J Plant Growth Regul

DOI 10.1007/s00344-016-9592-3

Exogenous Application of Selenium Mitigates Cadmium Toxicity

in Brassica juncea L. (Czern & Cross) by Up-Regulating
Antioxidative System and Secondary Metabolites
Parvaiz Ahmad1 E. F. Abd Allah2 Abeer Hashem3,4 Maryam Sarwat5
Salih Gucel6

Received: 20 December 2015 / Accepted: 18 January 2016

 Springer Science+Business Media New York 2016

Abstract The main aim of the present study was to

examine the role of selenium (Se) in ameliorating the toxic
effect of cadmium (Cd) in mustard (Brassica juncea)
plants. The plants exposed to elevated levels of Cd
exhibited reduced biomass, pigment content, and relative
water content (RWC). However, supplementation of Se
restores the negative effect of Cd and increases biomass,
pigment content, and RWC. Osmolyte (proline and glycine
betaine) and sugar content were increased under Cd stress
and further increase was observed with addition of Se. Cd
decreased protein content and supplementation of Se
increases it to appreciable levels. Cd also increased production of H2O2 and lipid peroxidation, electrolyte leakage,
and the activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, and glutathione
reductase. Supplementation of Se decreased accumulation
of H2O2 and lipid peroxidation, increased the activities of

& Parvaiz Ahmad

parvaizbot@yahoo.com
1

Department of Botany, S.P. College, Srinagar,

Jammu and Kashmir 190001, India

Department of Plant Production, Faculty of Food &

Agricultural Sciences, King Saud University, Riyadh, Saudi
Arabia

Department of Botany and Microbiology, Faculty of Science,

King Saud University, Riyadh 11451, Saudi Arabia

Mycology and Plant Disease Survey Department, Plant

Pathology Research Institute, Agriculture Research Center,
Giza, Egypt

Pharmaceutical Biotechnology, Amity Institute of Pharmacy,

Amity University, Noida, Uttar Pradesh 201303, India

Centre for Environmental Research, Near East University,

Lefkosa (Nicosia), The Northen Cyprus

antioxidant enzymes to greater levels, and regulates Cd

accumulation in roots and shoots. Ascorbic acid (AsA) and
flavonoids decreased with elevated concentrations of Cd;
however, tocopherol and total phenols were increased with
the same concentrations of Cd. Se application maintains
AsA and flavonoid content, and further increase in tocopherol and total phenols were observed with Se in the
present study. Overall the results confirm that exogenous
application of Se mitigates the negative effects of Cd stress
in mustard plants through the regulation of osmoprotectants, antioxidant enzymes, and secondary metabolites.
Keywords Cadmium stress  Selenium  Biochemical
attributes  Lipid peroxidation  Antioxidants  Flavonoids

Introduction
Selenium (Se) acts as a cofactor for the enzymes of many
biochemical pathways in animals and plants (Watts 1994).
In humans, Se deficiency is directly associated with disorders, like, Keshan disease, KashinBeck diseases, cancer,
cardiovascular diseases, liver diseases, and cataracts
(Tapiero and others 2003; Cox and Bastiaans 2007). In plant
species, supplementation of Se has shown enhanced tolerance to abiotic stresses (Hasanuzzaman and others 2011;
Hasanuzzaman and Fujita 2011, 2012b; Hasanuzzaman and
others 2014). Selenium was also reported to counter the
deleterious effects of cadmium toxicity in plants and restores
the damage (Battin and Brumaghim 2009). Se has been
shown to inhibit oxidative DNA damage caused by iron in
presence of hydrogen peroxide (Ramoutar and Brumaghim
2007), thereby enhancing tolerance to different types of
abiotic stresses, including metal stress. Selenium with the
help of selenoproteins is able to neutralize reactive oxygen

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J Plant Growth Regul

species and, therefore, protects cellular components from

oxidative damage. These selenoproteins have glutathione
peroxidase and thioredoxin reductase activities. Other systems independent of glutathione peroxidase activity are also
concerned with the defense action of selenium against
metal-induced oxidative stress (Valko and others 2006).
This evidence reveals that selenium plays an important role
in decreasing the detrimental effects of environmental stress
in plants (Kumar and others 2012).
Cadmium (Cd) is an important metal that is highly toxic
due to its detrimental impact on animals, humans, and plants
(Ahmad and others 2011a, 2012; Hasanuzzaman and Fujita
2012a, 2013). The non-ferrous metal industry, fossil fuel
combustion, and waste incineration contribute to Cd pollution. When this cadmium enters the human body through
agricultural produce and other ways, it causes deleterious
effects to kidneys, pulmonary functions, neurotoxicity,
endocrine disruption, and so on (Godt and others 2006).
Cadmium is also a reported human carcinogen (Stohs and
others 2000). Plants exposed to high concentrations of cadmium exhibit multifarious negative responses including
impairment of seed germination, growth inhibition, and
inhibition in the synthesis of photosynthetic pigments thus
lowering photosynthesis as well as reduction in yield (Hernandez and Cooke 1997; Ahmad and others 2011a, 2012),
inhibition of the plant water relationship and mineral nutrition
has also been reported (Mishra and others 2006). Cd toxicity
easily induces the generation of reactive oxygen species
(ROS), which in turn causes lipid peroxidation and other
biomolecular dysfunctions through oxidative stress (Ahmad
and others 2008, 2010, 2011b). Activities of enzymatic
antioxidants (superoxide dismutase, SOD; ascorbate peroxidase, APX; glutathione reductase GR and catalase CAT) and
non-enzymatic antioxidants (ascorbic acid, AsA and a-tocopherol) are up- and down-regulated in plants during oxidative
stress (Ahmad and others 2008, 2010, 2011b, 2015; Jaleel
2009). These antioxidants have the ability to protect plants
from oxidative damage. Phenolics have been observed to
accumulate under different environmental stress conditions in
plants (Diaz and others 2001; Sakihama and Yamasaki 2002).
Phenols have been reported to have the antioxidant property
that could reduce the formation of ROS (Jung and others
2003). Hashem and others (2015) have also reported the
accumulation of high levels of phenol in Solanum lycopersicum under Cd stress. Ahmad and others (2015) also reported
a positive correlation between the accumulation of phenol
content and Cd stress in Cannabis sativa.
Brassica juncea (Indian mustard) belongs to family
Brassicaceae is one of the most important oil seed crops of
the world. Its productivity is decreasing due to the negative
effects of different abiotic stresses. Cd adversely affects the
growth and physiobiochemical attributes of mustard plants,
which ultimately lead to decreased crop production

123

(Ahmad and others 2015). The present study deals with the
amelioration of cadmium toxicity by the exogenous
application of selenium in mustard plants. This new strategy could be used in bringing the marginal lands affected
with metals under cultivation to meet the needs of future
generations.

Materials and Methods

Seeds of B. juncea (L.) Czern. & Coss. cv. Varuna were
surface sterilized in a 5 % solution of sodium hypochlorite
(NaOCl) for 10 min. The sterilized seeds were sown as 10
pot-1 in plastic pots containing 5 kg sand plus vermicompost
(3:1) under glasshouse conditions. The mean day temperature was 28.6 5.1 C, night temperature 16.3 7.6 C,
photoperiod from 8 to 11 h, and average relative humidity
(RH) 35.9 6.5 during the entire growth period. When the
seedlings emerged from the sand, they were thinned to three
per pot. Hoaglands nutrient solution (full strength) was
supplied to all pots for 10 days and then different cadmium
(CdSO4 8H2O) treatments (0, 100 and 200 mg L-1) in
Hoaglands nutrient solution were initiated. The Cd treatment solutions were applied every alternate day after
leaching well the metal and nutrient solution already present
in the sand. To maintain the moisture content of the sand,
200 ml of distilled water was applied to each pot every day.
Se (50 lM) mixed with Tween-20 was sprayed in the evening with a manual sprayer (10 ml plant-1) to plants every
alternate day from the 7th day of treatment up to day 45. The
experiment was laid out in a completely randomized design
with five replicates. The plant leaves were collected for
analyses at 45 days after treatment (DAT). The treatments
were given as follows:
0: Nutrient solution alone (control);
0 ? Se: 0 mg L-1 Cd ? 50 lM Se;
100: 100 mg L-1 Cd;
100 ? Se: 100 mg L-1 Cd ? 50 lM Se;
200: 200 mg L-1 Cd;
200 ? Se: 200 mg L-1 Cd ? 50 lM Se.
Determination of Fresh Weight and Dry Weight
Leaves and roots were separated and their fresh weights
(FWs) were directly determined. For dry weight (DW)
determination, the leaves and roots were dried at 70 C in
oven for 48 h and then weighed.
Measurement of Gas Exchange Parameters
Net CO2 assimilation rate (A), stomatal conductance (gs),
and transpiration rate (E) of a fully expanded youngest leaf

J Plant Growth Regul

of each plant were measured using a portable infrared gas

analyzer (LCA-4 model, Analytical Development Company, Hoddesdon, England).

The chromatophore containing toluene was then aspirated

from the aqueous phase, and its absorbance was determined
spectrophotometrically at 520 nm (Beckman 640 D, USA)
using toluene as a blank.

Determination of Electrolyte Leakage

Determination of Protein
Twenty leaf disks were taken in a test tube containing
10 ml of deionized water and electrical conductivity (EC)
was measured (ECa) following the method of DionisioSese and Tobita (1998). The contents were heated at 50
60 C for 25 min in a water bath and EC values were
measured (ECb). Later, the contents were boiled at 100 C
for 10 min and again EC was recorded (ECc). The electrolytic leakage was calculated using the formula:
Electrolyte leakage % ECb  ECa = ECc  100:

Proteins were estimated by the method of Bradford (1976).

Fresh leaves (0.5 g) were homogenized in 1 ml phosphate
buffer (pH 7.0). The crude homogenate was centrifuged at
50009g for 10 min. A half ml of freshly prepared trichloroacetic acid (TCA) was then added and the mixture
was centrifuged at 80009g for 15 min. The precipitate was
dissolved in 1 ml of 0.1 N NaOH and 5 ml Bradford
reagent was then added. Absorbance was recorded spectrophotometrically at 595 nm (Beckman 640 D, USA)
using bovine serum albumin as the blank.

Determination of Leaf Relative Water Content

Determination of Glycine Betaine
Leaf disks of 10 mm diameter were excised from the
interveinal areas of each plant for the measurement of
relative water content. For each replicate, 20 disks were
pooled and their FW was determined. They were floated on
distilled water in Petri dishes for 4 h to regain turgidity,
then thawed and reweighed (TW). The samples were dried
at 80 C for 48 h to determine the DW. Tests showed that
complete hydration of leaf disks occurred within 4 h. RWC
was calculated using the following formula (Smart and
Bingham 1974):
RWC % FW DW=TW DW  100:
Determination of Chlorophyll Content
Chlorophyll (chl) content was determined by the method of
Hiscox and Israelstam (1979). Fresh material (100 mg) was
kept in dimethyl sulfoxide (DMSO). Tubes were kept in an
oven at 65 C for 40 min. A 1-ml aliquot was further
mixed with 2 ml DMSO and then vortexed. Absorbance
was determined spectrophotometrically at 645, and 663 nm
(Beckman 640 D, USA) using DMSO as a blank.
Determination of Proline
The proline content was determined using the method of
Bates and others (1973). Fresh material (300 mg each
sample) was homogenized in 10 ml of 3 % aqueous sulfosalicylic acid. The homogenate was centrifuged at
90009g for 15 min. A 2-ml aliquot of supernatant was
mixed with an equal volume of glacial acetic acid and acid
ninhydrin and incubated for 1 h at 100 C. The reaction
was terminated by putting it on an ice bath and extracted
with 4 ml of toluene. The extract was vortexed for 20 s.

Glycine betaine was measured according to Grieve and

Grattan (1983). Dried and finely ground plant material
(500 mg) was mechanically shaken with 20 ml deionized
water for 24 h at 25 C. The samples were then filtered and
the filtrates were diluted (1:1) with 2N H2SO4. Aliquots of
0.5 ml were taken in centrifuge tubes and cooled in an ice
bath for 1 h. Cold KII2 reagent (0.20 ml) was added and
then reactants were gently stirred. The tubes were stored at
4 C for 16 h and then centrifuged at 10,000 rpm for
15 min at 0 C. The supernatant was carefully aspirated
with a fine-tipped glass tube. The periodide crystals were
dissolved in 9.0 ml of 1, 2-dichloroethane and mixed vigorously. After 2 h, the absorbance was measured at 365 nm
using a spectrophotometer. A reference standard of glycine
betaine (50200 lg ml-1) was prepared in 1 N H2SO4.
Determination of Sugar
Sugar was estimated by the method of Dey (1990). Fresh
leaves of 500 mg were extracted twice with hot ethanol
(90 %). The two extracts were then mixed and the volume
was made to 25 ml with double-distilled water (DDW). To
the aliquot, 1 ml 5 % phenol and 5 ml concentrated sulfuric acid were added and the final volume was made to
10 ml with DDW. Absorbance was measured at 485 nm
using a UVVis spectrophotometer (Beckman 640 D,
USA).
Determination of H2O2
The hydrogen peroxide content was determined according
to Velikova and others (2000). A fresh fully expanded
youngest leaf from top (500 mg) was taken and

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J Plant Growth Regul

homogenized with 5 ml of 0.1 % (w/v) trichloroacetic acid

(TCA). The extract was centrifuged at 12,000 rpm for
15 min, and 0.5 ml of the supernatant was added to 0.5 ml
of 10 mM potassium phosphate buffer (pH 7.0) and 1 ml of
1 M potassium iodide (KI) solution. The absorbance of the
supernatant was read at 390 nm.
Determination of Lipid Peroxidation
Malondialdehyde (MDA) content (a measure of lipid peroxidation) was determined by the method of Rao and
Sresty (2000). A leaf sample (500 mg) was homogenized
with 2.5 ml of trichloroacetic acid (0.1 %) and the homogenate was centrifuged at 10,0009g for 10 min. One ml of
the aliquot was mixed with 4 ml of 20 % trichloroacetic
acid and 0.5 % of thiobarbituric acid (TBA). The mixture
was heated at 95 C for 30 min, cooled in an ice bath and
then centrifuged at 10, 0009g for 15 min. The absorbance
was measured at 532 nm. Measurements were corrected for
unspecific turbidity by subtracting the absorbance at
600 nm.
Antioxidant Enzymes
Enzyme Extraction
Leaves (0.5 g) were homogenized in 50 mM sodium phosphate
buffer (pH 7.0) containing 1 % soluble polyvinylpyrrolidine
(PVP). The homogenate was centrifuged at 20,0009g for
15 min at 4 C and the supernatant was used for the assays of
activities of SOD (EC 1.15.1.1), APX (EC 1.1.1.11), CAT (EC
1.11.1.6) and GR (EC 1.6.4.2).
Enzyme Assay
Superoxide Dismutase
The activity of SOD was assayed by monitoring its ability
to inhibit the photochemical reduction of NBT (Van Rossum and others 1997). SOD activity was expressed as unit
mg-1 protein. One unit of SOD was defined as the amount
of protein causing a 50 % decrease of NBT photoreduction
inhibition rate.
Catalase
The method of Luck (1974) was employed for the assay of
catalase (CAT, EC 1.11.1.6). The enzyme extract (50 ll)
was added to 3 ml of 20 mM hydrogen peroxide and
50 mM phosphate buffer (pH 7.0) solution. The decrease in
absorbance was measured at 240 nm. The enzyme activity
was calculated using the extinction coefficient of
36 9 103 mM-1 cm-1 and expressed as unit mg-1 protein.

123

Ascorbate Peroxidase
The method of Nakano and Asada (1981) was used for the
assay of APX (EC 1.11.1.11) activity. The assay mixture
contained 1.0 ml of reaction buffer (potassium phosphate
(pH 7.0) with 0.1 mM EDTA, 0.5 mM ascorbate, 0.1 mM
H2O2, and 0.1 ml of enzyme extract. APX was assayed as a
decrease in absorbance at 290 nm of ascorbate. APX
activity was expressed as unit mg-1 protein. For the calculation of APX enzyme activity, the extinction coefficient
of 2.8 mM-1 cm-1 was used. One unit of enzyme was
considered as the amount necessary to decompose 1 lmol
of substrate per min at 25 C.
Glutathione Reductase
Glutathione reductase activity was assayed as per the
method of Foster and Hess (1980). The reaction mixture
consists of enzyme extract, 100 mM potassium phosphate
buffer (pH 7.0) containing 1.0 mM EDTA, 150 lM
NADPH, and 500 lM oxidized glutathione. The enzyme
activity was measured at 340 nm. Activity was calculated
using the extinction coefficient for NADPH of 6.22 mM-1
cm-1 and expressed as lmol NADPH oxidized mg-1
protein min-1.
Estimation of Ascorbic Acid and a-Tocopherol
Fresh leaves (0.5 g fresh weight) were homogenized in
3 ml ice-cold acidic extraction buffer (5 % meta-phosphoric acid containing 1 mM EDTA) using a mortar and
pestle. Homogenates were centrifuged at 11,500 9 g for
15 min at 4 C and the supernatants were collected for
ascorbate analysis following the method of Huang and
others (2005).
The method of Backer and others (1980) was used for
the estimation of a-tocopherol. Fresh leaves (0.5 g) were
homogenized in presence of petroleum ether and ethanol
(2:1, v/v). The homogenate was centrifuged at 10,000 rpm
for 20 min. The mixture containing 1 ml of supernatant
and 0.2 ml of 2 % 2, 2-dipyridyl in ethanol was placed in
the dark for 5 min. After the mixture turned red, 4 ml of
distilled water was added and the absorbance was measured at 520 nm. The a-tocopherol was calculated using a
standard curve with known concentrations of atocopherol.
Estimation of Total Phenols and Flavonoids
The leaf sample (5 g) was ground and extracted with
methanol at room temperature. The extract containing
phenolic content was reduced by the FolinCiocalteu
reagent using the method of Chun and others (2003). The

J Plant Growth Regul

total phenolic content was expressed as mg gallic acid

equivalent (GAE) g-1 of extract (mg g-1).
The flavonoid content was estimated as per the colorimetric method of Zhishen and others (1999). Catechin was
used as standard for the calibration curve. The absorbance
was measured at 510 nm and flavonoid content was
expressed as mg catechin equivalents g-1 of extract.
Estimation of Cd and Se Accumulation
The shoot and root material (100 mg) were ground to
powder and digested in H2SO4/HNO3 mixture (1/5, v/v) for
24 h. After that, the mixture was subjected to nitric-perchloric acid HNO3/HClO4 mixture (5/1, v/v) treatment. The
Cd and Se in the solution were determined using a PerkinElmer (Analyst Model 300) atomic absorption spectrophotometer. The metal content was expressed as lmol g-1 DW.

of Se minimizes the negative effect of Cd and the decrease

in root length was observed to be only 23.38 % at
100 mg L-1 Cd ? Se and 41.90 % at 200 mg L-1
Cd ? Se concentrations as compared to control (Table 1).
The fresh and dry weights of shoots and roots were also
decreased with the increase in Cd concentration and the
maximum decrease was observed at 200 mg L-1 Cd concentration as compared to the control. Application of Se in
combination with Cd restores the negative effect of Cd
(Table 1). A non-significant increase in growth was
observed by the application of Se to control plants.
Total chlorophyll content was decreased by 26.43 and
42.29 % at 100 and 200 mg L-1 Cd stress, respectively, in
comparison to control. Application of Se showed minimum
decreases of 14.53 % at 100 mg L-1 Cd ? Se and
25.11 % at 200 mg L-1 Cd ? Se as compared to the
control plants (Table 2) indicating that Se restores the
negative effect of Cd on chlorophyll content.

Statistical Analysis
Statistical analysis was performed using one-way analysis
of variance (ANOVA) followed by Duncans Multiple
Range Test (DMRT). The values are mean S.D. for five
samples in each group. P values B0.05 were considered as
significant.

Results
Se Improved Growth, Biomass, and Pigment
Content of Mustard Plants Under Cd Stress
The effect of Cd and Se on shoot and root growth and their
biomass is shown in Table 1. The length of shoots was
decreased by 25.27 and 33.07 % at 100 and 200 mg L-1 Cd
concentrations, respectively. Root length also showed a
decrease of 35.55 % at 100 mg L-1 Cd and 56.51 % at
200 mg L-1 Cd concentrations. However, when Cd-stressed
plants were supplemented with Se, the length of both the
shoot and root was significantly increased. Supplementation

Se Maintains CO2 Assimilation, Stomatal

Conductance, and Transpiration Rate in Mustard
Plants Suffering from Cd Stress
The assimilation of CO2 (A) was decreased by 37.11 and
48.45 % at 100 and 200 mg L-1 Cd concentrations, respectively. However, supplementation with Se in these Cd-stressed plants improved the CO2 assimilation. The decrease was
observed only 16.77 % at 200 mg L-1 Cd ? Se (Table 2).
Stomatal conductance (gs) showed 13.95 % and 37.28 %
increases at 100 and 200 mg L-1 Cd treatments respectively,
whereas the increment was only 5.12 % at 100 mg L-1 Cd
?Se and 27.45 % at 200 mg L-1 Cd ? Se treatments
(Table 2). An increase of 21.46 and 36.34 % in transpiration
rate (E) was observed in mustard seedlings subjected to 100
and 200 mg L-1 Cd stress, respectively. However, when the
seedlings were treated with Se, the increase was only 5.64 %
at 100 mg L-1 Cd ? Se and 28.06 % at 200 mg L-1
Cd ? Se (Table 2). The results clearly showed a positive role
of Se in alleviating the negative effects of Cd on the physiological attributes of B. juncea.

Table 1 Effect of Se (50 lM) on growth and biomass yield in mustard plants under different concentrations of Cd stress
Treatments
(mg L-1 Cd)

Shoot length
(cm)

Root length
(cm)

Shoot FW (g
plant-1)

Shoot DW (g
plant-1)

Root FW (g
plant-1)

Root DW (g
plant-1)

44.71 1.71a

24.72 1.33a

42.44 1.69a

10.22 0.72a

18.94 1.1a

5.12 0.39a

0 ? Se

45.33 1.72a

25.71 1.34a

43.96 1.7a

10.51 0.73a

19.36 1.1a

5.43 0.39a

100

33.41 1.50c

15.93 0.85c

30.17 1.40c

6.73 0.47c

9.40 0.65c

3.37 0.15c

100 ? Se

36.51 1.62b

18.94 1.1b

38.91 1.55b

8.95 0.61b

13.92 0.93b

4.58 0.29b

200

29.92 1.31e

10.75 0.61d

24.74 1.25d

4.51 0.24e

7.34 0.52d

2.52 0.19d

200 ? Se

31.26 1.40d

14.36 0.79c

29.72 1.39c

5.54 0.35d

10.37 0.69c

3.75 0.21c

Data are mean SE (n = 5). Different letters next to the numbers indicate significant difference (P \ 0.05)

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J Plant Growth Regul

Table 2 Effect of Se (50 lM) on total chlorophyll, CO2 assimilation (A), stomatal conductance (gs), transpiration rate (E), electrolyte leakage,
and RWC in mustard plants under different concentrations of Cd stress
Treatments
(mg L-1
Cd)

Total
chlorophyll
(mg g-1 FW)

CO2 assimilation
(A) (lmol CO2
m-2 S-1)

Stomatal conductance
(gs) (mmol CO2 m-2
S-1)

Transpiration
rate (E) (mmol
H2O m-2 S-1)

Electrolyte
Leakage %

2.27 0.15a

12.61 0.74a

0.37 0.003e

5.31 0.38a

0 ? Se

2.33 0.15a

12.91 0.74a

0.39 0.003e

5.53 0.39a

9.21 0.65e

92.11 2.79a

100

1.67 0.08c

7.93 0.58c

0.43 0.006c

4.17 0.29b

31.74 1.35c

62.73 2.53c

9.33 0.65e

RWC %

91.50 2.79a

100 ? Se

1.94 0.11b

9.72 0.63b

0.39 0.003d

5.01 0.37a

20.11 1.22d

71.44 2.61b

200

1.31 0.03d

6.50 0.50d

0.59 0.009a

3.38 0.15d

51.34 2.52a

39.12 1.64e

200 ? Se

1.70 0.09c

7.81 0.57c

0.51 0.005b

3.82 0.21c

39.42 2.13b

48.81 1.93d

Data are mean SE (n = 5). Different letters next to the numbers indicate significant difference (P \ 0.05)

Se Restores Electrolyte Leakage and Relative Water

Content of Mustard Plants Under Cd Stress
Cd stress increases the electrolyte leakage in mustard
seedlings by 81.82 % at 200 mg L-1 Cd stress (Table 2).
Supplementation of Se with Cd decreases this effect and
the maximum increase was only 76.33 % as compared to
the control. RWC decreased by 31.44 % at 100 mg L-1 Cd
and 57.24 % at 200 mg L-1 Cd stress; however, Se in
combination with Cd improves this effect and the decrease
was only 21.92 and 46.65 % at 100 mg L-1 Cd ? Se and
200 mg L-1 Cd ? Se treatments, respectively, in comparison to the control (Table 2). The data indicated that Se
improved the electrolyte leakage and RWC in mustard
plants under Cd stress.
Effect of Se on Proline, Protein, Glycine Betaine,
and Soluble Sugars in Cd-Treated Mustard Plants
Proline (Pro) increases with the increase in Cd concentration and the maximum increase was 52.87 % at 200
mg L-1 Cd stress. Application of Se further increases the
Pro content by 49.68 and 60.80 % at 100 mg L-1 Cd ? Se
and 200 mg L-1 Cd ? Se treatments, respectively, as
compared to control (Fig. 1a). The increase in protein
content with respect to 200 mg L-1 Cd stress is 63.98 %
and 200 mg L-1 ? Se treatment increases the protein
accumulation by 35.42 % as compared to 200 mg L-1 Cd
(Fig. 1b). The minimum decrease of 48.06 % was observed
at 100 mg L-1 Cd; however, Se treatment in combination
with Cd (100 mg L-1 Cd ? Se) showed less decrease of
only 16.97 %.
Glycine betaine (GB) showed an increase of 2.3-fold
and 3-fold at 100 and 200 mg L-1 Cd treatments, respectively, as compared to control, and the results are presented
in Fig. 1c. Supplementation of Cd with Se further increases
the GB accumulation by 3.7-fold at 100 mg L-1 Cd ? Se
and 4-fold with 200 mg L-1 Cd ? Se concentrations. The

123

sugar content increased by 42.74 % at 200 mg L-1 Cd

stress; however, a further increase of 55.35 % was
observed at 200 mg L-1 Cd ? Se (Fig. 1d). The results
collectively indicate that Se supplementation maintains the
protein level and confers tolerance to mustard seedlings by
increasing Pro, GB, and sugar content under Cd stress.
Se reduces Hydrogen Peroxide and Lipid
Peroxidation in Cd-Stressed Mustard Plants
The results related to the effect of Cd on hydrogen peroxide
(H2O2) and lipid peroxidation (MDA) in mustard plants are
depicted in Fig. 2a, b. The mustard plants treated with 200
mg L-1 Cd showed an increase of 66.23 % in H2O2 over
the control. Cd treatment of 100 mg L-1 showed only
53.84 % increase in H2O2. However, plants treated with Se
in the presence of Cd showed less accumulation of H2O2 as
compared to control (Fig. 2a).
Malondialdehyde (MDA), a product of lipid peroxidation, showed an increase with the increasing concentration
of Cd. An increase of 34.99 and 51.31 % was observed at
100 and 200 mg L-1 Cd stress, respectively, as compared
to control (Fig. 2b). However, supplementation of Se to
Cd-treated plants significantly reduced the accumulation of
MDA content. Only 22.07 and 37.07 % of MDA accumulation was observed at 100 mg L-1 ? Se and 200
mg L-1 ? Se treatments, respectively, over the control
(Fig. 2b). The results showed that Se helps in decreasing
the H2O2 level and also minimizes the negative effect of
Cd stress on membranes.
Se Modulates Activities of Antioxidant Enzymes
in Cd-Stressed Mustard Plants
SOD activity showed an increase of 17.26 and 28.12 % at
100 and 200 mg L-1 Cd stress, respectively, as compared
to control (Fig. 3a). Cd-treated plants supplemented with
Se showed a further increase of 28.12 % at 100 mg L-1

J Plant Growth Regul

Fig. 2 Effect of Se (50 lM) on a H2O2 and b MDA content in

mustard plants under Cd stress. Data presented are the mean SE
(n = 5). Different letters indicate significant difference (P \ 0.05)
among the treatments

Fig. 1 Effect of Se (50 lM) on a proline, b protein, c glycine

betaine, and d sugar content in mustard plants under Cd stress. Data
presented are the mean SE (n = 5). Different letters indicate
significant difference (P \ 0.05) among the treatments

Cd ? Se and 34.65 % at 200 mg L-1 Cd ? Se treatments

over the control.
The activity of CAT decreases by 13.63 % at 100
mg L-1 Cd and 22.72 % at 200 mg L-1 Cd stress with
respect to control. Supplementation of Se to Cd-treated
plants increases the CAT activity by 8.90 and 7.03 % at
100 mg L-1 Cd ? Se and 200 mg L-1 Cd ? Se concentrations, respectively, as compared to 100 and 200 mg L-1
Cd treatments (Fig. 3b).
Regarding APX (Fig. 3c), the minimum increase was
found to be 18.91 % at 100 mg L-1 Cd stress over the
control. Application of Se to Cd-stressed plants further
increased the APX activity by 23.08 and 37.51 % at 100
mg L-1 Cd ? Se and 200 mg L-1 Cd ? Se treatments,
respectively, over the control.
The GR activity increased by 22.20 and 31.82 % at 100
and 200 mg L-1 Cd stress, respectively. Se supplementation further increased the GR activity by 34.87 % at 100

mg L-1 Cd ? Se and 38.08 % at 200 mg L-1 Cd ? Se

treatments as compared to control (Fig. 3d). An insignificant increase in the antioxidant enzyme activity was
observed with the application of Se to control plants. The
collective data suggested that Se has a positive impact on
quenching of ROS through increased antioxidant enzyme
activity and thus helps mustard plants to tolerate Cd stress.
Se Enhances the Accumulation of Ascorbic Acid
and a-Tocopherol in Cd-Stressed Plants
Plants treated with 200 mg L-1 Cd showed maximum
accumulation of 56.12 % in AsA content as compared to
control (Fig. 4a). Supplementation of Se to Cd-stressed
plants further increases the AsA content by 25.45 and
17.70 % at 100 mg L-1 Cd ? Se and 200 mg L-1
Cd ? Se, respectively.
Cd concentrations of 100 and 200 mg L-1 increase the atocopherol by 76.37 and 80.87 %, respectively, with respect
to control (Fig. 4b). Addition of Se to Cd-stressed plants
further enhanced the a-tocopherol content by 11.91 % at
100 mg L-1 Cd ? Se and 11.39 % at 200 mg L-1 Cd ? Se
over the plants treated with Cd (100 and 200 mg L-1) alone.
Se Increases Total Phenol Content and Flavonoids
in Cd-Treated Plants
Cd stress increases the total phenol content by 49.59 % at
100 mg L-1 Cd and 68.67 % at 200 mg L-1 Cd with

123

J Plant Growth Regul

Fig. 4 Effect of Se (50 lM) on a AsA and b tocopherol in mustard

plants under Cd stress. Data presented are the mean SE (n = 5).
Different letters indicate significant difference (P \ 0.05) among the
treatments

Fig. 3 Effect of Se (50 lM) on a SOD, b CAT, c APX, and d GR in

mustard plants under Cd stress. Data presented are the mean SE
(n = 5). Different letters indicate significant difference (P \ 0.05)
among the treatments

respect to control. Supplementation of Se further increased

the phenol content by 39.23 and 40.45 % at 100 mg L-1
Cd ? Se and 200 mg L-1 Cd ? Se concentrations,
respectively, as compared to plants treated with Cd alone
(Fig. 5a).
Flavonoid content showed a decrease by 30.94 and
50.93 % at 100 and 200 mg L-1 Cd, respectively, compared to control. Application of Se to Cd-stressed plants
improves the flavonoid content and the maximum increase
was 30.40 % at 200 mg L-1 Cd ? Se over the plants
treated with Cd (200 mg L-1 Cd) alone (Fig. 5b).
Effect of Se on Accumulation of Cd and Se
in Mustard Plants Under Cd Stress
An accumulation of 49.00 % Cd was observed in the roots
as compared to shoots at 100 mg L-1 Cd and the maximum
accumulation of 60.60 % was observed in roots as

123

Fig. 5 Effect of Se (50 lM) on a total phenol and b flavonoids in

mustard plants under Cd stress. Data presented are the mean SE
(n = 5). Different letters indicate significant difference (P \ 0.05)
among the treatments

compared to shoots at 200 mg L-1 Cd. Supplementation of

Se decreased the Cd uptake by 32.96 and 43.50 % in roots
at 100 mg L-1 Cd ? Se and 200 ? Se treatments,
respectively, as compared to 100 and 200 mg L-1 Cd stress
alone (Table 3). Se minimizes the Cd uptake and may
partly contribute to the growth of mustard plants under Cd
stress.

J Plant Growth Regul

Table 3 Effect of Se on the Cd
and Se accumulation in shoots
and roots of mustard plants
under different concentrations
of Cd stress

Cd (mg L-1)

Se (50 lM)

Concentration of Cd and Se in different organs (lmol g-1 dry wt)

Shoot

Root

Cd

Se

Cd

Se

nd

nd

nd

nd

se

nd

0.17 0.002b

nd

0.16 001a

100

7.93 0.31c

nd

15.55 0.81c

nd

100

se

5.24 0.25d

0.49 0.006a

9.07 0.49d

0.40 006c

200

18.53 0.93a

nd

31.42 1.43a

nd

200

se

8.31 0.43b

0.58 0.007a

13.71 0.65b

0.33 004b

Data are mean SE (n = 5). Different letters next to the numbers indicate significant difference
(P \ 0.05)

Plants treated with Cd supplemented with Se showed an

increase in root Se accumulation by 60.00 and 51.51 % at
100 Cd ? Se and 200 Cd ? Se, respectively, as compared
to Se-fed control plants (0 mg L-1 Cd ? Se) (Table 3).
Accumulation of Se by the shoots was more pronounced as
compared to roots in the present study. The increase was
observed to be 65.30 % at 100 Cd ? Se and 70.68 % at
200 Cd ? Se over the plants treated with Se only (0
mg L-1 Cd ? Se).

Discussion
Selenium often exerts a dual effect on plant growth and in
higher concentrations it acts as a pro-oxidant and causes
damage to plants. At low concentrations, it stimulates the
growth of plants and counteracts many types of environmental stresses including metal stress (Feng and others
2013). The possible mechanism of this protective effect of
Se for plants under abiotic stress is not fully understood.
The role of Se in regulation of ROS and antioxidants,
inhibition of uptake and translocation of Cd, changes in the
speciation of metals and rebuilding of the cell membrane
and chloroplast structure, and recovery of the photosynthetic systems is very well reported (Feng and others 2013).
Reduction of plant growth and dry matter is a common
phenomenon of metal toxicity. Cd-induced growth reduction is also reported in various plants and it may be due to
the inhibition of cell division and cell growth (Jegu and
others 2000; Irfan and others 2014). The decline in the
biomass yield under Cd stress in the present study is in tune
with the similar reports of Rady (2011) in Phaseolus vulgaris and Fernandez and others (2013) in Dittrichia viscosa. Nevertheless, the increase in the fresh weight of root
and shoot due to addition of Se reported in the present
study has also been reported in previous studies by Filek
and others (2008), Lyons and others (2009) Akladious
(2012), and Barrientos and others (2012). Potato plants

undergoing chilling stress showed improvement in the

growth after supplementation with Se (Chu and others
2010). Hawrylak-Nowak and others (2010) also reported
that cucumber plants treated with Se and subjected to low
temperature generally showed better growth as compared
to the plants grown without Se. The protection exhibited by
selenium in plants growing under stressful conditions
might be due to increased starch accumulation in their
chloroplasts (Xue and others 2001).
Cd stress causes a decline in pigment content, as
reported in many other plants like mustard (Ahmad and
others 2012), Vicia faba (Siddiqui and others 2012),
Phaseolus vulgaris (Rady 2011), Artemisia annua (Li and
others 2012a, b), and Dittrichia viscosa (Fernandez and
others 2013). The decrease in chl content may be attributed
to (a) inhibition of important enzymes, such as daminolevulinic acid dehydratase (ALA-dehydratase) (Padmaja and others 1990) and protochlorophyllide reductase
(Van Assche and Clijsters 1990) associated with chl
biosynthesis, and (b) impairment in the supply of Mg2?,
Fe2?, Zn2?, and so on (Kupper and others 1996). Se
application restores chlorophyll content in plants under Cd
stress in the present study and the results corroborate with
the findings of Akladious (2012) in wheat. Kumar and
others (2012) also reported that Cd diminished the chl and
carotenoid content to half as compared to controls in
Gracilaria dura. However, application of Se in combination with Cd restores the pigment content to appreciable
level suggesting that Se has a positive effect on chl content.
This might be because Se stimulates the respiration rates
and the flow of electrons in the respiratory chain and
accelerates chl biosynthesis (Germ and others 2005).
Increased chl content in mustard seedlings may also be
attributed to the role of Se in protecting the chloroplast
enzymes and thus increasing the biosynthesis of photosynthetic pigments (Pennanen and others 2002).
The decrease in CO2 assimilation (A) and transpiration
rate (E) in the present study corroborates with the findings

123

J Plant Growth Regul

of Ahmad and others (2011a) and Habibi (2013). Stomatal

conductance (gs) was also altered due to Cd treatment.
Application of Se restores the A and E in the present study
and may be attributed to the regulation of Cd uptake in
presence of Se.
Like other abiotic stresses, Cd is responsible for structural damage to cellular organelles, like increased membrane permeability thus leading to leakage of intercellular
contents (Ahmad and others 2010, 2011a). Rady (2011),
Ahmad and others (2011a), and Agami and Mohamed
(2013) demonstrated that Cd stress is responsible for
electrolyte leakage in tomato, mustard, and wheat plants,
respectively. Exogenous application of Se minimizes
electrolyte leakage and is also reported by Mervi and others
(2003) in lettuce, Akladious (2012) in wheat, Abbas (2012)
in sorghum under low-temperature stress, and Stanislawa
and others (2011) in Acer saccharium seeds under drought
stress. Electrolyte leakage has been thought to be an
important index of physiological functions of the cell. Cd
induced generation of ROS, acts on polyunsaturated fatty
acids (PUFA), and increases lipid peroxidation leading to
increased membrane permeability, thus leakage of intracellular contents takes place. Se acting as an antioxidant
helps in quenching of ROS thus lowering the lipid peroxidation and hence minimizing the electrolyte leakage (Feng
and others 2013).
Relative leaf water content (RWC) was determined as
an efficient factor for evaluating plants for tolerance to
osmotic stress. Decrease in RWC in the present study
corroborates with the findings of Sepehri and Golparvar
(2011). Similar results have been obtained by Ahmad and
others (2011a, 2012) in mustard and Rady (2011) in
tomato. Bayoumi and others (2008) reported that a
decrease in RWC might be linked to water-absorbing
capacity with varying levels and also its ability to prevent
water loss through stomata. In the present study, application of Se improves the RWC because Se has the potential
to maintain the efficient water uptake system and therefore
acts as a safeguard against abiotic stress (Kuznetsov and
others 2003). A positive role of Se might also be due to
maintenance of osmoprotectants and osmolytes, thus
maintaining the internal water balance of the cell. Other
reasons might be that Se acts as an antioxidant and thus
decreases the level of H2O2 and lipid peroxidation.
Our results of increased Pro content with the increasing
concentration of Cd corroborate with the findings of Zhao
(2011). Proline accumulation has been reported by many
workers against Cd stress (Ahmad and others 2011a, 2012;
Zhao 2011; Pandey and Singh 2012). Proline acts as a
natural mechanism that helps in protecting the plants
against stress and maintaining nutrient balance through
water transport (Chu and others 1974). In the present study,
application of Se stimulates the accumulation of proline,

123

indicating that exogenous Se enhances the production of

Pro and reduces oxidative stress, thereby improving Cd
tolerance in mustard seedlings. Similar results were
observed by Xu and others (2010). Akladious (2012) and
Abbas (2012) also demonstrated that Se increases proline
accumulation in wheat seedlings under low-temperature
stress. Addition of Se might stimulate the activity of
enzymes of proline metabolism thus favoring better growth
of these plants and hence providing a better defense
mechanism.
Our results of increased levels of soluble sugar under Cd
stress are in accordance with the results of Rahoui and
others (2010) who showed that increased Cd concentration
is responsible for increased sugar content in Vicia faba.
John and others (2008) also reported that lower levels of
Cd and Pd increase the sugar content in Lemna polyrrhiza.
Accumulation of soluble sugar content in plants has been
observed under various stresses (Abbas 2012; Ahmad and
others 2007; Ahmad and Sharma 2010). Soluble sugar
serves as an osmoprotectant and plays a pivotal role in
protecting the organelles from damage due to osmotic and
oxidative stress. Akladious (2012) reported that soaking of
seeds in Se solution enhanced soluble sugar content in
wheat. Application of Se promoted growth due to increased
starch accumulation in chloroplasts (Pennanen and others
2002). Se plays a leading role in the protection of chlorophyll pigments thus enhancing photosynthesis.
The decline in protein content in the present study
corroborates with the findings of John and others (2009)
who demonstrated that increased Cd concentrations
decrease the soluble protein content in mustard. Singh and
Sinha (2005) also reported a decrease in soluble protein
content in B. juncea under metal stress. Decreased protein
content causes reduced synthesis of protein and increased
proteolytic activity (Palma and others 2002). Application
of Se has been found to increase the protein content under
Cd stress. Protein oxidation is widespread and often used as
a diagnostic marker for oxidative stress. Cd-induced
oxidative stress causes damage to proteins in the form of
fragmentation of peptide chains, aggregation of crosslinked reaction products, proteolysis, and so on. It is evident from the present study that application of Se restores
the protein content and may be due to the role of Se as an
antioxidant that quenches ROS and thus minimizes protein
damage.
The effects of toxic metal/metalloids including Cd on
plants are known to be mediated, at least partially, through
generation of ROS and subsequent lipid peroxidation
(Sharma and Dietz 2009). Although H2O2 accumulation is
responsible for oxidative damage, however it also plays a
significant role in signaling pathways. The accumulation of
H2O2 after Cd exposure has been detected in the leaves of
Vigna radiata (Anjum and others 2011), Pisum sativum

J Plant Growth Regul

(Romero-Puertas and others 2004), and Brassica juncea

(Mobin and Khan 2007; Ahmad and others 2011a).
Schutzendubel and Polle (2002) reported that stimulation
of oxidant-producing enzymes induces transient loss in
antioxidative capacity that leads to internal H2O2
accumulation.
MDA acts as an indicator of oxidative stress, as it disrupts the membrane of plant cells by decreasing membrane
fluidity, increasing membrane leakage, enzyme inhibition
and damaging ion channels including proteins (Garg and
Manchanda 2009). All these changes are the consequences
of excessive production of ROS in presence of Cd (Ahmad
and others 2011a, b; Anjum and others 2011; Hossain and
others 2010). MDA content and H2O2 level increase with
the increasing concentrations of Cd and damage the cellular membrane integrity. The present study has demonstrated that Se alleviates Cd-induced oxidative stress as
reflected by reduced O2 , H2O2, and MDA. Selenium acts
as an antioxidant, as it decreases lipid peroxidation in
Pteris vittata (Srivastava and others 2009). The protective
role of Se in decreasing the levels of H2O2 and MDA has
also been reported by Filek and others (2008) in rape
seedlings. Hasanuzzaman and others (2011) and Hasanuzzaman and Fujita (2011) also demonstrated that Se pretreatment decreases the level of H2O2 and MDA content in
rape seedling under drought and salt stress, respectively.
The amelioration of Cd toxicity with Se may be due to (1)
Se could reactivate membrane enzymes and restore the
transport of metabolites and (2) Se could compete with Cd
for specific binding sites, such as thiol groups of cysteine,
in membrane proteins (Feng and others 2013).
The generation of ROS and increased activity of many
antioxidant enzymes during Cd stress have been reported in
different plant species. The activity of antioxidant enzymes
of Cd-tolerant genotypes increased in response to Cd,
whereas Cd-sensitive species failed to do so (Hasanuzzaman and Fujita 2012a). An increase in antioxidant activities (SOD, APX, and GR) has also been reported in many
plants like tomato (Cherif and others 2011), Phaseolus
vulgaris (Rady 2011), mustard (Ahmad and others 2011a),
and Vicia faba (Siddiqui and others 2012). Application of
Se further increased the activity of antioxidant enzymes
(SOD, APX, and GR) in the present study, and the results
corroborate with the findings of Kumar and others (2012)
who also reported that Se increased the activity of
antioxidants to several fold in red seaweed Gracilaria dura
under Cd stress. Hasanuzzaman and Fujita (2012a, b) also
demonstrated the elevated activities of antioxidants in
rapeseed seedlings in the presence of Se. CAT activity
decreases in the presence of Cd stress in the present study.
Se restores the CAT activity and is also confirmed by
Hasanuzzaman and Fujita (2011) in rapeseed. Selenium
may act as an antioxidant and reduce the levels of ROS

through its transportation to selenite and then to volatile

dimethylselenide (Pilon-Smits and others 1998). Selenium
treatment shows various changes in oxidationreduction.
This may increase the plants capacity to endure various
types of stress (Vikhreva and others 2002). The enhanced
activities of these enzymes have been demonstrated in
rocket plants (Khattab 2004). Increased APX activity helps
the plants to grow better by scavenging ROS radicals.
Reduction in activities of CAT enzymes demonstrates the
conversion of selenate-induced superoxide radicals to
H2O2 through SOD (Khattab 2004). In the present study,
increased SOD activity suggests a rapid breakdown of O2
radicals and thus regulates their production. APX and GR
also helps the plant to combat oxidative stress induced by
Cd. Hence the present study provides strong evidence of
the protective role of Se against Cd-induced oxidative
stress. The reduced CAT activity is also attributed to H2O2
accumulation and has been observed to trigger APX, SOD,
and GR to fight against H2O2.
Ascorbic Acid and a-Tocopherol
Ascorbic acid (AsA) is known as an important non-enzymatic antioxidant, which increases the tolerance of plants
to oxidative stress (Shao and others 2008). Apart from this
function, it is involved in many important physiological
functions (Barakat 2003). AsA increases with the increasing concentration of Cd in the present study and the results
corroborate with the findings of Goncalves and others
(2007) in cucumber. AsA has been reported to cause Cd
tolerance in different plant species (Wu and Zhang 2002;
Mendoza-Cozatl and others 2002). Anjum and others
(2011) have reported the role of the AsA-GSH cycle in Cd
stress tolerance in mung bean. APX detoxifies H2O2 into
water using AsA as the major substrate where GSH continuously regenerates AsA from DHA (oxidized form), via
the AsAGSH cycle. In this process, GSH is oxidized into
GSSG, which is subsequently recycled by GR (Anjum and
others 2011). Supplementation of Se further enhances the
AsA content in the Cd-stressed mustard seedlings.
The present study revealed that Cd stress increases
tocopherol under Cd stress and the results coincide with the
findings of Namdjoyan and others (2012) in Carthamus
tinctorius. Supplementation of Se to Cd-treated mustard
plants enhanced its accumulation. Genes responsible for
tocopherol biosynthesis in plants showed upregulation
under oxidative stress (Yusuf and others 2010). Tocopherols are non-enzymatic antioxidants and quench ROS
and lipid radicals under different environmental stress
conditions (Jaleel 2009; Yusuf and others 2010). Se
enhances the activity of certain enzymes and induces further accumulation of AsA and tocopherols so that the
plants can quench more and more ROS.

123

J Plant Growth Regul

Total Phenol and Flavonoids

Our results of increased total phenol and flavonoid content
corroborate with the findings of Marquez-Garca and others
(2012) in Erica andevalensis under Cd stress. Kapoor and
others (2014) have also reported increased total phenol and
flavonoid content in Cd-stressed mustard plants. Cd stress
has also been reported to increase total phenol and flavonoid content in different species (Hashem and others 2015;
Ahmad and others 2015; Abd_Allah and others 2015).
Addition of Se further increases the accumulation of total
phenols and flavonoids in mustard plants. The reason
behind this is still unknown. However, it might be because
of (i) Se acts as an antioxidant agent (Hartikainen 2005) or/
and (ii) Se activates enzyme activity in the phenol and
flavonoid biosynthesis pathway. It has been reported that
the phenols and flavonoids present in plants have a protective nature against the toxic effects imposed by different
stresses (Alqarawi and others 2014; Abd_Allah and others
2015). According to Winkel-Shirley (2001), the plants
growing on soils polluted with toxic metals accumulate
more flavonoids to protect the plants from stress. According to Michalak (2006), phenolic compounds are electrondonating agents, so they act as antioxidants. They also act
as reducing agents, hydrogen donors, and free radical
scavengers and prevent the generation of ROS (Lopes and
others 1999; Jung and others 2003).
In our study, Cd content in shoots and roots increased
significantly in a dose-dependent manner. However, the
content of Cd was higher in roots compared to shoots
(Table 3). The reason is that Cd is retained in roots and
subsequently a pinch of it is transported to shoots (Caltado
and others 1983). Li and others (2012a, b) also reported
more Cd accumulation in roots in comparison to shoots.
The difference in the positive effect of selenium for different plant parts might be due to less cadmium accumulation in the upper plant part. Similar results were obtained
by Ebbs and Leonard (2001), He and others (2004), and
Yathavakilla and Caruso (2007) in Hordeum vulgare,
Brassica rapa, and Glycine max, respectively.

Conclusion
Cadmium is a metal with no beneficial role in plants and it
causes detrimental effects to growth and metabolism. Cd in
higher concentrations (depending on the plant species) is
responsible for generation of ROS directly through Fentontype/HaberWeiss reactions, which leads to oxidative
damage to biomolecules. In the present study, Cd alters the
growth and other physiobiochemical attributes in mustard;
however, application of Se restored the negative effect of Cd
stress. Se is considered an essential trace element for plants

123

and animals. At higher concentrations, it becomes harmful

and at low levels it exerts beneficial effects. These positive
effects of Se are however dependent on the Se concentration
and the age of the plant. Cd stress increases the H2O2 concentration and lipid peroxidation, but application of Se
protects the plant from further increase in H2O2 and thus
minimizes lipid peroxidation. Cd also increases the antioxidant enzyme activity in the present study and Se supplementation further increases their activity and also enhances
non-enzymatic antioxidants, thus helps the mustard plants to
tolerate the Cd stress. Thus, Se fertilization is very useful to
make the plants adaptive to Cd-polluted areas.
Acknowledgments The authors would like to extend their sincere
appreciation to the Deanship of Scientific Research at King Saud
University for its funding of this research through the Research Group
Project No RGP- VPP-271.
Compliance with Ethical Standards
Conflict of interest

The authors declare no conflict of interest.

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