ORIGINAL PAPER

Accelerated Reactive Oxygen Scavenging System and MembraneIntegrity of Two Panicum Species Varying in Salt Tolerance

Jitha Bhaskaran • Rajaram Panneerselvam

� Springer Science+Business Media New York 2013

Abstract Plant exhibits various patterns of survival under

salinity and their growth and development depend on their

capacity to overcome the stress. Present investigation was

focused on the response and regulation of the antioxidant

defense system and the level of lipid peroxidation in

Panicum miliacium and Panicum sumatrense under salt

treatments. NaCl stress was imposed for 20 days after

sowing of two Panicum species. The changes in the anti-

oxidant enzyme activity like superoxide dismutase, cata-

lase, peroxidase, and ascorbate peroxidase and the rate of

lipid peroxidation level in terms of malondialdehyde

(MDA) were recorded in both Panicum species. A great

correlation exists between the antioxidant enzymes and

lipid peroxidation. The defense mechanism activated in

Panicum species studied was confirmed by the increased

antioxidant enzyme activities under progressive NaCl

stress. MDA content remained close to control at moderate

NaCl concentrations and increased at higher salinities.

Although lipid peroxidation increased in both Panicum

species under salt stress the percent of increase was low in

P. sumatrense indicating its salt-tolerant nature. Another

possible conclusion is that improved tolerance to salt stress

may be accomplished by increased capacity of antioxida-

tive system.

Keywords Antioxidant enzymes � Lipid peroxidation �Osmotic stress � Salt tolerance � Singlet oxygen

Introduction

In signal transduction pathway, reactive oxygen species

(ROS) can be regarded as secondary messengers [1] but over

ROS production is harmful to the plant causing oxidative

stress leading to oxidation of photosynthetic pigments,

membrane lipids, proteins, and nucleic acids [2]. ROS

include free radicals such as superoxide anion (O2-),

hydroxyl radical (OH-), as, as well as non-radical molecules

like hydrogen peroxide (H2O2), singlet oxygen (1O2). Highly

unstable ROS is formed as a by-product of stepwise reduc-

tion of molecular oxygen by electron transport reaction or by

high-energy exposure. When ROS reacts with various bio-

molecules, it causes lipid peroxidation and disturbs mem-

brane integrity and ultimately causing programmed cell

death [3]. One among such abiotic stress is salt stress [4]

which causes ROS production within minutes of salt appli-

cation [5].

Plant cell is evolved with a cascade of antioxidant sys-

tem to withstand the risk caused by the presence of ROS to

protect cells from oxidative damage [6]. Antioxidant

defense system also keeps a check over the levels of active

oxygen species (AOS) [7]. They have the ability to scav-

enge and remove ROS within the plants, and it is this

difference in plant protection mechanisms that decide their

tolerance to stress conditions associated with ROS toxicity.

Antioxidant enzyme activity is greatly affected by salinity

that caused oxidative stress [8]. There is a strong correla-

tion between the antioxidant capacity and NaCl tolerance

which has been demonstrated in some plant species [9, 10].

In ROS detoxification cascade, superoxide dismutases

(SODs) are the first antioxidant defense enzymes, cata-

lyzing the dismutation of superoxide anions by reacting

with superoxide radicals (O2-) to produce H2O2 [11]. In

the absence of natural scavengers such as catalase (CAT)

J. Bhaskaran � R. Panneerselvam (&)

Department of Botany, Annamalai University, Annamalai

Nagar, Chidambaram 608002, Tamilnadu, India

e-mail: rpselvam9@gmail.com

123

Cell Biochem Biophys

DOI 10.1007/s12013-013-9576-x

and peroxidase (POD), H2O2 accumulates in tissues to high

levels. Ascorbate peroxidases (APXs) play a crucial role in

the detoxification of cellular H2O2, the toxic product of

superoxide dismutation. APX removes H2O2 through the

Halliwell–Asada pathway [12, 13]. In general, it is well

accepted that plants with high levels of activity of the

antioxidant systems, both constitutive and induced, have

greater resistance to oxidative damage. However, data on

the effects of salt stress in roots are scarce [14–17].

Lipid peroxidation requires active uptake of O2 and

produces superoxide radical (O2-). Besides O2, the other

highly reactive chemical species involved that initiated

lipid peroxidation 1O2, OH-, and H2O2 [7]. There is a great

relation between antioxidant enzymes and lipid peroxida-

tion. As the level of antioxidant enzymes increase the level

of lipid peroxidation considerably gets reduced. There are

enough evidences that support the alleviation of oxidative

damage and increased resistance to salinity and other

environmental stresses which are often correlated with an

efficient antioxidative system [18–20].

Several studies have shown that salt-tolerant species

increased their antioxidant enzyme activities and antioxidant

contents in response to salt treatment, whereas salt-sensitive

species fails to exhibit the same [21, 22]. As reported by

Foolad et al. [23], Munns and Tester [24], and Grewal [25] at

the whole plant and cellular levels tolerance to biotic stresses

is very complex. This is partly due to the complexity of

interactions between stress factors and various molecular,

biochemical and physiological phenomena affecting plant

growth and development [4]. There are many findings in the

literature which supports the fact that there is close rela-

tionship between the increased enzymatic oxygen scaveng-

ers and increased tolerance to environmental stresses as in

foxtail millet [26], rice [27], and wheat [28].

Materials and Methods

Two landraces namely Panicum miliacium and Panicum

sumatrense seeds were collected from Kolimalai, of Salem

district, Tamilnadu, India and were identified by Tamil

Nadu Agricultural university, Coimbatore, Tamilnadu,

India. The experiment was laid out in a completely ran-

domized block design (CRBD). Pot cultures and the

treatment procedures were carried out in the month of

June–August (2012) in the Botanical Garden and the bio-

chemical analysis was conducted in Stress Physiology

Laboratory, Department of Botany, Annamalai University,

Tamil Nadu, India. The pots were filled with soil con-

taining mixture of red soil, sand, and farm yard manure at

1:1:1 ratio. Four concentrations of NaCl used for the

treatment were 50, 100, 150, and 200 mM, and 0 mM

served as control. For each treatment five replicates were

maintained. Treatments were imposed on the plant on 5,

10, and 15 DAS (days after sowing). On 20 days after,

sowing samples were collected for further analysis.

Measurement of Antioxidant Enzyme Activities

For extraction of enzymes, fresh samples (0.5 g) were

homogenized with 1.5 cm3 of 100 mM potassium

phosphate buffer solution (pH 7.0) containing 2 mM

phenylmethylsulfonyl fluoride (PMSF), and centrifuged at

14,0009g at 4 �C for 20 min.

SOD (EC 1.15.1.1) activity was assayed as described by

Beauchamp and Fridovich [29]. The reaction mixture

contained 1.17 9 10-6 M riboflavin, 0.1 M methionine,

2 9 10-5 M potassium cyanide (KCN), and 5.6 9 10-5 M

nitroblue tetrazolium salt (NBT) dissolved in 3 ml of

0.05 M sodium phosphate buffer (pH 7.8). 3 ml of the

reaction medium was added to 1 ml of enzyme extract. The

mixtures were illuminated in glass test tubes by two sets of

Philips 40 W fluorescent tubes in a single row. Illumination

was started to initiate the reaction at 30 �C for 1 h, iden-

tical solutions that were kept under dark served as blanks.

The absorbance was read at 560 nm in the spectropho-

tometer against the blank. SOD activity was expressed in

units. One unit (U) is defined as the amount of change in

the absorbance by 0.1 h-1 mg-1 protein.

CAT (EC 1.11.1.6) was measured according to the

method of Chandlee and Scandalios [30] with small modi-

fication. The assay mixture contained 2.6 ml of 50 mM

potassium phosphate buffer (pH 7.0), 0.4 ml of 15 mM

H2O2, and 0.04 ml of enzyme extract. The decomposition of

H2O2 is followed by the decline in absorbance at 240 nm.

The enzyme activity is expressed in U mg-1 protein

(U = 1 mM of H2O2 reduction min-1 mg-1 protein).

Peroxidase (EC 1.11.1.7) activity was determined by the

method of Reddy et al. [31]. 20 % homogenate was pre-

pared in 0.1 M phosphate buffer (pH 6.5). The reaction

mixture contained 3.0 ml of pyrogallol solution and 0.1 ml

of the enzyme extract. To the test cuvette, 0.5 ml of H2O2

was added and mixed. The change in absorbance was

recorded at 430 nm every 30 s up to 3 min. One unit of

peroxidase is defined as the change in absorbance/minute at

430 nm.

APX (EC 1.11.1.11) activity was determined as descri-

bed by Asada and Takahashi [32]. The reaction mixture

(1 ml) contained 50 mM potassium phosphate buffer (pH

7.0), 0.5 mM ascorbic acid, 0.1 mM H2O2, and 200 mM of

enzyme extract. The absorbance was read as decrease at

290 nm against the blank, correction was done for the

low, non-enzymatic oxidation of ascorbic acid by H2O2.

The enzyme activity was expressed in U mg-1 protein

(U = change in 0.1 absorbance min-1 mg-1 protein).

Cell Biochem Biophys

123

Determination of Lipid Peroxidation Malondialdehyde

(MDA)

The end-product of preoxidation of polyunsaturated fatty

acids, i.e., malondialdehyde (MDA) was estimated fol-

lowing the thiobarbituric (TBARS) reaction as described

by Heath and Packer [33]. It is the system most prone to

oxidative stress. One gram of tissue (FW) was homoge-

nized in 5 ml of 0.1 % (w:v) TCA. The homogenate was

centrifuged at 10,0009g for 5 min and 4 ml of 20 % TCA

containing 0.5 % (w:v) TBA was added to 1 ml of the

supernatant. The mixture was heated at 95 �C for 30 min

and then quickly cooled on ice. The contents were centri-

fuged at 10,0009g for 15 min and the absorbance was

measured at 532 nm in spectrophotometer. The concen-

tration of MDA was calculated using a extinction coeffi-

cient of 155 mM/1 cm/1. MDA content expressed as

nmol g-1 FW-1.

Results and Discussion

There were remarkable variations in antioxidant enzyme

activities between the two species with increasing NaCl

concentration. P. sumatrense exhibited increased SOD and

CAT activity when compared to P. miliacium at all levels

of NaCl stress.

Antioxidant enzymes such as SOD, CAT, POD, and

APX showed variations in their activities under NaCl

stress. The present results demonstrate a differential effect

of salt stress according to the duration of salt treatments on

shoot and root of two Panicum species. Root and shoot

exhibited large fluctuations with different salinity levels.

There was a significant (P B 0.05) increase in SOD, CAT,

and APX in shoots and roots of both the species of Pani-

cum. We observed greater activities of antioxidant enzymes

in roots than in shoots which suggest the existence of an

effective scavenging mechanism to remove ROS in roots

because roots are the first organs that come in contact with

salt and are thought to play a critical role in plant salt

tolerance. Similar variations were observed in previous

work carried out such as in pea [34], wheat [35], and

tomato [36]. Recently, Jiang et al. [37] analyzed the pro-

teome of Arabidopsis roots under NaCl stress and showed

that detoxifying enzymes such as APXs, glutathione per-

oxidases, and SODs are up-regulated by salt stress.

In our investigation, we found to a larger extend the

amount of ROS is modulated by SOD activity. The only

enzyme able to dismutate O2- to H2O2 and O2 is SOD.

SOD activity was found to be high in both shoot and root of

P. sumatrense, whereas there were no remarkable increase

in P. miliacium (Figs. 1 and 2). There was very little or no

changes in SOD activity in shoot and root of P. miliacium,

with the values being 112.75 % in shoot and 111.47 %

over control in root. In P. sumatrense, the values were

found to be high in comparison with P. miliacium, and it

was 156.81 % and 144.35 % over control in root and shoot

respectively. An increase in SOD activity under diverse

abiotic stresses has been shown in several plants [38]. As

reported earlier by Tsang et al. [39], SOD expression is

known to be substrate inducible. An increase in the SOD

activity may be due to an increased formation of AOS as

substrate that leads to increased expression of genes

encoding SOD. Our results does match with the findings of

Mallik et al. [40], where they observed accelerated amount

of SOD and CAT activity during NaCl stress in diverse

group of plants.

CAT activity was recorded to increase in both the

Panicum species and at high NaCl concentrations their

activity also increased significantly in P. sumatrense, but in

P. miliacium the percentage of increase was not much

visible. In shoot and root of both the species, maximum

values were recorded at 200 mM of NaCl treatment and it

was found to be 121.67 and 200.34 % over control in

shoots (Fig. 3) and 131.57 and 258.19 % in roots (Fig. 4)

of P. miliacium and P. sumatrense, respectively. Almost it

was double the activity of CAT enzymes in P. sumatrense.

In our study, we found considerable decrease in SOD

activity and increase in CAT and POD activity in P. mil-

iacium. This result goes hand in hand with the findings of

Rahnama and Ebrahimzadeh [41], who reported that in

potato cultivars salt stress caused reduction in SOD activity

and increase in the activity of CAT and POD. Kukreja et al.

[42] reported increase of CAT activity in Cicer arietinum

roots under salinity stress.

POD activity showed negative correlation with the level

of NaCl treatment in P. sumatrense. As and when the

Fig. 1 Superoxide dismutase (SOD) activity in shoots of P. milia-cium (blue) and P. sumatrense (green) under NaCl stress. Values are

mean ± SE based on six replicates. Bars with different letters are

significantly different at P B 0.05 (Color figure online)

Cell Biochem Biophys

123

concentration of NaCl stress increased POD activity con-

siderably got reduced in P. sumatrense, the values were

-79.56 % in shoot and -63.06 % in root (minus value

denotes the % of reduction). Unlike POD activity in

P. sumatrense, P. miliacium showed an increase in POD

activity (Fig. 5) and with the concentration of NaCl, POD

activity also increased. In P. miliacium, POD activity

reached the value 142.16 and 138.83 % over control in

shoot and root, respectively. At 200 mM of NaCl stress

maximum activity of POD was observed in P. miliacium,

and minimum values were recorded in P. sumatrense

(Figs. 5 and 6). Sreenivasulu et al. [43] reported that in

salt-tolerant Setaria italica, POD activity was found to be

higher to protect plants against the stress. In many cases, it

has been proposed that salt stress tolerance is related to a

higher activity of antioxidant enzymes such as APX, CAT,

and SOD, and that lower activity is found in sensitive

species [21].

An increase in APX activity was found in both the

Panicum species (Fig. 7), but the increase was considerably

less in P. miliacium when compared with P. sumatrense roots

and shoots (Fig. 8). As CAT and SOD activity, APX activity

also increased considerably in both the species. In root the

activity was found to be high and among the species,

P. sumatrense had high APX activity. In P. miliacium, it was

171.53 and 189.97 % over control in shoot and root,

respectively. Whereas in P. sumatrense, APX activity was

recorded to be 225.87 % over control in shoot and 251.06 %

over control in root under 200 mM of NaCl treatment.

Considering the fact that P. sumatrense have a higher activity

of CAT and APX enzymes (both inherent and salt-induced),

we suggest that both the enzymes contribute equally in H2O2

detoxification in P. sumatrense. These findings were in good

support with the findings of Shalata et al. [21], where SOD

and CAT activities considerably increased in salt-tolerant

cultivar where as it decreased in roots of a salt-sensitive

Fig. 2 Superoxide dismutase (SOD) activity in roots of P. miliacium(blue) and P. sumatrense (green) under NaCl stress. Values are

mean ± SE based on six replicates. Bars with different letters are

significantly different at P B 0.05 (Color figure online)

Fig. 3 Catalse (CAT) activity in shoots of P. miliacium (blue) and P.sumatrense (green) under NaCl stress. Values are mean ± SE based

on six replicates. Bars with different letters are significantly different

at P B 0.05 (Color figure online)

Fig. 4 Catalase (CAT) activity in roots of P. miliacium (blue) and P.sumatrense (green) under NaCl stress. Values are mean ± SE based

on six replicates. Bars with different letters are significantly different

at P B 0.05 (Color figure online)

Fig. 5 Peroxidase (POD) activity in shoots of P. miliacium (blue)

and P. sumatrense (green) under NaCl stress. Values are mean ± SE

based on six replicates. Bars with different letters are significantly

different at P B 0.05 (Color figure online)

Cell Biochem Biophys

123

tomato cultivar. These results are similar in part to results

obtained by Ghorbanli et al. [44]. Overall, the activities of

leaf SOD, POD, CAT, and APX in P. sumatrense were

greater than that of P. miliacium.

The increased antioxidant enzyme activities were cor-

related with decreased lipid peroxidation in the salt-stres-

sed Panicum species (Fig. 1). The data suggest that a rise

of SOD, CAT, and APX activity is stress-induced ROS

scavenging mechanism preserving membrane integrity. All

together, the data obtained from this study indicate that the

responses of oxidative stress and the related enzymes

strongly depend on the severity of NaCl stress. With age of

the plant, APX, CAT, and SOD activity increased gradu-

ally in the control shoot and root (0 mM), but rapidly in the

treated ones. It is visible that the enzyme activity was

continuously enhanced with the NaCl concentration. In the

roots of C. arietinum, under NaCl stress the enzymes such

as SOD, POD, CAT, and APX and their activity got ele-

vated [42]. In two maize genotypes, namely PEHM 3

(tolerant) and Navjot (susceptible), SOD, APX, and CAT

activity was exhibited high in PEHM 3 when compared to

Navjot [45]. These results are parallel to some earlier

studies [9, 46] where enhanced activities of CAT and POD

were related to salt tolerance in different crop species, e.g.,

strawberry [47] and canola [48].

Due to the decreased CAT activity, H2O2 accumulation

was enhanced in P. miliacium. Singha and Choudhuri [49]

reported that H2O2 accumulation in the leaves of Vigna and

Oryza seedlings under salinity stress was related to a

decrease in CAT activity. Decreased CAT activities, in

turn, might have promoted H2O2 accumulation in P. mili-

aceum, which could result in a Haber–Weiss reaction to

form hydroxyl radicals [11]. Since OH� radicals are known

to damage biological membranes and react with most

compounds present in biological systems [50], they might

have hastened lipid peroxidation and membrane damage in

P. miliacium. Since both SOD and CAT are inactivated by

singlet oxygen and peroxyl radicals [51], these enzymes

might have been deactivated in root and shoot of

P. miliacium by the increased levels of ROS.

MDA, a decomposition product of polyunsaturated fatty

acids hydroperoxides, has been utilized very often as a

suitable biomarker for lipid peroxidation [52], which is an

effect of oxidative damage. Salinity has a profound effect

on lipid peroxidation of plasma membrane, thus affecting

membrane permeability modulating the pattern of ion

leakage [35, 42]. A small ‘‘age-depended’’ increase in lipid

peroxidation was apparent at all stages of plant growth

under normal condition and it was found to be compara-

tively less under various levels of salinity in P. suma-

trelnse, but the change was much apparent in shoots and

roots of P. miliacium. The level of membrane damage in

P. miliacium increased significantly under all of the treat-

ments compared to the control and the values reached the

Fig. 7 Ascorbate peroxidase (APX) activity in shoots of P. miliacium(blue) and P. sumatrense (green) under NaCl stress. Values are

mean ± SE based on six replicates. Bars with different letters are

significantly different at P B 0.05 (Color figure online)

Fig. 8 Ascorbate peroxidase (APX) activity in roots of P. miliacium(blue) and P. sumatrense (green) under NaCl stress. Values are

mean ± SE based on six replicates. Bars with different letters are

significantly different at P B 0.05 (Color figure online)

Fig. 6 Peroxidase (POD) activity in roots of P. miliacium (blue) and

P. sumatrense (green) under NaCl stress. Values are mean ± SE

based on six replicates. Bars with different letters are significantly

different at P B 0.05 (Color figure online)

Cell Biochem Biophys

123

peak at 150 and 200 mM of NaCl concentration (Table 1).

On a whole, contrast to P. miliacium, shoots and roots of

P. sumatrense showed no much significant change in lipid

peroxidation with increased NaCl treatments.

At 200 mM of NaCl treatment, maximum of MDA

content was noted in both shoot and root of both Panicum

species. Among them, root displayed more MDA content

when compared to leaf. In roots of P. miliacium the values

were 215.94 % over control and in P. sumatrense it was

found to be 184.31 % over control. The increase was steep

in P. miliacium, whereas MDA content increased slowly in

P. sumatrense. The data of the present investigation shows

the increase in MDA content is in positive correlation with

the increase in concentration of NaCl which is in terms

with the previous findings of Aghaleh et al. [53]. This

depicts the membrane damage due to the peroxidation of

lipids which enhances ROS production leading to oxidative

stress. These finding coincides with the previous reports,

where salinity causes drastic lipid peroxidation in wheat

[54] and sugarcane [55]. The lower level of lipid peroxi-

dation in leaves and roots of P. sumatrense suggests that,

these cultivars are better protected from oxidative damage

under drought stress than P. miliacium. Valentovic et al.

[56] showed salinity increase MAD content in corn salt-

sensitive cultivar but in tolerant cultivar, MAD remained

unchanged. These finding coincides with the previous

reports, where salinity causes drastic lipid peroxidation in

wheat [54] and sugarcane [55].

Our finding highlights the relevance of antioxidant

system to protect the cell against the oxidative damage.

P. sumatrense had much lower lipid peroxidation (MDA)

and higher antioxidant enzyme activity when compared to

P. miliaceum. From the data obtained we can say that a

significant elevation in antioxidant enzymes (SOD, CAT,

POD, and APX) occurred and this was inversely related

with the level of lipid peroxidation. Furthermore, the

degree of increase of antioxidant enzymes were more and

the level of lipid peroxidation was relatively less and this

relation between antioxidant enzymes and lipid peroxida-

tion were more visible in P. sumatrense when compared to

P. miliacium. Therefore, P. sumatrense can be considered

as better tolerant species than P. miliacium. Our findings

are in concordance with the findings of Demiral and Tur-

kan [22] and Hernandez et al. [57].

Conclusion

In view of the results observed in this work, the enzymatic

antioxidative system in two Panicum species was highly

affected by salinity. Salt-stressed Panicum species exhib-

ited specific responses of increased SOD, CAT, POD, and

APX activities, accompanied by decreased lipid peroxida-

tion and their responses vary between species. The signif-

icant increase in the activities of SOD, CAT, POD, and

APX in NaCl-stressed Panicum species to some extend

could overcome the damage caused to the membranes due

to lipid peroxidation in terms of MDA and these two fac-

tors were highly correlated with the regulation and main-

tenance of the normal functioning of the cell under salinity

stress. It is clear that the application of saline irrigation

resulted in increase in membrane injury (MDA content) in

P. miliacium and the level of increase was less in P. su-

matrense. Together with a relative importance of CAT,

POD, and APX in H2O2 detoxification, the results suggest

that significance of tissue- and age-specific activities of

antioxidant enzymes are responsible in determining the

membrane integrity, which is in turn dependent on the

species, the development and the metabolic state of plant as

well as the duration and intensity of stress.

However, a direct correlation cannot always be found

between salt stress tolerance and the induction of antioxi-

dant enzyme as proved by Munns and Tester [24] in

transgenic plants where overexpressing these enzymes did

not always induce salt tolerance. Hence, in view of con-

siderable variations in the protective mechanisms against

ROS in different plant species or cultivar, further work is

needed to establish the general validity of this phenomenon

in salt tolerance.

Table 1 NaCl effect on lipid peroxidation in terms of malonyladehyde (MDA) in two Panicum species

NaCl treatments P. miliacium P. sumatrense

Shoot Root Shoot Root

Control 2.44 ± 0.04a 2.76 ± 0.21a 1.43 ± 0.43a 2.04 ± 0.16a

50 mM 3.75 ± 0.14b (153.69) 4.33 ± 0.33b (156.88) 1.44 ± 0.03a (100.70) 2.47 ± 0.12b (121.08)

100 mM 3.79 ± 0.43c (155.32) 4.35 ± 0.06b (157.61) 1.52 ± 0.02b (106.29) 2.49 ± 0.09b (122.06)

150 mM 4.32 ± 0.36d (177.05) 4.63 ± 0.09c (167.75) 2.51 ± 0.39c (155.94) 3.35 ± 0.53c (164.21)

200 mm 4.76 ± 0.07e (195.08) 5.96 ± 0.17d (215.94) 2.54 ± 0.61c (177.62) 3.76 ± 0.37d (184.31)

Values are mean ± SE of four replications. The mean with the same letters do not differ statistically by Duncan’s multiple range test (P B 0.05).

Values in parenthesis denote the percent (%) over control

Cell Biochem Biophys

123

Acknowledgments The authors take this opportunity to thank

UGC-BSR for the financial support and the management of

Annamalai university for providing the facility to carry out the

present work.

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