The Growth and Antioxidative Responses of Sonchus oleraceus ( Linnaeus , 1753 ) Under Cu ( II ) , Pb ( II ) , Cd ( II ) and Cr ( VI ) Stress Condition

Plants growing in soil containing heavy metal polutan such as chromium (Cr), lead (Pb), cadmium (Cd) and copper (Cu) will be stunted, and increase production of Reactive Oxygen Species (ROS). In dealing with the excess amount of ROS, plants have an enzymatic defense system, using superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxide (APX). The aim of this study was to determine and analyze the Sonchus oleraceus (Linnaeus, 1753) plant response to heavy metals stress, seen from the growth and antioxidative defense enzymatically. Research carried out using a Completely Randomized Design (CRD) with four treatments and five replicates. The metal treatment was 10 mg ⋅ L−1. The presence of heavy metals in the growing medium significantly decreased the plant height and leaf area, so the impact is on the weight of wet and dry weight. The metal treatments of Cr, Cd, Pb and Cu increase the activity of SOD and APX enzymes but decrease the activity of the CAT enzyme. Chromium is a metal that has a significant influence on the growth and activity of SOD, APX, and CAT enzymes in S. oleraceus.


Introduction
Heavy metal pollution is one of the problems of the environment in which the presence of a metal in the soil, air and waters exceeds the threshold and being toxic.Heavy metal is an element that has a density of more than 5 g ⋅ cm −3 [1].Heavy metals are naturally contained in the soil but can also come from agricultural and industrial waste as well as the burning of transportation.On soil, heavy metal pollution lead to various problems including the disruption of the growth of soil organisms and plants as well as the problems associated with aesthetics (discoloration and odor) [2].
Heavy metals can be essential for plants for example copper (Cu), but Cu is only needed in very small amounts, the exessive concentration can be toxic to plants.According to Rokhmah research, 0.3 mg ⋅ L −1 dissolved Cu concentrations disrupt the growth of rice plants [3].Heavy metals that are non-essential for plants such as lead ICBS Conference Proceedings (Pb), cadmium (Cd) and chromium (Cr).The metals are toxic to plants despite in the low amount.In plants, high concentrations of Pb can interfere the process of photosynthesis, chlorophyll synthesis and synthesis of antioxidant enzymes [4].Cd is very reactive and can activate a lot of enzyme activity that needed by the cell [5].Cd accumulates in potato plants inhibit the formation of chlorophyll thereby reducing biomass [6].Cr accumulation can increase the activity of antioxidative enzymes and decrease the rate of growth in Sorghum plants [7,8].
The plasma membrane is the first part that is in direct contact with heavy metals, so that it will disrupt the absorption of other essential elements.Along with the heavy metal absorption of nutrients and water uptake by plant roots, then the compound will accumulate to plant's parts such as roots, stems, and leaves [9].The presence of heavy metals can disrupt metabolic processes and developmental germination [10].Heavy metals cause the decrease of leaf area and plant dry weight, root plasma membrane damage, a decrease in photosynthetic activity and the growth of bean plants [11].Heavy metal also affects the lowered spinach plant leaf area, causing the death of cells in the leaf tips and leaf growth rate slows [12].
Heavy metal toxicity in plants are also associated with the producer of ROS (Reactive Oxygen Species) which reached 30 times higher than normal conditions [13].ROS are oxygen derivatives oxidizing compounds that are highly reactive.ROS can be H 2 O 2 (hydrogen peroxide), O 2 (superoxide radicals), OH (hydroxyl radical), HO 2 (hydrogen oxide radicals), etc. [14].Under normal conditions, ROS are produced in low quantities in the cell organelles, but when the amount exceeds the amount of ROS increases antioxidative defense, it can cause a condition called oxidative stress.ROS can damage the membrane and react with biomolecules, thereby disrupting normal cellular structure and function are indicated by abnormalities (symptoms) [15].
Antioxidant defenses needed to reduce oxidative damage from ROS.The defense can be enzymatic and non-enzymatic [16].In enzymatically for example using enzymes superdioxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX).Balance the activity of SOD, CAT and APX are important in suppressing ROS in cells.SOD is an initial defense against ROS, followed by CAT and APX work [17] [18].Oxidative defense becomes an important mechanism for plants tolerant to fight heavy metal stress, because the plant cannot move so susceptible to heavy metal stress in their habitat.
Sonchus oleraceus (Linnaeus, 1753) or known as tempuyung is a weed that is included in the annual plant that grows wild in the area with an altitude of 200 m to 2 200 m above sea level in areas exposed to sunlight and a little shade.This plant is used as food and medicine.S. oleraceus can be used as test plants as easily obtained and grown, its life cycle is relatively short at around 3 mo and widely used as a medicinal plant for diseases such as inflammation, kidney stones and blood purifier.
The research using S. oleraceus the treatment of heavy metal is still rare.Most of it only research on the content of S. oleraceus for use as a medicine.The aim of this study was to determine and analyze the plant response to stress S. oleraceus heavy metals, in terms of the growth of and enzymatic antioxidative defense.

Materials and Methods
Research carried out experimentally using a Completely Randomized Design (CRD) with four treatments and five replications.The research carried out with four treatments and five replications.Treatment given is heavy metal stress Cr (VI), Cd (II), Cu (II) and Pb (II) with a concentration of 10 mg ⋅ L −1 were determined by a preliminary test.Data were analyzed using one way ANOVA at 5% significance level to determine the effect of treatment on each parameter.Turkey continued testing to determine the mean or average mutually significantly different.The program used was SPSS 17.0 software.

The planting of seeds of S. oleraceus and preparation of test plants
The germinated seeds in one pot using a mixture of soil and compost medium with a ratio of 1 : 1.After 30 d, the test plants were selected based on height and number of leaves uniform.Test plant was moved in polythene bags containing soil medium, one polybag one plant, then acclimatized for 7 d.

The treatment of heavy metal stress
Cr, Cd, Cu and Pb concentrations of 10 mg ⋅ L −1 are given at the beginning of treatment.Heavy metal compounds used are K 2 Cr 2 O 7 , CdCl 2 , CuSO 4 and Pb (CH 3 COO) 2 obtained from the laboratory of biochemistry and Molecular Cell Biology, Faculty of Biology, Satya Wacana Christian University, Salatiga.The solution of heavy metals are provided on each polybag (500 g soil) as many as 50 mL.Treatment was given for 30 d.During the treatment period, each of the plants are watered using tap water 20 mL.

The measurement of plant growth
The growth measured by plant height, number of leaves, leaf area, dry weight and wet weight.Measurements of plant height and number of leaves is done during the treatment, while the leaf area, fresh weight and dry weight at the end of the treatment time.

The height of plant
Plant height measurements performed every 2 d.Plant measured from the base of the stem to the highest branches of plants.

Numbers of leaves and leaf area
Observation of the number of leaves every 2 d.Whole leaf is calculated including dried and withered, except the leaves are still buds.Leaf area was measured by gravimetric methods, principally through the leaf area estimated weight ratio.The measures undertaken which is draw on the fifth leaf buds on a piece of paper which produces replicas of leaves.Then cut leaf replica leaf area was estimated based on the following Equation (( 1)) [19]: Description: LD = Broad Leaf, Wr = weight paper replicas of leaves, Wt = total weight of paper, LK = the total area of the paper.

Dry weight and wet weight
The fresh weight of plant was measured by means of a whole plant except the root is cleaned and weighed.Plants put in a petri dish and then dried in an oven at 70 ∘ C for 2 d to obtain a constant weight, then weighed to determine the plant dry weight.

Antioxidative enzyme activity measurements 2.7.1. Enzyme sample preparation
Extraction of antioxidative enzymes using methods Sunkar [20].A total of 200 mg of wet second and third leaves from the shoots were mixed with 1.5 mL of extraction buffer, which is 0.2 M sodium phosphate (pH 7.8) containing 0.1 mM EDTA.Pulverized using a mortar and pestel, then centrifuged at 15.000 g for 20 min at 4 ∘ C. Supernatant was used for determination of the activity of SOD, CAT and APX.

Superoxide Dismutase (SOD)
A total of 20 L enzyme extract and 20 L of 1 mM riboflavin was added in 1.5 mL of the reaction mixture.The reaction mixture in the form of 50 mM sodium phosphate buffer (pH 7.8) containing NaH2PO4.2H2O and Na2HPO4.I2H2O; 0.2 mL EDTA 2mM; methionine 9.9 mM; Nitroblue tetrasolium (NBT) 55 M and 0.025% Triton X100.The reaction begins when riboflavin is added to the mixture, then placed under fluorescent light 15 Watt at a distance of 20 cm for 10 min.The reaction was stopped by turning off the lights and closing the tube with less black plastic.Shells and controls used are the same reaction mixture, but the sample was replaced with phosphate buffer, and then placed in a dark place for the blank and the light to control.Absorbance was measured at a wavelength of 560 nm using a spectrophotometer (Shimadzu UV-vis mini 1240).SOD activity (2) is determined by the amount of enzyme that produces 50% inhibition of NBT reduction photo formazen formed by O 2 − .One unit of enzyme activity is expressed by the amount of enzyme that reduces the absorbance of up to 50% of control.Enzyme activity is expressed in units per gram of wet weight (WW).

Catalase (CAT) and Ascorbate Peroxydase (APX)
The assay mixture (3 mL) contained 1.99 mL sodium phosphate buffer (pH 7.0), 10 L enzyme extract and 1 mL H 2 O 2 12.058 mM, for CAT measurement.Absorbance was measured at a wavelength of 240 nm using a spectrophotometer (Shimadzu UV-vis mini 1240).The extinction coefficient of H 2 O 2 (40 mM −1 cm −1 at 240 nm) was used to calculate the enzyme activity that was expressed in terms of millimoles of H 2 O 2 per minute per gram fresh weight.
APX activity was determined from the decrease in absorbance at 290 nm due to oxidation of ascorbate in the reaction.The 3 mL assay mixture contained 2.7 mL 50 mM potassium phosphate buffer (pH 7.0), 150 L 10 mM ascorbate, and 30 L enzyme extract.150 L H 2 O 2 was added last to initiate the reaction, and the decrease in absorbance was recorded for 3 min.The extinction coefficient of H 2 O 2 (2,8 mM −1 cm −1 at 290 nm) for reduced ascorbate was used in calculating the enzyme activity that was expressed in terms of millimoles of ascorbate per minute per gram fresh weight.
CAT and APX enzyme activity (3) was determined using the following Equation (3).

Result and Discussion
S. oleraceus leaf morphology after exposure to heavy metals for 30 d showed different results (Figure 1).Visually, the leaves on the plant by exposure to Cu look normal and green, not much different from the metal leaves that are not given heavy metal (control).While the leaf is exposed to a yellowish-green colored metals Pb, Cd metal exposed leaves are pale green and wrinkled.In contrast to the leaves by exposure to Cr, green-purple color of the leaves and the leaves are dried.
The height growth of plants S. oleraceus with heavy metals Cu treatment was not significantly different to the control plants, while the plants are both significantly different from that given exposure to Pb, Cd and Cr (Figure 2).The provision of heavy metals Cu, Pb, Cd, and Cr in plants S. oleraceus not lead to a decrease in the number of leaves significantly compared to controls (Figure 3).The treatments of heavy metals showed significantly different results in leaf area S. oleraceus.Control plants had an average of greatest leaf area followed by treated plant with Cu, Pb, Cr and Cd was smallest (Figure 4).
The fresh and dry weight of plants S. oleraceus were significantly different at each treatment of heavy metals, except control and Cu on a dry weight parameters (Figure 5 and 6).Both have the same downward trend.Cr exposure causes decreased S. oleraceus high, followed by exposure to Cd, Pb and Cu.
The treatment of heavy metals Cu, Pb, Cd and Cr overall have significant effects on the enzyme activity of SOD.SOD activity in plants treated with heavy metals all increased compared to control plants (Figure 7).The SOD activity is in Sonchus oleraceus with exposure to Cr increased 97.3%, followed by the plant with exposure to Pb, Cd and Cu with the percentage increase respectively by 27.2%, 13.4% and 6.8%.APX enzyme activity in plants S. oleraceus with exposure to heavy metals Cu, Pb, Cd and Cr significantly increased compared to control plants (Figure 8).Treatment of Cu and Pb give different results significant to the entire treatment with increased activity of APX of 19.8% and 62.3%, while plants exposed metal against metal Cd,Cr and the reverse of it are not significantly different, but both are significantly different from other treatments.Increased activity of APX in Sonchus oleraceus with exposure to Cd metal by 79.20%, while those exposed to Cr amounting to 78.90%.
Unlike the enzyme SOD and APX, CAT enzyme activity in plants by exposure to heavy metals had a significant reduction compared to the control.Sonchus oleraceus plants with exposure to Cr has the smallest CAT enzyme activity with a decrease of 85.9%.Followed by a decline in CAT activity in plants exposed to Pb, amounted to 38.8%, and Cd by 26.5% and amounted to 21.9% Cu.Plant growth and development (Figure 1 to Figure 6) is the initial expression that can describe the environment.Cu were exposed for 30 days not affect the morphological appearance of S. oleraceus.This is possible because Cu including essential metals (micronutrients) needed by plants in the process of metabolism, in addition to the translocation of Cu from root to stem relatively low [21].Pb causes the S. oleraceus leaves are green-yellowish, the same result is also shown Eichornia crassipes leaves exposed metal Pb [22].Pb can cause damage to photosynthetic pigments such as chlorophyll, chloroplasts and carotenoids [23].Cd inhibits the absorption of plant essensiat elements such as P, K, S, Ca, Zn and Mn so as to cause a reduction of nutrients in plants [24].Cd metal causes the leaves S. oleraceus wrinkled and dwarf plants (stunting), because nutrition is not balanced so that growth is not optimal.The worst appearance of the leaves is the leaf S. oleraceus with exposure to Cr. Purple leaves and dry at the edges.Cr inhibit the uptake of P element, P element that decreases will lead to increased anthocyanin so that the leaves will be purple.Cr also causes root undergo plasmolysis and wilt, it is consistent with the results of research that found some dead plants.
The treatment of heavy metals Cu, Pb, Cd and Cr with a concentration of 10 mg ⋅ L −1 in the growing medium become barriers the growth of S. oleraceus plants.The presence of heavy metals in the plant growing medium so that the roots can damage the membrane will disrupt the process of absorption of water and nutrients, resulting in the need for water and nutrients are not being met so that the plants do not grow well.This can be seen from plant height, leaf area, as well as wet weight and dry weight of the treatment plant is lower than the control plants.Overall treatment had no significant effect on the number of S. oleraceus.Leaf formation occurs in the early phase of growth sprouts, thus the control plants and plants treated have the same capability in the production of the leaf.Giving the metal in the growth phase relative does not affect the number of leaves [25].A decrease in leaf area due to its reduced parenchyma cells palisade and spongy parenchyma by heavy metals so that the size of a small leaf.
Antioxidative enzymes have an important role in the adaptation of plants when subjected to stress.Antioxidative defense responses of S. oleraceus towards heavy metal stress indicated by an increase or decrease in the activity of antioxidative enzymes.Presence of heavy metals in plants not only causes growth inhibition and oxidative damage in tissues of plants but also induces the activity of antioxidative enzymes to counteract the toxicity of heavy metals.Heavy metals into plants will react with proteins, lipids and other macromolecular membranes thus increasing membrane permeability to heavy metal.Increased permeability of the membrane leads to the income levels of heavy metals are more into the cells and the production of ROS is greater than the ability to detoxify the oxidative enzymes [26].SOD enzyme activity in S. oleraceus with exposure to heavy metals has increased compared to controls.SOD is the first enzim working against ROS.SOD works transform superoxide (O 2 ) into H 2 O 2 and O 2 with donating 2H + .Results showed that the most significant increase in SOD occurs in plants with exposure to Cr, SOD activity increased by 97.3% compared to controls.Cr is a strong oxidant that rapidly produce ROS in large numbers, so that the plant will produce more antioxidative enzymes to fight it.
S. oleraceus exposed to heavy metals also increased APX enzyme activity, but CAT enzyme activity decreased.APX and CAT enzymes convert H 2 O 2 into non-toxic compounds, namely water and oxygen.CAT decline is expected because O 2 can directly inhibit the action enim CAT [27].APX is more effective in detoxify H 2 O 2 than CAT.It is caused APX contained in the various cell organelles and cytoplasm and has a higher affinity due to ascorbic acid as a reduction agent, while CAT is limited only contained in the micro entities and the mitochondria and has a smaller affinity.

Conclusion
Stress heavy metals Cu, Pb, Cd and Cr in growing media causes impaired growth and S. oleraceus not optimal.The existence of these heavy metals cause high decrease in plant height and leaf area so wet weight and dry weight go down.The most significant decline in growth occurred in S. oleraceus exposed is Cr.Heavy metal stress also has significant impact on the activity of antioxidative enzymes in S. oleraceus.The enzyme Enzyme activity (mol/mg protein) = (Δ absorbance : a) × (1 : molar extinction coefficient) × (total volume : volume of enzyme) × (total volume of enzyme extract : WW sample) × total protein × 1000 (3)

Figure 1 :
Figure 1: The second leaf of S. oleraceus on heavy metal stress conditions.

Figure 2 :
Figure 2: The height of S. oleraceus plants in heavy metal stress conditions.The notation a, b show a significant difference (p ≤ 0.05) between treatments.

Figure 3 :
Figure 3: Number of leaves S. oleraceus on heavy metal stress conditions.

Figure 4 :
Figure 4: Leaf area S. oleraceus on heavy metal stress condition.The notation a, b, c, d show a significant difference (p ≤ 0.05) between treatments.

Figure 5 :
Figure 5: The weight of wet plant Sonchus oleraceus on heavy metal stress conditions.The notation a, b, c, d show a significant difference (p ≤ 0.05) between treatments.

Figure 6 :
Figure 6: The dry weight of the plant Sonchus oleraceus on heavy metal stress conditions.The notation a, b, c, d show a significant difference (p ≤ 0.05) between treatments.

Figure 7 :
Figure 7: The SOD activity S. oleraceus plants in heavy metal stress conditions.The notation a, b, c, d, e shows a significant difference (p ≤ 0.05) between treatments.

Figure 8 :
Figure 8: Activity APX S. oleraceus plants in heavy metal stress conditions.The notation a, b, c, d show a significant difference (p ≤ 0.05) between treatments.

Figure 9 :
Figure 9: CAT activity S. oleraceus plants in heavy metal stress conditions.The notation a, b, c, d, e shows a significant difference (p ≤ 0.05) between treatments.
. SOD functions to change the O 2 into H 2 O 2 .CAT works to change the H 2 O 2 into H 2 O and O 2 .APX function converts H 2 O 2 into H 2 O and MDHA (monodehydroascorbate)