activity during recovery. Malondialdehyde (MDA) content was significantly ( p < 0.05) increased, and
Root vigor was lowest in the 240 kg N ha1 N treatment during water stress. At the 10th day after soil
re-watering, MDA content of 240 kg N ha1 was similar to that of 480 kg N ha1, but less than
that of 0 kg N ha1. The root vigor, which was debased by water stress, was the highest at
240 kg N ha1. After soil re-watering, N application promoted root vigor. The trends of net
photosynthetic rate were the same as that of root vigor during water stress. These results
suggest that appropriate N supply (240 kg N ha1 in this investigation) may contribute to
drought resistance of cotton plants by adjusting the antioxidant enzyme activities of root,
debasing lipid peroxidation and boosting root vigor during short-duration water stress
(withholding water for 8 days in this investigation), however, excessive N supply
(480 kg N ha1) had a deleterious effect on plant drought resistance.
# 2008 Elsevier B.V. All rights reserved.

1. Introduction during this stage significantly affects various physiological

and biochemical characters in cotton plants, such as leaf
The flowering and boll development stage is the key yield expansion, photosynthesis, carbon and nitrogen metabolism,
determinant period of upland cotton (Gossypium hirsutum L.) and antioxidant metabolism (Ball et al., 1994; Potikha et al.,
(Han and Kang, 2001). Short-duration water stress occurring 1999; Pettigrew, 2001; Ennahli and Earl, 2005), which leads to a

* Corresponding author. Present address: Department of Agronomy, Nanjing Agricultural University, Weigang Road 1, Nanjing 210095,
Jiangsu Province, PR China. Tel.: +86 25 84396813; fax: +86 25 84396813.
E-mail addresses: giscott@njau.edu.cn, lrxjll@163.com (Z.-G. Zhou).
0378-3774/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.agwat.2008.05.002
1262 agricultural water management 95 (2008) 1261–1270
reduction in cotton development and final productivity.
Irrigation is a key agricultural solution to this problem, but
water supplies are limited due to human increased demands,
and high financial and environmental costs (Wu and Cosgrove,
2000). Hence, it is important to consider alternative strategies
for ameliorating the detrimental effects of water deficit, such
as by improving the drought resistance of crop plants (Apse
et al., 1999).
Nitrogen is an essential mineral nutrient required in large
amounts by plants (Feil et al., 2005). N application could
improve drought resistance in plants (Halvoson and Reule,
1994; Fife and Nambiar, 1997). Halvoson and Reule (1994)
found that wheat (Triticum aestivum L.), maize (Zea mays L.) and
barley (Horrdeum vulgare L.) yield in dry lands were increased
with increased N supply. For wheat, N fertilizer increased dry
matter, grain production and water use efficiency under
drought, and the effect was due to increased leaf area index
and the maintenance of leaf area duration (Latiri-Souki et al.,
1998). Saneoka et al. (2004) found that malondialdehyde
(MDA) content in creeping bentgrass (Agrostis palustris Huds.)
leaves was greater in water-stressed treatments than in the
control and decreased with increasing N supply. This showed
that higher levels of N contributed to drought resistance of
bentgrass by preventing cell membrane damage and enhan-
cing osmoregulation. However, in contrast, Stroup et al. (2003)
showed that switchgrass (Panicum virgatum L.) plants grown
with high N showed a greater reduction in photosynthesis
and leaf water potential under water stress. In winter wheat,
Shangguan et al. (2000) found that water use efficiency of the
plants in the high-N treatment 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 under drought conditions. Increasing soil N supply
decreased pearl millet (Pennisetum glaucum (L.) R.Br.) growth
under soil water deficit (Ashraf et al., 2001). In view of these
reports it is clear that N does not always play a positive role in
alleviating the adverse effects of dry soil on plant develop-
ment.
Root plays an important role in the plant’s ability to
explore the available soil volume for water and nutrients.
Several researchers (Sharp et al., 1988; Spollen and Sharp,
1991) have hypothesized that the ability of a plant to change
root physiological and biochemical processes is an impor-
tant mechanism for drought resistance. Benjamin and
Nielsen (2006) showed that chickpea (Cicer arietinum L.)
was able to change root distribution in the soil because of
soil water deficit stress and, therefore, extract water to
deeper depths. It is common for the root/shoot ratio of
plants to increase when water availability is limiting (Ball
et al., 1994; Noctor et al., 2002). Wu and Cosgrove (2000)
found maize seedlings adapted to low soil water potential by
increasing flexibility of cell walls in the apical part of root.
Compared with well-watered plants, drought-sensitive
spring wheat increased root respiration rate (used a
relatively higher amount of glucose) in order to absorb
water in a dry soil treatment, especially with severely dry
soils (Liu et al., 2004). However, few studies have focused on
the root response of plants to lowered water content (Adda
et al., 2005), because the basis for the relative drought
resistance of root growth, in comparison with shoot growth,
is complicated and probably involves both nutrition meta-
bolism and osmotic adjustment.
To our knowledge, there is little shown on the root
physiological and biochemical changes in N metabolism,
protein synthesis, and antioxidant metabolism under low soil
water availability. Previous studies have shown that N
significantly affects the responses of plants to soil water
stress (Halvoson and Reule, 1994; Fife and Nambiar, 1997;
Latiri-Souki et al., 1998; Shangguan et al., 2000; Stroup et al.,
2003). However, related research on root growth is scarce, and
the effects of N on root physiological and biochemical
characters needs to be examined. This investigation was
designed to study the physiological and biochemical mechan-
ism of cotton root in response to soil N status with short-
duration water shortage. The goal was to ascertain whether
high levels of N supply are beneficial or deleterious to drought
resistance of cotton plants.
2. Materials and methods
2.1. Plant materials and growth conditions
Pot experiments were conducted in the summer seasons of
2005 and 2006 in the field at the experimental station of the
Nanjing Agricultural University, located at Nanjing (328020 N
and 1188500 E), Jiangsu Province of China. Cotton seeds (cv.
NuCOTN 33B) were planted on 25 April 2005 and 23 April 2006,
respectively. When the seedlings had three true leaves,
individual healthy, uniform plants were transplanted into
plastic pots, 55 cm high and 60 cm wide, filled with 40 kg soil.
The yellow brown soil (Dystrudept) was collected from 0 to
30 cm topsoil layer from the experimental station. The
chemical properties of the soil are shown in Table 1. The
field water capacity (FWC) of the soil was 0.31 g water g1 dried
soil in 2005 and 0.30 g water g1 dried soil in 2006. The pots
were randomly rearranged fortnightly to minimize possible
positional effects.
2.2. Experimental designs and experimental treatments
2.2.1. Experimental designs
The experiment was arranged in a completely random design,
with two levels of soil water management and three levels of
applied N fertilizer. Each treatment had 15 replications where
one pot with single plant represented one replication.
2.2.2. Soil water stress treatments
Two soil water management treatments were maintained: (i)
the well-watered control with soil-relative water content of
70–80%, and (ii) the slowly applied soil water stress where
plants were watered to FWC, after which water was
withhold for 8 days. Plants were re-watered on the evening
of the 8th day, and watered daily for a ten days recovery
period. The drying soil treatments were begun at the
flowering stage (83 days after planting in 2005, 88 days after
planting in 2006).
Soil water content was monitored daily by weighing and
watering the pots. When soil water content of the well-
watered control was smaller than the lower limit (soil-relative
 agricultural water management 95 (2008) 1261–1270
Table 1 – Chemical properties of soil used for the
experiment in 2005 and 2006
2005 (mg kg1) 2006 (mg kg1)
Organic matter (g kg1) 15.3  0.1 17.8  0.1
Total N (g kg1) 0.80  0.0 0.92  0.1
Alkali hydrolysable N 63.5  1.1 74.6  1.7
Available P 40.0  0.7 37.1  0.5
Available K 238  3 214  4
The data given are mean  S.E. of three replicates.
water content of 70%), water was added to the upper limit (soil-
relative water content of 80%).
2.2.3. Nitrogen treatment
The three N levels were 0 kg N ha1 (N0), 240 kg N ha1 (N1),
480 kg N ha1 (N2), equivalent to 0, 4.5, 9.0 g N pot1, respec-
tively. N fertilization was split into two equal applications as
urea and applied before transplanting and at the early
flowering stage (15 days before the drying soil treatment) in
every treatment.
2.3. Soil-relative water content assays
Soil water content (SWC) was measured according the method
of Liang et al. (1997). Soil samples at 0–30 cm depth were
harvested daily with a punch from the three plants of every
treatment during water stress period. The fresh weight of soil
samples was determined then samples were oven-dried at
80 8C for 12 h. SWC was expressed as g water g1 dried soil.
The soil-relative water content (SRWC) = SWC/FWC.
2.4. Measurements of photosynthesis
At the end of water stress period and 10th day after soil re-
watering, net photosynthetic rate (Pn), stomatal conductance
(Gs) and internal CO2 concentration (Ci) of leaves were
measured with a photosynthesis system (Li-6400, Li-COR
Inc., NE, USA) under 1500 mmol m2 s2 light intensity at 9:30–
11:00 h. Photosynthesis measurements were taken on five
fully expanded main-stem leaves, fourth node from the
terminal, per treatment.
2.5. Determination of plant dry matter partition and N
accumulation
After measurement of photosynthesis, three plants from
every treatment were cut at the cotyledonary node. The root
was separated from the soil by washing. Samples of 0.5 g of
white young root were used to assay root activity immedi-
ately, and samples of 2.0 g of white young root were
immediately frozen in liquid N2 and stored at 70 8C for
enzyme activity analysis. Leaf area was measured with a
portable area meter (Li 3000-A, Li-COR Inc., NE, USA), then
root, leaves, stems, branches, flowers and bolls were dried in
an oven at 80 8C to a constant weight, and the dry matter was
determined.
N content was measured on samples of root, leaf, stem,
branch, flower and boll for each replicate using the Kjeldahl
method (Feil et al., 2005).
1263
2.6. Determination of antioxidant enzyme activity and
MDA content
Frozen root segments (0.5 g) were crushed into fine power in a
mortar in an ice-bath. 5.0 ml of 0.05 mol l1 phosphate buffer
(pH 7.8) with 1% polyvinylpyrrolidone (PVP) was used as an
extraction buffer. The homogenate was centrifuged at
15,000  g for 15 min at 4 8C, then the liquid were used to
measure antioxidant enzyme activities and malondialdehyde
(MDA) content.
Superoxide dismutase (SOD) activity was assayed accord-
ing the method of Li (2000). One unit of SOD activity was
defined as the amount of enzyme required to cause 50%
inhibition of nitro blue tetrazolium (NBT) reduction, mea-
sured with a UV–vis recording spectrophotometer (UV-2401,
Shimadzu Corporation, Japan) at 560 nm. Catalase (CAT)
activity was determined by potassium permanganate
titration (Gisnnopolitis and Nries, 1977). The action mixture
contained 2.9 ml of 50 mM phosphate buffer (pH 7.0),
1.0 ml 10 mM H2O2 and 100 ml enzyme extract in tubes.
Peroxidase (POD) activity was analyzed in 2.9 ml of
0.05 mol l1 phosphate buffer containing 1.0 ml of
0.05 mol l1 guaiacol and 1.0 ml 2% H2O2 (Amalo et al.,
1994). The increase in absorbance at 470 nm was recorded
after adding 2.0 ml 20% chloroacetic acid. All the above
procedures of enzyme extraction were carried out at 0–4 8C.
Measurement of MDA content was according to the method
of Zhang (1992).
2.7. Root vigor assays
Measurement of root vigor was according to the triphenylte-
trazolium chloride (TTC) method (Li, 2000). The surface liquid
of white young root was blotted with tissue paper and their
fresh weights measured. Root with weights <0.5 g were
placed in tubes, filled with 5 ml of 0.4% triphenyltetrazolium
chloride (TTC), 5 ml phosphate buffer (0.06 mol l1, pH 7.0).
The tubes were incubated at 37 8C for up to 3 h. The chemical
reaction was stopped by adding 2 ml of 1 mol l1 sulfuric acid
in the tubes. This step was followed by extraction with
triphenylformazan (TPF), which consisted of taking the root
out of the tubes and placing them in a pestle, filled with 3–4 ml
of ethyl acetate and a little quartz sand and ground. The liquid
phase was removed into a test tube. Ethyl acetate was added
to the 10 ml level and recorded OD values with a UV–vis
recording spectrophotometer at 485 nm. The OD values were
used to calculate equivalent TPF concentrations with which
the root activity was determined for each fresh root weight as
follows:
root vigorðTPF mg g1 FW hour1 Þ
¼ TPF reductionðmgÞ=fresh weight ðgÞ=time ðhÞ
2.8. Statistical analysis
Each determination was carried out with three or four
replicates. Statistical analysis was performed using the
GLM analysis of variance from the SAS Institute and Inc.
(1999).
1264 agricultural water management 95 (2008) 1261–1270
3. Results
3.1. Soil-relative water content (SRWC) of cotton with
different N levels during the soil water stress period
The soil-relative water content (SRWC) was measured every
day from the third day to the end of water stress period (Fig. 1).
Because there was no difference of SRWC among three N
levels, only the SRWC of N1 was shown and ranged from 70%
to 75% in the well-watered control. The SRWC of the water-
stressed plants declined gradually in a similar fashion during
water stress period to 34.1% (N0), 30.3% (N1), 27.3% (N2) in 2005
and 38.9% (N0), 33.8% (N1), 30.0% (N2) in 2006 at the end of
water stress period, respectively. The SRWC was markedly
different among three N levels. The reduction of SRWC
increased with enhancing N levels, indicating that the
application of N decreased the available water for cotton
plants during water stress.
3.2. Plants growth and dry matter partitioning with
different N levels during water stress and re-watering
At the end of water stress period, leaf area, root dry weight and
shoot dry weight were significantly ( p < 0.05) lower during
water stress compared to the control (Table 2). Leaf area was
increased with increasing N under both water stress and the
control. The root dry weight and shoot dry weight were higher
in N2 than in N1 and N0 during water stress, but the root/shoot
ratio (R/S) was smaller in N1 than in N0 and N2.
Soil water affected plant growth not only during the soil
water stress period but also after relief of the stress. At the 10th
day after soil re-watering, the root dry weight showed a quick
recovery in water stress treatments, and exceeded the root
weight of the well-watered control. Root dry weight in the
water stress treatments were 0.5, 0.5 and 1.3 g more than the
well-watered control in N0, N1, and N2 in 2005, and 0.3, 0.9 and
2.1 g in 2006, respectively. However, the leaf area and shoot dry
weight during the soil water stress period were still sig-
nificantly ( p < 0.05) lower compared to the control. Leaf area,
Fig. 1 – Changes in soil-relative water content of cotton with different N levels during water stress.
dry weight of root and shoot were increased with increasing N
application in both water stress treatments and the control,
but the R/S ratio in N1 was the smallest under water stress.
3.3. N accumulation of plants with different N levels
during water stress and re-watering
Cotton plants exhibited a significant ( p < 0.05) reduction of N
accumulation in root, shoots and total plant under water stress
(Table 3). There were significant ( p < 0.05) differences in N
accumulation among the three N levels for root, shoots and total
plant. N application resulted in increasing N accumulation. The
root-N/shoot-N ratio (RN/SN) was markedly ( p < 0.05) increased
under water stress. At the end of water stress period, the RN/SN
were 0.16 and 0.01 lower in N1 than in N0 and N2 in 2005, and
0.24 and 0.02 lower in N1 than in N0 and N2 in 2006, respectively.
At the 10th day after soil re-watering, the root N
accumulation during the soil water stress period was
dramatically ( p < 0.05) higher, but the shoot N accumulation
was still lower than in the soil well-watered control, which
was the reason that RN/SN was higher during the soil water
stress period. The effects of N treatment on N accumulation in
root and shoots were not changed with N2 > N1 > N0, but the
RN/SN of N1 was similar to that of N2, and significantly
( p < 0.05) smaller than that of N0.
3.4. Antioxidant enzyme activity of root with different N
levels during water stress and re-watering
The activities of SOD and POD in water-stressed cotton root
were decreased compared to soil well-watered treatments,
while the CAT activity behaved contrarily (Fig. 2). N application
played a significant role in adjusting the antioxidant enzyme
activity. As shown in Fig. 2, N application significantly
( p < 0.05) increased the activities of POD and CAT, but
decreased the SOD activity in both the water-stressed and
well-watered control.
At the 10th day after soil re-watering, there was no
difference of SOD activity between water-stressed root and
Table 2 – Leaf area and dry matter partitioning of cotton plants with different N levels during water stress period and re-watering in 2005 and 2006
Year Water regimes Nitrogen levels The end of water stress period The 10th day after soil re-watering

Leaf area Dry weight (g plant1) Root/ Leaf area Dry weight (g plant1) Root/shoot
(m2 plant1) shoot (m2 plant1)
Root Shoot Total plant Root Shoot Total plant

2005 Water stress N0 0.19 e 12.6 c 59.4 c 72.0 c 0.21 a 0.19 e 14.1 d 61.9 c 76.1 c 0.23 a
N1 0.21 de 14.5 b 77.9 b 92.3 b 0.19 b 0.20 e 18.3 c 96.2 b 115 b 0.19 b
N2 0.23 cd 15.5 ab 77.6 bc 93.1 b 0.20 a 0.27 d 20.1 a 102 b 123 b 0.20 b
Control N0 0.25 c 13.8 bc 70.4 bc 84.1 bc 0.20 a 0.30 c 13.6 d 80.3 b 93.9 b 0.17 c
N1 0.30 b 16.5 a 103 a 119 a 0.16 c 0.38 b 17.8 b 123 a 141 a 0.14 e

agricultural water management 95 (2008) 1261–1270

N2 0.40 a 17.2 a 109 a 126 a 0.16 c 0.42 a 18.8 b 130 a 149 a 0.14 e

2006 Water stress N0 0.24 f 14.1 d 31.0 c 45.1 d 0.45 a 0.26 e 17.3 d 38.8 d 56.1 c 0.45 a
N1 0.32 d 15.1 cd 51.4 b 66.4 c 0.29 bc 0.33 cd 23.3 ab 90.3 c 114 b 0.26 c
N2 0.34 c 16.4 c 54.5 b 70.9 c 0.30 b 0.35 c 24.6 a 87.7 bc 112 ab 0.28 c
Control N0 0.28 e 15.3 cd 35.1 c 50.4 d 0.43 a 0.30 de 17.0 d 48.8 d 65.8 c 0.35 b
N1 0.40 b 18.9 b 66.6 ab 85.5 b 0.28 c 0.42 b 21.4 c 111 ab 132 ab 0.19 d
N2 0.48 a 20.9 a 77.7 a 98.6 a 0.27 c 0.52 a 22.5 bc 116 a 139 a 0.19 d

Means in the same column followed by the same letter are not significantly different at P = 0.05. Values are mean of three replicates.

Table 3 – N accumulation of plants with different N levels during the soil water stress period and re-watering in 2005 and 2006
Year Water regimes Nitrogen levels The end of water stress period The 10th day after soil re-watering
1
N accumulation (g plant ) Root-N/shoot-N N accumulation (g plant1) Root-N/shoot-N

Root Shoot Total plant RN/SN Root Shoot Total plant RN/SN

2005 Water stress N0 0.16 b 0.42 e 0.58 d 0.39 a 0.19 d 0.44 e 0.63 d 0.44 a
N1 0.23 a 1.00 d 1.22 c 0.23 c 0.26 c 1.09 c 1.35 c 0.24 b
N2 0.28 a 1.15 c 1.42 b 0.24 c 0.35 a 1.34 b 1.68 b 0.26 b
Control N0 0.17 b 0.48 e 0.65 d 0.35 b 0.15 e 0.53 d 0.69 d 0.28 b
N1 0.26 a 1.30 b 1.55 b 0.20 d 0.26 c 1.28 bc 1.54 b 0.21 c
N2 0.28 a 1.66 a 1.94 a 0.17 e 0.30 b 1.67 a 1.97 a 0.18 c

2006 Water stress N0 0.11 d 0.22 d 0.33 e 0.49 a 0.11 e 0.26 e 0.37 d 0.42 a
N1 0.15 c 0.59 c 0.74 d 0.25 c 0.20 b 0.88 c 1.08 b 0.23 b
N2 0.21 a 0.77 b 0.98 b 0.27 b 0.26 a 1.09 a 1.35 a 0.24 b
Control N0 0.10 d 0.23 d 0.34 e 0.45 a 0.10 e 0.33 d 0.43 c 0.29 b
N1 0.17 b 0.68 bc 0.84 c 0.25 c 0.16 d 0.97 b 1.13 b 0.16 c
N2 0.22 a 0.97 a 1.18 a 0.23 c 0.19 c 1.25 a 1.44 a 0.15 c

Means in the same column followed by the same letter are not significantly different at P = 0.05. Values are mean of three replicates.

1265
1266 agricultural water management 95 (2008) 1261–1270
Fig. 2 – Antioxidant enzyme activity of roots with different N levels during the soil water stress period and re-watering.
the well-watered control, whereas the POD activity of water-
stressed root were smaller than that of the well-watered
control, and the CAT activity of water-stressed root were
significantly ( p < 0.05) increased. N application notably
( p < 0.05) enhanced the POD and CAT activities, whereas
the SOD activity of the N1 treatment was similar to that of N2,
but markedly ( p < 0.05) smaller than that of N0.
3.5. MDA content of root with different N levels during the
soil water stress and re-watering
The MDA content of the water-stressed cotton root at the end
of water stress period increased by 48.6%, 64.7%, 214.9% for N0,
N1, N2 in 2005, and 16.5%, 37.2%, 98.6% for N0, N1, N2 in 2006,
respectively (Fig. 3). However, the MDA content was greatest in
N2 and least in N1, indicating that the rates of lipid
peroxidation in water stress were N1 < N0 < N2.
At the 10th day after soil re-watering, the MDA content of
water-stressed cotton root was similar to that of the well-
watered control (Fig. 3). The MDA content of N1 was the
same as that of N2 under both water stress and control
condition, but less than that of N0, indicating that N
application reduced the rates of lipid peroxidation of cotton
root.
3.6. Root vigor of cotton plants with different N levels
during water stress and re-watering
Root vigor decreased significantly ( p < 0.05) during the soil
water stress period (Fig. 4), with decreases of 39.5%, 31.7%,
60.2% for N0, N1, N2 in 2005, and 44.7%, 45.3%, 62.1% for N0, N1,
N2 in 2006, respectively. However, the root vigor of the water-
stressed cotton plants in N1 was the highest among the three
N levels, but the root vigor of the control increased with
increasing N levels.
At the 10th day after soil re-watering, the root vigor showed
a quick recovery in the water-stressed treatments which
markedly ( p < 0.05) exceeded root vigor in the well-watered
control. Root vigor of the stressed cotton were enhanced by
5.7%, 33.4%, 35.7% for N0, N1, N2 in 2005, and 30.7%, 42.6%,
 agricultural water management 95 (2008) 1261–1270 1267

Fig. 3 – MDA content of cotton roots with different N levels during the soil water stress period and re-watering.

Fig. 4 – Roots vigor of cotton plants with different N levels during water stress and re-watering.

44.2% for N0, N1, N2 in 2006, respectively, illustrating that N
promoted root vigor.
3.7. Photosynthesis of cotton plants with different N levels
during water stress and re-watering
Net photosynthetic rate (Pn), stomatal conductance (Gs) and
internal CO2 concentration (Ci) of water-stressed cotton plants
were markedly ( p < 0.05) decreased under water stress
condition (Table 4). At the end of water stress period, the Pn
in N1 was the highest among the three N levels during water
stress, however, Pn increased with enhancing N levels in the
well-watered control. This trend was the same as that for root
vigor (Fig. 4).
At the 10th day after soil re-watering, there were no
significant ( p < 0.05) difference between water-stressed and

Table 4 – Photosynthesis of cotton plants with different N levels during the soil water stress period and re-watering in
2005 and 2006
Year Water Nitrogen The end of water The 10th day after soil re-watering
regimes levels stress period

Pna (mmol CO2 Gs (mmol Ci (mmol CO2 Pn (mmol CO2 Gs (mmol Ci (mmol CO2
m2 s2) m2 s2) mol2) m2 s2) m2 s2) mol2)

2005 Water stress N0 9.4cb 0.32 b 223 bc 15.5 b 0.51 a 266 a

N1 12.1bc 0.26 bc 225 bc 18.2 a 0.52 a 250 a
N2 8.6c 0.22 c 214 c 20.1 a 0.55 a 253 a
Control N0 15.7 ab 0.56 a 263 a 14.3 b 0.50 a 270 a
N1 18.5 a 0.63 a 270 a 18.8 a 0.56 a 252 a
N2 19.8 a 0.59 a 259 ab 19.4 a 0.57 a 248 a

2006 Water stress N0 13.4 c 0.52 b 250 c 18.1 b 0.85 a 256 a

N1 15.7 b 0.43 c 258 bc 22.1 a 0.82 a 260 a
N2 12.5 c 0.43 c 237 d 22.1 a 0.85 a 263 a
Control N0 16.3 b 0.90 a 265 ab 17.0 b 0.90 a 260 a
N1 20.6 a 0.95 a 274 a 22.9 a 0.86 a 262 a
N2 23.1 a 0.94 a 266 ab 22.6 a 0.87 a 257 a
a
Pn, net photosynthetic rate; Gs, stomatal conductance; Ci, internal CO2 concentration.
b
Means in the same column followed by the same letter are not significantly different at P = 0.05. Values are mean of three replicates.
1268 agricultural water management 95 (2008) 1261–1270
well-watered control plants for Pn, Gs and Ci. Pn of N1 was the
same as that of N2, but markedly ( p < 0.05) higher than that of
N0. No significant N effect was noticed for Gs and Ci.
4. Discussion
Numerous studies have shown that N can alter drought
resistance of plants (Halvoson and Reule, 1994; Fife and
Nambiar, 1997; Latiri-Souki et al., 1998; Shangguan et al., 2000;
Ashraf et al., 2001; Stroup et al., 2003; Saneoka et al., 2004). This
investigation was designed to study the responses of cotton
root with different N levels to gradually developing water
stress and re-watering.
The different soil-relative water content lost for each water
stress treatment (Fig. 1) supported the idea that the effect of
water stress was related to the intensity of the N treatment
(Bradford and Hsiao, 1982). Adding N fertilizer increased
biomass and leaf area. But during water stress, the high
demand for transpiration water in the large canopy cultivar
resulted in reduced water status (Prasertsak and Fukai, 1997).
Smaller leaf area and shorter crop duration are desirable to
decrease water transpiration by a crop. In this study, the leaf
area increased with increasing N levels (Table 2), which caused
the soil-relative water content to decrease more rapidly
(Fig. 1).
When the soil dries, plants respond in different ways. One
response could be to shed leaves to decrease water transpira-
tion and transfer a larger relative proportion of assimilates to
the root to enhance the water and nutrient uptake capacity,
thereby increasing the R/S ratio to maintain the water balance
in plants (Passioura, 1983; Shangguan et al., 2000; Liu et al.,
2004). This was observed in this study where leaf area, shoot
dry matter, root dry matter, shoot N accumulation and root N
accumulation were all decreased under water-stressed con-
dition, but the R/S and RN/SN were all increased. N nutrition has
been shown to have significant effects on the relationship
between root and shoots of plants (Lioert et al., 1999). Maranov
et al. (1998) found that increasing N application increased root
growth, but decreased shoots growth when plants grew during
severe water stress because the limited water extracted by
root was mainly consumed by the root system itself and only a
small amount of water was transported to shoots. However, in
the present study, the R/S and RN/SN of the water-stressed
cotton plants were smaller at 240 kg N ha1 than at 0 and
480 kg N ha1 (Tables 2 and 3). This may be due to the
restricted amount of shoot biomass relative to root biomass
under water stress which was greater at 480 kg N ha1 than at
240 kg N ha1. This indicated that cotton plants suffered more
water stress at 480 kg N ha1 than at 240 kg N ha1. After re-
watering, the trends were not changed.
Drying soils have been reported to trigger increased
radicals and reactive oxygen species (O2, 1O2, H2O2, OH)
formation in plants (Foryer and Noctor, 2000), which affect
plant metabolism in a variety of ways and result in cellular
damage. The abilities of crops to resist drought have been
connected to the activity of antioxidant enzymes scavenging
reactive oxygen species (ROS) (Ge et al., 2006). These
mechanisms may be the main physiological action and
defense from injury of crops during the soil water stress
period. Shao et al. (2005) reported that wheat with higher
activities of SOD, POD, and CAT displayed higher antioxidative
ability, reflecting higher drought resistance. In this investiga-
tion, SOD and POD activities were increased and CAT activity
was decreased during the soil water stress period (Fig. 2).
Similar results were reported by Iturbe-Ormaetxe et al. (1998).
The effects of N on antioxidant enzymes activity have been
previously reported By Zhang et al. (2002). These researchers
found that activities of SOD, POD, CAT and soluble protein
content in the ear leaf of maize were higher in the treatment
with added N than in the treatment without N, which
enhanced drought resistance. In this study, it was found that
N increased the activities of POD and CAT in cotton root, but
decreased the activity of SOD under water stress (Fig. 2).
Cell membrane stability has been shown to be affected by
lipid peroxidation caused by ROS under stress conditions
(Sudhakar et al., 2001). In our study, lipid peroxidation
evaluated by the MDA content in root tissue increased with
water stress, which suggested that active oxygen species
accumulated in root during the soil water stress period (Fig. 3).
Saneoka et al. (2004) reported that MDA decreased with
increased N in the leaf of water-stressed plants. However,
MDA content was the lowest in 240 kg N ha1 and the most in
480 kg N ha1 in our experiment, indicating that appropriate N
supply (240 kg N ha1 in this investigation) reduce, but
excessive N supply (480 kg N ha1) aggravate, lipid peroxida-
tion under water stress. After soil re-watering, the MDA
content of 240 kg N ha1 was similar to that of 480 kg N ha1,
but less than that of 0 kg N ha1. This suggested that lipid
peroxidation was smaller in 240 and 480 kg N ha1 than in
0 kg N ha1.
Root vigor has been shown to be a reliable and sensitive
indicator to evaluate drought resistance (Wang et al., 2006),
and reflects water and nutrition absorbing abilities of drought-
stressed plants. Zhai et al. (2003) suggested that root vigor was
affected by relative enzyme activity, and appropriate N levels
enhanced root growth. In our study, drought decreased root
vigor during the water stress period, and the root vigor at
240 kg N ha1 was the highest among the three N levels (Fig. 4).
However, root vigor in water-stressed plants was higher than
that of the well-watered control after soil re-watering, and
increased with increasing N levels. All results indicated that
appropriate N was beneficial to the drought resistance of
cotton plants, however excessive N was deleterious. After
relief of water stress, root vigor recovered quickly and
increased with increasing N levels.
The extent to which photosynthetic capability is main-
tained during soil water stress plays an important role in a
plants’ acclimation to a dry environment (Niu et al., 2005). The
root and shoot systems are interdependent and because the
root are the source for water and all inorganic nutrients, an
adjustment of cotton root to drying soil would change the
response of shoot physiological functions, such as photo-
synthesis. In our results, the trends in net photosynthetic rate
were the same as that of root vigor under water stress
(Table 4). It appeared that appropriate N may have contributed
to the extraction of more water and mineral nutrients to
maintain photosynthesis. The photosynthesis of cotton plant
with increased soil N under soil water stress might be
associated with increases in activity of photosynthetic carbon
 agricultural water management 95 (2008) 1261–1270
reduction cycle enzymes, such as ribulose bisphosphate
carboxylase/oxygenase (Rubisco) (Lawlor, 2002), and chlor-
ophyll content in the leaf, but additional research is needed.
5. Conclusions
This study showed that N was involved in physiological and
biochemical functions in cotton root during soil water stress
period and re-watering. The results also suggested that
240 kg N ha1 was the best rate for cotton growth under water
stress, and that excessive N (480 kg N ha1) was deleterious to
the drought resistance of cotton. However, more N application
was attributed to compensate the root growth, antioxidant
enzyme activity, root vigor and photosynthesis after stress
alleviation. Therefore, the application of N should be reduced
to decrease stress when drought occurred, but after stress
alleviation, farmers could apply additional N fertilizer to
compensate for lost cotton growth.
Droughts from global warming and environmental pollu-
tion, as well as judicious N rates will be of great concern in the
future. Thus, it will be extremely important to understand the
plants’ physiological and biochemical processes under water
stress and the plants ability to recover after re-watering.
However, there is a still need to confirm those plant responses,
obtained from a pot study, in a field environment. Experiments
should also investigate short- to long-term responses and
acclimation of cotton after alleviation of water stress.
Acknowledgements
This work was funded by the National Natural Science
Foundation of China (Grant Nos. 30571095, 30771279,
30771277), and the Jiangsu Province Natural Science Founda-
tion (Grant Nos. BK2005091, BK2006141).
references
Adda, A., Sahnoune, M., Kaid-Harch, M., Merah, O., 2005. Impact
of water deficit intensity on durum wheat seminal roots. C.
R. Biol. 328, 918–927.
Amalo, K., Chen, G.X., Asade, K., 1994. Separate assays specific
for ascorbate peroxidase and guaiacal peroxidase and for
the chloroplastic and cytosolic isozymes of ascorbate
peroxidase implants. Plant Cell Physiol. 35, 497–504.
Apse, M.P., Aharon, G.S., Snedden, W.A., Blumwald, E., 1999.
Salt tolerance conferred by over expression of a vacuolar
Na+/H+ antiport in arabidopsis. Science 285, 1256–1258.
Ashraf, M., Shabaz, M., Ashraf, M.Y., 2001. Influence of nitrogen
supply and water stress on growth and nitrogen,
phosphorus, potassium and calcium contents in pearl
millet. Biol. Plantarum 44, 459–462.
Ball, R.A., Oosterhuis, D.M., Mauromoustakos, A., 1994. Growth
dynamics of the cotton plant during water-deficit stress.
Agron. J. 86, 788–795.
Benjamin, J.G., Nielsen, D.C., 2006. Water deficit effects on root
distribution of soybean, field pea and chickpea. Field Crops
Res. 97, 248–253.
Bradford, K.J., Hsiao, T.C., 1982. Physiological responses to
moderate water stress. In: Lange, O.L., Nobel, P.S., Osmond,
1269
C.B., Ziegler, H. (Eds.), Encyclopedia of Plant Physiology,
New Series, vol. 12B. Springer-Verlag, New York, pp. 263–
324.
Ennahli, S., Earl, H., 2005. Physiological limitation to
photosynthetic carbon assimilation in cotton under water
stress. Crop Sci. 45, 2374–2382.
Feil, B., Moser, S.B., Jampatong, S., Stamp, P., 2005. Mineral
composition of the grain of tropical maize varieties as
affected by pre-anthesis drought and rate of nitrogen
fertilization. Crop Sci. 45, 516–523.
Fife, D.N., Nambiar, E.K.S., 1997. Changes in the canopy and
growth of Pinus raiata in response to nitrogen supply. For.
Ecol. Manage. 93, 137–152.
Foryer, C.H., Noctor, G., 2000. Oxygen processing in
photosynthesis: regulation and signaling. New Phytol. 146,
359–388.
Ge, T.D., Sui, F.G., Bai, L.P., Lu, Y.Y., Zhou, G.S., 2006. Effects of
water stress on the protective enzyme activities and liquid
peroxidation in root and leaves of summer maize. Agric. Sci.
China 5, 291–298 (in Chinese, with English abstract).
Gisnnopolitis, C., Nries, S.K., 1977. Superoxide dismutases
occurrence in higher plants. Plant Physiol. 59, 309–314.
Halvoson, A.D., Reule, C.A., 1994. Nitrogen fertilizer
requirements in an annual dryland cropping system. Agron.
J. 86, 315–318.
Han, H.L., Kang, F.J., 2001. Experiment and study on effect of
moisture coerce on cotton producing. Trans. CSAE 17, 37–40
(in Chinese, with English abstract).
Iturbe-Ormaetxe, I., Escuredo, R.P., Arrese-Igor, C., Becana, B.,
1998. Oxidative damage in pea plant exposed to water
deficit or paraquat. Plant Physiol. 119, 173–181.
Latiri-Souki, k., Nortcliff, S., Lawlor, D.W., 1998. Nitrogen
fertilizer can increase dry matter, grain production and
radiation and water use efficiencies for durum wheat under
semi-arid conditions. Eur. J. Agron. 9, 21–34.
Lawlor, D.W., 2002. Limitation to photosynthesis in water
stressed leaves: stomata vs. metabolism and the role of
ATP. Ann. Bot. 89, 1–15.
Liang, J., Zhang, J., Wong, M.H., 1997. Can stomatal closure caused
by xylem ABA explain the inhibition of leaf photosynthesis
under soil drying? Photosynth. Res. 51, 149–159.
Li, H.S., 2000. Principles and Techniques of Plant Physiological
Experiment. Higher Education Press, Beijing, pp. 119–120 (in
Chinese).
Lioert, F., Casanovas, C., Penuelas, J., 1999. Seedling survival of
Mediterranean shrub land species in relation to root: shoot
ratio, seed size and water and nitrogen use. Funct. Ecol. 13,
210–216.
Liu, H.S., Li, F.M., Xu, H., 2004. Deficiency of water can enhance
root respiration rate of drought-sensitive but not drought-
tolerant spring wheat. Agric. Water Manage. 64, 41–48.
Maranov, A., Samedovam, A., Shirvany, T., 1998. Root–shoot
relationships in plant adaptation to nitrogen deficiency
develop. Plant Soil 82, 147–154.
Niu, S.L., Jiang, G.M., Wan, S.Q., Liu, M.Z., Gao, L.M., Li, Y.G.,
2005. Ecophysiological acclimation to different soil moisture
in plants from a semi-arid sandland. J. Arid Environ. 63,
353–365.
Noctor, G., Gomez, L., Helene, V., Foyer, C.H., 2002. Interactions
between biosynthesis, compartmentation and transport in
the control of glutathione homeostasis and signaling. J. Exp.
Bot. 53, 1283–1304.
Passioura, J.B., 1983. Root and drought resistance. Agric. Water
Manage. 7, 265–280.
Pettigrew, W.T., 2001. Environmental effects on cotton fiber
carbohydrate concentration and quality. Crop Sci. 41, 1108–
1113.
Potikha, T.S., Collins, C.C., Johnson, D.I., Delmer, D.P., Levine, A.,
1999. The involvement of hydrogen peroxide in the
1270 agricultural water management 95 (2008) 1261–1270
differentiation of secondary walls in cotton fibers. Plant
Physiol. 119, 849–858.
Prasertsak, A., Fukai, S., 1997. Nitrogen availability and water
stress interaction on rice growth and yield. Field Crop Res.
52, 249–260.
Saneoka, H., Moghaieb, R.E.A., Premachandra, G.S., Fujita, K.,
2004. Nitrogen nutrition and water stress effects on cell
membrane stability and leaf water relations in Agostis
palustris Huds. Environ. Exp. Bot. 52, 131–138.
SAS Institute Inc., 1999. SAS/STAT Guide for Personal
Computers. Version 7. Cary, NC.
Shangguan, Z.P., Shao, M.A., Dyckmans, J., 2000. Nitrogen
nutrition and water stress effects on leaf photosynthetic gas
exchange and water use efficiency in winter wheat.
Environ. Exp. Bot. 44, 141–149.
Shao, H.B., Liang, Z.S., Shao, M.A., 2005. Changes of anti-
oxidative enzymes and MDA content under soil water
deficits among 10 wheat (Triticum aestivum L) genotypes at
maturation stage. Colloid Surf. B 45, 7–13.
Sharp, R.E., Silk, W.K., Hsiao, T.C., 1988. Growth of the maize
primary root at low water potentials. I. Spatial distribution
of expansive growth. Plant Physiol. 87, 50–57.
Spollen, W.G., Sharp, R.E., 1991. Spatial distribution of turgor
and root growth at low water potentials. Plant Physiol. 96,
438–443.
Stroup, J.A., Sanderson, M.A., Muir, J.P., McFarland, M.J., Read,
R.L., 2003. Comparison of growth and performance in
upland and lowland switchgrass types to water and
nitrogen stress. Bioresour. Technol. 86, 65–72.
Sudhakar, C., Lakshmi, A., Giridarrakumar, S., 2001. Changes in
the antioxidant enzyme efficacy in two high yielding
genotypes of mulberry (morus alba L.) under NaCl salinity.
Plant Sci. 161, 613–619.
Wang, H.F., Zhu, Y.H., Sun, H.J., 2006. Determination of drought
tolerance using root activities in Robina pseudoacacia ‘Idaho’
transformed with mtl-D gene. For. Stud. China 8, 75–81. (in
Chinese, with English abstract).
Wu, Y.J., Cosgrove, D.J., 2000. Adaptation of root to low water
potentials by changes in cell wall extensibility and cell wall
proteins. J. Exp. Bot. 51, 1543–1553.
Zhai, B.N., Sun, C.M., Wang, J.R., Li, S.X., 2003. Effects of nitrogen
deficiency on the growth and development of winter wheat
root. Acta Agron. Sin. 29, 913–918. (in Chinese, with English
abstract).
Zhang, J.W., Wang, K.J., Hu, C.H., Dong, S.T., Liu, P., 2002. Effects
of different nitrogen application stages on forage nutritive
value of summer maize. Sci. Agric. Sin. 35, 1337–1342. (in
Chinese, with English abstract).
Zhang, X.Z., 1992. Crop Physiology Research Methods. China
Agricultural Press, Beijing, pp. 131–207 (in Chinese).