Cadmium Stress Induced Changes in Antioxidant Enzymes , Lipid Peroxidation and Hydrogen Peroxide Contents in Barley Seedlings

Cadmium pollution is a problem of increasing significance for ecological, nutritional and environmental reasons. Different plant species and varieties show a wide range of plasticity in Cadmium tolerance, from a high degree of sensitivity to the hyper-accumulating phenotype of some tolerant plants. To avoid Cadmium toxicity, plants adopt various defense strategies. The present study was undertaken to assess and investigate the antioxidant responses of barley (Hordeum vulgare L.) to cadmium treatment, seedlings of barley were grown in increasing concentrations of CdCl2 ranging from 25-100 μM, for up 14 days in a hydroponic system. The results showed that CdCl2 reduce pigment content and caused oxidative damage as characterized by increased total soluble protein, Malondialdehyde (MDA) and Hydrogen peroxide (H2O2) contents. Under cadmium stress, the activities of antioxidative enzymes, including Ascorbate Peroxidase (APX), Peroxidase (POD) and Catalase (CAT) were increased considerably in plant tissues. The present results allow us to conclude that the barley plants showed a negative response to cadmium toxicity. The physiological and biochemical process in plants was significantly affected by stress of CdCl2. To deal with the cadmium induced oxidative stress, barley plants activated antioxidant enzymes to diminish the Reactive Oxygen Species (ROS).


INTRODUCTION
Heavy metal contamination in soil mainly due to industrial emission, the application of sewage sludge and phosphate fertilizers containing Cadmium (Davis, 1984), has become a serious problem in crop and vegetable production.It is widely recognized that cadmium taken up by plants is the main source of cadmium accumulation in food (López-Millán et al., 2009).Cadmium can be easily absorbed by plant roots and transported to shoots.Roots are likely to be firstly affected by heavy metals since much more metal ions are accumulated in roots than shoots (Sanita di Toppi and Gabbrielli, 1999).Numerous studies have indicated that cadmium causes nutrient deficiency (Boulila et al., 2006;López-Millán et al., 2009) and induces inhibition of chlorophyll biosynthesis and a decline in the photosynthetic rate (Tukaj et al., 2007;Lopez-Millan et al., 2009).Cadmium toxicity also causes oxidative damages, such as cell membrane lipids and protein damages to plants, directly or indirectly via Reactive Oxygen Species (ROS) formation (Lesser, 2006).ROS can be quenched by the induction of enzymatic defense systems to alleviate the oxidative damage in Cadmium stressed plants (Wu et al., 2003).
Cadmium is a toxic element traces causing serious problems in cereal crops (Prasad, 1995).Among the oldest and most important crops, we expect Barley (Hordeum vulgare L.).It now ranks fifth among all cultures in the production of cereals in the world behind corn (Zea mays L.), wheat (Triticum spp.), Rice (Oryza sativa L.) and soybeans.The importance of barley stems from its ability to grow and be productive in marginal environments, which are often, characterized by drought, low temperatures, conditions of high salinity (Maas and Hoffman, 1997) and pollution of heavy metals (Dionisio-Sese and Tobita, 1998).
Therefore, the barley plants (Hordeum vulgare cv.Saida) were chosen as a biological model in this study.The objective of this study was to evaluate the responses of the defense system and the tolerance of barley cultivar Saida under stress induced by cadmium.

MATERIALS AND METHODS
Plant material and treatments: Seeds of "Hordeum vulgare cv.Saida" were provided by the Algerian Office Inter Cereals (AOIC) El Hadjar Annaba, Algeria.Barley seeds were sterilized by 5% sodium hypochloride solution for 10 min and then rinsed thoroughly with distilled water, submerged in deionized water in the dark overnight and germinated on filter paper in dishes petri.Twelve-days-old uniform seedlings (second leaf stage) were transplanted on to 3 L pots.The composition on the basic nutrient solution consisted of the following compounds (mg/L): (NH 4 ) 2 SO 4 48.2,MgSO 4 65.9, K 2 SO 4 15.9, KNO 3 18.5, Ca (NO 3 ) 2 59.9, KH 2 PO 4 24.8,Fe citrate 6.8, MnCl 2 •4H 2 O 0.9, ZnSO 4 •7H 2 O 0.11, CuSO 4 •5H 2 O 0.04, H 3 BO 3 2.9 and H 2 MoO 4 0.01.Different concentrations of CdCl 2 (25, 50, 75 and 100 µM, respectively) were added to the nutrient solution.The pH of the culture solution in each pot was adjusted every other day with 1 M HCl or NaOH as required.The solution was continuously aerated with an air pump and renewed every 4 days.

Determination of chlorophyll and carotenoid contents:
The concentrations of chlorophyll a, b and carotenoids (mg/g FW) were evaluated by adopting the method given by Arnon (1949).The absorbance was measured at 663, 645 and 470 nm, respectively.The concentration of pigments was calculated according to the formulas of Lichtenthaler and Wellburn (1985).

Lipid peroxidation and hydrogen peroxide determinations:
The level of lipid peroxidation in plant tissues was expressed as 2-Thiobarbituric Acid (TBA) reactive metabolites, mainly Malondialdehyde (MDA) and was determined according to Hodges et al. (1999).Fresh samples (leaves and roots) of around 0.5 g were homogenized in 4.0 mL of 1% Trichloroacetic Acid (TCA) solution and centrifuged at 10,000×g for 10 min.The supernatant was added to 1 mL 0.5% (w/v) TBA made in 20% TCA.The mixture was heated in boiling water for 30 min and the reaction was stopped by placing the tubes in an ice bath.The samples were centrifuged at 10,000×g for 10 min and the absorbance of the supernatant was recorded at 532 nm.Correction of non-specific turbidity was made by subtracting the absorbance value read at 600 nm.The level of lipid peroxidation was expressed as nmol/g fresh weight, with a molar extinction coefficient of 0.155/mM/cm.

The Hydrogen peroxide (H 2 O 2 ):
The Hydrogen peroxide (H 2 O 2 ) contents in the leaves and roots were assayed according to the method of Velikova et al. (2000).Leaves and roots were homogenized in ice bath with 0.1% (w/v) TCA.The extract was centrifuged at 12,000×g for 15 min, after which to 0.5 mL of the supernatant was added 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI and the absorbance was read at 390 nm.The content of H 2 O 2 was given on a standard curve.

Determination of total soluble protein content:
Protein contents were assayed by following Bradford's method (Bradford, 1976) with BSA as standard.The absorbance was measured at 595 nm.

Antioxidant enzyme activity determinations:
The leaves and roots of barley were collected for enzyme analysis.Fresh samples (1.0 g) were homogenized in ice-cold 50 mM phosphate buffer (pH 7.5).The homogenate was centrifuged at 12 000 g for 20 min at 4°C and the supernatants were used for the various enzymatic assays.
Catalase (CAT) activity was determinated according to Cakmak and Horst (1991).The reaction mixture for catalase in a total volume of 3 mL contained 50 mM Na-phosphate buffer (pH 7.2) and 300 mM H 2 O 2 .The reaction was started by adding enzyme extract and the activity was determined by monitoring the initial rate of H 2 O 2 disappearance at 240 nm (ε = 39.4 /mM/cm).

Statistical analysis:
All values reported in this study are the mean of at least three replicates.For each parameter, data were subjected to a one-way ANOVA analysis.When the effect was significant (p = 0.05), differences between means were evaluated for significance by using Tukey's (HSD) test (MINITAB software version 16.0).

Lipid peroxidation (MDA) and Hydrogen peroxide (H 2 O 2 ) contents:
The MDA and H 2 O 2 contents in both the roots and leaves of barley plants are shown in Table 2.The application of CdCl 2 (25, 50, 75 and 100 µM, respectively) of barley plants results in very significant (p<0.001) for the production of MDA in leaves and roots in all treated plants compared to the control.Treatment with cadmium in the roots compared to the control product a change in the synthesis of lipoperoxides.These increases relative to the control from the first concentration CdCl 2 and they multiply from 50 µM and strongly accumulate at the 100 µM concentration.The H 2 O 2 content in both the roots and leaves of barley plants are shown in Table 2.The H 2 O 2 content in the leaves and roots of barley plants increased markedly (p<0.001) when the plants were exposed to cadmium stress.The accumulation of H 2 O 2 increased in leaves of barley at higher cadmium concentrations, H 2 O 2 peaked at 100 µM in leaves.The content of H 2 O 2 in roots increased and peaked at different concentrations of CdCl 2 .Thus, we see that the increase of the synthesis of H 2 O 2 is higher in roots than in the barley leaves.
Total soluble protein content: Table 3 shows the change in total soluble protein content in the roots and leaves of barley plants.From the results, we observe a very highly significant increase in total soluble protein in the roots and leaves of the treated plants compared to the control (p<0.001).

Antioxidant enzyme activity:
The changes in antioxidative enzymatic activities in barley roots and leaves, including APX, POD and CAT, induced by Cadmium at different concentrations are shown in (Table 4).
Cadmium caused a marked induction in APX activity in leaves and roots of barley.APX was increased significantly in leaves and roots barley with 100 µM CdCl 2 treatment as compared to the control.The changes in APX activity were also statistically significant among the different concentrations of CdCl 2 (p<0.001).
Catalase is a key enzyme in protecting cells against oxidative stress.In the present work, CAT activities in leaves and roots barley were increased at all CdCl 2 treatments compared with the control (Table 4).The highest Catalase activity of the barley roots and leaves occurred at 100 µM CdCl 2 .Furthermore, the changes in CAT activities were statistically significant among all treatments (p<0.001).
Exposure of plants to toxic metals can cause many physiological and biochemical disorders.Concentrations of photosynthetic pigments are often measured to assess the impact of many environmental stresses.According to our results, Cadmium induces a lowering concentrations of chlorophyll (a, b, a+b) and carotenoids.This is consistent with numerous studies that report a reduction in the concentration of chlorophyll in the presence of cadmium (Wang and Zhou, 2006;Groppa et al., 2007;Belkhadi et al., 2010;Agami and Mohamed, 2013) and heavy metals in general (Mysliwa-Kurdziel and Strzalka, 2002;Lei et al., 2007).The decrease in chlorophyll is a primary event in plants subject to metal stress and results from the inhibition of the enzymes responsible for the biosynthesis of the chlorophyll (Mysliwa- Kurdziel and Strzalka, 2002).The stress induced by cadmium decreases the rate of assimilation of CO 2 causing disruption in the process of photosynthesis and the chlorophyll degradation and inhibition of its biosynthesis; which could lead to disruptions in the transport of electron flow PSI and PSII leading to the reduction of O 2 and the generation of ROS (Moussa, 2004).
Cadmium also induces a decrease in the concentration of carotenoids; this is consistent with many studies on various plants (Mysliwa-Kurdziel and Strzalka, 2002;Belkhadi et al., 2010).The high accumulation of cadmium in leaves of barley plants is probably responsible for the production of ROS; this can cause partial destruction of antioxidants such as carotenoids.
According to our results, we also note that the leaves and roots of plants exposed to cadmium have high levels of H 2 O 2 and MDA as a result of the generation of ROS.The increase of MDA production indicates increased lipid peroxidation.It is well known that the peroxidation of polyunsaturated fatty acids of membrane phospholipids and causes a deficit of membrane functions, in particular through reduction of the fluidity and the inactivation of enzymes and receptors located at the membranes (Lagadic et al., 1997).This can then participate in a change in membrane permeability.It is also possible that partial exclusion of Cadmium results from a change of the capacity of the cell walls to bond to the metal or an increased excretion of chelating substances as reported by Ghosh and Sigh (2005) and Kirkham (2006).
As regards hydrogen peroxide, we have observed a significant increase of H 2 O 2 in leaves and roots; this may be related to oxidative damage the membrane.Foyer et al. (1997) indicates that H 2 O 2 is a strong oxidant that can initiate localized oxidative damage leading to disruption of function and loss of metabolic cell integrity at sites where it accumulates.H 2 O 2 and other ROS may be responsible for lipid peroxidation.H 2 O 2 can release at relatively long distances causing changes in the redox status of tissues and surrounding cells to which relatively low concentrations or triggers an antioxidant response.These results may indicate that the harmful impact of toxic metals on plants is probably exerted by ROS production.
Numerous studies have demonstrated an increase in total protein in plants under different stress (heavy metals, pesticides and drought) (Haiyan et al., 2005;Zhiqiang et al., 2009).We observed similar results in our work, especially in the roots of barley treated with cadmium.Moreover, the accumulation of total protein in the leaves and roots of barley may be related to oxidative damage generated by the toxicity of cadmium.Indeed, the variation of the levels of total protein is linked firstly to the variation of the enzymatic activities (Zhou et al., 2004).On the other hand, in response to the substantial ROS production plants develop antioxidant defense mechanisms including the induction of stress protein (Sanita di Toppi and Gabbrielli, 1999;Shah et al., 2001).
The accumulation of intracellular ROS in situations of environmental stress leads to the activation of defense mechanisms by increasing antioxidant enzyme activities or mechanisms to repair oxidative damage (Ramel et al., 2009).Indeed, according to our results we found that cadmium tends to stimulate the synthesis of antioxidant enzymes (APX, POD and CAT) in leaves and roots of barley.Similar results have been reported by many studies (Hegedüs et al., 2001;Aravind and Prasad, 2003;Tiryakioglu et al., 2006;Touiserkani et al., 2012).Levels of APX, CAT and POD increased under certain environmental conditions such as the presence of high concentrations of salt or heavy metals (Sugimoto and Sakamoto, 1997).In response to increased ROS, the antioxidant defense system including POD, APX and CAT plays an important role in ROS scavenging (Sandalio et al., 2001;Ali et al., 2013;Issaad et al., 2013).However, according to our results we note that the defense system could not regulate the concentration of H 2 O 2 produced by the stress induced by cadmium was strongly accumulated in leaves and roots of barley.
These results are consistent with previous reports by Chou et al. (2012) for rice and Mohamed et al. (2012) for Indian mustard, indicating that antioxidant enzymes are not a sufficient defense system.A drastic increase in H 2 O 2 may consequently have a lower extensibility of plant cell walls, which can rapidly terminate growth (Shutzendubel and Polle, 2002).

CONCLUSION
The results related to the effects of the toxicity of cadmium on enzyme activities are very controversial.The reason for such inconsistent results on the effects of cadmium seems to be related to some differences in the study material: • Plant organ studied (root, leaf) • Duration of exposure and concentration of cadmium used • Cultivars and genotypes (or plant species) considered in the studies (Tiryakioglu et al., 2006) The results obtained in the present work showed that the barley Saida is a sensitive and non-tolerant cultivar to cadmium toxicity.The sensitivity of this cultivar is associated with increased enzyme activity and levels of hydrogen peroxide and registered trapping defense mechanisms.In perspective, in the future we should pay particular attention to the study of the impact of cadmium on different cultivars of barley to upgrade the culture of barley in Algeria.

Table 1 :
Changes of chlorophyll and carotenoid contents (mg/g FW) in leaves of barley seedlings induced by CdCl2

Table 2 :
Effect of CdCl2 on the Malondialdehyde (MDA) and Hydrogen peroxide (H2O2) concentrations of barley plants

Table 3 :
Data are presented as mean±S.E. of three replicates for each parameter; The same letters after the data indicate that there is no significant difference at a probability level of 95% (Tukey's test) Effect of CdCl2 on the total soluble protein content of barley plants