Antioxidative defense mechanism of the ruderal Verbascum olympicum Boiss. against copper (Cu)-induced stress

Ayşegül Uğar Akpınar; Hülya Arslan; Gürcan Güleryüz; Serap Kırmızı

doi:10.1515/biol-2016-0002

Article Open Access

Antioxidative defense mechanism of the ruderal Verbascum olympicum Boiss. against copper (Cu)-induced stress

Ayşegül Uğar Akpınar , Hülya Arslan , Gürcan Güleryüz and Serap Kırmızı

Published/Copyright: February 16, 2016

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From the journal Open Life Sciences Volume 11 Issue 1

Abstract

The endemic Verbascum olympicum has characteristics that allows it to live in degraded areas of Uludağ Mountain, Turkey and therefore known as a ruderal species. In this study, V. olympicum seeds collected from Uludağ Mountain were grown in the Hoagland nutrient solution, under hydroponic conditions. The activity of antioxidative enzymes (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX) were examined to demonstrate the role of antioxidative mechanism in the seedlings exposed to different Cu concentrations (0, 50, 250, 500 μM) for seven days. Also, certain growth parameters (such as the water content, biomass production, soluble protein), the level of lipid peroxidation, cell membrane injury and permeability were investigated. As a result, some toxic effects are observed following the application of 500 μM Cu, after the seedlings growing in 50 and 250 mM Cu concentrations showed high resistance and survived in hydroponic conditions. Our findings provide information about the resistance of V. Olympicum seedlings to oxidative stress caused by excessive Cu concentrations.

Keywords: Copper; Superoxide dismutase; Ascorbate peroxidase; Catalase; V. olympicum

1 Introduction

Destroyed areas such as the roadsides, developed building areas, rubbish tips, mining areas are increasing because of rapid industrialization and urbanization. It has become an increasingly serious environmental problem [1], as these disturbances give rise to heavy metal contamination of ecosystems.

Copper is the third most used metal in the world [2]. The upper limit of Cu content in unpolluted soil is 15 mg/kg [3]. When it is above this limit, it is a source of stress for many plant species, mostly ending with the death of the plant species [4]. Disruption of membrane permeability leading to ion leakage, inhibition of photosynthesis and respiratory processes, inhibition in enzyme activity and increased levels of oxidative stress are negative effects of high Cu concentrations in plants [5-7]. Also, excess Cu concentrations induce overproduction of reactive oxygen species (ROS) such as superoxide (O₂^-1), hydroxyl radical (OH^-) and hydrogen peroxide (H₂O₂) [8]. Antioxidative defence systems, including enzymes such as superoxide dismutase (SOD); catalase (CAT) and ascorbate peroxidase (APX), play an important role in preventing damage created by ROS. These protective capacity of the antioxidative defence systems change according to species, Cu concentrations and exposure durations [9].

Ruderal species typically dominant in degraded areas and have a relatively great potential to use limited resources in degraded areas. These plant species were endemic and have adapted to contaminated soils by developing tolerance mechanisms to metal-induced stress. Verbascum olympicum has become a dominant ruderal plant in the local flora of destroyed areas in Uludağ Mountain (Bursa-Turkey) [10]. At the same time, V. Olympicum has some functional properties like high nitrate assimilation capacity [11] and high organic matter production [12]. In addition, heavy metal accumulation in different plant parts of V. Olympicum have been determined by Güleryüz et al. [13,14].

In this study, we aimed to determine whether variations in antioxidant defence system play a role in the Cu tolerance of V. Olympicum and to investigate the levels of oxidative damage caused by excess Cu concentrations as the Cu content in the destroyed areas of Uludağ Mountain is about twice the upper limit of unpolluted soil [15]. Thus, we investigated the effects of excess copper concentrations (50, 250 and 500 μM) in the V. Olympicum seedlings growing under hydroponic conditions and identified both physiological and biochemical changes induced by culture in the presence of copper. We analyzed a number of plant growth parameters (such as the water content, biomass production and soluble protein), the cell membrane integrity and permeability, the level of lipid peroxidation and the activity of antioxidative enzymes (SOD, CAT and APX) in both leaves and roots of the seedlings.

2 Material and Methods

2.1 Plant material and Culture conditions

Seeds were collected from 1850-1900 m elevation of Uludağ Mountain, Turkey and were stored in paper bags in room conditions. To avoid variations between individual plants due to genetic characteristics, seeds from only one individual plant were used in the experiments. The seeds were sterilised with 5% sodium hypochlorite for 5 min and rinsed with double distilled water. Sterilised seeds were planted in Petri dishes with the help of water-moistened filter paper (Figure 1). Petri dishes were enclosed with aluminium foil for germination in dark conditions and incubated at 20°C for 48 h. For the development of cotyledons in germinating seeds, Petri dishes were wrapped with stretch film and placed in a growth chamber (Heraeus Vötsch HPS500) at 15°C/25°C day/night temperature with a 16 h photoperiod. On the 10^th day after sowing, seedlings with two cotyledons were transferred to polyvinyl chloride plates floating on 10% Hoagland's nutrient solution [16], (Figure 2). This solution was renewed every other day and concentration of the solution was increased by 10% every week. The seedlings were grown in these conditions for eight weeks (Figure 3). Then uniform plants with 8-leaves were selected from among seedlings growing in the presence of Cu, which was added in the form of CuSO₄ (E. Merck, Darmstadt, Germany, 1027901000). Here, either 0 (control), 50, 250 or 500 μM Cu was added to 80% Hoagland nutrient solution. Five plants per treatment were used and three replications Seeds were collected from 1850-1900 m elevation of Uludağ Mountain, Turkey and were stored in paper bags in room conditions. To avoid variations between individual plants due to genetic characteristics, seeds from only one individual plant were used in the experiments. The seeds were sterilised with 5% sodium hypochlorite for 5 min and rinsed with double distilled water. Sterilised seeds were planted in Petri dishes with the help of water-moistened filter paper (Figure 1). Petri dishes were enclosed with aluminium foil for germination in dark conditions and incubated at 20°C for 48 h. For the development of cotyledons in germinating seeds, Petri dishes were wrapped with stretch film and placed in a growth chamber (Heraeus Vötsch HPS500) at 15°C/25°C day/night temperature with a 16 h photoperiod. On the 10^th day after sowing, seedlings with two cotyledons were transferred to polyvinyl chloride plates floating on 10% Hoagland's nutrient solution [16], (Figure 2). This solution was renewed every other day and concentration of the solution was increased by 10% every week. The seedlings were grown in these conditions for eight weeks (Figure 3). Then uniform plants with 8-leaves were selected from among seedlings growing in the presence of Cu, which was added in the form of CuSO₄ (E. Merck, Darmstadt, Germany, 1027901000). Here, either 0 (control), 50, 250 or 500 μM Cu was added to 80% Hoagland nutrient solution. Five plants per treatment were used and three replications per plant were performed. Plants were harvested on the 1^st, 3^rd and 7^th days of treatment. Plant samples were washed thoroughly with de-ionised water and separated into root and leaf portions. Fresh plant materials were used for the determination of growth parameters such as the water content, biomass production and cell membrane injury and permeability; the rest were stored at 70°C after freezing in liquid nitrogen.

Fig. 1

The sowed seeds of V. olympicum in sterilised petri dishes.

Fig.2

The seedlings with two cotiledone in 10% Hoagland solution

Fig.3

Eight week old seedlings, before Cu application

2.2 Plant biomass and water content

Plant biomass was measured on dry weight basis (mg dry weight / plant). Fresh weight of plant samples was measured. Then, they were dried at 80°C until the weight became constant and their dry weight was measured. Water content (%) and the biomass of roots and leaves were determined by assessing differences between fresh and dry weights.

2.3 Cell membrane injury and permeability

For an estimation of the cell membrane permeability, electrolyte leakage was determined according to the method described by Masood et al. [17]. Fresh leaves (0.5 g) and root samples (0.25 g) were cut into pieces and immersed in 20 ml distilled de-ionised water. They were incubated in a water bath at 32°C for 2 h and the initial electrical conductivity of the medium (EC₁) was measured. Then, the samples were autoclaved at 121°C for 20 min to release all the electrolytes. After cooling to 25°C, the final electrical conductivity (EC₂) was measured. Electrolyte leakage was calculated as the percentage of the initial value (EC₁) over the final value (EC₂)

The cell membrane permeability (%):(EC₁/EC₂)*100

Also, cell membrane injury was expressed according to the formula above expressed as a percentage,[18]:

The cell membrane injury (%): 1 - (1 - EC₁/EC₂) / (1–EC₁^*/EC₂^*)

where, E^* is the electrical conductivity of the control sample.

2.4 Lipid peroxidation

Lipid peroxidation was measured as the amount of malondialdehyde (MDA) determined by thiobarbituric acid (TBA) reaction and was expressed as nmol/g fresh weight. MDA content was determined described by Heath & Packer [19] with some modifications. Leaf samples (0.1 g fresh weight) were homogenised in 0.5 ml of 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 15,000 g for 10 min. Then, the reaction mixture containing 0.5 ml of the supernatant and 1.5 ml of the mixture of 20% TCA and 0.5% TBA was added into new tubes. After incubation at 95°C for 30 min, the tubes were cooled with ice and centrifuged (15,000 g, 4°C for 5 min). The absorbance of the mixture was read at 532 and 600 nm (Novaspec II, LKB Biochrom). The MDA concentration was calculated using the extinction coefficient of 155 mM^-1cm^-1

2.5 Total soluble protein (TSP) content

TSP content was determined according to the method described by Bradford [20] and expressed as mg g^-1 fresh weight. To prepare the stock dye solution, 100 mg Coomassie Brillant Blue G250 was dissolved in 50 ml of 95% ethanol, then 100 ml of 85% phosphoric acid was added. When the dye had completely dissolved, it was diluted to 1 litre and filtered through Whatman #1 paper. The dye solution was stored in a dark bottle at 4°C. BSA (bovine serum albumin) was used as a standard.

2.6 Extraction of plant material for antioxidative enzyme assay

1 g fresh mass was homogenised with 3 ml of buffer solution containing 50 mM Na-phosphate buffer (pH 7.8), 1 mM EDTA and 2% (w/v) polyvinylpolypyrrolidone (PVP) in an ice bath. Homogenised materials were centrifuged at 14,000 g for 40 min at 4°C. The supernatants were transferred into Eppendorf tubes for the determination of enzymatic activities. For the APX activity assay, 2 mM ascorbate was added to the extraction buffer solution described above.

2.7 Superoxide dismutase (SOD) assay

The SOD (EC 1.15.1.1) activity was determined by the method of Beauchamp & Fridovich [21]. This method is based on the inhibition of the nitroblue-tetrazolium at 560 nm. The total mixture (3 ml) in the SOD activity assay contained 0.5 ml of the enzyme extract, 2.5 ml of an ice-cold buffer containing 20 mM sodium phosphate buffer (pH 7.5), 0.1 mM EDTA, 10 mM methionine, 0.1 mM p-Nitro Blue Tetrazolium (NBT) and 5 μM riboflavin. Control test tubes (without samples) and sample test tubes were placed under 300 μmol m^-2s^-1 fluorescent light for 15 minutes. Then, the reaction was stopped by switching off the light. The absorbance of the solution was measured at 560 nm (Novaspec II LKB Biochrom). The SOD assay kit (SOD S7446, Sigma-Aldrich, USA) was used in the preparation of standards. After the calculation of % inhibition, the enzyme activity was determined according to the linear equation which is obtained from the curve and expressed as units per mg protein.

2.8 Catalase (CAT) assay

The CAT (EC 1.11.1.6) activity was assayed as described by Lester et al. [22] with some modifications. The reaction was initiated by adding 0.1 ml of the enzyme extract to the assay mixture containing 20 mM sodium phosphate buffer (pH 6.8) and 15 mM H₂O₂. The change in absorbance was measured at 240 nm (extinction coefficient 40 mM^-1cm^-1) for 3 minutes (Shimadzu UV-2100). The activity of enzyme was expressed as units per mg protein.

2.9 Ascorbate peroxidase (APX) assay

The APX (EC 1.11.1.11) activity was determined according to the method described by Lester et al. [22]. This enzyme activity was stated from the decrease in absorbance at 290 nm as ascorbate was oxidised. The reaction mixture was comprised of 50 mM potassium phosphate (pH 6.6), 0.25 mM ascorbate and 1 mM H₂O₂(3% H₂O₂) and 1 ml of the enzyme extract. The change in absorbance was measured at 290 nm (extinction coefficient of 2.8 mM^-1cm^-1) for 3 minutes (Shimadzu UV-2100). The activity of enzyme was expressed as units per mg protein.

2.10 Statistical analysis

Results were expressed in figures and tables as mean values ± SD. The experiments were set up in a completely randomised design. According to different Cu concentrations and exposure periods, data were subjected to two-way ANOVA using SPSS 16.0 for Windows (SPSS Inc. 2007). For statistical analyses significance of the results was noted at P < 0.05 levels.

3 Results

3.1 Effects of Cu on plant growth and morphology

The growth of V. Olympicum seedlings was decreased depending on the time of exposure to Cu and the concentration of Cu. The root and leaf biomass values began to decline on the 3^rd day and reached a minimum value on the 7^th day (P < 0.05). The lowest biomass values were measured in the seedlings exposed to 500 μM Cu on the 7^th day for both the leaves (366.6 ± 24.5 mg dry weight / plant) and roots (158.8 ± 9.1 mg dry weight / plant) (Table 1). The water content (%) in the leaves and roots decreased with as growth conditions induced increasing Cu concentrations on all the sampling days (P < 0.05) Whereas the highest root and leaf water content was observed in control seedlings on the 7^th day, the lowest root and leaf water content values were obtained in the seedlings exposed to 500 μM Cu on the 7^th day (Table 1)

Table 1

Biomass (mg dry weight / plant) and water contents (%) in plant parts of V. Olympicum seedlings exposed to different Cu concentrations for seven days (Mean ± Standard Deviation; n = 5, α = 0.05)

Biomass (mg dry weight/plant)					Water Contents(%)
	Concentrations	1st day	3rd day	7th day	1st day	3rd day	7th day
Leaves	Control	696.0±41.3	721.6±30.1	764.5±29.3	88.6±7.3	87.2±5.0	90.6±5.4
	50μM Cu	689.9±35.5	663.3±33.0	641.9±37.9	88.0±4.8	71.8±3.3	64.6±3.6
	250μM Cu	672.8±24.5	548.6±29.5	513.6±24.5	78.2±3.0	57.6±3.0	48.8±2.3
	500μM Cu	679.8±25.7	463.6±30.7	366.6±24.5	69.2±3.3	39.4±2.4	23.8±1.8
Roots	Control	364.9±50.5	440.8±24.7	449.8±26.7	79.6±3.8	79.6±3.6	81.6±2.6
	50μM Cu	382.6±25.3	347.7±32.9	294.7±28.1	79.6±6.0	73.0±2.0	70.2±1.8
	250μM Cu	357.2±26.6	280.6±15.1	191.2±28.9	78.8±3.6	64.4±3.4	47.8±3.2
	500μM Cu	360.7±38.2	226.9±22.3	158.8±9.1	77.6±3.8	50.4±2.6	28.4±2.7

[All parameters were analysed for concentration (Cu) x duration at α; 0.05 significance level according to Two-way ANOVA.Root Biomass, F_{Concentration(3,48)} = 100.79, p = 0.000, F_{Duration(2,48)} = 50.69, p = 0.000, F_{Concentration} X _{Duration(6,48)} = 25.39, p = 0.000.Leaves BiomassF_{Concentration(3,48)} = 150.15, p = 0.000, F_{Duration(2,48)} = 72.09, p = 0.000, F_{Concentration x Duration(6,48)} = 35.70, p = 0.000.Root Water content, F_{Concentration(3,48)} = 194.19, p = 0.000, F_{Duration(2,48)} = 203.33, p = 0.000, F_{Concentration x Duration(6,48)} = 55.14, p = 0.000.Leaves Water content, F_{Concentration(3,48)} = 327.02, p = 0.000, F_{Duration(2,48)} = 183.69, p = 0.000, F_{Concentration x Duration (6,48)} = 29.54, p = 0.000].

In addition to growth restriction, chlorosis occurred in the margins of the leaves of the seedlings and a reduction in the expansion of leaves was observed at 500 mM Cu treatment (Figure 4). Also, the formation of roots in the seedlings exposed to 500 mM Cu treatment on the 7^th days was poor, resulting in decreased root length and root volume. In the plants treated with 50 mM Cu only root length reduced (Figure 5).

Fig. 4

The 500 μM Cu-treated seedlings on the 7^th day

Fig.5

The 50 μM Cu-treated seedlings on the 7^th day

The total soluble protein content was significantly decreased in the roots and leaves of seedlings treated with 250 μM and 500 μM Cu (Figure 6; P<0.05). The lowest TSP content was found in the 500 μM Cu-treated seedlings at 7 days for both the leaves (1.30 ± 0.03 mg/g YA) and roots (1.01 ± 0.04 mg/g YA). However, the TSP content was increased in the roots and leaves of seedlings with 50 μM Cu treatment (Figure 6). The highest TSP content in the seedlings treated with 50 μM Cu was found on the 7^th day for the roots and on the 3rd day for the leaves.

Fig.6

Total Soluble Protein (TSP) contents in leaves and roots of Verbascum olympicum seedlings exposed to different Cu concentrations for seven days (mg protein, n = 5). Values are mean of five replicates ±SD and bars indicate standart error (SE). [All parameters were analysed for concentration (Cu) × duration at α; 0.05 significance level according to Two-way ANOVA. Root TSP Content, F_{Concentration(3,48)} = 640.62, p = 0.000, F_{Duration(2,48)} = 269.91, p = 0.000, F_{Concentration × Duration(6,48)} = 328.31, p = 0.000 Leaves TSP Content, F_{Concentration(3,48)} = 499.92, p = 0.000, F_{Concentration(2,48)} = 166.58, p = 0.000, F_{Concentration(6,48)} = 111.09, p = 0.000].

3.2 Changes in MDA (lipid peroxidation) content and cell membrane injury and permeability

The MDA contents in the leaves of seedlings increased markedly when growth conditions included Cu concentrations at 250 μM and 500 μM (Figure 7; P < 0.05). The highest MDA content was determined in the leaves of the seedlings exposed to 500 μM Cu for 7days. However, in the seedlings treated with 50 μM Cu, no changes relating to lipid peroxidation were observed.

Fig.7

Malondialdehyde (MDA) contents in leaves of V. olympicum seedlings exposed to different Cu concentrations for seven days (nmol/gYA, n = 5). Values are mean of five replicates ±SD and bars indicate standart error (SE). [All parameters were analysed for concentration (Cu) × duration at α; 0.05 significance level according to Two-way ANOVA. Leaves MDA Content, F_{Concentration(3,48)} = 512.33, p = 0.0000, F_{Duration(2,48)} = 312.38, p = 0.0000, F_{Concentration× Duration(6,48)} = 132.8, p = 0.000].

Electrolyte leakage, which was representative of the cell membrane permeability, tended to increase significantly in both the leaves and roots with increasing Cu concentrations and increasing durations of exposure (Table 2; P < 0.05). The percentage of electrolyte leakage at 500 μM Cu concentration on the 7^th day exhibited the highest value for both the leaves and roots. The highest electrolyte leakage value in the roots was approximately 30% higher than the value of the control seedlings on the 7^th day, whereas electrolyte leakage approximately doubled in the leaves compared to the control seedlings after 7 days of treatment.

A similar trend was also observed for the cell membrane injury and this level of injury increased in both the leaves and roots with exposure to increasing levels of Cu on all sampling days (Table 2; P < 0.05). The highest value was found in the seedlings exposed to 500 μM Cu treatment for 7 days. This value was 75% for the leaves, 65% for the roots.

Table 2.

The electrolyte leakage(%) and cell membrane injury (%) in plant parts of V. Olympicum seedlings exposed to different Cu concentrations for seven days (Mean ± Standard Deviation; n = 5, α = 0.05).

		Electrolyte leakage(%)			Cell Membrane Injury(%)
	Concentrations	1st day	3rd day	7th day	1st day	3rd day	7th day
Leaves	Control	44.2±3.0	46.7±3.1	43.6±2.4	0	0	0
	50μM Cu	66.8±1.9	69.4±1.1	69.2±2.1	37.8±3.4	45.8±2.0	45.5±3.7
	250μM Cu	69.6±1.4	74.6±1.8	81.5±1.9	46.3±2.4	55.0±3.1	67.3±3.3
	500μM Cu	74.3±1.4	79.9±1.6	86.0±2.1	54.6±2.6	64.5±2.9	75.2±3.8
Roots	Control	54.5±3.2	54.4±1.6	53.7±2.9	0	0	0
	50μM Cu	63.2±0.5	64.7±0.9	67.0±0.3	19.2±1.0	22.5±1.9	27.6±0.8
	250μM Cu	66.8±0.5	70.9±1.1	74.6±0.8	27.3±1.0	36.2±2.4	44.3±1.8
	500μM Cu	69.5±0.8	76.6±1.1	84.1±1.1	33.2±1.9	48.7±2.3	65.2±2.4

[All parameters were analysed for concentration (Cu)× duration at α; 0.05 significance level according to Two-way ANOVA. Root Electrolyte leakage percentage F_{concentration(3,48)} = 606.89, p = 0.000, F_{Duration(2,48)} = 87.46, p = 0.000, F_{concentration}×_{Duration(6,48)} = 23.42, p = 0.000. Leaves Electrolyte leakage percentage, F_{Concentration(3,48)} = 851.31, p = 0.000, F_{Duration(2,48)} = 55.70, p = 0.000, F_{concentration×Duration(6,48)} = 12.06, p = 0.000. Root Cell Membrane Injury percentage, F_{concentration(3,48)} = 2592.07, p = 0.000, F_{Duration(2,48)} = 406.28, p = 0.000, F_{concentration}×_{Duration(6,48)} = 92.7, p = 0.000. Leaves Cell Membrane Injury percentage F_{Conentration(3,48)} = 1735.23, p = 0.000, F_{Duration(2,48)} = 106.82, p = 0.000, F_{concentration×Duration(6,48)} = 20.08, p = 0.000]

3.3 Alteration in the antioxidant enzymes activities

The SOD enzyme activity in both the roots and leaves was elevated by growth conditions with added Cu; this elevation of SOD enzyme activity was proportional to the increasing Cu concentrations and durations of exposure (Figure 8; P < 0.05). The highest SOD activity was determined in the leaves and roots of seedlings exposed to 500 μM Cu for 7 days. Whereas this value for the roots was approximately 6 times higher than the control seedlings; for the leaves, it was approximately 3 times higher than the control seedlings.

The Cu treatments led to remarkable elevation of the APX activity in both the leaves and roots (Figure 8). The APX activity was significantly increased in the roots and leaves of seedlings depending on both the Cu concentration and duration of exposure (P < 0.05). The mean APX activity reached 2.52 ± 0.04 units /mg protein in the leaves of seedlings treated with the highest Cu concentration on the 7^th day, whereas it was 1.20 ± 0.05 units /mg protein in control seedlings on the first day. A similar trend was also observed for the APX activity for the roots samples. The highest APX activity was observed in the roots of seedlings exposed to 500 μM Cu for 7 days.

Fig.8

Enzyme activities of Superoxide dismutase (SOD), Askorbate peroxidase (APX) and Catalase (CAT) in leaves and roots of V. olympicum seedlings exposed to different Cu concentrations for seven days (U/mg protein, n = 5). Values are mean of five replicates ± SD and bars indicate standart error (SE).[All parameters were analysed for concentration (Cu)×duration at α; 0.05 significance level according to Two-way ANOVA. Root SOD Activity, F_{Concentration(3,48)} = 594.48, p = 0.000, F_{Duration(2,48)} = 204.57, p = 0.0000, F_{Concentration×Duration(6,48)} = 56.52, p = 0.000.Leaves SOD Activity, F_{Concentration(3,48)} = 246.71, p = 0.000, F_{Duration(2,48)} = 121.93, p = 0.000, F_{Concentration}×_{Duration(6,48)} = 13.13, p = 0.000 Root APX ACtivity, F_{Concentration(3,48)} = 1767.20, p = 0.000, F_{Duration(2,48)} = 516.54, p = 0.000, F_{Concentration × Duration(6,48)} = 89.95, p = 0.000, Leaves APX Activity F_{Concentration(3,48)} = 1707.99, p = 0.000, F_{Duration(2,48)} = 409.64, p = 0.000, F_{Concentration}×_{Duration(6,48)} = 91.73, p = 0.000,Root CAT Activity, F_{Concentration(3,48)} = 2943.23, p = 0.000, F_{Duration(2,48)} = 829.50, p = 0.000, F_{Concentration × Duration(6,48)} = 137.60, p = 0.000,Leaves CAT activity, F_{Concentration(3,48)} = 1208.40, p = 0.000, F_{Duration(2,48)} = 544.20, p = 0.000, F_{Concentration × Duration(6,48)} = 76.13, p = 0.000]

The leaf and root CAT activity of seedlings varied significantly with treatments of increasing Cu concentrations and increasing durations of exposure (Figure 8; P < 0.05). The highest values for CAT activity were observed on the 7^th day with growth conditions of 250 μM Cu for both leaves and roots. This value reached 2.80 ± 0.04 units /mg protein for the roots of seedlings and 2.28 ± 0.09 units /mg protein for the leaves. The CAT activity in both the roots and leaves of seedlings decreased when growth conditions included 500 μM Cu.

4 Discussion

These findings indicate that the water content (%) and biomass value (mg dry weight / plant) in the leaves and roots of V. olympicum were significantly decreased by addition of increasing levels of Cu in the nutrient medium (Table 1). It is seen that the growth of V. Olympicum seedlings slowed due to increasing Cu concentrations and duration of exposure to elevated Cu concentrations. Plant growth and development are negatively affected by oxidative stress, which presumably occurred due to the generation of large amounts of ROS induced by the treatments with elevated Cu concentrations. Moreover, it is known that oxidative stress damages a range of specific plant hormones that play important roles in the growth and development [23].

In addition to growth restriction, chlorosis and poor root formation occurred in the margins of leaves of seedlings exposed to 500 mM Cu treatment indicating that the seedlings of V. Olympicum are negatively affected by elevated Cu concentrations (Figure 4). It is possible that these observed changes in the seedlings may be due to competition between elevated Cu ions and other metal ions present in the nutrient solution, such as Mn, Zn, and Fe, which serve as micronutrients [24]. Indeed, it has been reported that similar growth related changes in the plant morphology occur due to Fe deficiency [25]. These effects resulted in a decrease of the water content of plants due to a reduction in the absorption surface and similar observations have been reported by studies using radishes [25] and sunflowers [26]

Growth of plants in the presence of elevated levels of Cu is able to directly generate ROS through the Haber-Weiss reaction and the increased levels of ROS initiate oxidative stress-related effects [27, 28]. Lipids and proteins are the main target of ROS, such as H₂O₂, OH^• and O2^• and lipid peroxidation is the most obvious indicator of oxidative stress. For example, when the seedlings of V. Olympicum were treated with 250 μM and 500 μM Cu in the nutrient medium marked increase in malondialdehyde (MDA) content were observed; these effects were also dependent on treatment duration (Figure 7). However, in the 50 μM Cu treatments, no such changes were observed. The increased capacity of lipid peroxidation observed in treatments with ≥ 250 μM Cu is probably due to the oxidizing properties of Cu⁺², which promotes the catalytic formation of extremely reactive hydroxyl radicals [29]. Similar results have also been reported in leaves of Peganum harmala [30], Withania somnifera [31] and Astragalus neo-mobayenii [32]. Furthermore, Olga et al. [33] reported that elevated levels of copper stimulated lipid peroxidation and enhanced membrane permeability in Hydrilla verticillata.

Electrolyte leakage which is representative of cell membrane permeability is in accordance with the findings in MDA content (Table 2). Electrolyte leakage was markedly increased as a function of Cu treatment in our study. The obtained results in cell membrane injury were similar to increase in the level of electrolyte leakage. The highest value in the cell membrane injury was found in the leaves and roots of seedlings exposed to 500 μM Cu for 7 days. In support of these current findings it has been found that electrolyte leakage was increased by the generation of ROS due to decreasing the membrane stability in Avicennia germinans and W.somnifera [31, 34]

The TSP content is another important indicator in understanding the responses to metal induced-stress responses [35] and these effects occur because ROS generation induced by heavy metals causes disturbances in protein synthesis pathways. In the current study, treatment in the presence of 50 μM Cu caused an increase in the TSP content. These results suggest that these growth conditions in the presence of 50 μM Cu was not the cause of structural damage in V. Olympicum seedlings in the short-term. However, TSP content in all plant parts (leaves and roots) remarkably decreased in treatments with 250 μM Cu (Figure 6). These alterations are related to nitrogen assimilation and also to the inhibition of enzymes involved in protein synthesis in Verbascum olympicum [14]. Similar results have been observed in Cu-tolerant E. Haichowensis [36] and also in both tolerant and sensitive species of Silene vulgaris [37].

Boojar and Goodarzi [27] reported that increases in antioxidative enzyme activities in plants is related to the level of tolerance against Cu toxicity. In this current study, growth condition including elevated Cu concentrations increase the antioxidative enzyme activity. The levels of SOD activity in both the roots and leaves are directly related to both the increasing Cu concentrations and durations of exposure (Figure 8). The highest SOD activity in all plant parts was found in the seedlings exposed to 500 μM Cu for seven days. Since SOD acts as a primary agent in defence against oxidative stress, the changes observed herein emphasise that exposure to Cu induces stress responses that generate superoxide radicals in V. Olympicum seedlings. Furthermore, our observations are consistent with those advanced by Thounaojam et al. [38] and Devi & Prasad [39]. In this current study, the levels of SOD activity in the roots was greater than that in the leaves of V. Olympicum seedlings suggesting that there is a higher level of oxidative stress in roots.

The Cu treatments led to a remarkable elevation in APX activity in both the leaves and roots (Figure 8). The increased levels of APX activity in V. Olympicum seedlings indicates the importance of the ascorbate-glutathione (ASH-GSH) cycle in reducing the toxic effect of elevated Cu concentrations [40]. Thounaojam et al. [38] reported that elevated APX activity acts to prevent the accumulation of toxic levels of H₂O₂ in photosynthetic organelles and enhancement of APX activity has been observed in various plants such as rice, tea and mulberry under different treatment conditions with elevated Cu concentrations [38, 41, 42]. In addition to these changes in APX activity, the CAT activity in both the roots and leaves of V. Olympicum seedlings decreased in growth condition with 500 μM Cu concentration (Figure 8). The present study showed that CAT enzyme activity in V. Olympicum seedlings is sensitive to high Cu concentrations and its activity is inhibited in treatments with such high Cu concentrations. In fact, it has been reported that elevated Cu levels have an inhibitory effect on CAT activity [43]. Similar responses in CAT activity have also been described in several studies [44-46].

5 Conclusion

Responses of V. Olympicum to Cu-induced stress are important indicators for reversible and irreversible changes in metabolic homeostasis. In this study, the effect of elevated Cu concentrations was investigated due to the fact that V. Olympicum is a species that can live in degraded areas. The V. Olympicum seedlings growing in the presence of 50 and 250 mM Cu concentrations were largely resistant to the potential adverse toxic effects of Cu and survived under these hydroponic growth conditions. It is important to note that at Cu concentrations of 50 mM no negative effects on the growth parameters and cell membrane integrity were observed. The long-term effects of the growth conditions including 250 μM Cu is a research topic for future studies. Furthermore, treatments with 500 mM Cu concentrations for seven days appeared to be the most toxic conditions for V. Olympicum seedlings and all measured parameters reached maximal levels. Our current findings suggest that V. Olympicum seedlings exhibit resistance to oxidative stress promoted by elevated Cu concentrations. It is proposed that the resistance to Cu-induced stress demonstrated by V. Olympicum seedlings is related to the plants ability to maintain a balance between the generation of ROS and their detoxification by SOD, CAT and APX.

Previous studies investigating the physiological and antioxidative responses to heavy metal toxicity have generally been on cultivated plants [1,5,39,47]. There are very few studies that investigate Cu-induced stress responses in wild plants. However, from an ecological perspective it is increasingly important to understand the mechanisms that maintain the life of wild plants due to the increasing problems associated with metal pollution worldwide.

Acknowledgements.

This study was supported by the Scientific Research Commission of Uludağ University [to G.GULERYUZ, Project No UBAP(F)-2009/27] and is part of the PhD thesis of A. AKPINAR.

Conflict of Interest: Dr.Akpinar has nothing to disclose.

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Received: 2015-11-12

Accepted: 2015-12-21

Published Online: 2016-2-16

Published in Print: 2016-1-1

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