ARTICLE IN PRESS

Journal of Plant Physiology ] (]]]]) ]]]—]]]

www.elsevier.de/jplph

Photosynthetic activity, pigment composition and

antioxidative response of two mustard (Brassica
juncea) cultivars differing in photosynthetic
capacity subjected to cadmium stress
Mohammad Mobin, Nafees A. Khan

Plant Physiology and Biochemistry Division, Department of Botany, Aligarh Muslim University,
Aligarh (U.P.) 202002, India

Received 17 November 2005; accepted 14 March 2006

KEYWORDS Summary
Anthocyanins;
Photosynthetic performance, contents of chlorophyll and associated pigments,
Antioxidant;
cellular damage and activities of antioxidative enzymes were investigated in two
Brassica;
mustard (Brassica juncea L.) cultivars differing in photosynthetic capacity subjected
Cadmium;
to cadmium (Cd) stress. Exposure to Cd severely restricted the net photosynthetic
Photosynthesis
rate (PN) of RH-30 compared to Varuna. This corresponded to the reductions in the
activities of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase
(Rubisco) in both the cultivars. Decline in chlorophyll (Chl) (a+b) and Chl a content
was observed but decrease in Chl b was more conspicuous in Varuna under Cd
treatments, which was responsible for higher Chl a:b ratio. Additionally, the relative
amount of anthocyanin remained higher in Varuna compared to RH-30 even in the
presence of high Cd concentration, while percent pheophytin content increased in
RH-30 at low Cd concentration. A higher concentration of Cd (100 mg Cd kg
1 soil)
resulted in elevated hydrogen peroxide (H2O2) content in both the cultivars.
However, Varuna exhibited lower content of H2O2 in comparison to RH-30. This was
reflected in the increased cellular damage in RH-30, expressed by greater
thiobarbituric acid reactive substances (TBARS) content and electrolyte leakage.
The enhanced activities of antioxidative enzymes, ascorbate peroxidase (APX),

Abbreviations: AOS, active oxygen species; APX, ascorbate peroxidase; CA, carbonic anhydrase; CAT, catalase; Cd, cadmium; Chl,
chlorophyll; DMSO, dimethyl sulfoxide; E, transpiration rate; GR, glutathione reductase; gS, stomatal conductance; PN, net
photosynthetic rate; Rubisco, ribulose-1, 5-bisphosphate carboxylase; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive
substance
Corresponding author.
E-mail address: mohammad_mobin@hotmail.com (M. Mobin).

0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2006.03.003
 ARTICLE IN PRESS
2 M. Mobin, N.A. Khan

catalase (CAT) and glutathione reductase (GR) and also lower activity of superoxide
dismutase (SOD) in Varuna alleviated Cd stress and protected the photosynthetic
activity.
& 2006 Elsevier GmbH. All rights reserved.

Introduction lase (Rubisco), net photosynthetic rate (PN), stoma-

tal conductance (gS), transpiration rate (E) and
Cadmium (Cd) can be accumulated to higher contents of chlorophyll (Chl), pheophytin and
levels in the aerial organs (Pence et al., 2000), relative amount of anthocyanin, associated changes
preferentially in the chloroplasts and disturbs the in the contents of H2O2, thiobarbituric acid reactive
chloroplast function by inhibiting the activities of substances (TBARS), electrolyte leakage and the
enzymes of chlorophyll biosynthesis and CO2 fixa- capacities of antioxidative enzymes SOD, APX, CAT
tion (Böddi et al., 1995; Krupa and Baszynski, 1995; and GR under Cd stress.
Siedlecka et al., 1997) or the aggregation of
pigment protein complexes of the photosystems
(Horvath et al., 1996). The Cd-induced formation of
Material and methods
active oxygen species (AOS), superoxide anion
(O2
), hydroxyl (OH
) radicals and H2O2 result in
Plant material and growth conditions
the damage of chloroplast. The presence of H2O2 in
the chloroplasts restricts Calvin-cycle enzymes
The seeds of mustard (B. juncea L. Czern & Coss)
reducing carbon assimilation (Takeda et al.,
cultivars Varuna (high photosynthetic capacity) and
1995). It induces changes in the functions of
RH-30 (low photosynthetic capacity) were surface
membranes by initiating peroxidation of polyunsa-
sterilized with 0.5% NaOCl for 20 min, rinsed and
turated fatty acids (De Vos et al., 1993), oxidative
soaked overnight in sterile water for 12 h at 4 1C for
damage by formation of oxygen free radicals or by
uniform germination. The seeds were transferred
the reduction in the status of enzymatic and non-
to 23-cm-diameter earthen pots filled with 5 kg of
enzymatic antioxidants (Shaw, 1995; Somashekar-
reconstituted soil (sand:clay:peat; 70:20:10, by dry
aiah et al., 1992).
weight) in the green house of the Botany Depart-
Plants appear to possess a wide array of defense
ment, Aligarh Muslim University, Aligarh, India,
strategies to protect the photosynthetic apparatus
under semi-controlled condition. A polythene plas-
and cellular membranes from AOS (Foyer and
tic film was used to thwart the effects of rainfall,
Harbinson, 1994). Production of antioxidative en-
which allowed the transmittance of 90% of visible
zymes is one part of the defense system that plants
wavelength (400–700 nm) under natural day and
require to protect against stress. Superoxide dis-
night conditions with a day/night temperature 25/
mutase (SOD; EC 1.15.1.1) constitutes the primary
2074 1C and relative humidity of 7075%. Cd at a
step of cellular defense. It dismutates O2
to H2O2
concentration of 0, 25, 50 and 100 mg kg
1 was
and O2. Further, the accumulation of H2O2 is
added to the soil as CdCl2  6H2O and thoroughly
restricted through the action of catalase (CAT; EC
mixed. The soil pH was 7.5. Plants (4 per pot) were
1.11.1.6) or by the ascorbate–glutathione cycle,
watered every alternate day with half strength of
where ascorbate peroxidase (APX; EC 1.11.1.11)
Hoagland nutrient solution. Watering schedule was
reduces it to H2O. Finally, glutathione reductase
adjusted throughout the experimental duration in
(GR; EC 1.6.4.2) catalyzes the NADPH-dependent
order to avert leaching. The treatments were
reduction of oxidized GSSG to the reduced GSH
arranged in a randomized complete block design,
(Noctor et al., 2002).
and each treatment was replicated five times.
Mustard (Brassica juncea L. Czern & Coss) is
recognized as an accumulator of heavy metals. It
is, therefore, postulated that the mustard cultivars Photosynthetic and gas exchange
with diverse photosynthetic capacity detoxifies the measurements
AOS and protects the chloroplast functions differ-
ently from oxidative damage. In the present Photosynthetic parameters, PN, gS and E were
investigation, two mustard cultivars, Varuna and recorded on fully expanded leaves of second
RH-30 (Khan, 2004), were used to study carbonic youngest nodes at 30 days after sowing using an
anhydrase (CA), ribulose-1,5-bisphosphate carboxy- infra-red gas analyzer (IRGA, LICOR, 6200, Lincoln,
 ARTICLE IN PRESS
Photosynthesis and oxidative stress in cadmium-treated mustard
NE, USA) between 11:00 and 13:00 h at light
saturation intensity. These observations were re-
corded on five plants in a treatment.
Determination of enzymes of carbon
assimilation and reduction pathway
Activity of CA in leaf was estimated according to
the method described by Makino et al. (1992).
Sampled leaves were homogenized in 10 mL of
buffer containing 50 mM HEPES-NaOH (pH 7.5),
10 mM DTT, 0.5 mM EDTA and 10% (v/v) glycerol.
Triton X-100 was added to the homogenate to a
final concentration of 0.1% (v/v). The homogenate
was centrifuged (15,000g, 10 min), and the super-
natant was used for the determination of CA.
Activity of CA was determined in Wilbur–Anderson
unit following time-dependent reduction in pH
from 8.25 to 7.45 at 0–3 1C. The Unit [U] of enzyme
activity was calculated according to the formula
U ¼ 10ðT
T 0 Þ=T 0 , where T and T0 represent the
time required to change the pH from 8.25 to 7.45,
with and without the extract of crude enzyme,
respectively. The enzyme activity was presented as
U per mg protein.
Rubisco activity was determined spectrophotome-
trically by monitoring NADH oxidation at 30 1C and
A340 (Usuda, 1985). Leaf samples were homogenized
in a chilled mortar with ice-cold extraction buffer
solution containing 0.25 M Tris–HCl (pH 7.8), 0.05 M
MgCl2, 0.0025 M EDTA and 37.5 mg DTT. The homo-
genate was centrifuged at 4 1C for 10 min at 10,000g.
The resulting supernatant was used for assay of the
enzyme. The reaction mixture contained 100 mM
Tris–HCl (pH 8.0), 40 mM NaHCO3, 10 mM MgCl2,
0.2 mM NADH, 4 mM ATP, 0.2 mM EDTA, 5 mM DTT, 1 U
of glyceraldehyde 3-phosphodehydrogenase and 1 U
of 3-phosphoglycerate kinase. The activity was
estimated after the addition of enzyme extract
and 0.2 mM ribulose-1,5-bisphosphate (RuBP). En-
zyme activity was expressed as mmol CO2 fixed
min
1 mg
1 protein. Estimation of protein was done
according to Bradford (1976) using bovine serum
albumin as standard.
Pigment analysis
Determination of chlorophyll content
Chlorophyll was extracted from freshly sampled
leaves using the DMSO method based on Barnes
et al. (1992). Chl absorption in the extract was
measured using UV-VIS spectrophotometer. Content
of the Chl was calculated from the following
formulae taken from Barnes et al. (1992):
Chl a ¼ 14:85A664:9
5:14A648:2 ,
3
Chl b ¼ 25:48A648:2
7:36A664:9 ,
Chl a þ b ¼ 7:49A664:9 þ 20:3A648:2 .
Determination of anthocyanin and pheophytin
contents
The relative amount of anthocyanin was esti-
mated spectrophotometrically after extraction in
acidified methanol (methanol:water:HCl: 79:20:1)
as described by Mancinelli (1984) using A530–
0.25A657 to correct for Chl and non-specific
degradation products.
For determination of pheophytin, leaf samples
were extracted in 80% acetone and the percentage
of Chl converted to pheophytin was estimated by
an increase in absorbance at A553 relative to the
absorbance at A665 (Bowler et al., 1991).
Determination of TBARS content, electrolyte
leakage and H2O2 content
Contents of TBARS were measured according to
Cakmak and Horst (1991) by recording absorbance
at A532 and corrected for non-specific turbidity by
subtracting the absorbance at A600. The TBARS
content was calculated using its extinction coeffi-
cient of 155 mM
1 cm
1.
For measuring electrolyte leakage, samples were
thoroughly washed with sterile water to get rid of
surface adhered electrolyte and then kept in closed
vials containing 10 mL of deionized water and
incubated at 25 1C for 6 h on a shaker and
consequently electrical conductivity was deter-
mined (C1). Samples were then kept at 90 1C for
2 h and the electrical conductivity was obtained
after attaining equilibrium at 25 1C (C2). Electrolyte
leakage was calculated using the following equa-
tion:
Percent electrolyte leakage ð%Þ ¼ ðC1 =C2 Þ  100.
The assay of H2O2 was made following Okuda
et al. (1991). Leaves (0.5 g) were ground in ice-cold
200 mM perchloric acid. After centrifugation at
1200g for 10 min, perchloric acid of the supernatant
was neutralized with 4 M KOH. The insoluble
potassium perchlorate was eliminated by centrifu-
gation at 500g for 3 min. The reaction was started
by the addition of peroxidase and increase in the
absorbance was recorded at A590 for 3 min.
Extraction and estimation of antioxidative
enzymes
The overall procedures were carried out at 4 1C
unless otherwise mentioned. Leaf tissue was
ground in chilled mortar using different specific
buffers and pH values for each enzyme.
 ARTICLE IN PRESS
4 M. Mobin, N.A. Khan

Homogenates were squeezed through four layers of Data analysis

cheesecloth and centrifuged at 15,000g in a cooling
centrifuge at 4 1C for 15 min. Supernatants ob-
tained were used for enzyme determinations. SOD
activity was assayed by monitoring the inhibition of
photochemical reduction of nitroblue tetrazolium
(NBT) according to Giannopolitis and Ries (1977).
The extraction buffer consisted of 50 mM phosphate
buffer (pH 7.8) containing 0.1% (w/v) BSA, 0.1% (w/
v) as ascorbate and 0.05% (w/v) b-mercaptoetha-
nol. The photoreduction of NBT (production of blue
formazan) was measured at A560 and an inhibition
curve was made against different volumes of the
extract. One unit of SOD was defined as the amount
of enzyme required to cause 50% inhibition of the
reduction of NBT at A560. CAT activity was assayed
following the degradation of H2O2 (extinction
coefficient 39.4 mM
1 cm
1) at 25 1C according to
Chaparro-Giraldo et al. (2000) with minor modifica-
tions. Leaf samples were homogenized in a buffer
composed of 100 mM potassium phosphate buffer
(pH 7.5), containing 1 mM EDTA, 3 mM DTT and 5%
insoluble PVP (w/v). The reaction was initiated by
the addition of leaf extract and the activity was
measured by monitoring degradation of H2O2 at
A240 over 1 min, against a leaf extract free blank.
For determination of APX activity, leaf samples
Analysis of variance (ANOVA) for all the measured
variables was performed by SPSS Ver. 10, Inc.,
Chicago, USA. The treatment means were sepa-
rated using Duncan’s multiple range test (DMRT)
taking Po0:05 as significant.
Results
The Cd concentration in roots and leaves was
greater in RH-30 than Varuna at all Cd treatments
(Fig. 1A and B).
Significant reductions were found in photosyn-
thetic parameters with all Cd treatments in both
the cultivars (Table 1). PN was 3.5%, 30.9% and
35.5% less in Varuna and 4.7%, 35.0% and 50.0% less
in RH-30 with 25, 50 and 100 mg Cd kg
1 soil,
respectively, compared to the control. gS and
E were significantly enhanced at 50 and
100 mg Cd kg
1 soil in RH-30, but in Varuna the
changes in gS and E were non-significant (Table 1).
Increasing Cd concentration depressed the activ-
ities of CA and Rubisco in general, but the
600 Root (A) a
0 Cd
Cadmium content (µg g-1DW)

25 Cd
were extracted in the buffer containing 50 mM 500 50 Cd
potassium phosphate buffer (pH 7.0), 1 mM EDTA 100 Cd
and 1% PVP (w/v) with the addition of 1 mM 400
b
ascorbic acid. APX activity was estimated according
a
to Nakano and Asada (1981) by following the 300
b
reduction in a reaction mixture at A290 (extinction
200
coefficient 2.8 mM
1 cm
1). The reaction was c
c
started by the addition of enzyme extract. For 100
non-enzymatic oxidation of ascorbate, correction d
d
was made by H2O2. The leaf samples for estimation 0
of GR were extracted in 100 mM potassium phos- 100
phate buffer (pH 7.0) containing 1 mM Na2-EDTA Leaf (B)
a
a
Cadmium content (µg g-1DW)

and 4% polyclar AT (w/v). Activity of GR was

80
determined following the method of Sgherri et al.
(1994) by measuring decrease in absorbance at A340 a
and using extinction coefficient of 6.2 mM
1 cm
1. 60
The reaction was started with the addition of
NADPH. This method does not require correction 40 b
b
for GSSH-independent NADH oxidation.
20 c
Cadmium determination
d c
Root and leaf samples were immersed in 5 mM 0
CaCl2 solution for 5 min, rinsed in distilled water, Varuna RH-30
desorbed and blotted (Meuwly and Rauser, 1992) Figure 1. Cadmium contents of root (A) and leaf (B) of
for Cd determination. Cd concentration was esti- Varuna (high photosynthetic capacity) and RH-30 (low
mated after digesting the sample in nitric:perchlo- photosynthetic capacity) cultivars of mustard (Brassica
ric acid (3:1, v/v). Cd concentration was juncea L.) treated with cadmium concentrations. Results
determined by atomic absorption spectrophoto- are means of five replications7SE. The data followed by
meter (Perkin-Elmer A, Analyst, 300). different letters are significantly different at Pp0:05.
 ARTICLE IN PRESS
Photosynthesis and oxidative stress in cadmium-treated mustard 5

Table 1. Chlorophyll (Chl) a, Chl b, Chl a:b, anthocyanin, percent pheophytin, carbonic anhydrase (CA) activity, ribulose-1,5-bisphosphate carboxylase (Rubisco), rate

0.1470.01d

0.9570.03d

3.3370.23d
0.5670.04c

4.070.22c

0.2670.01c
8.6170.44c

4.6970.25c
reduction in the activity of Rubisco was greater in

33.7971.7c
RH-30 at 50 and 100 mg Cd kg
1 soil compared to

44979d
Varuna. Reduction in the activity of CA was 11.9%,
34.9% and 45.0% in Varuna, while 18.9%, 38.1% and

100
62.9% in RH-30 with 25, 50 and 100 mg Cd kg
1 soil,
of photosynthesis (PN), stomatal conductance (gS) and transpiration rate (E) of two cultivars of mustard (Brassica juncea L.) exposed to cadmium stress

respectively, compared to the control. The inhibi-

Each value represents mean of five replicates7SE. Means were compared using ANOVA. Data followed by different letters in a row are significantly different at Pp0:05.
0.7470.05b

11.2670.54b

4.3370.23b
0.2070.01c

1.1670.03c
29.6972.27c
5.0770.29c
0.3870.03c
3.6670.2c
tion in the activity of Rubisco was 22.7%, 48.6% and

39079c
55.0% in Varuna and 30.7%, 50.0% and 72.0% in RH-
30 as the Cd concentration increased from 25 to
RH-30 (Low photosynthetic capacity)

50

100 mg Cd kg
1 soil in comparison to the control

(Table 1).
0.8470.04b

2.9970.08b
0.6870.01b
9.17371.22b
6.6170.43b
0.5670.04b
16.5270.42a

3.8470.22a
0.287.02b

Total Chl decreased with increasing Cd concen-

35477b
trations in the soil. The reduction in Chl content
was 20.0%, 35.0% and 50.0% in Varuna and 31.0%,
43.0% and 57.0% in RH-30 at 25, 50 and
25

100 mg Cd kg
1 soil, respectively, compared to the

0.4970.009a

control. Decrease in Chl a content was almost

1.1170.05a
0.5370.02a
2.0970.03a
0.4970.01a

9.9670.54a
0.7770.05a
17.3570.29a

3.6270.16a
similar in both the cultivars. The reduction in Chl a
31376a

was 15.0%, 29.0% and 56.0% in Varuna and 24.0%,

33.0% and 49.0% in RH-30 with 25, 50 and
100 mg Cd kg
1 soil, respectively, in comparison to
0

the control. The decline in Chl b was more than Chl

4.6470.31d

12.6370.39b
0.7970.03c

4.6270.32c
1.4570.04c
35.8372.88c

0.2770.01c

4.0670.30c
0.177.02d

a. It decreased to 32.0%, 48.0% and 70.0% in

3327 9c

Varuna, and 46.0%, 62.0% and 74.0% in RH-30 with

25, 50 and 100 mg Cd kg
1 soil, respectively, in
100

comparison to the control. Cd treatment affected

Chl b more than Chl a, and therefore Chl a:b ratio
2.9770.22b

16.1671.11b

13.5370.43b

3.8270.27b
0.9070.04c
0.3170.02c

1.3570.07c

7.0270.29c
0.4170.04c

was higher at higher Cd concentration in the soil.

30078b

Chl a:b ratio in Varuna was 2.1, 2.7, 2.9 and 4.6,
while in RH-30, the ratio was 2.9, 2.0, 3.6 and 4.1
Varuna (High photosynthetic capacity)

with 0, 25, 50 and 100 mg Cd kg
1 soil (Table 1). The

50

relative amount of anthocyanin increased signifi-

cantly up to 50 mg Cd kg
1 soil in both the cultivars.
1.0870.04b
0.4070.03b

0.8570.03b
10.5070.79b
8.0970.39b
0.6270.06b
18.8870.26a

3.4470.30a
2.717.1ab

The absolute value was equal when there was no Cd

27278a

but the relative amount of anthocyanin increased

by 38.0%, 136.0% and 53.0% in Varuna and 44.0%,
25

82.0% and 28.0% in RH-30 with 25, 50 and

100 mg Cd kg
1 soil in comparison to the control.
1.2770.02a
0.5970.01a

0.5270.01a
0.5270.01a
11.2870.45a
0.8070.04a
19.5870.48a

3.1670.29a
2.157.04a

An increase in pheophytin was observed in both the

27679a

cultivars with increasing Cd concentration. How-

ever, no significant increase was noted beyond
50 mg Cd kg
1 soil in RH-30 (Table 1).
0

The TBARS content increased by 19.7%, 217.0%

Cadmium (Cd) treatments (mg kg
1 soil)

Rubisco (mmol CO2 mg
1 protein min
1)

and 230.0% in comparison to the control in Varuna

with 25, 50 and 100 mg Cd kg
1 soil. However, the
increase in the TBARS in RH-30 was 53.0%, 282.0%
Anthocyanin (A530–0.25A667 g
1)

and 307.0% in the respective Cd treatments in

comparison to the control (Fig. 2A). The degree of
membrane disruption as represented by electrolyte
leakage increased by 187.0% and 372.0% in Varuna
CA (U mg
1 protein)
Percent pheophytin

gs (mmol m
2 s
1)

PN (mmol m
2 s
1)

E (mmol m
2 s
1)

and 83.0% and 308.0% in RH-30 with 25 and

Chl b (mg g
1)

50 mg Cd kg
1 soil in comparison to the control,

Chl a (mg g
1)

respectively. However, the release of electrolytes

was less at 100 mg Cd kg
1 soil, which was 47.0% in
Chl a:b

Varuna and 96.0% in RH-30 (Fig. 2B). The produc-

tion of H2O2 in the Varuna increased by 120.0%,
 ARTICLE IN PRESS
6 M. Mobin, N.A. Khan

50 0 Cd (A) a 100 mg Cd kg
1 soil, respectively, in comparison to

TBARS content (nmolg-1 FW)
a
25 Cd the control plants (Fig. 3B). The activity of APX in
50 Cd a
40 100 Cd
a the control plants was higher in Varuna compared
to RH-30. APX activity was enhanced by 215.0%,
30 369.0% and 328.0% in Varuna and 161.0%, 230.0%
and 303.0% in RH-30 in comparison to the control
20 b
b with 25, 50, 100 mg Cd kg
1 soil (Fig. 3C). GR
b c
activity was higher in Varuna than RH-30 even
10
without Cd treatment. The increase in GR activity
0 was 122.0%, 144.0% and 136.0% with 25, 50 and
100 mg Cd kg
1 soil for Varuna, and 124.0%, 132.0%
8
(B) a and 79.0% for RH-30, respectively, in comparison to
7 the control (Fig. 3D).
Electrolyte leakage (%)

6
a
5
4
3
b b Discussion
2 c
c
Plant species and genotypes significantly differ in
1 d
d the uptake of Cd and its subsequent translocation
0 from roots into shoots (Metwally et al., 2005; Salt
et al., 1995). In our study, Varuna accumulated less
(C) a a
300 Cd in both roots and leaves than RH-30 (Fig. 1A and
B). The accumulation of Cd in roots and shoots
250
H2O2 (µmol g-1 FW)

depends on binding to extracellular matrix (Horst,

200 ab
a 1995), complexing inside the cell (Cobbett et al.,
b
150
100
c
50
0
Varuna
Figure 2. TBARS content (A), % electrolyte leakage (B)
and H2O2 content (C) in leaves of Varuna (high photo-
synthetic capacity) and RH-30 (low photosynthetic
capacity) cultivars of mustard (Brassica juncea L.)
treated with cadmium concentrations. Results are means
of five replications7SE. The data followed by different
letters are significantly different at Pp0:05.
131.0% and 146.0% in 25, 50 and 100 mg Cd kg
1
soil, while the increase in H2O2 in RH-30 was
134.0%, 248.0% and 250.0%, respectively, in com-
parison to the control (Fig. 2C).
Cd treatment of plants enhanced the SOD activity
in Varuna as well as in RH-30. The enzyme activity
was increased by 118.0%, 145.0% and 142.0% in
Varuna, whereas the increase was 169.0%, 190.0%,
and 193.0% in RH-30 with 25, 50 and 100 mg Cd kg
1
soil, respectively, in comparison to the control
(Fig. 3A). CAT and APX mediate in scavenging high
levels of H2O2. The level of induction in CAT activity
was 141.0%, 142.0% and 134.0% in Varuna and
104.0%, 128.0% and 176.0% in RH-30 with 25, 50 and
b
1998) and on the transport efficiency (Marchiol
et al., 1996). Further, the transport efficiency
c
relies on transpiration rate and thereby on stomatal
conductance. It was observed that exposure to Cd
influenced the stomatal conductance and transpira-
tion rate in both the cultivars, but to a significantly
RH-30 greater extent in RH-30. Likewise, net photosyn-
thetic rate was affected by Cd stress. It has been
shown that light and dark reactions of photosynth-
esis are suppressed by heavy metals at different
target sites (Krupa and Baszynski, 1995). In RH-30,
the decrease in the rate of photosynthesis in
response to Cd treatment was accompanied by an
increase in the stomatal conductance but in Varuna
it remains unaltered (Table 1). The present result
indicates that activities of CA and Rubisco declined
in both the cultivars, but Varuna maintained higher
levels of these enzymes than RH-30 at all Cd
treatments. Hence, the mechanism of photosyn-
thetic response involved both stomatal and non-
stomatal effects under Cd stress. Siedlecka et al.
(1997) reported that Cd affects photosynthesis by
inhibiting different reaction steps of Calvin-cycle.
However, Di Cagno et al. (2001) observed significant
reduction in CO2-assimilation rate and Rubisco
activity while stomatal conductance and Fv:Fm
ratio remain unchanged when sunflower plants
were grown in the presence of Cd.
The decrease in total Chl, Chl a and Chl b was in a
dose-dependent manner in both the cultivars. The
 ARTICLE IN PRESS
Photosynthesis and oxidative stress in cadmium-treated mustard 7

16 240
(A) (B)
Varuna Varuna
RH-30 220 RH-30
SOD Units (mg-1protein min-1)
14

CAT Units (mg-1protein min-1)

200
12
180
10
160
8
140

6 120

4 100
0 25 50 100 0 25 50 100
Cadmium (mg kg-1 soil) -1
Cadmium (mg kg soil)

(C) (D)
2.8 Varuna Varuna
RH-30 0.24
RH-30
APX Units (mg-1protein min-1)

2.4

2.0 GR Units (mg-1protein min-1) 0.20

1.6
0.16
1.2

0.8
0.12
0.4

0.0 0.08
0 25 50 100 0 25 50 100
-1 -1
Cadmium (mg kg soil) Cadmium (mg kg soil)

Figure 3. SOD (A), CAT (B), APX (C) and GR (D) activities in leaves of Varuna (high photosynthetic capacity) and RH-30
(low photosynthetic capacity) cultivars of mustard (Brassica juncea L.) treated with cadmium concentrations. Results
are means of five replications7SE. The data followed by different letters are significantly different at Pp0:05.

reduction in Chl b was extremely sharp in Varuna
which resulted in higher Chl a:b ratio as
the concentration of Cd increased in the soil
(Table 1). The increases in the ratio of Chl a:b
have been linked with the change in pigment
composition of photosynthetic apparatus which
possesses lower level of light harvesting chlorophyll
proteins (LHCPs) (Loggini et al., 1999). The reduc-
tion in LHCPs content is an adaptive defense
mechanism of chloroplasts, leaves and plants which
allows them to endure the adverse conditions
(Asada et al., 1998).
Induction of higher level of relative amount of
anthocyanin in response to Cd stress was observed
in both the cultivars (Table 1), although it increased
greatly in Varuna in comparison to RH-30. Several
attempts have been made to explain the accumula-
tion of anthocyanin in leaves under stress (Gould
et al., 1995, 2002; Krupa et al., 1996). The
anthocyanin synthesis takes place in cytoplasm,
rapidly transported into the cell vacuoles (Marrs
et al., 1995) and is not in direct contact with the
stress-generated activated oxygen species. In fact,
cytosolic and organelle-bound antioxidants act as a
primary cellular defense system rather than the
vacuolar anthocyanins. Our findings indicate that
accumulation of anthocyanins may be only of
secondary importance in living cells. Nonetheless,
induction of anthocyanin accumulation might help
in the protection of photosynthetic apparatus by
screening it from deleterious effects of stress-
generated superoxide radicals without limiting
photosynthesis. Percent pheophytin remained rela-
tively much higher in RH-30 than Varuna at higher
Cd concentration in the soil, but at lower Cd level
percent conversion of chlorophyll to pheophytin
 ARTICLE IN PRESS
8 M. Mobin, N.A. Khan
was almost identical in both the cultivars (Table 1).
Küpper et al. (1996) carried out experiments with
submerged water plants and observed that the
substitution of Mg2+ ion in the chlorophyll molecule
by toxic heavy metals, such as Cu, Zn, Cd or Hg,
resulted in an abrupt cessation of photosynthesis.
Although Cd is not a transition metal like Fe and Cu,
and therefore, it is not capable of generating AOS
by catalyzing Haber–Weiss or Fenton type reactions
(Deckert, 2005). Nevertheless, Cd toxicity results
from the alteration of oxidant levels in plants
(Foyer and Noctor, 2005). Accumulation of Cd was
correlated with the generation of AOS in sensitive
clones of Holcus lanatus (Hendry et al., 1992). Cd
has been shown to elevate lipid peroxidation via
AOS formation in plants (Halliwell and Gutteridge,
1989). Cd-dependent induction of TBARS was
observed in the leaves of both the cultivars
(Fig. 2A), which is an index of lipid peroxidation
and thereby oxidative stress. It has been observed
that exposure to Cd increased lipid peroxidation in
Pisum sativum (Chaoui et al., 1997; Metwally et al.,
2005), rice (Chien et al., 2001) and sunflower
seedlings (Gallego et al., 1996). The production of
lipid peroxides was lower in Varuna compared to
RH-30 at all Cd concentrations. This could be
explained in terms of higher degree of protection
against oxidative damage in Varuna by fast removal
of H2O2 or by other scavenging systems. The
enhancement in the activities of antioxidative
enzymes was negatively correlated with the level
of TBARS content.
As a sequel to Cd-catalyzed AOS generation,
disruption of membrane stability, enhanced perme-
ability and inactivation of proteins take place
(De Vos et al., 1993). The increase in TBARS
content in the present study induced electrolyte
leakage in a dose-dependent manner (Fig. 2B). The
enhanced cellular damage in RH-30 seems to reflect
deterioration on the equilibrium between genera-
tion of AOS and defense mechanisms towards
removal of AOS. The electrolyte leakage through
plasmalemma has been associated with depressed
photosynthetic and mitochondrial activity (Ristic
et al., 1996).
H2O2 generation is induced in plants following
exposure to a wide variety of abiotic and biotic
stimuli (Karpinski et al., 1999; Lamb and Dixon,
1997). The level of H2O2 was lower in Varuna than
RH-30 even in the presence of Cd, which implies
that the generation of H2O2 was quenched by the
efficient antioxidative mechanism of Varuna. In RH-
30, the level of H2O2 increased in a dose-dependent
manner (Fig. 2C), which is indicative of higher
oxidative stress imposed by Cd in soil. It is most
likely that the presence of Cd inhibited the H2O2-
scavenging system, i.e. GR, CAT and APX and
increased activity of SOD (Fig. 3A–D), which
catalyzes the conversion of superoxide anion to
O2 and H2O2 (Sudhakar et al., 2001). There was
more pronounced increase in the SOD activity in
RH-30 with the increase in Cd levels, which possibly
generated higher level of superoxide radicals and
resulted in higher cellular damage in comparison to
Varuna. Gossett et al. (1994) reported that higher
SOD activity without complementary increase in
the ability to scavenge the formed H2O2 can result
in the increased cellular damage.
The detoxification of H2O2 takes place by
involvement of ascorbate–glutathione cycle, where
H2O2 sends a systemic signal for the induction of
APX (Karpinski et al., 1999). In the present study,
the comparison of the absolute enzymatic values in
the Cd stressed RH-30 plants indicated that APX
level kept on increasing with the increasing levels
of Cd while in Varuna the sub-lethal concentration
of Cd in the soil induced the maximum level of APX.
The increase in CAT activity is considered as an
indirect evidence of an enhanced oxidative damage
(Smirnoff, 1995). We found dose-dependent in-
crease of CAT activity, which further indicated the
oxidative damage from Cd treatments in both the
cultivars. Varuna was found to possess higher level
of CAT activity both constitutively and inductively.
However, the CAT activity was found to be induced
nearly 1.5 times more in RH-30 than in Varuna
at 100 mg Cd kg
1 soil, which further pointed
towards increased oxidative stress experienced
by RH-30.
GR catalyzes the last rate-limiting step of
ascorbate–glutathione cycle. This enzyme main-
tained high ratio of GSH/GSSG which is required for
the regeneration of ascorbate and for the activa-
tion of several CO2-fixing enzymes (Noctor and
Foyer, 1998). Contrary to APX gene expression,
which is activated by H2O2, GR is stimulated by
different stimuli (Schützendübel et al., 2001). This
is well correlated in the present study, where the
constitutive and Cd-induced GR activity was re-
markably higher in Varuna. The reduction in GR
activity of RH-30 was prominent at 100 mg Cd kg
1
soil. The cooperative action of APX and GR in
Varuna suggested the presence of more efficient
ascorbate–glutathione cycle, which resulted in the
development of higher tolerance to Cd.
It is concluded that deleterious effect of Cd-
generated AOS on photosynthetic characteristics
was more conspicuous in RH-30 than Varuna.
The greater tolerance in Varuna to Cd was due to
better synergies between the antioxidative
enzymes which help to protect the photosynthetic
apparatus.
 ARTICLE IN PRESS
Photosynthesis and oxidative stress in cadmium-treated mustard
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