Environmental and Experimental Botany 60 (2007) 318–323

The effects of rate and timing of N fertilizer on growth,

photosynthesis, N accumulation and yield of mustard
(Brassica juncea) subjected to defoliation
P.M. Lone, N.A. Khan ∗
Department of Botany, Aligarh Muslim University, Aligarh 202002, India
Received 1 January 2006; received in revised form 21 July 2006; accepted 28 December 2006

Abstract
Mustard (Brassica juncea L.) is characterized by large number of broad oblong shaped leaves in the lower layers. Our earlier studies have shown
that removal of these shaded lower leaves on mustard plant axis enhanced growth, photosynthetic capacity and yield of the crop. We now present
evidence that soil-applied nitrogen (N) at pre- or post-flowering stage following defoliation of lower leaves influences plant growth, photosynthesis
and assimilation balance. Following defoliation at pre-flowering, i.e. 40 d after sowing (DAS) and N applied at the rate of 100 kg ha−1 at the time of
sowing and 50 kg ha−1 at post-flowering (60 DAS) enhanced the characteristics maximally. The defoliation treatment together with N combinations
and the time of its application, N at 150 kg ha−1 applied as single dose at the time of sowing or N applied in split; 100 kg ha−1 at the time of sowing
and 50 kg ha−1 at 40 DAS or 75 kg ha−1 at the time of sowing or 75 kg ha−1 at pre- or post-flowering time proved less effective. The plants which
were not defoliated and received 75 kg N ha−1 at the time of sowing and 75 kg ha−1 at 60 DAS showed lowest values. Furthermore, N assimilation
was more efficient in plants following defoliation at 40 DAS. The results suggest that split N application (100 kg ha−1 at sowing and 50 kg ha−1
at post-flowering) enhances substantially growth, photosynthesis, N assimilation and yield of mustard following defoliation. This management
practice could be adopted in mustard culture for increasing seed yield together with minimizing N loss.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Brassica juncea; Defoliation; Nitrogen

1. Introduction
The productivity of crops depends on soil inputs and leaf area
index and efficiency of leaves for conversion of light and car-
bon dioxide absorbed into biomass. Photosynthetic photon flux
density decreases from top to lower leaves in an axis. Therefore,
the photosynthetic potential of lower leaves is less compared
to upper leaves (Nobel et al., 1993). Moreover, adequate sup-
ply of nitrogen (N) is required for compensating N loss by crop
removal or leaching (Tabachow et al., 2001). This is normally
attained by a judicious supply of input and its timing (Rice et
al., 1995). Earlier research has shown that removal of 50% of
leaves on lower regions at pre-flowering stage, i.e. 40 d after sow-
ing (DAS) improved photosynthetic efficiency of the remaining
leaves and increase in dry mass and yield of mustard (Khan
∗ Corresponding author. Tel.: +91 571 2702016; fax: +91 571 2702016.
E-mail address: naf9@lycos.com (N.A. Khan).
and Ahsan, 2000; Khan, 2002, 2003; Khan and Lone, 2005).
However, defoliation at post-flowering stage (60 DAS) was less
effective (Khan and Lone, 2005). Assuming that N is used more
efficiently by the crop after defoliation, it was decided to test
the effect of the rate and timing of N application to the crop
following defoliation on growth, photosynthesis, N assimilation
and yield of mustard.
2. Material and methods
2.1. Plant material and treatments
Seeds of mustard (Brassica juncea L. Czern and Coss. cv.
Alankar) were sown in 10 m2 (5 m × 2 m) experimental plots of
Aligarh Muslim University, Aligarh, India (27◦ 52 N, 78◦ 51 E
and 187.4 m asl) in the winter season of 2002–2003. Plant pop-
ulation of 12 m−2 was maintained by keeping distance of 30 cm
between rows and 15 cm between plants. The soil was sandy
loam (Alfisols with Ustochrept type). Available soil N measured

0098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2006.12.013
 P.M. Lone, N.A. Khan / Environmental and Experimental Botany 60 (2007) 318–323
at the depth of 30 cm was 100 kg N ha−1 . The plots were treated
with 150 kg N ha−1 as a single dose or in its split application.
The treatment of 150 kg N ha−1 was based on earlier experi-
ence (Lone, 2004) in which plants grown with 250 kg N ha−1
(100 kg N ha−1 as available soil N and 150 kg N ha−1 as addi-
tional N treatment) increased growth and yield maximally. Split
application of 150 kg N ha−1 was given as 100 kg N ha−1 at the
time of sowing (BN100) and 50 kg N ha−1 as top dressing at 40
DAS or 60 DAS, and 75 kg N ha−1 at the time of sowing and
75 kg N ha−1 as top dressing at 40 DAS or 60 DAS. A sufficient
amount of P and K fertilizers were applied as single super phos-
phate and muriate of potash, respectively as these nutrients were
non-limiting. Defoliation of 50% leaf number on lower layers on
plant axis was done at 40 DAS (pre-flowering). The details of the
defoliation procedure have been described elsewhere (Khan et
al., 2002a; Khan, 2005). This stage of defoliation has been found
more effective than the other stages (Khan and Lone, 2005).
Intact plants also received these N treatments and served as the
control. The treatments were arranged in a randomized block
design with five replications. Data on growth, photosynthesis, N
assimilation were recorded at 80 DAS (pod-fill), and yield char-
acteristics at 120 DAS (maturity). The environmental conditions
during the crop growth monitored at 40, 60 and 80 DAS were:
PAR, 1035 ± 10, 1021 ± 7 and 1016 ± 6 ␮mol m−2 s−1 ; humid-
ity, 61 ± 3, 59 ± 2 and 60 ± 3%; temperature, 23 ± 2, 22 ± 1 and
20 ± 2 ◦ C, respectively.
2.2. Determination of growth characteristics
Number of functional leaves on plant axis was counted and
recorded as leaf number per plant, and area of these leaves
was determined with a leaf area meter (LA21, Systronics,
Ahmedabad, India). The dry mass of the leaves and plants was
determined after drying them in an oven at 80 ◦ C till constant
weight.
2.3. Photosynthetic characteristics
Carbonic anhydrase (CA) activity was determined in leaves
used for photosynthesis measurement. Leaves were homoge-
nized in 5 mM Tris–HCl (pH 8.5), containing 1 mM MgCl2 ,
1 mM EDTA, and 1% polyvinylpyrrolidone. Homogenate
was passed through Whatman 42 filter paper and cen-
trifuged first at 1000 × g for 10 min and then at 5000 × g
for 30 min. The CA activity was determined by an electro-
metric method (Rickli et al., 1964) in the supernatant. Net
photosynthetic rate (PN ) and stomatal conductance (gS ) in
fully expanded uppermost leaves (four plants in each treat-
ment) were measured at light saturating intensity between
11:00 and 12:00 h using a portable photosynthesis sys-
tem (LiCOR-6200, NE, USA). The atmospheric conditions
during measurement were PAR, 1016 ± 6 ␮mol m−2 s−1 ; rel-
ative humidity 60 ± 3%, atmospheric temperature 20 ± 2 ◦ C
and atmospheric CO2 360 ± 4 ␮mol mol−1 . The ratio of
atmospheric CO2 to intercellular CO2 concentration was
constant. The data on PN and gS were used for calculat-
319
ing water use efficiency (WUE) as described by Dudley
(1996).
2.4. N assimilation
For N assimilation, the activities of nitrate reductase (NR),
nitrite reductase (Ni R) and glutamine synthetase (GS) were
assayed. Leaves used for photosynthetic measurements were
selected for the assay of enzyme activities. Plant N content was
determined as a product of plant dry mass and its N concentra-
tion, determined in acid-peroxide digested leaf sample according
to Lindner (1944).
Activity of NR in leaves was estimated by the method
of Jaworski (1971). Fresh 200 mg leaf tissue was incubated
for 2 h at 30 ◦ C in reaction mixture containing 2.5 ml phos-
phate buffer (pH 7.5), 0.5 ml of 0.2 M potassium nitrate
solution and 2.5 ml of 5% isopropanol. To 0.4 ml reac-
tion mixture, 0.3 ml each of 1% sulphanilamide and 0.02%
NED-HCl were added and absorbance was read on spec-
trophotometer at 540 nm (SL-164 UV–vis, Elico, Hyderabad,
India).
The activity of Ni R was determined by the Methyl Vio-
logen method (Lillo, 1984). Enzyme extract was prepared
by homogenizing 5 g leaf tissue in 50 ml Tris–HCl buffer
(pH 7.5) in blender. The homogenate was passed through
multilayered cheese cloth. To 0.3 ml enzyme extract, a reac-
tion mixture of 6.25 ml of Tris–HCl buffer, 2 ml of sodium
nitrite, 2 ml of methyl viologen solution and 14.75 ml of dis-
tilled water were added. Freshly prepared 0.2 ml of 0.29 M
dithionite sodium bicarbonate solution was added to start the
reaction and incubated at 30 ◦ C for 15 min. To stop the reac-
tion, the mixture was vigorously shaken on vortex mixer till
the blue colour disappeared. To 20 ␮l aliquot, 1.0 ml each of
sulphanilamide and NED-HCl was added, and the absorbance
was noted spectrophotometrically at 540 nm (SL-164 UV–vis,
Elico).
The enzyme extract for GS assayed by the method of Farnden
and Robertson (1980) was prepared by homogenizing 1.0 g leaf
material in 5 ml of 50 mM inidazole–acetate buffer (pH 7.8) con-
taining 0.5 mM EDTA, 1 mM dithiothreitol, 2 mM MnCl2 and
20% glycerol. The enzyme extract was centrifuged at 10,000 rpm
at 4 ◦ C (CPR 24 Remi, New Delhi, India) for 3 min. For purifi-
cation the enzyme was precipitated with (NH4 )2 SO4 at 60%
saturation and the precipitate was resuspended in extraction
buffer and then desalted over sephadex G25. To 0.2 ml enzyme
extract, 2.0 ml of 0.2 M l-glutamine, 0.5 ml of 20 mM sodium
arsenate and 0.3 ml of 2 mM MnCl2 were added followed by the
addition of 0.5 ml of 1 mM ADP and 50 mM hydroxylamine and
then incubated at 37 ◦ C for 30 min. The reaction was stopped by
adding 1.0 ml ferric chloride to the reaction mixture, and the
absorbance was read on spectrophotometer at 540 nm (SL-164
UV–vis, Elico).
2.5. Yield characteristics
At maturity, plants in 1 m2 land area were harvested, sun-
dried and pod number per plant was counted. Seeds were
320 P.M. Lone, N.A. Khan / Environmental and Experimental Botany 60 (2007) 318–323

Table 1
Leaf number, leaf area, leaf dry mass and plant dry mass of mustard (Brassica juncea) at pod-fill, i.e. 80 d after sowing (DAS) following 50% defoliation of lower
leaves at 40 DAS and treated with single or split N at 40 (pre-flowering) or 60 (post-flowering) DAS
Treatment Leaf number Leaf area (cm2 plant−1 ) Leaf dry mass (g plant−1 ) Plant dry mass (g plant−1 )

No defoliation
N levels (kg ha−1 )
BN150 57fg 1142f 9.1g 28.7g
BN100 + N50 (40 d) 63cd 1285d 9.6e 30.7ef
BN75 + N75 (40 d) 59ef 1189e 9.4ef 30.0f
BN100 + N50 (60 d) 63cd 1292d 10.0d 32.3d
BN75 + N75 (60 d) 52h 1063g 7.9h 27.7h
Defoliation
N levels (kg ha−1 )
BN150 62de 1317c 10.4c 33.7c
BN100 + N50 (40 d) 66b 1348b 11.1b 35.7b
BN75 + N75 (40 d) 64bc 1339bc 10.9b 35.2b
BN100 + N50 (60 d) 70a 1425a 12.8a 38.7a
BN75 + N75 (60 d) 55g 1181e 9.3fg 31.7de
P <0.001 <0.001 <0.001 <0.001

The statistical evaluation using analysis of variance (ANOVA). Data followed by the same letter within a column are significantly not different at P < 0.05 as
determined by LSD.

collected after thrashing pods to record 1000 seed weight and
seed yield.
2.6. Data analysis
Data presented are mean of five replicates per treatment. As
four plants per treatment were analyzed for photosynthetic char-
acteristics and N assimilation, the total number of samples for
these purposes was twenty. Statistical analysis was carried out
by analysis of variance (ANOVA) using SPSS (10.0 for Win-
dows). The least significant difference (LSD) at p < 0.05 was
calculated for the significant data to identify significant differ-
ence in the mean of the treatment. The treatment mean was
separated using Duncan’s multiple range test. Different letters
indicate significant difference at p < 0.05.
3. Results and discussion
Application of 100 kg N ha−1 at the time of sowing and
50 kg N ha−1 at 60 DAS [BN100 + N50 (60 d)] to plants sub-
jected to defoliation proved more effective in enhancing growth,
photosynthesis, N assimilation and yield than other N doses
applied to plants irrespective of defoliation at 40 (pre-flowering)
or 60 (post-flowering) DAS (Tables 1–4). Other splits of N given
at pre-flowering or post-flowering proved less effective.
3.1. Growth characteristics
The growth of plants was maximally increased when
defoliated and given 100 kg N ha−1 at sowing followed by
50 kg N ha−1 at post-flowering [BN100 + N50 (60 d)]. The treat-

Table 2
Carbonic anhydrase (CA) activity, net photosynthetic rate (PN ), stomatal conductance (gS ) and water use efficiency (WUE) of mustard (Brassica juncea) at pod-fill,
i.e. 80 d after sowing (DAS) following 50% defoliation of lower leaves at 40 DAS and treated with single or split N at 40 (pre-flowering) or 60 (post-flowering) DAS
Treatment CA activity (m mol m−2 s−1 ) PN (␮ mol m−2 s−1 ) gS (m mol m−2 s−1 ) WUE (␮ mol mol−1 )

No defoliation
N levels (kg ha−1 )
BN150 18.6f 24.9d 454ef 55.0g
BN100 + N50 (40 d) 19.6de 25.6c 458bc 56.0ef
BN75 + N75 (40 d) 19.4e 25.4c 456cd 55.8f
BN100 + N50 (60 d) 20.0c 26.1b 460b 56.9cd
BN75 + N75 (60 d) 18.2g 24.2e 453f 55.1g
Defoliation
N levels (kg ha−1 )
BN150 19.8cd 25.8c 457c 56.4de
BN100 + N50 (40 d) 20.5b 26.5b 459b 57.8b
BN75 + N75 (40 d) 20.4b 26.1b 458bc 57.1bc
BN100 + N50 (60 d) 22.5a 29.2a 464a 63.0a
BN75 + N75 (60 d) 19.5e 25.2cd 455d 56.2e
P <0.001 <0.001 <0.01 <0.001

The statistical evaluation using analysis of variance (ANOVA). Data followed by the same letter within a column are significantly not different at P < 0.05 as
determined by LSD.
 P.M. Lone, N.A. Khan / Environmental and Experimental Botany 60 (2007) 318–323 321

Table 3
Activities of nitrate reductase (NR), nitrite reductase (Ni R), glutamine synthetase (GS) and plant N content of mustard (Brassica juncea) at pod-fill, i.e. 80 d after
sowing (DAS) following 50% defoliation of lower leaves at 40 DAS and treated with single or split N at 40 (pre-flowering) or 60 (post-flowering) DAS
Treatment NR activity (␮ Ni R activity GS activity (␮ mol ␥-glutamyl Plant N content
mol NO2 g−1 (f.m.) h−1 ) (␮ mol NH4 g−1 (f.m.) h−1 ) hydroxamate g−1 (f.m.) h−1 ) (mg per plant)

No defoliation
N levels (kg ha−1 )
BN150 8.0f 21.7d 38.4g 809.3f
BN100 + N50 (40 d) 8.5cd 23.6c 40.0e 919.7e
BN75 + N75 (40 d) 8.4d 21.9d 39.8ef 874.7e
BN100 + N50 (60 d) 8.6bc 24.3c 42.2d 974.3cd
BN75 + N75 (60 d) 7.9f 19.3f 36.2h 773.9f
Defoliation
N levels (kg ha−1 )
BN150 8.4d 24.0c 42.5d 1005.7c
BN100 + N50 (40 d) 8.7b 25.6b 45.1b 1104.6b
BN75 + N75 (40 d) 8.6bc 23.7c 43.8c 1065.1b
BN100 + N50 (60 d) 8.9a 27.6a 48.7a 1245.8a
BN75 + N75 (60 d) 8.2e 20.6e 39.1fg 924.8de
P <0.001 <0.001 <0.001 <0.001

The statistical evaluation using analysis of variance (ANOVA). Data followed by the same letter within a column are significantly not different at P < 0.05 as
determined by LSD.

ment increased leaf number by 22.8 and 11.1%, leaf area by 24.7 mass. It has been reported that defoliation at 40 DAS markedly
and 10.3%, leaf dry mass by 40.6 and 28.0% and plant dry mass increased the growth of new leaves that were photosyntheti-
by 34.8 and 19.8% compared to application of total N (BN150) cally more active (Khan and Lone, 2005). New leaves emerging
plus no-defoliation and BN100 + N50 (60 d) plus no-defoliation, after defoliation have been found to have greater efficiency for
respectively (Table 1). CO2 assimilation (Alderfer and Eagles, 1976; Caemmerer and
The extent of leaf formation and expansion influence the light Farquhar, 1984; Khan et al., 2002a,b; Khan and Lone, 2005).
absorption by the individual leaves within a plant. The plants Goulas et al. (2002) suggested that regrowth of new leaves after
after defoliation require more assimilates for regrowth which is defoliation depends upon the supply of carbon and N reserves.
balanced by the increased leaf assimilatory capacity and effi- Similarly, carbon compounds are also remobilized after defoli-
cient N use (Lone, 2004). This leads to the enhancement in the ation to support leaf growth (Ourry et al., 1988; Thornton et al.,
photoassimilate synthesis in leaf increasing leaf and plant dry 1993, 1994; Thornton and Millard, 1996). Nitrogen is required
in sufficient amount to sustain growth after defoliation. There-
Table 4 fore, any change in source–sink relationships is considered as
Pod number per plant, 1000 seed weight and seed yield of mustard (Brassica dependent on N reserve accumulation (Lone, 2004). Hilbert et
juncea) at harvest, i.e. 120 d after sowing (DAS) following 50% defoliation of al. (1981) and McPherson and Williams (1998) have reported
lower leaves at 40 DAS and treated with single or split N at 40 (pre-flowering)
that remobilization of stored carbohydrate influenced regrowth.
or 60 (post-flowering) DAS
The enhanced leaf area has also been reported to be the major
Treatment Pod number 1000 seed Seed yield mechanism leading to compensatory growth (McNaughton et
weight (g) (g m−2 )
al., 1983). The increase in leaf area is brought about by large N
No defoliation supply by causing the expansion of individual leaves and branch-
N levels (kg ha−1 ) ing or tillering in grasses (Gastal and Lemaire, 2002; Trapani and
BN150 191de 4.2f 147.2fg
Hall, 1996; Taylor et al., 1993; Vos and Biemond, 1992; Vos et
BN100 + N50 (40 d) 196d 4.4e 148.6ef
BN75 + N75 (40 d) 180gh 4.1h 144.1g al., 1996) presumably through its effect on cell division and cell
BN100 + N50 (60 d) 182fg 4.4e 156.0c expansion (Lemaire, 2001). Increase in biomass accumulation is
BN75 + N75 (60 d) 174h 4.1g 132.5h attributed to the increased CO2 assimilation due to higher rates
Defoliation of photosynthesis by younger leaves (Khan and Lone, 2005).
N levels (kg ha−1 ) Thus, the increased photosynthetic CO2 assimilation in defo-
BN150 198cd 4.7c 151.2de liated plants leads to enhanced leaf and plant dry mass. Gold
BN100 + N50 (40 d) 212b 4.8b 169.3b and Caldwell (1990) and Anten and Ackerly (2001) suggested
BN75 + N75 (40 d) 205bc 4.9b 166.6b
BN100 + N50 (60 d) 229a 5.6a 180.5a
an increase in light interception by the crop canopy enhanced
BN75 + N75 (60 d) 189ef 4.5d 153.3cd nutrient availability due to defoliation, which leads to increased
P <0.001 <0.05 <0.001 photosynthetic rate and unit leaf rate. The increase in unit leaf
The statistical evaluation using analysis of variance (ANOVA). Data followed
rate contributed more to compensate for the losses in growth
by the same letter within a column are significantly not different at P < 0.05 as (Anten et al., 2003). Thus, the physiological changes that are
determined by LSD. related to enhancement of unit leaf rate, including increase in
322 P.M. Lone, N.A. Khan / Environmental and Experimental Botany 60 (2007) 318–323
leaf photosynthesis, are important for enhancing plant growth
and dry mass.
3.2. Photosynthetic characteristics
Photosynthetic characteristics significantly increased after
defoliation and split N application of 100 kg ha−1 at sow-
ing and 50 kg ha−1 at post-flowering [BN100 + N50 (60 d)]
increased CA activity, photosynthesis, stomatal conductance
and WUE. The treatment increased CA activity, photosynthe-
sis and WUE by 20.9, 17.2 and 14.5% over no-defoliation
plants and 150 kg N ha−1 at sowing (BN150), and 12.5, 11.8
and 10.7% over no-defoliation plants treated with BN100 + N50
(60), respectively (Table 2). Defoliation at early stage of growth
reduces the competition between the leaves for efficient uti-
lization of light, water and nutrients. The increase in the
photosynthetic characteristics of plants defoliated at 40 DAS
was due to the fact that leaf growth following defoliation at this
stage of growth had higher requirement of food reserves for
growth and development. The other possibility is of accumula-
tion of nutrients like potassium, which helped in maintaining the
rate of photosynthesis by improving the relative water content
of leaf through osmotic adjustment. Khan et al. (2000) reported
that potassium accumulation increased with N supply, causing
an increase in photosynthetic rate and dry mass. It is empha-
sized that an allocation of leaf N due to N application in suitable
package [BN100 + N50 (60)] increased the photosynthetic char-
acteristics. The increased N supply has been found to enhance the
activities of CA and RuBP carboxylase (Terashima and Evans,
1988; Burnell et al., 1990; Khan et al., 1996). Thus, the plants
after defoliation required larger amount of N in compensatory
mechanisms, which influenced enzymes of photosynthesis. An
increase in CA and ribulose 1–5 bisphosphate (RuBP) carboxy-
lase following defoliation has been observed (Khan, 2002).
Considering the effect of defoliation and split N application on
stomatal and mesophyll processes, it emerges from the data that
the latter were mainly responsible for the substantial increase in
photosynthesis.
3.3. N assimilation
The activity of N assimilation enzymes and plant N con-
tent were significantly increased following defoliation and N
treatments. Maximal activities of N assimilating enzymes were
recorded in defoliated plants treated with 100 kg N ha−1 at sow-
ing and 50 kg N ha−1 at 60 DAS [BN100 + N50 (60 d)]. The
activities of enzymes NR, Ni R and GS and N content were
found significantly higher in defoliated plants treated with
BN100 + N50 (60 d) compared to no-defoliation plants treated
with BN150 or BN100 + N50 (60 d). The increase in NR activ-
ity due to the treatment BN100 + N50(60 d) was 11.2 and 3.4%,
and plant N was 53.8 and 27.9% in comparison to no-defoliation
plants treated with BN150 or BN100 + N50(60 d), respectively
(Table 3).
Nitrogen is important for overall growth, physiology and
development of plants. At initial stages of growth, the soil-
applied N is liable to losses by volatilization and leaching, etc.
Secondly, further depletion occurs due to a greater demand by
the crop at that time. The plants after defoliation required N in
large amount to compensate growth. Therefore, application of N
partly at 60 DAS stage might have been timely. Higher rates of
N incorporation have been reported to increase the activities of
N enzymes like NR (Vyas et al., 1995; Khan et al., 1996; Wang
et al., 2000).
3.4. Yield characteristics
Defoliation and N application significantly increased yield
characteristics. Number of pods per plant, 1000 seed weight
and seed yield were found greatest in defoliated plants treated
with 100 kg N ha−1 at sowing and 50 kg N ha−1 at 60 DAS
[BN100 + N50 (60 d)]. An increase of 19.9% in pod number,
33.3% in 1000 seed weight and 22.6% in seed yield over no-
defoliation plants treated with 150 kg N ha−1 at sowing (BN150)
was recorded. Similarly, these characteristics also showed an
increase of 25.8, 27.2 and 15.7%, respectively over the intact
plants treated with soil-applied N as [BN100 + N50 (60 d)]
(Table 4). Increased seed yield was due to the higher percent
increase in pod number and seed weight in defoliated plants.
The size of N pools in vegetative parts determines seed set, seed
growth and finally seed yield (Marschner, 1995). It can be argued
that timely application of N at post-flowering (60 DAS) met the
N requirement for seed set, which increased seed yield. The con-
current enhanced regrowth following utilization of soil N more
efficiently resulted in higher dry mass and finally seed yield. The
increase in seed yield characteristics after defoliation finds sup-
port from similar findings of Bruening and Egli (2000), Khan and
Ahsan (2000); Khan et al. (2000); Khan (2003) and Khan and
Lone (2005). However, the effect of defoliation together with
split N application on mustard has not been reported earlier.
4. Conclusion
Loss of soil-applied N in terms of leaching, volatilization
and surface run off minimizes N use efficiency and causes eco-
nomic loss together with environmental degradation. It can be
concluded from the study that the amount and timing of N appli-
cation together with defoliation has an important influence in
augmenting growth, photosynthesis and yield of mustard. The
quantum of increase in the characteristics with defoliation and
split N application of 100 kg N ha−1 at sowing and 50 kg N ha−1
as top dressing at 60 DAS (post-flowering) was much higher than
no-defoliation and one time N application as 150 kg N ha−1 or
no-defoliation followed by split N application at pre- or post-
flowering.
References
Alderfer, R.G., Eagles, C.F., 1976. The effect of partial defoliation on the growth
and photosynthetic efficiency of bean leaves. Bot. Gaz. 137, 351–355.
Anten, N.P.R., Ackerly, D.D., 2001. Canopy-level photosynthetic compensation
after defoliation in a tropical understorey palm. Funct. Ecol. 15, 252–262.
Anten, N.P.R., Martinez-Ramos, M., Ackerly, D.D., 2003. Defoliation and
growth in an understory palm: quantifying the contributions of compensatory
responses. Ecology 84, 2905–2918.
 P.M. Lone, N.A. Khan / Environmental and Experimental Botany 60 (2007) 318–323
Bruening, W.P., Egli, D.B., 2000. Leaf starch accumulation and seed set at
phloem-isolated nodes in soybean. Field Crops Res. 68, 113–120.
Burnell, J.N., Suzuki, I., Sugiyama, T., 1990. Light induction and the effect of
nitrogen status upon the activity of carbonic anhydrase in maize leaves. Plant
Physiol. 94, 384–387.
Caemmerer, S.V., Farquhar, G.D., 1984. Effects of partial defoliation, changes
of irradiance during growth, short-term water stress and growth at enhanced
p(CO2 ) on the photosynthetic capacity of leaves of Phaseolus vulgaris L.
Planta 160, 320–329.
Dudley, S.A., 1996. Differing selection on plant physiological traits in response
to environmental water availability: a test of adaptive hypothesis. Evolution
50, 92–102.
Farnden, K.J.S., Robertson, J.G., 1980. Methods for studying enzymes involved
in metabolism related to nitrogenase. In: Bergersen, F.J. (Ed.), Methods for
Evaluating Biological Nitrogen Fixation. John Wiley & Sons, New York,
pp. 265–315.
Gastal, F., Lemaire, G., 2002. N uptake and distribution in crops: an agronomical
and ecophysiological perspective. J. Exp. Bot. 53, 789–799.
Gold, W.G., Caldwell, M.M., 1990. The effects of the spatial pattern of defoli-
ation on regrowth of a tussock grass. III. Photosynthesis: canopy structure
and light interception. Oeocologia 82, 12–17.
Goulas, E., Le Dily, F., Simon, J.C., Ourry, A., 2002. Morphological pattern
of development affects the contribution of nitrogen reserves to regrowth of
defoliated white clover (Trifolium repens L.). J. Exp. Bot. 53, 1941–1948.
Hilbert, D.W., Swift, D.M., Detling, J.K., Dyer, M.I., 1981. Relative growth
rates and the grazing optimization hypothesis. Oecologia 51, 14–18.
Jaworski, E.G., 1971. Nitrate reductase assay in intact plant tissues. Biochem.
Biophys. Res. Commun. 43, 1274–1279.
Khan, N.A., 2002. Activities of carbonic anhydrase and ribulose-1,5-
bisphosphate carboxylase, and dry mass accumulation in Brassica juncea
following defoliation. Photosynthetica 40, 633–634.
Khan, N.A., 2003. Changes in photosynthetic biomass accumulation, auxin and
ethylene level following defoliation in Brassica juncea. Food Agric. Environ.
1, 125–128.
Khan, N.A., 2005. The influence of exogenous ethylene on growth and photo-
synthesis of mustard (Brassica juncea) following defoliation. Sci. Hort. 105,
499–505.
Khan, N.A., Ahsan, N., 2000. Evaluation of yield potential of defoliated mustard
cultivars. Tests Agrochem. Cult. 21, 33–34.
Khan, N.A., Ansari, H.R., Mobin, M., 1996. Effect of gibberellic acid and nitro-
gen on carbonic anhydrase activity and mustard biomass. Biol. Plant. 38,
601–603.
Khan, N.A., Khan, M., Samiullah, 2002b. Changes in ethylene level associated
with defoliation and its relationship with seed yield in rapeseed-mustard.
Brassica 4, 12–17.
Khan, N.A., Khan, M., Ansari, H.R., Samiullah, 2002a. Auxin and defoliation
effects on photosynthesis and ethylene evolution in mustard. Sci. Hort. 96,
43–51.
Khan, N.A., Lone, N.A., Samiullah, 2000. Response of mustard (Brassica juncea
L.) to applied nitrogen with or without ethrel spray under non-irrigated
conditions. J. Agron. Crop Sci. 183, 1–4.
Khan, N.A., Lone, P.M., 2005. Effects of early and late season defoliation on
photosynthesis, growth and yield of mustard (Brassica juncea L.). Brazilian
J. Plant Physiol. 17, 181–186.
Lemaire, G., 2001. Ecophysiology of grassland: dynamic aspects of forage
plant populations in grazed swards. In: Proceedings of the XIX International
Grassland Congress, Brazil 01, pp. 29–37.
Lillo, C., 1984. Diurnal variations of nitrite reductase, glutamine synthetase,
alanine aminotransferase and aspartate aminotransferase in barley leaves.
Physiol. Plant 61, 214–218.
323
Lindner, R.C., 1944. Rapid analytical methods for some of the more com-
mon inorganic constituents of plant tissues. Plant Physiol. 19, 70–
89.
Lone, P. 2004. Growth and metabolism of mustard (Brassica juncea) following
defoliation and nitrogen treatment. Ph.D. Thesis. Aligarh Muslim University,
Aligarh, India.
Marschner, H., 1995. Mineral Nutrition of Higher Plants, second ed. Academic
Press, New York.
McNaughton, S.J., Linda, L., Wallace, L.L., Coughenour, M.B., 1983. Plant
adaptation in an ecosystem context: effects of defoliation, nitrogen and water
on growth of an African sedge. Ecology 64, 307–318.
McPherson, K., Williams, K., 1998. The role of carbohydrate reserves in growth,
resilience and persistence of cabbage palm seedlings (Sabal palmetto).
Oecology 117, 460–468.
Nobel, P.S., Forseth, I.N., Long, S.P., 1993. Canopy structure and light inter-
ception. In: Hall, D.O., Scurlock, J.M.O., Bolhar-Nordenkampf, Leegood,
R.C., Long, S.P. (Eds.), Photosynthesis and Production in a Changing Envi-
ronment. Chapman and Hall, London, pp. 79–90.
Ourry, A., Boucaud, J., Salette, J., 1988. Nitrogen mobilization from stubble
and roots during regrowth of defoliated perennial ryegrass. J. Exp. Bot. 39,
803–809.
Rice, C.W., Havlin, J.L., Schepers, J.S., 1995. Rational nitrogen fertilization in
intensive cropping systems. Fertil. Res. 42, 89–97.
Rickli, E.E., Ghazanfar, S.A.S., Gibbons, B.H., Edsall, J.T., 1964. Carbonic
anhydrase from human erythrocytes: preparation and properties of two
enzymes. J. Biol. Chem. 239, 1065–1078.
Tabachow, R.M., Jeffrey Pierce, J., Richter, D.D., 2001. Biogeochemical models
relating soil nitrogen losses to plant-available N. Environ. Eng. Sci. 18,
81–89.
Taylor, G., McDonald, A.J.S., Stadenberg, I., Freer-Smith, P.H., 1993. Nitrate
supply and the biophysics of leaf growth in Salix viminalis. J. Exp. Bot. 44,
155–164.
Terashima, I., Evans, J.R., 1988. Effects of light and nitrogen nutrition on the
organization of the photosynthetic apparatus in spinach. Plant Cell Physiol.
29, 143–155.
Thornton, B., Millard, P., 1996. Effects of severity of defoliation on root func-
tioning in grasses. J. Range Mange. 49, 443–447.
Thornton, B., Millard, P., Duff, E.I., 1994. Effects of nitrogen supply on the
source of nitrogen used for regrowth of laminae after defoliation of four
grass species. New Phytol. 128, 615–620.
Thornton, B., Millard, P., Duff, E.I., Buckland, S.T., 1993. The relative contribu-
tion of remobilization and root uptake in supplying nitrogen after defoliation
for regrowth of laminae in four grass species. New Phytol. 124, 689–
694.
Trapani, N., Hall, A.J., 1996. Effects of leaf position and nitrogen supply on the
expansion of leaves of field-grown sunflower (Helianthus annuus L.). Plant
Soil 184, 331–340.
Vos, J., Biemond, H., 1992. Effects of nitrogen on the development and growth
of the potato plant. I. Leaf appearance, expansion, growth, life span of leaves
and stem branching. Ann. Bot. 70, 27–35.
Vos, J., Biemond, H., Struik, P.C., 1996. Dynamics of change of leaf attributes
of brussels sprouts in response to switches between high and low supply of
nitrogen. Netherlands J. Agric. Sci. 44, 31–42.
Vyas, S.P., Kathju, S., Garg, B.K., Lahiri, A.N., 1995. Influence of nitrogen on
Indian mustard grown under different levels of stored soil moisture. J. Arid
Environ. 29, 173–184.
Wang, R., Guegler, K., Labrie, S.T., Crawford, N.M., 2000. Genomic analysis
at a nutrient response in Arabidopsis reveals diverse expression pattern and
novel metabolic and potential regulatory genes induced by nitrate. Plant Cell
12, 1491–1509.