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Plant Physiology and Biochemistry 51 (2012) 129e138

Contents lists available at SciVerse ScienceDirect

Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Research article

Selenium and spermine alleviate cadmium induced toxicity in the red seaweed
Gracilaria dura by regulating antioxidants and DNA methylation
Manoj Kumar, A.J. Bijo, Ravi S. Baghel, C.R.K. Reddy*, Bhavanath Jha
Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientic and Industrial Research (CSIR), Gijubhai Badheka Marg,
Bhavnagar 364021, India

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 2 August 2011
Accepted 24 October 2011
Available online 10 November 2011

The protective role of exogenously supplied selenium (Se) and polyamines (PAs) such as putrescine (Put)
and spermine (Spm) in detoxifying the cadmium (Cd) induced toxicity was studied in the marine red alga
Gracilaria dura in laboratory conditions. The Cd exposure (0.4 mM) impede the growth of alga while
triggering the reactive oxygen species (ROS viz. O

2 and H2O2) generation, inhibition of antioxidant
system, and enhancing the lipoxygenase (LOX) activity, malondialdehyde (MDA) level and demethylation
of DNA. Additions of Se (50 mM) and/or Spm (1 mM) to the culture medium in contrast to Put, efciently
ameliorated the Cd toxicity by decreasing the accumulation of ROS and MDA contents, while restoring or
enhancing the level of enzymatic and nonenzymatic antioxidants and their redox ratio, phycobiliproteins
and phytochelatins, over the controls. The isoforms of antioxidant enzymes namely superoxide dismutase (Mn-SOD, w 150 kDa; Fe-SOD w120 kDa), glutathione peroxidase (GSH-Px, w120 and 140 kDa),
glutathione reductase (GR, w110 kDa) regulated differentially to Se and/or Spm supplementation.
Furthermore, it has also resulted in enhanced levels of endogenous PAs (specially free and bound
insoluble Put and Spm) and n-6 PUFAs (C20-3, n-6 and C20-4, n-6). This is for the rst time wherein Se
and Spm were found to regulate the stabilization of DNA methylation by reducing the events of cytosine
demethylation in a mechanism to alleviate the Cd stress in marine alga. The present ndings reveal that
both Se and Spm play a crucial role in controlling the Cd induced oxidative stress in G. dura.
2011 Elsevier Masson SAS. All rights reserved.

Keywords:
Cadmium
Selenium
Spermine
DNA methylation
Gracilaria dura
Oxidative stress

1. Introduction
Among the resident organism living in the coastal and estuarine
environment, the marine macroalgae (seaweeds) are the most
threatened by chemical toxicants of which metals are of signicant
component because of their persistence, acute toxicity and high
mobility in food chain [1]. Seaweeds despite being prominent
primary producers in near-shore waters, are also key ecosystem
engineers providing conducive habitat for a variety of marine
organisms. Cd is a metal without any known biological function,
except as a cofactor for carbonic anhydrase in a diatom [2], is most
common environmental toxicants with potential for bioaccumulation and persistence in the body of aquatic organisms, and
produce versatile biotic changes in the aquatic ecosystem [3]. Cd
being a redox inactive metal incapable of generating reactive
oxygen species (ROS) directly through Fenton-type/HabereWeiss

* Corresponding author. Tel.: 91 278 256 5801/256 3805x614; fax: 91 278 256
6970/256 7562.
E-mail address: crk@csmcri.org (C.R.K. Reddy).
0981-9428/$ e see front matter 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.plaphy.2011.10.016

reactions, at higher concentrations (such as 5e200 mM or higher)

can cause oxidative damage by generating free radicals and active
oxygen species [4].
Acclimation of seaweeds to heavy metal induced oxidative
stress involves induction of complex antioxidant machinery that
functions in a much coordinated manner to abate the cellular
osmolarity, ion disequilibrium and detoxication of ROS [3,5e7].
Most of the metals including Cd enter algal cells via energydependent transport across the plasma membrane [8]. Algae
employ the cytosolic binding substrates such as glutathione and
their derived peptidesephytochelatins (PCs) for Cd cheltaion
besides antioxidant system in reducing the Cd toxicity. Among the
different fundamental metabolic processes, photosynthesis,
cellular respiration and lipid peroxidation are the most affected by
Cd toxicity [5]. Moreover, Cd may also affect the cellular redox
homeostasis by promoting ROS accumulation and concomitantly
the oxidative stress, but the processes whereby disruption of
cellular activities is prevented and metal-homeostasis is maintained are not well elucidated.
In the recent years, Selenium (Se) has been recognized for its
potential antioxidant property and ability to increase the

Author's personal copy

1.66b,c,d
1.20a,b
14.43c
6.02d
5.80c





40.79
19.28
288.25
120.87
87.30
1.34c,d
1.25c
9.19c
7.14d,e
4.55c





38.78
16.45
293.02
117.04
84.41
2.81a,b
1.17a
13.67a
2.86bc
2.93a





42.89
21.22
342.73
159.63
119.12
1.73e
0.82d
8.89d
4.80d,e
6.79d





27.42
11.25
246.35
113.02
75.09
2.77e
0.86d
8.57d
8.19e,f
3.74d





26.30
12.62
234.01
107.29
70.60
2.03a,b,c
1.34a,b
7.47a,b
6.94a
8.56a,b





42.25
20.65
333.58
174.92
111.07
1.90d
1.19b,c
10.19b
3.45c
4.19b





aef

Different lowercase letters in a row indicate signicance limits at p < 0.05.

37.78
18.37
321.70
150.36
105.87
3.94a,b
2.77a
9.37a,b
8.86a
5.98a





44.29
21.44
336.30
176.45
119.90
1.84f
1.13d
12.70e
9.49f
4.09d





21.54
10.61
190.44
105.67
73.37
2.38a
1.47a,b
6.25a,b
11.73a,b
4.70b






0.47  0.04a
0.09  0.03d,e
0.10  0.02e
0.12  0.02de

45.02
20.38
334.93
164.94
109.66

0.21  0.02d
0.24  0.02b
0.29  0.03c,d
0.11  0.01d,e
0.11  0.01e
0.16  0.03c
0.33  0.02c
0.20  0.02b
0.39  0.03b
0.13  0.03c,d
0.11  0.02e
0.10  0.02d,e
0.20  0.03d
0.16  0.02c
0.13  0.02e
0.28  0.01a

0.39c
0.74d,e
0.02d
0.0.5c




6.26
3.80
0.33
0.88
0.58b,c
0.62c
0.04c
0.09c




6.73
5.01
0.41
0.94

Cd Spm

0.53a
0.47e,f
0.02e
0.07d




Spm

7.59
3.05
0.20
0.69
0.42d
1.05b
0.04b
0.15b




4.67
7.52
0.59
1.98

Cd Put Se

0.37d
0.89
0.05a
0.18a




4.39
8.75
0.67
2.45

Cd Put

0.45b,c
0.64e,f
0.03e
0.04c




6.80
3.14
0.21
0.73

Put

0.38b,c
0.30c,d
0.04d
0.13c




6.41
4.27
0.29
0.87

Cd Se

0.55a
0.43d,e,f
0.02e,f
0.12c,d




7.85
3.58
0.16
0.81
Se
0.52d
1.02a
0.06a
0.12a




4.32
9.35
0.69
2.28

Cd

0.20a
0.33f
0.03f
0.09d




7.17
2.85
0.14
0.66

Cadmium exposure resulted in a signicant growth decrease

by almost 40% (p < 0.05) in the plants treated with either Cd alone
(4.32% DGR) or with Cd Put (4.39% DGR) and Cd Put Se
(4.67% DGR) when compared to controls (7.17% DGR). The
decreased growth rate observed in these treatments was accompanied with a signicant higher lipid peroxidation and ROS
generation when compared to control (Table 1). However, both
MDA and ROS contents were signicantly lower (p < 0.05) in the
thalli grown in Se and/or Spm supplemented media during Cd
exposure and displayed a growth rate almost similar to controls. A
dense blue formazan formed due to the reduction of NBT by O

2
appeared distributed all over the tissue and particularly impregnated in the epidermal and cortical cells, clearly evidenced the
generation of superoxide radicals in Cd, Cd Put and
Cd Put Se treatments (Fig. 1a). On the contrary, a slight formazan deposition conned to the epidermal cells was observed in
Cd Se, Cd Spm and Cd Spm Se treatments, but was
completely absent in the thalli treated with Se alone and in the
control. Similarly, a signicant accumulation of H2O2 dependent
brown precipitates were also observed in the treatments of Cd,
Cd Put and Cd Put Se compared to the other treatments
(Fig. 1b). Furthermore, the LOX activity markedly enhanced in
thalli exposed to Cd (2.28), Cd Put (2.45) and Cd Put Se
(1.98) compared with the control activity (0.66 U mg1 protein)
(Table 1).

Control

2.1. Growth rate, lipid peroxidation, ROS generation, histochemical

detection of O

2 and H2O2, metal uptake and photosynthetic
pigments

Parameters

2. Results

DGR (%)
MDA (nmol g1 FW)
ROS (mmol g1 FW)
LOX (U mg1 protein)
Metal uptake
(mg g1 DW)
Cd
Se
Pigments
(mg g1 FW)
Chlorophyll
Carotenoids
PE
PC
APC

glutathione content in plant cell. A Se dependent glutathione

peroxidase activity has been conrmed in aquatic organism
Mytilus edulis preventing the Cu toxicity [9]. The role of Se in
reducing the bioavailability of toxic metal to the plant cell has also
been established [10,11]. Further, the role of exogenous polyamines (PAs) such as putrescine (Put), spermine (Spm) and spermidine (spd) has been speculated in cell division, and
development of reproductive structures in seaweeds [12]. There
are few studies looking at salinity and metal effects on PA
metabolism in algae. Hyposalinity and Cd metal toxicity has been
shown to stimulate the accumulation of endogenous free Put, Spd,
and Spm in the green macroalga Ulva fasciata and Ulva lactuca
respectively, suggesting that the accumulation of PAs in particular
Put and Spm may be a crucial factor for protecting the seaweeds
from hyposaline and metal injury [3,13].
The epigenetic regulatory strategy such as DNA methylation
has been employed by plants in adverse milieu to maintain the
genomic integrity [14]. The previous studies have shown the
alteration in cytosine methylation throughout the genome and at
specic loci as a result of abiotic stress [15]. In the present study,
we hypothesised the ameliorating effects of exogenous Se and PAs
(Put and Spm) when applied individually or in combinations
against Cd induced oxidative damage in industrially important
agarophytic marine red alga Gracilaria dura. To test our hypothesis,
we constructed the experiment whereby the growth rate, histochemical detection of ROS, regulation of antioxidant system,
polyamines, lipoxygenase (LOX), photosynthetic pigments, polyunsaturated fatty acids (PUFAs), non-protein thiols (NP-SHs) and
PCs were examined to evaluate the possible role of Se and/or PAs
in combating the Cd toxicity. This is the rst report wherein the
role of Se and/or PAs in stabilization of DNA methylation in G. dura
was investigated following the exposure to Cd metal.

Cd Spm Se

M. Kumar et al. / Plant Physiology and Biochemistry 51 (2012) 129e138

Table 1
Changes in growth, lipid peroxidation, reactive oxygen species generation, lipoxygenase activity and photosynthetic pigments in G. dura following its exposure to Cd (0.4 mM), Se (50 mM) and PAs (1 mM) individually or in
different combinations for 4 d. DGR, MDA, ROS and LOX indicate daily growth rate, malondialdehyde, reactive oxygen species and lipoxygenase respectively. PE, PC and APC indicate phycobiliproteins namely phycoerythrin,
phycocyanin and allophycocyanin respectively.

130

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M. Kumar et al. / Plant Physiology and Biochemistry 51 (2012) 129e138

131

Fig. 1. Reactive oxygen species generation in G. dura following the Cd (0.4 mM) exposure for 4 d (a) O

2 and (b) H2O2 radicals were detected with nitroblue tetrazolium (NBT) and
3,3-diaminobenzidine (DAB) staining, respectively. A-H represents the various treatments viz. control, Cd, Se, Cd Se, Cd Put, Cd Put Se, Cd Spm and Cd Spm Se
respectively.

A considerable accumulation (p < 0.05) of cadmium metal was

found in the thalli exposed to the treaetments of Cd, Cd Put and
Cd Spm with values 0.47, 0.39 and 0.29 mg g1 DW over the
control with 0.10 mg g1 DW However, supplementation of Se in
these treatments signicantly lowered (p < 0.05) Cd uptake
(Table 1). Further, Cd exposure diminished the chlorophyll and
carotenoids contents to half the value of their contents in control.
Among the phycobiliproteins, phycoerythrin (PE) was most
affected under Cd treatment and decreased to 190.44 mg g1 FW
(p < 0.05) (Cd) in comparison with controls (334.93 mg g1 FW).
However, a moderated but statistically signicant decrease for PE
was recorded in other treatments. The treatment of Put or Spm
alone did not result any oxidative damage as evident by higher
growth rate and photosynthetic pigments content with lower
lipid peroxidation and ROS generation. Henceforth, these treatments were omitted in subsequent experiments though their
effects in combination with Se were examined during Cd
exposure.

2.2. Polyamines, non-protein thiols (NP-SHs), water soluble

antioxidants and phytochelatins
Among the investigated PAs such as putrescine (Put), spermidine (Spd) and spermine (Spm), Put was the predominant PA
in both free (F) and bound insoluble (BI) forms followed by
spermine and spermidine. Following the Cd exposure (Cd) the
content of the three endogenous PAs declined signicantly in
both the forms (F and BI) (p < 0.05) by 26e66% being Put the
most effected one. The corresponding values for F and BI-PAs
(Put, Spd and Spm) in control were 9.73, 2.71, 2.21 and 23.29,
4.46, 4.17 mmol g1 FW respectively. A similar decrease in PAs
content was also observed in plants grown with the exogenous
application of Put alone or with combination of Se during Cd
exposure (Fig. 2). The exogenous application of Se protects the FPAs from Cd toxicity but could not guard the BI-PAs. Nevertheless, exogenous application of Spm in case of Cd Spm and
Cd Spm Se led to the remarkably increase in endogenous Put

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132

M. Kumar et al. / Plant Physiology and Biochemistry 51 (2012) 129e138

Fig. 2. Changes in the endogenous polyamines level (mmol g1 FW) in G. dura following the Cd (0.4 mM) exposure for 4 d. Put, Spd and Spm represents putrescine, spermidine and
spermine respectively. Different lowercase on the similar bars indicate signicance limits (p < 0.05).

and Spm contents specically in their F form by 1.6e2.4 folds

(p < 0.05) over the control. The contents of free and bound
insoluble PAs for each endogenous Put, Spd and Spm have also
been compared using non-parametric ManneWhiteney Wilcoxon test and were found signicantly different for endogenous
Put (P  0.05, z 1.96, U 49), Spd (P < 0.05, z 2.94, U 60)
and Spm (P < 0.01, z 2.20, U 53).
The increase for NP-SH level was markedly higher (p < 0.01) in
the presence of Spm in the treatments of Cd Spm
(3.45 mmol g1 FW), Cd Spm Se (3.29 mmol g1 FW) when
compared to control (1.29 mmol g1 FW) and other treatments
(Table 2). The contents of GSH and PCs were greatly inuenced by
Cd exposure and showed a considerable increase by 2e3 folds
(p < 0.01) in Cd Se, Cd Spm, Cd Spm Se treatments when
compared to their corresponding contents in control with value
0.26 and 1.02 mmol g1 FW respectively, while their accumulation
was greatly inhibited in Cd, Cd Put, Cd Put Se treatments.
The glutathione redox ratio also increased markedly (p < 0.05)
from 1.03 (control) to 1.99 (Cd Spm) and 2.74 (Cd Spm Se)
in response to Cd exposure. The contents of AsA, DHA and their
redox ratio also showed the similar response as that of glutathione. The levels of AsA and ascorbate redox ratio (AsA/DHA)
registered more than two fold increase p < 0.01 in plants exposed
to Cd Spm Se and Cd Spm treatment while decreased
signicantly in the treatments of Cd, Cd Put and Cd Put Se,

over the control with value 0.73 mmol g1 FW and redox ratio 2.03
respectively (Table 2).
2.3. Antioxidative enzymes activities and their in-gel assay
All the studied antioxidative enzymes exhibited enhanced
activities in the plants exposed to Cd together with Se and/or Spm
treatments. The specic activities of SOD, APX, GR and GSH-Px
except POX increased by 1.4e1.9 fold (p < 0.05) in Cd Se,
Cd Spm, Cd Spm Se treatments while decreased considerably
in Cd, Cd Put, Cd Put Se treatments, over the control activities
with 87, 0.63, 1.25 and 0.99 U mg1 protein respectively (Fig. 3(a)).
Native PAGE analysis supported the spectrophotometric measurements of these enzymes activities (Fig. 3(b)). Apparent higher
activity of SOD observed in Se and Spm treatments was solely
attributed to Mn-SOD (SOD-1,w150 kDa) and Fe-SOD (SOD2,w120 kDa) conrmed by using H2O2/KCN as inhibitors, while
a partial or complete inhibition of Zn-SOD (SOD-3,w100 kDa) was
also observed in Cd, Cd Put and Cd Spm and Cd Spm Se
treatments. Both spectrophotometric analysis as well as in-gel
assay for POX activity indicated its maximal inhibition in the
thalli exposed to Cd, Cd Put, Cd Put Se treatments, while its
activity restored signicantly in plants grown in Se and Spm supplemented media. GR and GSH-Px in particular revealed the
specic response to the Cd exposure and exhibited three isoforms

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M. Kumar et al. / Plant Physiology and Biochemistry 51 (2012) 129e138

133

Table 2
Changes in the level of water soluble antioxidants in G. dura following the exposure to Cd (0.4 mM) Se (50 mM) and PAs (1 mM) individually or in dif ferent combinations for 4 d.
NP-SH, PCs, GSSG and GSH (each in mmol g1 FW) indicates non-protein thiols, phytocheletins, oxidised and reduced glutathione respectively. DHA and AsA indicate oxidised
and reduced ascorbate respectively.
Treatments

NP-SH

Control
Cd
Se
Cd Se
Cd Put
Cd Put Se
Cd Spm
Cd Spm Se
LSD (5%)

1.29
1.92
2.00
3.41
2.07
2.21
3.45
3.29
0.43

a-e










GSSG
0.15c
0.37b
0.15b
0.18a
0.30b
0.28b
0.31a
0.33a

0.22
0.47
0.33
0.34
0.48
0.41
0.30
0.25
0.04










GSH
0.04e
0.03a
0.02c
0.03c
0.04a
0.02b
0.02c,d
0.03d,e

0.26
0.17
0.37
0.51
0.21
0.23
0.60
0.68
0.07

GSH/GSSG









0.05d
0.03e
0.08c
0.01b
004d,e
0.01d,e
0.03a
0.08a

1.03
0.37
1.10
1.51
0.45
0.56
1.99
2.74
0.45










0.31d
0.08e
0.29c,d
0.13c
0.12e
0.03e
0.15b
0.23a

PCs
1.02
1.75
1.64
2.90
1.86
1.98
2.86
2.62
0.46

DHA









0.20c
0.40b
0.12b
0.19a
0.28b
0.28b
0.30a
0.40a

0.36
0.45
0.34
0.54
0.43
0.59
0.36
0.25
0.07

AsA









0.02c,d
0.07b
0.06d
0.04a
0.03b,c
0.05a
0.04c,d
0.03e

0.73
0.49
0.74
1.24
0.47
0.51
1.50
1.43
0.30

AsA/DHA









0.21d
0.15c
0.19d
0.24b
0.19c
0.13c
0.21a
0.13a

2.03
1.15
2.23
2.30
1.10
0.86
4.15
5.70
0.81










0.63c
0.54d
0.80c
0.32c
0.46d
0.19d
0.20b
0.52a

Different lowercase letters in a row indicate signicance limits (p < 0.05).

GSH-Px-2 (w125 kDa), GSH-Px-5 (w60 kDa), GSH-Px-6 (w40 kDa)

in control. Plants cultured with Spm supplemented media also
showed the similar GPX isoenzymes prole with intense activity of
GSH-Px-2. An additional isoform GSH-Px-3 (w100 kDa) was
demonstrated in plants exposed to Cd Put and Cd Put Se
treatments. Two other isoforms of GSH-Px-1 (w140 kDa) and GSHPx-4 were also observed only in plants treated with Se and Cd Se
(Fig. 3(b)). The activity gel of GR displayed four isoforms GR-1
(w125 kDa), GR-2 (w110 kDa), GR-3 (w85 kDa) and GR-4
(w60 kDa) in both the control and Se treatements, though the

intensity of GR-2 was higher in Se exposed plants. The Cd exposure

had induced an isoform GR-1 (w180 kDa) and its expression was
consistently observed in all the plants grown in Cd supplemented
media together with Se and or PAs.
2.4. Fatty acids and DNA methylation pattern
Table 3 summarizes the variation in fatty acid composition of G.
dura in response to metal exposure in terms of percentage of total
fatty acids (% TFA). Plants exposed to the treatments of Cd, Cd Put

Fig. 3. Effect of Cd (0.4 mM) exposure on (a) antioxidative enzymes activities (U mg1 protein) and (b) their isoenzyme analysis in G. dura in various treatments index from A-H
according to Fig. 1. Different lowercase on the bars indicate signicance limits (p < 0.05).

Author's personal copy

134

M. Kumar et al. / Plant Physiology and Biochemistry 51 (2012) 129e138

Table 3
Changes in the fatty acids composition (% total fatty acids) in G. dura following the exposure to Cd (0.4 mM), Se (50 mM) and PAs (1 mM) individually or in different
combinations for 4 d. UFA and SFA indicates unsaturated and saturated fatty acids respectively.
Fatty acids

Control

C14:0
C15:0
C16:0
C18:0
C16:1(n-7)
C18:1(n-9) cis
C18:1(n-9) trans
C20:3(n6)
C20:4(n-6)
UFA/SFA

1.00
e
34.48
3.15
e
0.45
3.19
6.15
51.59
1.50

aef

Cd

 0.11d
 1.31c
 0.20d






0.05e,f
0.15d
0.31a
2.75b
0.10b

1.97
e
56.93
5.30
3.00
2.72
4.14
2.69
27.18
0.47

Se
 0.15a









1.55a
0.52b
0.17a
0.21a
0.33c
0.28d
1.97d
0.02d

1.34
e
34.01
2.54
e
0.62
2.53
6.64
52.37
1.56

 0.11c
 1.48c
 0.19e






0.06e
0.20e
0.33a
2.26b
0.11b

Cd Se

Cd Put

e
e
30.17
2.22
e
0.33
3.36
6.33
56.74
1.95

2.04
1.12
57.37
6.34
2.15
2.46
5.21
3.69
24.18
0.42

 1.60d
 0.15e






0.06f
0.21d
0.23a
0.58a
0.09a












016a
0.07a
1.69a
0.40a
0.26b
0.15b
0.29b
0.39c
3.10d
0.05d

Cd Put Se
1.54
0.56
56.53
5.89
e
0.61
2.36
4.81
25.73
0.47






0.08b
0.08b
1.17a
0.41a







0.07e
0.11e
0.41b
0.90d
0.01d

Cd Spm

Cd Spm Se

LSD (5%)

e
e
37.23
5.14
e
2.10
5.05
6.34
46.90
1.26

e
e
38.98
3.77
e
1.65
6.80
6.44
43.81
1.18

0.16
0.06
2.36
0.53
0.17
0.22
0.38
0.52
3.77
0.12

 1.21b
 0.24b






0.24c
0.23b
0.38a
2.48c
0.08c

 1.62b
 0.35c






0.08d
0.30a
0.21a
3.21c
0.08c

Different lowercase letters in a row indicate signicance limits at p < 0.05.

and Cd Put Se exhibited a signicant increase (p < 0.05) in

saturated fatty acids such as C16:0 and C18:0 acid, with a concomitant decrease in n-6 PUFAs such as C20-3, n-6 and C20-4, n-6 acids
when compared with their corresponding values of 34.48% TFA,
3.15% TFA, 6.15% TFA and 51.59% TFA in control. On the contrary, the
contents of both the n-6 PUFAs and C16:0 were found either similar
or marginally differ in plants cultured with the supplementation of
Se and/or Spm. Further, a phenomenal increase (2e5 folds,
p < 0.01) in the content of C18:1, n-9 cis and C18:1, n-9 trans was
observed in Cd Spm, Cd Spm Se treatments. Overall, the
observed uctuation in fatty acid composition resulted a substantial decrease in the ratio of unsaturated to saturated fatty acids
(UFA/SFA) to almost 0.4 (Cd, Cd Put and Cd Put Se) from the
ratio 1.5 in control. Nevertheless, a relatively higher UFA/SFA ratio
was experienced in the plants treated with Se and/or Spm during
Cd exposure.
The MSAP analysis employed to assess the cytosine methylation
at specic restriction sites throughout the genome using
EcoRI HpaII/MspI enzymatic systems resulted in a total of 116
amplication products (Table 4). Cadmium exposure (Cd) was
found to stimulate the demethylation events and resulted in
hypomethylation (18.1%). The exogenous Put application either
alone or in combination with Se during Cd exposure also induced
remarkable demethylation events. On the contrary, the addition of

Se and Spm individually or in combination signicantly reduced

hypomethylation represented by 12.06% and 9.48% in the case of
Cd Spm and Cd Spm Se respectively, over the control events
(7.7%).
3. Discussion
ROS and MDA level are the well known indices for determining
the degree of oxidative stress and considered to be one of the main
contributors for growth retardation [5,6]. The Cd exposed plants
had enhanced level of ROS and MDA contents which together
affected the cell membrane functionality and integrity. These were
in turn interfering with the biosynthesis of photosynthetic
machinery and impaired the subsequent growth. Further, the
photosynthetic pigments which adversely got affected during Cd
exposure did not resume even after the supplementation of Put
indicating the Cd induced down regulation of genes involved
biosynthesis of chlorophyll, proteins of PSI and PSII, electron
transporters, H-ATPase as well the enzymes of Calvin cycle [16].
Moreover, the enhanced LOX activity recorded in Cd, Cd Put and
Cd Put Se treatments reveals the involvement of LOXdependent MDA accumulation besides their direct formation
from ROS. The LOX-dependent lipoperperoxide production has also
been apparent in Lessonia nigrescens and Scytosiphon lomentaria

Table 4
Variation in DNA methylation pattern of G. dura following the exposure to Cd (0.4 mM), Se (50 mM) and polyamines (1 mM) individually or in different combinations for 4 d. Put,
Spm, Cd and Se indicate putrescine, spermine, cadmium and selenium respectively. and  indicate the presence and absence of bands respectively. Class AeP represents the
different conditions of methylation patterns where Class AeC represents unchanged events with no polymorphic bands, Class DeH represents hypomethylation events and
class IeM represents hypermethylation events. Class NeP represents cases of methylation status of the CCGG sequence between control and treated samples showing
unambiguous information.
Parameter

Class

Methylation pattern
Control
HpaII

Banding pattern
Not polymorphic bands

Demethylation events
(Hypomethylation)

Methylation events
(Hypermethylation)

Not informative

Total bands 116

A

B
e
C

E
e
F
e
G
e
H
e
I

N
e
O
e
P
e

Number of sites
Treated

Msp I

HpaII

Msp I

Cd

Se

Cd Se

Put Cd

Put Cd Se

Spm Cd

Spm Cd Se

e
e
e

e
e
e
e
e
e
e

e
e
e

e
e

2
3
68
2
5
9
3
2
2
5
1
3
1
4
2
4

3
3
76
1
2
4
1
0
2
7
1
3
3
4
1
5

2
5
72
2
2
6
1
2
2
8
2
4
3
4
1
4

3
2
66
3
4
8
2
2
3
6
2
4
1
4
4
2

4
3
66
2
3
7
2
2
3
9
1
2
2
5
1
4

3
1
71
4
2
6
1
2
1
8
2
3
3
3
4
2

2
2
74
2
1
5
3
0
2
11
3
4
1
2
4
3

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M. Kumar et al. / Plant Physiology and Biochemistry 51 (2012) 129e138

following the exposure to Cu [6]. On the contrary, additions of Se to

culture media decreased the accumulation of ROS, MDA contents
and counteract the Cd induced changes in fatty acid unsaturation.
When added alone, Se had a similar impact while retaining the
higher content of unsaturated fatty acids without any negative
effects on plant growth. Selenate is thought to enter the plant cell
through sulfate transporters in the plasma membrane and it can be
assumed that Se ions are co-transported with Cd ions by the same
protein carriers. As shown previously, both Cd and Se are bound to
thiol groups of cysteine, an amino acid present in some proteins
[17]. Perhaps competition for the specic binding sites in proteins
partly explains the protective effect of Se against Cd toxicity.
Additionally, the water soluble antioxidants (GSH, AsA, NP-SH
and PCs) were got enhanced by 2e2.5 fold in plants treated with
Se and Spm signies their role for combating the Cd induced
oxidative damage. The tripeptide GSH (L-g-glutamyl- L-cysteinyleglycine) is an important antioxidant that neutralizes ROS,
detoxies Cd directly and is also the direct precursor of PCs [18].
The observed higher level of NP-SHs in these treatments implies
the stimulation of sulfate reduction pathway such as APS reductase
and serine acetyltransferase [19]. The elevated level of PCs in these
treatments could be required to form the Cd-PCs complexes and
their sequestration in the vacuoles to reduce the free Cd concentration in the cytosol [20] Recently, Se based induction of phytochelatins and glutathione was reported in Chlorella vulgaris [21].
On the contrary, a signicant increase in total NP-SH level during
Cd, Cd Put and Cd Put Se with the depleted GSH content in
these treatments supposed to be due to decreased GR activity or its
consumption for PCs synthesis which was also not sufcient
enough to attenuate Cd toxicity. Furthermore, the decreased level
of reduced GSH and AsA level in these treatments accompanied
with a signicant increase in GSSG and DHA level, thus plants
under these treatments seemed inefcient to maintain the GSH/
GSSG and AsA/DHA redox balance to counteract the oxidative stress
[3]. However, the observed high GSH/GSSG and AsA/DHA ratio in
other treatments (Cd Se, Cd Spm, and Cd Spm Se) would
enable the plants to maintain the redox state of cell whilst keeping
the sulfhydryl groups of soluble and membrane proteins in reduced
state resulting to the alleviation of Cd toxicity. Also, as an indispensible strategy for overcoming ROS attack, plants deploy detoxifying enzymes to control the ROS level, preventing the cells from
oxidative stress [3,22]. In this study, it is interesting to note that
higher activities of SOD particularly Mn- and Fe-SOD isoforms in
the treatments of Cd Se, Cd Spm, and Cd Spm Se coincide
with the higher activities of other antioxidant enzymes. This
suggests a rapid breakdown of O

2 radicals by SOD to keep their
levels in control at the place of their generation and subsequently
followed by the action of APX, GR and GSH-Px that might have
allowed plants to combate oxidative stress efciently. Further,
a partial or complete inhibition of SOD-3 (Zn-SOD) in Cd, Cd Put,
Cd Spm and Cd Spm Se treatments shows its sensitivity for
O

radicals. However, the decreased activity of antioxidative
2
enzymes in case of Cd, Cd Put and Cd Put Se could be the
possible reason for inefcient buffering of ROS and collapsing
nature of plants. Also, the decreased GR activity in these treatments
may be attributed to the interaction of Cd with its active site thiol
groups [23]. A sizable increase in GR activity and the induction of
GR-1, 2 and 3 isoforms in plants grown in Se and Spm supplemented media accredit their potentials to mitigate the Cd toxicity.
The role of GR is crucial for the maintaining the higher GSH level
which is not only required for the synthesis of PCs and functioning
of ascorbateeglutathione cycle but also needed as a reductant in
many biochemical reactions [8].
The signicant higher activity of GSH-Px with the induction
of GSH-Px-1 and 4 isoforms specically in Se and Cd Se

135

treatments signied the existence of Se dependent GSH-Px

activities and may be attributed to the increased bioavailability
of Se following co-treatment with sodium selenite [14]. Further,
the up-regulation of GSH-Px and APX by Se in these treatments
may be required to detoxify the H2O2 radicals generated by the
enhanced SOD activity. Evidences for alleviation of Al induced
toxicity by Se through enhanced SOD, GSH-Px enzyme activities
and spontaneous detoxication of ROS has been demonstrated
recently [14]. Enhanced activity of either individual or combined
SOD, APX, GR and GSH-Px enzymes have also been observed in
seaweeds such as U. lactuca [3,24], Gracilaria tenuistipitata [5]
and U. fasciata [7] following their exposure to toxic metals and
industrial efuents.
The increase in SOD and APX activities were totally consistent
with less in situ histochemical staining of O

2 and H2O2 in plants
treated with Spm (Cd Spm and Cd Spm Se). Further, it is
worth-noting here that the exogenous application of Spm during Cd
exposure resulted higher level of both the F and BI forms of Put and
Spm. The mechanism for the PAs particularly Spm against heavy
metal toxicity has been suggested with the formation of ternary
complex with metal cations and the phospholipid polar head. The
highly protonated nature of Spm at physiological pH would have led
to the electrostatic binding of PAs to negatively charged components of membranes, resulting in membrane stabilization through
ionic interactions [3,25]. The membrane damage observed in
Cd Put and Cd Put Se treatments further suggest the Put
mediated unregulatory production of NO, which in turn generates
the peroxynitrite ions (ONOO) by reacting with excess O

2 ions.
These ions appeared to be involved in dis-functioning of many
proteins such as superoxide dismutase, ascorbate peroxidase and
glutathione synthetase, by modifying their Tyr nitration or
oxidizing the thiols residues [26,27].
The ability to adjust membrane uidity by modulating the
unsaturated fatty acids contents with enhanced activity of fatty acid
desaturases is a sign of stress acclimating plants. Fluidity in
membranes is required to maintain the diffusion of lipophilic
compounds and the activities of membrane bound enzymes [28]. A
consistent higher level of n-6 PUFAs (C20-3, n-6 and C20:4, n-6)
and UFA/SFA ratio with Se and Spm supplemention implies their
role in modulation and protection of D5 desaturase from fatty acid
peroxidase activities during oxidative stress. Enhanced desaturase
activity and higher lipid unsaturation has also been demonstrated
in maize seedlings exposed to Cd [22]. On the contrary, declined n-6
PUFAs and UFA/SFA ratio in Cd, and Put supplemented cultures
suggest the Cd induced activation of fatty acids peroxidase activities including dioxygenases, peroxidases and lipoxygenases, lead to
ROS accumulation and loss of membrane integrity. The methylation
and demethylation of DNA cytosines in specic regions of the genes
play a crucial role in regulating the genes expression during plant
development [14]. The Se or Spm added in the culture medium
during Cd exposure appeared to prevent the DNA demethylation
process, as also been demonstrated in animals cells [29]. Changes in
DNA methylation can be considered either a simple indirect effect
of metal stress or a precise defensive mechanism for regulating the
gene expression [30]. Since oxidative stress is known to generate
ROS, the present ndings suggest that the induction of demethylation under Cd stress may have mediated through oxygen radicals.
Participation of oxygen radicals in nucleic acid fragmentation has
been experienced in many stresses [16]. For DNA demethylation,
active or passive mechanisms have also been proposed. The former
mechanism mostly prevails during environmental stresses and
involves enzymes to excise methyl from cytosine residue [31]. The
protective role of Se and Spm in stabilizating the DNA methylation
patterns could be allied either with the elimination of Cd from
enzymes that are active in metabolic processes or with the removal

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136

M. Kumar et al. / Plant Physiology and Biochemistry 51 (2012) 129e138

of ROS and may also be due to the binding Se with metallothioneins

and the replacement of Cd at these sites [14].
4. Materials and methods
4.1. Algal culture and treatments
The vegetative thalli of G. dura were collected from intertidal
region from Veraval Coast (20 540 N, 70 220 E), Gujarat, India. The
fronds thus cleaned were acclimatized to laboratory conditions by
culturing in aerated at bottom round asks in ltered and autoclaved natural seawater (salinity, 32 psu) supplemented with Provasoli Enrichment Seawater medium and GeO2 (5 mg L1) for 10 d.
During the acclimatization period, the medium was replenished
every alternate day and maintained under white cool uorescent
tube lights at 50 mmoL photons m2 s1 with a 12:12 h light:dark
cycle at 22  1  C. For treatment, the acclimatized thalli were
incubated in articial seawater (ASW) having salinity 32 psu
(Accumix, Tulip Diagnostics Pvt. Ltd., Microxpress, India) (without
adding any nutrient and chelator), with CdCl2 (0.4 mM) and
without it (control) for 4 d in aerated cultures. The ameliorating
effect of Se (Na2SeO3) and/or PAs (Putrescine, Put; Spermine, Spm)
against Cd toxicity were also examined as described in the
following treatments:
(1) Control in ASW (C) (2) ASW- 0.4 mM CdCl2 (Cd) (3) ASW50 mM Se (Se) (4) ASW- 50 mM Se- 0.4 mM CdCl2 (Cd Se) (5) ASW1 mM Put (Put) (6) ASW-1 mM Pute0.4 mM CdCl2 (Cd Put) (7)
ASW- 1 mM Pute0.4 mM CdCl2- 50 mM Se (Cd Put Se) (8) ASW1 mM Spm (Spm) (9) ASW- 1 mM Spme0.4 mM CdCl2 (Cd Spm)
(10) ASW- 1 mM Spme0.4 mM CdCl2- 50 mM Se (Cd Spm Se).
Metals and PAs concentrations were chosen according to results
obtained in previous [3] and preliminary experiments carried out
under laboratory conditions.
4.2. Determination of growth, lipid peroxidation, ROS generation,
metal uptake and photosynthetic pigments
The daily growth rate (DGR) was measured as increase in fresh
weight (FW) after 4 days and calculated by using formula DGR
% [(W4/Wo)1/41]  100, where W4 represents fresh weigh after
4 days and Wo as initial fresh weight. The level of lipid peroxidation was determined by thiobarbituric acid reacting substances
(TBARS) assay [32] which is based on the production of malondialdehyde (MDA) during the oxidation of polyunsaturated fatty
acids. The thalli (0.5 g FW) were homogenized in 2 mL of 5% (w/v)
trichloroacetic acid (TCA) and the homogenate was centrifuged at
3500  g for 20 min at 4  C. To 1 mL of aliquot of the supernatant,
1 mL of 20% TCA containing 0.5% (w/v) TBA and 100 mL of 4%
butylated hydroxytoluene (BHT) prepared in ethanol were added.
The mixture was heated at 95  C for 30 min, quickly cooled in ice
and centrifuged. The concentration of TBARS was expressed in
MDA level while subtracting the non specic absorbance
measured at A600 from A532 and using the extinction coefcient
155 mM1 cm1. For ROS determination the individuals of G. dura
(0.5 g FW) were incubated for 1 h at 15  C in 100 mL of 5 mM 2, 7dichlorouoresceine diacetate (Calbiochem, San Diego, CA, USA),
dissolved in ltered seawater. After incubation, the tissue was
rinsed in seawater, blotted dry, weighed, and frozen in liquid
nitrogen. The tissue was then ground in liquid nitrogen, suspended in 5 mL of 40 mM Tris HCl buffer, pH 7.0, and centrifuged
at 15,000  g for 25 min. Fluorescence was determined using LS-5
spectro-uorometer (PerkineElmer, Norwalk, CT, USA) at an
excitation wavelength of 488 nm and at an emission wavelength of
525 nm. Fluorescence values were obtained using a standard
curve of 2, 7-dichlorouoresceine (DCF; Sigma, St. Louis, MO, USA)

[22]. Cd and Se contents were determined in the thalli dried to

constant weight at 60  C. Dried tissues were acid digested by
HNO3/HClO4 (5:1, v/v) and then analyzed by inductively coupled
plasma atomic emission spectroscopy (ICP-AES, PerkineElmer,
Optima 2000). The photosynthetic pigments such as chlorophyll
and carotenoids were extracted in 80% acetone whereas phycobiliproteins were extracted in 100 mM potassium phosphate
buffer, pH 6.5 [22].
4.3. Histochemical detection of O

2 and H2O2
The production of O

2 and H2O2 in response to Cd exposure
were analysed according to Castro-Mercado et al. [33]. For the
detection of O

2 radicals the hand cut sections of thalli (25 in
number) were immersed in 5 ml detection solution containing
0.05% nitroblue tetrazolium (NBT) in 50 mM potassium phosphate
buffer (pH 6.4) and 10 mM NaN3. The sections were inltrated
under vacuum and illuminated for 2 h till appearance of blue formazan. H2O2 production was visually detected by an endogenous
peroxidase-dependent staining procedure using 3, 30 -diaminobenzidine (DAB). Hand cut sections of thalli (25 in number
each) were immersed in DAB solution 1 mg mL1 (pH 5.0), vacuuminltrated for 5 min and illuminated for 4 h till the brown polymerization product formed by the reaction of DAB and H2O2
appeared.
4.4. Determination of polyamines, non-protein thiols, water soluble
antioxidants and phytochelatins
Polyamines were extracted in 5% TCA [12], the TCA extracts were
analysed for free polyamines (F-PAs) and pellet remained after
centrifugation was used for bound insoluble polyamines (BI-PAs)
following their dansylation in a mixture consisted of 200 mL TCA
extract, 400 mL dansylchloride (Sigma, 5 mg mL1 in acetone), and
200 mL saturated Na2CO3. After incubation at 70  C for 1 h, 100 mL
proline (100 mg mL1) was added to remove excess dansylchloride
and extracted in 0.5 mL of ethylacetate by vortexing for 15e30 s.
The organic layer containing the polyamines was separated by low
speed centrifugation (3000  g), removed and dried under
nitrogen. An aliquot of 20 mL was used for spotting onto LK 60
Whatman silica gel thin layer chromatography (TLC) plates. Identication of polyamines was accomplished by comparison of Rf
values with authentic polyamine standards (Put, Spd and Spm
obtained from Sigma as hydrochlorides) on TLC using the solvent
system: cyclohexane: ethylacetate (5:4, v/v) and chloroform: triethylamine (25:2, v/v). Dansyl-polyamines were scrapped off from
the TLC plates and redissolved in 500 mL acetone. The samples thus
obtained were shaken and centrifuged at 6000  g for 3 min, and
quantied using high-resolution spectro-uorometer (PerkineElmer, Norwalk, CT, USA) with excitation at 365 nm and
emission at 510 nm.
Extracts for non-protein thiols (NP-SHs) were prepared in 5%
(w/v) sulfosalicylic acid and measured spectrophotometrically at
412 nm, while adding 1 volume of plant extract prepared in sulfosalicylic acid with 9 volume of Ellmans reagent (containing 5 mM
EDTA, 0.6 mM DTNB in 125 mM phosphate buffer of pH 7.5). NP-SHs
content were calculated using GSH as standard and expressed in
mmol g1 FW. For determining the water soluble antioxidants such
as reduced (GSH) and oxidized glutathione (GSSG), the samples
(500 mg FW) were homogenized in 5 mL of 5% (w/v) sulfosalicylic
acid and centrifuged at 10,000  g for 15 min. The clear homogenate was neutralized with 1.25 M K2CO3 to bring down the pH to
7.0. Total glutathione (GSH GSSG) content was determined by
adding of 100 mL of neutralized homogenate to a reaction mixture
containing 100 mM potassium phosphate buffer (pH 7.0), 0.15 mM

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M. Kumar et al. / Plant Physiology and Biochemistry 51 (2012) 129e138

NADPH, 60 mM 5, 50 -dithio-bis-(2-nitrobenzoic acid) [DTNB] and

0.66 U of GR in a nal volume of 1 mL. The reaction mixture was
incubated for 1 h at 37  C, and the absorbance was determined at
412 nm. GSSG was determined following the same procedure
described above except that 100 mL of the neutralized homogenate
was previously incubated with 20 mL of 1 M 2-vynil pyridine for 1 h
at room temperature. The calibration curve was prepared with
standard GSH in the same reaction mixture. The reduced GSH
content was calculated by the subtraction of oxidized GSH content
from total glutathione content. For determining the reduced (AsA)
and oxidized (DHA) ascorbate, samples (500 mg FW) were
homogenized in 5 mL of 2.5 M perchloric acid and centrifuged at
10,000  g for 15 min. AsA was detected by addition of 100 mL of the
clear homogenate to a reaction mixture containing 2% (w/v) trichloroacetic acid (TCA), 8.8% orthophosphoric acid, 0.01% a,a0 dipyridyl, and 10 mM ferric chloride in a nal volume of 1 mL. The
reaction mixture was incubated for 1 h at 40  C and the absorbance
was recorded at 525 nm. Total ascorbate (AsA DHA) was determined following the same procedure described above except that
100 mL of the clear homogenate was previously incubated with 5 mL
of 100 mM dithiothreitol (DTT) for 1 h at room temperature.
Dithiothreitol was subsequently inactivated by addition of 5 mL of
5% (w/v) N-ethylmaleimide. The calibration curve was prepared
with standard AsA in the same reaction mixture. The oxidised DHA
content was calculated by the subtraction of reduced AsA content
from total ascorbate content. Total phytochelatins concentrations
were calculated by subtracting the amount of GSH from the total
amount of NP-SHs.

4.5. Determination of antioxidative enzymes, lipoxygenase and

their in-gel assay
Extracts for determining the activities of antioxidant enzymes
namely superoxide dismutase (SOD), ascorbate peroxidise (APX),
glutathione reductase (GR) and glutathione peroxidase (GSH-Px)
and lipoxygenase (LOX) were prepared under ice-cooled conditions in the extraction buffer at a proportion of 1:2 (w/v). For SOD,
thalli were homogenized in buffer [0.05 M potassium phosphate
buffer (PPB, pH 7.8), 1% (w/v) polyvinylpyrrolidone (PVP), 0.1 mM
Na2EDTA, and 1% (v/v) triton X-100], for APX [0.1 M PPB (pH 6.8),
1 mM Na2EDTA, 0.1 mM phenylmethylsulphonyl uoride (PMSF)
and 0.5 mM L-AsA (freshly prepared)], for GR 0.1 M PPB (pH 6.8),
3 mM Na2EDTA, 1% (w/v) PVP and 1% (v/v) Triton X-100], for GSHPx [100 mM Tris HCl (pH 7.9), 1% ascorbate and 1% (w/v) PVP]. The
homogenates were centrifuged at 12,000  g for 15 min at 4  C,
and supernatants were assayed for respective enzyme activity
according to Kumar et al. [3]. For SOD, one unit of enzyme activity
was dened as the amount of enzyme required for 50% inhibition
of NBT photoreduction rate and was expressed as U mg protein1.
One unit of enzyme activity was dened as 1 mmol min1 for APX,
GR, and GSH-Px. Extracts for LOX activity were prepared in
extraction buffer containing [50 mM PPB (pH 7.5), 0.2 mM CaCl2,
1 mM GSH, I mM PMSF, 0.3 mM DDT, 0.2 mM EDTA, 1% PVP and
0.1% Triton X-100]. The activity of antioxidant enzymes and LOX
was determined according to Kumar et al. [3], their isoenzyme
analysis and molecular mass were determined with 10% or 12%
non-denaturating polyacrylamide gels using their specic activity
staining procedures [3]. The enzyme activity and isoenzyme
prole of peroxidase (POX) was determined according to Un-Haing
and Kim [34], while preparing the enzyme extracts in buffer
containing 50 mM Tris HCl (pH 7.3), 0.5 M CaCl2 and 5 mM bmercaptoethanol. One unit of POX is dened as the amount of
enzyme releasing 1 mmol of guaiacol radical per min under the
assay conditions.

137

4.6. Determination of fatty acids and methylation sensitive

amplication polymorphism (MSAP) analysis
Fatty acids from lipids were converted to respective methyl
esters by trans-methylation using 1% NaOH in methanol and heated
for 15 min at 55  C, followed by the addition of 5% methanolic HCl
and again heated for 15 min at 55  C [3]. Fatty acid methyl esters
(FAMEs) were extracted in hexane and analyzed by GC-2010
coupled with GCMS-QP2010. FAMES peaks were identied by
comparing their retention time with those of standard mixture
(FAME mix C4-C24, Sigma-USA) by GCMS post-run analysis and
quantied by area normalization.
For MSAP analysis, total genomic DNA extracted from samples
using CTAB method was digested using the restriction enzyme
systems namely EcoR1/MspI and EcoR1/HpaII in two sets of
digestion reaction [35]. In the rst reaction, 400 ng DNA from
treated samples was digested at 37  C overnight with 3 U EcoRI/
HpaII in a 10 mL reaction volume. In the second reaction, 400 ng
DNA was digested with EcoRI/MspI under the same reaction
conditions. Subsequently, the digested DNA fragments from the
two reactions were ligated separately with an equal volume of the
ligation solution containing 10 U DNA ligase, 5 pmol L1 EcoRI
adapter, 50 pmol L1 HpaII-MspI adapter and 1 mol L1 ATP at 15  C
for overnight. The ligation mixture was diluted with TriseEDTA
buffer in ratio 1:10 (v/v) and used as template DNA in the preselective amplication. The sequence of adapters, preamplication and selective amplication primers used for MSAP
analysis is given in Supplementary Table 1. The PCR reaction was
performed in 25 mL of reaction mixture containing 1 mL of diluted
ligation mixture DNA, 0.5 mmol L1 of each forward and reverse
primers, 200 mmol L1 dNTPs, 1X polymerase buffers and 1 U Taq
DNA polymerase (Sigma-USA) for 21 cycle with 30 s denaturation at
94  C, 1 min annealing at 56  C and 1 min extension at 72  C. The
PCR product from the above pre-amplication was diluted to 20
times with TE buffer and used as template DNA for next selective
amplication [35]. The nal selective products were denaturated,
separated by electrophoresis on a 2% agarose gel and visualized
under UV light. The MSAP data originated from the electrophoresis
of PCR products were converted into binary matrix of 1 and 0 based
on presence or absence of band and analysed according to Zhong
et al. [30].

4.7. Statistical analysis

Results are expressed as mean of three replicates with standard
deviation. Statistical analyses were performed by one way analysis
of variance (ANOVA). Signicant differences among the mean
values were determined by least signicant difference (LSD) at
p < 0.05. Also, the polyamines levels have been compared using
non-parametric Mann-Whiteny Wilcoxon, two tailed test.

Acknowledgments
The rst author (MK) gratefully acknowledges the Council of
Scientic and Industrial Research (CSIR) for awarding the senior
research fellowship.

Appendix. Supplementary material

Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.plaphy.2011.10.016.

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M. Kumar et al. / Plant Physiology and Biochemistry 51 (2012) 129e138

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