Environ Sci Pollut Res

DOI 10.1007/s11356-015-4396-8

RESEARCH ARTICLE

Citric acid enhances the phytoextraction of chromium, plant

growth, and photosynthesis by alleviating the oxidative damages
in Brassica napus L.
Sehar Afshan 1 & Shafaqat Ali 1 & Saima Aslam Bharwana 1 & Muhammad Rizwan 1 &
Mujahid Farid 1 & Farhat Abbas 1 & Muhammad Ibrahim 1 &
Muhammad Aamer Mehmood 2 & Ghulam Hasan Abbasi 3

Received: 10 December 2014 / Accepted: 17 March 2015

# Springer-Verlag Berlin Heidelberg 2015

Abstract Chromium (Cr) toxicity is widespread in crops Cr treatments alone, CA supply reduced the MDA and EL
grown on Cr-contaminated soils and has become a serious values in both shoots and roots. Moreover, the activity of
environmental issue which requires affordable strategies for superoxide dismutase (SOD), guaiacol peroxidase (POD), cat-
the remediation of such soils. This study was performed to alase (CAT), and ascorbate peroxidase (APX) in shoots and
assess the performance of citric acid (CA) through growing roots markedly increased by 100 μM Cr exposure, while de-
Brassica napus in the phytoextraction of Cr from contaminat- creased at 500 μM Cr stress. CA application enhanced the
ed soil. Different Cr (0, 100, and 500 μM) and citric acid (0, activities of antioxidant enzymes compared to the same Cr
2.5, and 5.0 mM) treatments were applied alone and in com- treatment alone. Thus, the data indicate that exogenous CA
binations to 4-week-old seedlings of B. napus plants in soil application can increase Cr uptake and can minimize Cr stress
under wire house condition. Plants were harvested after in plants and may be beneficial in accelerating the
12 weeks of sowing, and the data was recorded regarding phytoextraction of Cr through hyper-accumulating plants such
growth characteristics, biomass, photosynthetic pigments, as B. napus.
malondialdehyde (MDA), electrolytic leakage (EL), antioxi-
dant enzymes, and Cr uptake and accumulation. The results Keywords Biomass . Brassica napus . Chromium . Citric
showed that the plant growth, biomass, chlorophyll contents, acid . Electrolyte leakage . Guaiacol peroxidase .
and carotenoid as well as soluble protein concentrations sig- Phytoextractions
nificantly decreased under Cr stress alone while these adverse
effects were alleviated by application of CA. Cr concentration
in roots, stem, and leaves of CA-supplied plant was signifi-
cantly reduced while total uptake of Cr increased in all plant Introduction
parts with CA application. Furthermore, in comparison with
The increasing concentration of heavy metals in agricultural
soil is an important environmental issue which can limit future
Responsible editor: Elena Maestri
land-use options as the heavy metals are toxic and nonbiode-
* Shafaqat Ali
gradable (Sharma and Pandey 2014; Habiba et al. 2015).
[email protected] Among various heavy metals, chromium (Cr) is the seventh
most abundant element of the earth’s crust (Katz and Salem
1
1994). Anthropogenic activities such as tanning, mining, and
Department of Environmental Sciences and Engineering,
Government College University, Allama Iqbal Road,
electroplating are the main sources of soil Cr contamination.
Faisalabad 38000, Pakistan Cr is a nonessential element for plants and induces toxicity in
2
Department of Bioinformatics and Biotechnology, Government
many plant species (Zeng et al. 2011; Singh et al. 2013; Gill
College University, Allama Iqbal Road, Faisalabad 38000, Pakistan et al. 2015). Higher concentration of Cr in plants caused re-
3
Department of Soil Science University College of Agriculture and
duction in root growth and biomass, chlorosis, photosynthetic
Environmental Sciences, The Islamia University of Bahawalpur, impairment, and finally plant death (Das et al. 2014).
Bahawalpur, Pakistan Recently, Singh et al. (2013) extensively reviewed the Cr toxic
 Environ Sci Pollut Res

effects in different plant species at morphological, physiolog- practical implications of CA-mediated phytoextraction and
ical, biochemical, and molecular levels. Thus, remediation of tolerance of metals so that successful field experiments can
Cr-contaminated soil has become an important environmental be conducted.
issue worldwide (Ali et al. 2012). The cultivation of high-biomass-producing crops along
Phytoremediation is an emerging technology which uses with solubilization through natural chelating agents may be
the green plants to remediate or sequester heavy metals from attractive for the remediation of contaminated soils. Recently,
contaminated soils (Raziuddin et al. 2011). It is widely accept- however, biofuel plants are being used as phytoextractors of
ed and is mainly considered for cleanup of polluted sites due heavy metals as the biomass produced can be converted to
to its visual advantages, extensive applicability, and cost- biofuel, and it may pose an economic return as well (Melo
effectiveness (Su and Wong 2004; Boonyapookana et al. et al. 2012). Brassica napus L. was chosen as the plant can-
2005). Phytoextraction is a promising treatment method, didate due to its inherent capability to hyper-accumulate
which uses high biomass-producing plants to hyper- metals and high biomass production (Vamerali et al. 2010;
accumulate metals from soils and water into plant harvestable Szczygłowska et al. 2011; Bareen 2012). It has also been
tissues. However, the degree of metal translocation from root reported that B. napus can be grown on contaminated soils
to aerial plant parts depends upon plant species, metals, and for remediation purposes, and the oil obtained from this plant
environmental conditions (Farooq et al. 2013; Anwaar et al. can be used as a source of biodiesel (Park et al. 2008) because
2015). Moreover, the efficiency of concerned metal uptake by this plant contains 40–44 % oil in its seeds (Laaniste et al.
plants is associated with the bioavailability of metals from soil 2004).
for root uptake. The present study was conducted in a wire house experi-
Although most of heavy metals have low bioavailability in ment to investigate the effect of CA on (1) Cr bioaccumula-
soils, there is a need to meet stringent cleanup targets. To tion; (2) growth and biomass and photosynthetic pigments;
overcome the limitations of natural phytoextraction has led and (3) electrolyte leakage and malondialdehyde (MDA) and
to several studies on different chelates that increase the bio- activities of the antioxidant enzymes: superoxide dismutase
availability of heavy metals (Evangelou et al. 2007). For this (SOD), catalase (CAT), guaiacol peroxidase (POD), and
purpose, different synthetic and natural chelators are used to ascorbate peroxidase (APX) in B. napus grown in Cr-
enhance the bioavailability of metals in contaminated medi- contaminated soil.
um. Among synthetic chelating agents, ethylenediaminetetra-
acetic acid (EDTA) and diethylene triamine pentaacetic acid
(DTPA) are commonly used as they are efficient in
complexing metals and increasing their concentration in the Materials and methods
upper plant parts (Kanwal et al. 2014). However, they are
nonbiodegradable and can cause ground water contamination Soil treatments and B. napus plant
due to uncontrolled leaching in the soil (Meers et al. 2005;
Anwer et al. 2012; Bareen 2012). Organic acids could be an A clay loam (25 % sand, 12 % silt, 51 % clay) soil was used in
interesting alternative to the persistent synthetic chelating this study and was collected at 0–15 cm depth from botanical
agents described above. Organic chelating agents are low- garden of the Government College University Faisalabad,
molecular-weight organic acids such as citric acid (CA) and Pakistan. Before pot experiment, the soil was air-dried and
can form complexes with heavy metals and have higher de- passed through a 2-mm diameter sieve to remove stones and
gree of biodegradability and less leaching hazard as compared crop residues. The soil was characterized chemically and
to synthetic chelating agents (Meers et al. 2005; Bareen 2012). physically (Table 1). Mature seeds of B. napus L. genotype
So synthetic chelating agents should be replaced by natural (Faisal Canola (RBN-03060) developed from a cross KS-75 x
organic acids for the mobilization and availability of metals Rainbow) were taken from Oilseeds Research Institute,
from the contaminated medium. Recently, it has been reported AARI, Faisalabad, Pakistan (Mahmood et al. 2012). The
that citric acid (CA) significantly enhances metal solubility seeds were washed with distilled water thoroughly, and they
and uptake by plants (Yeh and Pan 2012; Freitas et al. were sown in earthen pots each containing 5 kg soil (dry
2013). To date, many studies have reported the effects of or- weight) under wire house conditions. In each pot, ten seeds
ganic chelating agents on the extraction of heavy metals from were sown thinned to five individuals per pot after 15 days of
the solution cultures (Gunawardana et al. 2011; Das et al. germination, and the pulled-up plants were crushed carefully
2014; Ehsan et al. 2014); there are few reports on the role of into the same pot. Each soil pot was fertilized with a 500-mL
these chelating agents during phytoextraction of heavy metal solution containing 2.19 g L−1 N (as (NH2)2CO), 0.5 g L−1 P
from contaminated soils (Chigbo and Batty 2013). Thus, we (as (NH4)2HPO4), and 2.14 g L−1 K (as K2SO4). Half of the
need longer term (as compared to hydroponic cultures) and fertilizer solution was applied after 15 days of germination and
more realistic soil-based studies to better understand the remaining half after 30 days of germination. All plastic and
Environ Sci Pollut Res

Table 1 Properties of soil used for the pot experiment for the determination of antioxidant enzymes. After weighing,
Physicochemical properties dried plant samples were grounded and were used for further
analysis. The plants were safely disposed off after the comple-
Texture Clay loam tion of the experiment.
Sand (%) 27
Silt (%) 20 Pigment content assay
Clay (%) 53
pH (1/2.5 soil to water ratio) 6.7 After 8 weeks of treatments, the uppermost fully extended
ECe (dS m−1) 2.9 fresh leaves of B. napus plants were used for the determination
SAR (mmol−1)1/2 6.5 of chlorophyll (a and b) and carotenoid contents. The pigment
Organic matter (%) 0.31 contents were extracted in the dark with 85 % (v/v, Sigma)
Available P (mg kg−1) 2.17 aqueous acetone solution at 4 °C by continuous shaking until
HCO3 (mmol L−1) 3.55 color had completely disappeared from the leaves. Then, the
Cl− (mmol L−1) 2.34 assay mixture was centrifuged at 4000 rpm for 10 min at 4 °C,
SO42− (mmol L−1) 6.67 and supernatant was taken. Light absorbance at 663, 644, and
Ca2+ +Mg2+ (mmol L−1) 3.5 452.5 nm was determined by spectrophotometer (Halo DB-
Na2+ (mmol L−1) 3.7 20/ DB-20S, Dynamica Company, London, UK) (Metzner
K+ (mmol L−1) 0.06 et al. 1965). The concentrations of chlorophylls and caroten-
Available Cu2+ (mg kg−1) 0.35 oids were calculated by using the adjusted extinction coeffi-
Available Zn2+ (mg kg−1) 0.85 cients and equations (Lichtenthaler 1987).

Chromium content analysis

glasswares were rinsed with 10 % HNO3 and then washed
with distilled water till neutral pH. For Cr content determination, plant sample (0.5 g) was taken
in a 100-mL flask, and then 15 mL of concentrated HNO3
(70 %, Sigma) was added. Then, the flasks were put on a
Treatments
hot plate which temperature was gradually increased up to
275 °C, and dense yellow fumes appeared from the flask.
After 4 weeks of germination, uniform plants were treated
When the quantity of dense yellow fumes became low, then
with increasing concentrations of Cr (0, 100, and 500 μM)
hydrogen peroxide was added until fumes disappeared. When
along with increasing concentration of CA (0, 2.5, and
samples became colorless, the extracts were diluted to 25 mL
5.0 mM). In total, there were nine, T1: Cr (0 μM) + CA
with distilled water and were analyzed for Cr contents by
(0 mM); T 2 : Cr (100 μM); T 3 : Cr (500 μM); T 4 : CA
means of flame atomic absorption spectrometry (AAS) made
(2.5 mM); T 5 : CA (5 mM); T 6 : Cr (100 μM) + CA
by novA A400, Analytik Jena, Germany, followed by Ehsan
(2.5 mM); T 7 : Cr (100 μM) + CA (5 mM); T 8 : Cr
et al. (2013).
(500 μM)+CA (5 mM), T9: Cr (500 μM)+CA (5 mM), treat-
ments. Each treatment was triplicated, and the experimental
Determination of electrolyte leakage and MDA
pots were rotated randomly in the wire house and weeds were
removed when present. Each treatment was applied in the soil
Electrolyte leakage (EL) was measured according to the meth-
in solution form prepared in distilled water to the pots after
od of Dionisio-Sese and Tobita (1998). After 8 weeks of treat-
4 days interval for next 8 weeks.
ments, the uppermost fully expanded leaves were cut into
small parts of about 5 mm length and positioned in test tubes
Plant sampling and analysis containing 8 mL of distilled water. Then, the tubes were incu-
bated in water bath at 32 °C for 2 h, and then electrical con-
Plants were harvested after 8 weeks of treatments. Roots were ductivity of the initial medium (EC1) was measured. After
carefully separated from the soil. All the samples were thor- this, all samples were again placed in autoclave at 121 °C
oughly washed with deionized water, and excess water was for 20 min so that all electrolytes could release, then these
removed with the help of tissue paper. All samples were sep- samples were cooled to 25 °C, and then again electrical con-
arated into shoots, roots, and leaves. Then, growth variables ductivity (EC2) was measured by using pH/conductivity me-
including root and shoot length, no. of leaves per plant, area of ter (model 720, INCO-LAB Company, Kuwait) and computed
leaves, and root and shoot fresh weights were measured. After by using the following formula.
this, half of each of the sample was oven-dried at 70 °C for
EL ¼ ðEC1=EC2Þ  100:
about 3 days until constant weight, and the other half was used
 Environ Sci Pollut Res

The level of lipid peroxidation in leaf tissue was Statistical analysis

measured in terms of malondialdehyde (MDA; a product
of lipid peroxidation) content determined by the thiobar- All values described in this study are mean of three replicates.
bituric acid (TBA) reaction using the method of Heath Analysis of variance (ANOVA) was done by using a statistical
and Packer (1968), with minor modifications as de- package, SPSS version 16.0 (SPSS, Chicago, IL), followed by
scribed by Dhindsa and others (1981) and Zhang and Tukey’s post hoc test between the means of treatments to
Kirham (1994). A 0.25-g leaf sample was homogenized determine the significant difference.
in 5 mL of 0.1 % trichloroacetic acid (TCA). The ho-
mogenate was centrifuged at 10,000×g for 5 min. To
1 mL aliquot of the supernatant, 4 mL of 20 % TCA Results
containing 0.5 % TBA was added. The mixture was
heated at 95 °C for 30 min and then quickly cooled Effect of chromium and citric acid on growth and biomass
in an ice bath. After centrifugation at 10,000×g for
10 min, the absorbance of the supernatant at 532 nm Plant growth, estimated as plant height, root length, number of
was measured and the value for the nonspecific absorp- leaves per plant, and leaf area, significantly decreased at both
tion at 600 nm was subtracted. The MDA content was Cr levels (100 and 500 μM) alone as compared to control
calculated by using an extinction coefficient of (Fig. 1). The maximum decrease in growth parameters was
155 mM−1 cm−1. observed with highest Cr (500 μM) treatment alone. The ad-
verse effects of Cr stress on the B. napus plants were alleviated
when CA was applied along with Cr stress. CA application
Evaluation of antioxidant enzymes and protein content markedly increased the plant height, root length, number of
leaves per plant, and leaf area under Cr stress. The highest
Antioxidant enzymes including superoxide dismutases increase in growth parameters was observed with highest
(SOD), guaiacol peroxidase (POD), catalase (CAT), ascorbate CA (5 mM) treatment without Cr stress. Fresh and dry weights
peroxidase (APX), and soluble protein in roots and leaves of root, stem, and leaves significantly decreased at both levels
were evaluated spectrophotometrically. After 8 weeks of treat- of Cr stress alone as compared to those of control (Table 2).
ment, fully expanded leaves of plants and roots samples were When compared with control, this decrease in root, stem, and
taken for enzymatic analysis. The leaves and roots samples leaf dry biomass was 43, 44, and 39 % at 100 μM Cr treatment
(1.0 g) were quickly frozen in liquid nitrogen and grounded and was 72, 73, and 70 % at 500 μM Cr treatment, respec-
with precooled pestle and mortar. This pattern was stan- tively. Application of CA, at both levels, significantly in-
dardized in 0.05 M phosphate buffer (pH 7.8) and fil- creased the fresh and dry weights of plants as compared to
tered through four layers of muslin cloth and centri- the same treatment without CA application. Degree of in-
fuged at 12,000×g, at 4 °C for 10 min. The final su- crease in biomass with CA application was dose dependent
pernatant was for the estimation of SOD and POD ac-
tivities according to Zhang (1992). The soluble protein Cr concentration and uptake
content in the supernatant was measured according to
(Bradford 1976) using coomassie brilliant blue G-250 Chromium contents in roots stem and leaves are presented in
as dye and albumin as a standard. Table 3. Cr concentrations in all three plant parts of B. napus
Catalase (CAT; EC 1.11.1.6) activity was determined ac- seedlings were dramatically increased at both levels of Cr
cording to (Aebi 1984). The assay mixture (3.0 mL) consisted (100 and 500 μM) relative to control plant (Table 3). Cr con-
of 100 μL enzyme extract, 100 μL H2O2 (300 mM), and centration was higher in roots irrespective of Cr levels follow-
2.8 mL 50 mM phosphate buffer with 2 mM CA (pH 7.0). ed by stem and leaves. In the presence of 2.5 mM CA, how-
The CAT activity was assayed by measuring the decrease in ever, Cr concentrations in roots were significantly decreased
absorbance at 240 nm as consequence of H2O2 disappearance by 13.2 and 14.3 % compared with the 100 and 500 μM Cr
(ε=39.4 mM−1 cm−1). treatment alone, respectively, while in the presence of 5.0 mM
Ascorbate peroxidase (APX; EC 1.11.1.11) activity was CA, this decrease Cr in roots was 20.8 and 23.6 % compared
assayed according to the method of Nakano and Asada with the 100 and 500 μM Cr treatment alone, respectively.
1981. The reaction mixture consisted of 100 μL enzyme ex- Similarly, the application of 2.5 mM CA decreased 13.1 and
tract, 100 μL ascorbate (7.5 mM), 100 μL H2O2 (300 mM), 29.9 % Cr compared with 100 and 500 μM Cr treatment
and 2.7 mL 25 mM potassium phosphate buffer with 2 mM alone, respectively, while 5.0 mM CA under Cr stress, this
CA (pH 7.0). The oxidation activity of ascorbate was deter- decrease in Cr concentration was 23.9 and 42.7 % than those
mined by measuring the change in wavelength at 290 nm (ε= of 100 and 500 μM Cr treatment alone, respectively.
2.8 mM−1 cm−1). Moreover, 2.5 mM CA decreased 8.3 and 23.4 % Cr in leaves
Environ Sci Pollut Res

70
Fig. 1 Effect of various Cr (0, A) CA (0 mM) 25
B)
100, and 500 μM) and citric acid a CA (2.5 mM) a
60 b
(0, 2.5, and 5 mM) treatments on c d CA (5 mM)

Root length (cm plant-1)

b

Plant height (cm plant-1)

20
plant height (a), root length (b), 50 c
e d
number of leaves per plant (c), f
and leaf (d) of Brassica napus 40 g 15 e
seedlings grown in soil. Values h f
are expressed as means of three 30 i g
10 h
replicates with standard
20 i
deviations. Different letters
5
indicate that values are significant 10
different at P<0.05
0 0

7
B)
140 D)
a
120 a a
6 b a
c
No. of leaves plant-1 e d 100

Leaf area (cm-1)

5 d
f
4 80 e
g f
3 h 60 g
i h
2 40 i

1 20

0 0
0 100 500 0 100 500
[Cr] in soil in µM [Cr] in soil in µM

compared with 100 and 500 μM Cr treatment alone, respec- concentrations in different plant parts while increased total Cr
tively, and in the presence of 5.0 mM CA, this decrease in Cr uptake as compared to Cr treatments alone.
concentration was up to 19.5 and 35 % as compared to 100
and 500 μM Cr treatment alone, respectively. Photosynthetic pigments and protein contents
Total Cr uptake significantly increased in roots, stem, and
leaves of B. napus plants at both levels of Cr (100 and Chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll,
500 μM) relative to control plant (Table 3). Total Cr uptake and carotenoid and soluble protein contents are presented in
was highest in leaves followed by stem and roots. CA appli- Fig. 2. At both levels of Cr (100 and 500 μM), Chl a, Chl b,
cation, at both levels, further enhanced the total Cr uptake in total chlorophyll, and carotenoid concentrations in leaves of
all three plant parts as compared to respective Cr treatments B. napus significantly decreased as compared to those of
alone. These results depicted that CA application decreased Cr plants grown without Cr stress. CA application with and

Table 2 Effect of different concentration of chromium (Cr) (0, 100, and 500 μM) and citric acid (CA) (0, 2.5, and 5.0 mM) on biomass of B. napus
plants

Treatments Root weight (g plant−1) Stem weight (g plant−1) Leaf weight (g per plant−1)

Fresh Dry Fresh Dry Fresh Dry

Control 3.02±0.27a 0.72±0.04a 38.93±1.00a 2.52±0.21a 21.43±1.07a 12.95±0.56a

CA (2.5 mM) 4.08±0.21a 0.75±0.01a 40.64±1.29a 2.59±0.19a 22.58±0.80a 13.51±0.09b
CA (5.0 mM) 4.23±0.2a 0.78±0.03a 42.08±1.78a 2.67±0.24a 23.25±1.02a 14.86±0.58c
Cr (100 μM) 2.26±0.17b 0.41±0.02b 22.29±0.51b 1.41±0.10b 12.31±0.56b 7.87±0.38d
Cr100+2.5 CA 2.63±0.16c 0.48±0.03c 25.93±1.32c 1.64±0.10c 14.33±1.05c 9.16±0.58e
Cr100+5.0 CA 3.09±0.22d 0.56±0.04d 30.46±1.70d 1.93±0.11d 16.83±1.04d 10.76±0.36f
Cr (500 μM) 1.10±0.13e 0.20±0.03e 10.85±1.13e 0.67±0.03e 6.00±0.10 e 3.83±0.47g
Cr500+2.5 CA 1.50±0.20f 0.27±0.02f 14.82±1.54f 0.94±0.07f 8.19±0.71f 5.23±0.25h
Cr500+5.0 CA 1.90±0.16g 0.35±0.04g 18.70±1.68g 1.18±0.06g 10.33±0.58g 6.60±0.34i

Values are expressed as means of three replicates with standard deviations. Different letters indicate that values are significant different at P<0.05
 Environ Sci Pollut Res

Table 3 Effect of different concentration of chromium (Cr) (0, 100 and 500 μM) and citric acid (CA) (0, 2.5, and 5.0 mM) on Cr concentrations and
accumulation/uptake by B. napus plants

Treatments Cr concentrations (mg kg−1) Cr accumulation (mg plant−1)

Roots Stem Leaves Roots Stem Leaves

Control 0.00±0.00a 0.00±0.0a 0.0±0.0 a 0.0±0.0a 0.00±0.00a 0.00±0.00a

CA (2.5 mM) 0.47±0.36a 0.5±0.0a 0.0±0.0a 0.0±0.0a 0.001±0.00a 0.00±0.00a
CA (5.0 mM) 0.08±0.0a 0.7±0.0a 0.1±0.0a 0.0±0.0a 0.002±0.00a 0.00±0.00a
Cr (100 μM) 422.8±10.2b 175.6±13.1b 91.2±5.2b 0.173±0.002b 0.248±0.02b 0.718±0.012b
Cr100+2.5 CA 367.0±11.1c 152.6±8.0c 83.6±1.3c 0.176±0.003b 0.250±0.01b 0.766±0.018c
Cr100+5.0 CA 334.7±9.8d 133.6±4.8d 73.4±3.2d 0.187±0.004c 0.258±0.00b 0.790±0.006c
Cr (500 μM) 685.4±21.8e 378.4±24.7e 201.7±4.2e 0.137±0.004d 0.254±0.01b 0.772±0.020c
Cr500+2.5 CA 587.2±29.0f 265.3±13.1f 154.5±4.54f 0.159±0.006e 0.25±0.006b 0.808±0.010d
Cr500+5.0 CA 523.6±14.9g 216.7±11.8g 131.2±4.0g 0.183±0.009c 0.256±0.01b 0.866±0.024e

Values presented are means of three replicates with standard deviations. Different letters indicate that values are significant different at P<0.05

without Cr stress positively affected the photosynthetic pig- increased these pigment concentrations under Cr stress as
ment concentrations when compared with their corresponding compared to plants treated with Cr alone. The evaluation of
plants under Cr stress alone. CA application significantly protein contents also indicated a significant effect of CA

Fig. 2 Effect of various Cr (0, 30 A) CA (0 mM) 10 B)

100, and 500 μM) and citric acid a

[Chl b] in mg g-1 FW
[Chl a] in mg g-1 FW

a CA (2.5 mM)
(0, 2.5, and 5 mM) treatments on 25 a a 8 b
b CA (5 mM) c
chlorophyll a (a), chlorophyll b c
20
(b), total chlorophyll (c), d 6
carotenoids (d), and soluble e d
15 f e
proteins (e, f) of Brassica napus g 4 f
seedlings grown in soil. Values 10 g
h
are expressed as means of three i
5 2
replicates with standard
deviations. Different letters 0 0
indicate that values are significant
different at P<0.05 40 C) 10 D)
35 a a a
c b 8 a
[Chla + Chl b] in

30 b
[Carotenoids]
in mg g-1 FW

d c
mg g-1 FW

25 e 6 d
f e
20 g f
h 4 g
15 i
10
2
5
0 0

600 600
E) a F)
Protein content in root
Protein content in leaf

500 b 500
c
(mg g-1 FW)

400 400
(mg g-1 FW)

d
e a
300 f 300 c b
g d
200
h
200 f e g
i
i h
100 100

0 0
0 100 500 0 100 500
[Cr] in soil in µM [Cr] in soil in µM
Environ Sci Pollut Res

treatments on Cr stressed and unstressed plants (Fig. 2e, f). Both levels of Cr significantly affected the activities of anti-
Soluble protein concentrations significantly decreased in both oxidant enzymes in roots and leaves. Addition of 100 μM Cr
leaves and roots of plants under Cr stress alone relative to treatment alone significantly enhanced the activities of antiox-
control treatment. Both levels of CA under Cr stress signifi- idant enzymes as compared to control and 500 μM Cr alone.
cantly increased the soluble protein contents than those of Cr Addition of CA further enhanced the activities of antioxidant
treatments alone. The protein contents also significantly in- enzymes and exhibited a synergetic effect. The combination
creased in CA-treated plants without Cr stress relative to con- of CA with Cr treatments improved the activity of antioxidant
trol plants. enzymes. The increase of the antioxidant enzyme activities in
Cr+CA treatments was significantly higher than that of the
Effect of citric acid and Cr on electrolyte leakage respective Cr treatments alone.
and MDA contents

Malondialdehyde and EL concentration were measured to Discussion

evaluate the oxidative damages in leaves and roots of
B. napus (Fig. 3). MDA and EL concentrations were not sta- Our results showed that plant growth attributes and biomass
tistical different between the CA treatments alone and the were significantly reduced with increasing Cr supply in the
control. MDA and EL significantly increased with increasing soil (Fig. 1 and Table 1). Toxic effects of Cr on plant growth
Cr concentrations in the soil relative to control plants. The and biomass have already been observed in many plants
exogenous addition of both CA along with both Cr levels (Rodriguez et al. 2012; Ali et al. 2013a, b; Das et al. 2014).
significantly reduced MDA and EL contents in shoots and This decrease in plant growth might be due to ultrastructural
roots as compared to Cr treatment alone suggesting that CA changes in plant organs (Gill et al. 2015). In the present study,
application reduced oxidative stress induced by Cr in leaves however, decrease in plant growth may be due to reduction in
and roots. photosynthetic pigments (Fig. 2). CA application increased
plant growth and biomass under Cr stress as compared to
Antioxidant enzyme activities in shoots and roots plants treated with Cr alone (Fig. 1 and Table 2). In the liter-
ature, conflicting results have been reported related to CA
Four key (SOD, POD, CAT, and APX) antioxidant enzyme effect on plant growth and biomass. Freitas et al. (2013) re-
activities were evaluated to find out the effect of CA applica- ported that biomass of Sorghum bicolor decreased after the
tion on the enzyme activities in Cr-stressed B. napus (Fig. 4). application of 40 mmol kg−1 CA in lead (Pb)-contaminated

Fig. 3 Effect of various Cr (0, 12 12

100, and 500 μM) and citric acid
A) CA (0 mM) B)
[MDA] in leaves µM g-1 FW

g
[MDA] in roots µM g-1 FW

10 CA (2.5 mM) 10
(0, 2.5, and 5 mM) treatments on f g
CA (5 mM) f
malondialdehyde (MDA) and e
electrolyte leakage (EL) in 8 8 e
d
leaves and roots of Brassica c d
napus seedlings grown in soil (a– 6 6 c
b
d). Values are expressed as means b
a
of three replicates with standard 4 a a 4 a
deviations. Different letters a a
indicate that values are significant 2 2
different at P<0.05
0 0

140 140
C) D) g
g
Root electrolyte leakage (%)
Leaf electrolyte leakage (%)

120 120 d
d e
100 f 100 e
e c
80 c 80 b
b
60 a a a 60
a
a a
40 40

20 20

0 0
0 100 500 0 100 500
[Cr] in soil in µM [Cr] in soil in µM
 Environ Sci Pollut Res

Fig. 4 Effect of different 240 240

concentrations of chromium (Cr)
A) CA (0 mM) B)
CA (2.5 mM) b

(Units g-1 FW)

200 200

SOD in roots
(0, 100, and 500 μM) and citric c

SOD in leaves
(Units g-1 FW)
CA (5 mM)
160 160 d
c b
acid (CA) (0, 2.5, and 5 mM) on e
superoxide dismutase (SOD) of d 120 f
120 e g
leaf (a) and root (b), guaiacol g f a a a
peroxidase (POD) of leaf (c) and 80 80
a a a
root (d), catalase (CAT) of leaf (e) 40
40
and root (f), and ascorbate
peroxidase (APX) of leaf (g) and 0 0
root (h) of B. napus plants grown
4000 1500
in soil. Values are expressed as C) b D)
POD in leaves

(Units g-1 FW)

(Units g-1 FW)
means of three replicates with

POD in roots
c b
standard deviations. Different 3000 c
letters indicate that values are d 1000
e d
significant different at P<0.05 f f e
2000 g g
a a a a a a
500
1000

0 0

5000 5000
E) b F) b
c
CAT in leaves
(Units g-1 FW)

(Units g-1 FW)

4000 c 4000 d

CAT in roots
d e
3000 e 3000 f
f
g g
2000 2000 a
a a a a a
1000 1000
0 0

700 b 700 b
G) c H)
600 600 c
(Units g-1 FW)

(Units g-1 FW)

APX in leaves

APX in roots

d d
500 e 500
400 f 400 e
g f
300 300
a a a g
200 200 a a a
100 100
0 0
0 100 500 0 100 500
[Cr] in soil in µM [Cr] in soil in µM

soil for 7 days before harvesting. Similarly, Araújo and phytochelation (PCs) in plants (Muhammad et al. 2009). It
Nascimento (2010) also observed decrease in biomass of corn has been reported that sulfur (S) uptake decreased in plants
plants under Pb stress after the application of 30 mmol kg−1 of due to increase in Cr uptake by plants (Schiavon et al. 2008
CA in soil for 9 days before harvesting. Lesage et al. (2005) and 2012), so the increase in biomass might also be due to
also reported that growth of sunflower severally decreased by efficient S uptake and assimilation by plants in the presence of
the use of CA in calcareous soil moderately contaminated with CA. Overall, increase or decrease in plant growth and biomass
different (Cu, Pb, Zn, and Cd) metals. They suggested that this with CA application in different plants may be due to variation
reduction in biomass might be due to the chelant-induced in plant species and genotypes, time of CA applications, ex-
metal mobility, chelate toxicity itself, and the chelant-metal perimental conditions, and the duration of stress exposed.
complexes. However, it has been reported by several re- Excess Cr decreased plant photosynthetic pigment concen-
searchers that CA application increased plant growth and bio- trations of B. napus (Fig. 2). These results are in line with
mass under metal stress in Juncus effusus (Najeeb et al. 2009) previous findings that excess Cr decreased photosynthetic pig-
and in Zea mays (Anwer et al. 2012). This increase in plant ment contents in many plant species (Singh et al. 2013; Gill
growth and biomass might be due to enhanced nutrient uptake et al. 2015). Contrarily, pigment contents and carotenoids sig-
by plants (Najeeb et al. 2011) or due to synthesis of nificantly increased in Pisum sativum in response to Cr stress
Environ Sci Pollut Res

(Rodriguez et al. 2012). Inhibition in photosynthetic pigments may also be due to ameliorative effect of CA on root structure
was also observed in B. napus under other metal stress such as and shape (Najeeb et al. 2011). Meers et al. (2005) reported
Cd (Ali et al. 2013a, b). This decrease in photosynthetic pig- that mobility of heavy metals (Cu, Zn, Cd, and Pb) in the soil
ment content may be due to marked distortion of chloroplast increased with CA application for some time and then de-
ultrastructure under metal stress (Najeeb et al. 2011). In the creased which might be due to degradability of CA. In the
present study, reduction in biomass, chlorophyll, and caroten- present study, the decrease Cr concentration in different plant
oid contents may be due to oxidative stress as observed by the parts was concomitant with an increase in biomass (Table 2)
increased production of MDA and electrolyte leakage (Fig. 4). indicating a dilution effect (the same uptake but larger bio-
Photosynthetic pigment contents increased in the treatment of mass) and thus increase in plant growth characteristics
Cr+CA in comparison with the Cr exposure alone (Figs. 2 and (Fig. 1). Interestingly, application of CA+Cr increased Cr
3). In the present study, however, increase in plant growth and uptake (concentration×biomass) by the plants relative to Cr
biomass and photosynthetic pigments of B. napus with CA treatment alone (Table 3). Similar results have been reported
application under Cr stress may be due to enhanced antioxi- in different species under metal stress other than Cr (Najeeb
dant enzyme activity (Fig. 4) which decreased the production et al. 2009, 2011; Ehsan et al. 2014). Our results do not cor-
of MDA and electrolyte leakage (Fig. 3). roborate by the study of Lesage et al. (2005) who reported that
Chromium application induced oxidative stress in plants as CA was not effective as an amendment to enhance the extrac-
shown by increased production of MDA and electrolyte leak- tion of Cu, Cd, and Zn by sunflower from a calcareous soil.
age (Fig. 4). Similar results have already been reported in Increase in total uptake of Cr by B. napus is directly related to
many studies (Singh et al. 2013). By contrast, CA+Cd addi- increase in biomass in the presence of citric acid. Data regard-
tion significantly reduced the MDA and electrolyte leakage ing CA effect on Cr uptake and phytoextraction is scarce
compared with the Cr treatment alone, suggesting a protective (Diwan et al. 2008; Tsetimi and Okieimen 2011). The data
role of CA in preventing oxidative stress in B. napus. Plants that is provided in this article highlighted that B. napus plants
have developed a self-defense mechanism for the reduction of accumulated higher Cr in roots and leaves which is due to high
oxidative stress which is the production of antioxidant en- biomass production in the presence of CA which might justify
zymes (Mittler et al. 2004). Our results showed that activities potential applications of CA in Cr phytoremediation strategies
of key antioxidants such as SOD, CAT, POD, and APX in using B. napus plants.
both roots and leaves initially enhanced at 100 μM of Cr Moreover, half-life of heavy metal mobilization by CA
concentration and then reduced at 500 μM of Cr concentration varied between 1.5 and 5.7 days while metal mobilization
(Fig. 4). This overexpression of antioxidant enzymes at lower remained constant in time for soils treated with EDTA
Cr (100 μM) stress in plants might be a powerful tool for the (Meers et al. 2005). Therefore, environmental risks associated
survival of plants with the highest metal accumulation capac- with chelate-assisted mobilization and leaching of metals can
ity (Haouari et al. 2012; Muhammad et al. 2009). Decrease in also be minimized by the use of degradable CA as opposed to
antioxidant enzyme activity at larger Cr stress might be due to permanent mobilization by using persistent amendments such
severe oxidative stress in plants (Mishra et al. 2006). Cr+CA as EDTA.
application significantly increased the activities of these en-
zymes in shoots and roots as compared to Cr treatment alone
(Fig. 4). It has been reported that CA helped plants to over-
Conclusion
come oxidative stress by enhancing their antioxidant enzyme
activities under metal stress (Meng et al. 2009; Najeeb et al.
In conclusion, the results demonstrate that plant growth,
2009, 2011). These results suggest that CA application scav-
biomass, and photosynthetic pigments decreased under
enges oxidative stress, increase in electrolyte leakage and
Cr stress while Cr concentration increased in different
MDA accumulation, in B. napus seedlings by enhancing the
plant parts. CA application significantly increased plant
activity of antioxidant enzymes.
growth, biomass, and chlorophyll content by enhancing
Chromium application in the soil significantly increased Cr
antioxidant enzyme activity, and it also increased metal
uptake and accumulation in different parts of B. napus
uptake in B. napus. Our results highlighted the role of
(Table 3). Similar results have already been reported in differ-
CA in enhancing Cr accumulation and phytoremediation
ent B. napus cultivars under Cr stress (Gill et al. 2015). CA
of Cr-contaminated soils.
treatments decreased Cr concentrations in different plant parts
as compared to the same Cr treatments alone (Table 3). These
results do not corroborate by the study of Freitas et al. (2013)
who reported increase in Pb concentration in maize and veti-
Acknowledgments Authors thank the Higher Education Commission
ver plants with CA (40 mmol kg−1) in Pb-contaminated soil. of Pakistan for the financial support. The results presented in this paper
In the present study, decrease in Cr concentration with CA are a part of MPhil studies of Sehar Afshan.
 Environ Sci Pollut Res

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