Abstract: Background and Objective: The effect of lead on several metabolic reactions in the radish plant has been investigated. The present study was conducted to evaluate the potential role of enzymes activity, their isoenzyme profile pattern and free radical formation as biomarkers of Pb pollution in the radish (Raphanus sativus L.) plant. Materials and Methods: Varied concentrations of Pb(NO3)2 ranging from 25-500 ppm in the growth media were used. The plant samples were collected after 40 days growth period. Lead and micronutrients concentration, the profile of electron spins resonance determination and hydrolytic enzymes activities were analyzed. Results: Lead concentrations in roots and leaves increased with increasing Pb concentration in the tested media; however the most accumulation were observed in leaves. Generally, the concentration of micronutrients such as Fe, Zn, Mn and Cu declined in leaves compared to the roots. Results of the profile of Electron Spin Resonance (ESR) determination showed a decrease in unstable free radical level in the roots, followed by a significant increase with increasing Pb concentrations. The Pb induced changes in some enzymes activity and its isoenzyme profiles such as acid phosphatase (AP), esterase (EST) and polyphenol oxidase (PPO) in leaves and roots of radish plant. Results of isoenzymes suggested that the staining intensities of isoform patterns were consistent with the changes of the activities assayed in solutions. Conclusion: These results suggested that (Raphanus sativus L.) seedlings may have a better protection against oxidative stress by increasing antioxidant enzymes activity exposed to Pb toxicity.
INTRODUCTION
The continuous increase of heavy metal ions in the environment is imposing serious problems in agricultural yield and increases human health threats through accumulation in the food chain. Lead (Pb) is a highly toxic metal which can be potentially harmful to plants, animals and humans at elevated levels. Therefore, accumulation of Pb in plants poses a potentially serious health risk to humans through contamination of the food chain. It exerts direct and indirect effects on nutrient uptake, seedling growth and photosynthetic processes of plants and causes inhibition of enzyme activities, water imbalance and alterations in membrane permeability1. The Pb can cause changes in chloroplast ultra structure in plants, thereby reducing their growth2. Furthermore, deleterious effects of heavy metal stress in plants may be coupled to other physiological processes via the stimulation of some enzymatic activities that limit cell growth and consequently, accelerate tissue ageing. Several hydrolytic enzymes including glucosidases, esterases, acid and alkaline phosphatases were pro-posed to be involved in plant morphogenesis, especially in cell wall metabolism that is necessary for its turnover and plasticity3. In plants, Reactive Oxygen Species (ROS) such as superoxide anion (O2˙¯), singlet oxygen (1O2), hydrogen peroxide (H2O2) and the hydroxyl radical (˙OH) are produced continuously as byproducts of various metabolic pathways that are localized in different cellular compartments4,5. However, under stressful conditions, their formation might increase to exceed the antioxidant scavenging capacity, thus creating oxidative stress by reaction and damage to all biomolecules6,7. Many heavy metals, including Pb are known to induce overproduction of ROS and consequently enhance lipid peroxidation, decrease the saturated fatty acids and increase the unsaturated fatty acid contents of membranes of several plant species8. In addition, ROS are highly reactive to membrane lipids, protein and DNA9,10. They are believed to be the major contributing factors to stress injuries and to cause rapid cellular damage11,12. Acid Phosphatases (AP) form a group of enzymes catalyzing hydrolysis of a variety of phosphate esters in acidic environments. The AP is believed to increase orthophosphate (Pi) availability under phosphorous deficient conditions13. Salt, drought and osmotic stresses have also been reported to increase AP activity14-16. In addition, the activity of acid phosphatase (AP) increased in leaves and roots of tomato and radish plants grown at high zinc and cadmium concentration in the growth media17. Esterases, a group of hydrolases, catalyze the formation or cleavage of ester bonds of water soluble substrates. Generally, these enzymes have a broad spectrum of substrates and act on a variety of natural and xenobiotic compounds18. Polyphenol oxidase (PPO) has been the subject of several reviews because enzymatic browning in fruits and vegetables provokes unpleasant sensory qualities and losses in nutrient quality19. Stress-tolerant plants usually contain high constitutive levels of protective metabolites, while the more sensitive ones show their induction under different kind of stress. Isoenzymes are an easily obtained class of molecular markers, co-dominant, reliable and can provide valuable information about abiotic stress tolerance20. Esterase isoenzymes have been used as a genetic marker in barley crop, identification of esterase loci was reported to encode monomeric isoenzymes21. Free radicals and hydrogen peroxides formation were reported to cause the membrane damage, which is often related to lipid peroxidation22. The ESR techniques have been implicated to detect the formation of free radicals in plants during nutrient stress23. The ESR method can also be utilized for estimating the type and content of more stable free radicals that accumulated in cells as markers of oxidative stress due to O2 enrichment24. As Pb is one of the most abundant heavy metal pollutants in both aquatic and terrestrial environments, the present study was conducted to evaluate the potential role of enzymes activity, their isoenzyme profile pattern and free radical formation as biomarkers of Pb pollution in the radish (Raphanus sativus L.) plant.
MATERIALS AND METHODS
Plant material and stress conditions: Healthy, homogenous seeds of radish (Raphanus sativus L.) were subjected to surface sterilization with 0.1% sodium hypochlorite solution for 10 min and then rinsed with double distilled water. After 24 h imbibition of seeds in water, seeds were grown in sand cultures in plastic pots saturated with Hoagland nutrient solution25, which served as control or nutrient solutions supplemented with Pb(NO3)2 to achieve different concentrations of 0, 25, 50, 100, 150, 250 and 500 ppm Pb2+ which served as treatment solutions. The incubation temperature was set at 28°C with a 12 h photoperiod with 40-50 μmol–2 sec–1 irradiance. The experiment was arranged in a completely randomized design with three replicates, each replicate with about 10 radish seeds. The plant samples were collected after 40 days growth period and the following biochemical parameters were analyzed.
Pb accumulation and micronutrient contents: Lead and micronutrient content (Zn, Fe, Mn and Cu) were determined in the fine powdered dry matter of plants (leaves and roots). The samples were washed and then mineralized with perchloric acid. Metal concentrations (ppm) were estimated by atomic absorption spectrometry (Unicam Sp 1900 model) according to the method of Lu26.
Spectra of electron spin resonance (ESR): Representative sample composed of 10 roots from each treatment were frozen in liquid N2 and were then ground in a mortar. The powder material (500 mg) was packed into ESR flat quartz cuvette. The ESR spectra were recorded at room temperature using ESR spectrometer ELEXSYS E500 (Bruker-Germany). The experimental condition was as follows: Field mod. amplitude 0.001, field mod. frequency (Hz) 100000, microwave frequency (Hz) 9.8145e+09, microwave power (W) 0.0202637, receiver grain 40, receiver time constant (S) 0.00512. The standardization of "g" value was carried out using 1,1 Diphenyl-2-picryl-hydrazyl (DPPH) as a standard. The areas under ESR curve for each sample were calculated. The area under control sample was represented as 100% and was used to plot the relative radicals versus cadmium concentration27.
Preparation of crude enzyme extracts: The method described by Vitoria et al.28 was applied to prepare the crude enzyme extracts. In this method, 0.5 g roots and leaves tissues were homogenized in a chilled pastel and mortar with 100 mM potassium phosphate buffer (pH 7.5) containing 1 mM EDTA, 3 mM DL-dithiothreitol and 5% (w/v) insoluble polyvinyl pyrrolidone. The homogenates were centrifuged at 10000×g for 30 min and then the supernatants were kept stored in separate aliquots at -20°C until analysis.
Protein determination: Soluble protein was estimated by using the coomassie brillent blue G-250 reagent according to the method of Bradford29 with bovine serum albumin as standard.
Enzyme specific activity: Acid phosphatase (AP) specific activity was determined spectrophotochemically according to Yoneyama et al.30. The assay mixture contained sodium acetate buffer 50 mM, pH 5.5, 3 mM pNPP and 20 μL enzyme extract. After 10 min at 37°C, the reaction was stopped by adding 1 mL of 0.2 M NaOH. The increasing in absorbance at 410 nm was measured.
Esterase (EST) specific activity was assayed spectrophotochemically according to Brahimi-Horn et al.31. The reaction mixture contained sodium phosphate buffer 66 mM pH 7.5, ρ-nitrophenyl acetate (100 μM) and 20 μL enzyme extract. The change in absorbance was followed for 5 min with 30 sec intervals at 405 nm.
Polyphenol oxidase (PPO) specific activity was assayed by using photochemical method as described by Coseteng and Lee32. The reaction mixture contained 50 mM potassium phosphate buffer pH 6.2 and 250 mM catechol and 50 μL enzyme extract. The increasing in absorbance at 420 nm was measured.
Native gel electrophoresis and isoenzyme staining: Isoenzymes were separated using native-polyacrylamide gel electrophoresis (PAGE) technique with 6% PAA in tris-borate buffer 0.125 M, pH 8.9 on slab gel according to the method of Stegmann et al.33.
PPO isoenzyme staining: Immediately after stopping the electrophoretic run, the slabs were dipped and agitated in 15 mM catechol in 0.1-0.2 M phosphate buffer (pH 5) and 0.05 M p-phenylenediamine until bands showing PPO activity were evident (30-40 min). The gels were placed in deionized water overnight and transferred to 50% methanol for storage34.
AP isoenzyme staining: The staining solution was prepared according to Wendel and Weeden35. The Na-a naphthyl acid phosphate 100 mg, MgCl2 100 mg and 100 mg of fast garent GBG salt were dissolved in 100 mL of 50 mM Na-acetate buffer, pH 5.0. Gels were incubated in dark at room temperature until black bands appeared.
EST isoenzyme staining: After electrophoresis, the gels were removed from the glass plates and incubated for 30-40 min at 37°C in 100 mL 0.2 M sodium phosphate buffer (pH 6.5) containing 50 mg fast blue RR salt with the addition of 2 mL 1% α-naphtyl acetate in acetone at 50%33.
Statistical analyses: Statistical analysis was done using SPSS (version 10) program. Mean and standard error (SE) were descriptive measures of quantitative data using the analysis of variance test (ANOVA) for independent samples. The p≤0.05 were considered significant. Values reported here are means of three replicates.
RESULTS AND DISCUSSION
Pb accumulation and micronutrient contents: Lead is one of the heavy metal widely used in modern industry that has been recognized as highly toxic and carcinogenic. It can affect growth and metabolism of plant to varying degrees depending on the concentration and tissue types of plant species1.
| Fig. 1(a-b): | (a) Signals of free radical formation in Pb-treated radish roots 0, 100, 500 ppm and (b) Percent of free radical level in Pb-treated radish roots in relation the control |
Regarding to the data of Pb accumulation, which represented in Table 1, when radish seedlings were grown under increasing concentrations of lead in the growth medium, during a 40 days growth period increasing lead levels caused a continuous increase in the content of lead. The absorbed lead was localized to a greater extent in roots than in shoots. The Pb concentration was ranged between 20.3 and 207 ppm in leaves and 20.3-398 ppm in roots. It means a progressive accumulation of Pb in roots, which translocated to leaves. Lead increase was paralleled by a decline in the concentration of Fe, Zn, Mn and Cu was decreased by increasing Pb concentration in the growth media. The lowest concentration of micronutrients was found at 500 ppm Pb in both leaves and roots. Nevertheless this increase is not toxic to the plant and may be render to continuous addition of nutrient solution but its translocation from the roots to the leaves differ from one element to other. These results are in harmony with previous studies, who found an accumulation of lead in plants with increasing Pb concentration in the growth media in Sesbania exaltata and Jatropha curcas plants, respectively36-39.
Effect of Pb on stable free radical formation in radish roots: Electron Spin Resonance (ESR) spectra of radish untreated and treated roots grow under Pb treatments (100 and 500 ppm) were measured using X-band ESR spectrometer at room temperature and the results are represented in Fig. 1a and b. The ESR lines recorded with g-value 2.003. Under control condition, the spectrum area was lower than the high treated sample with Pb (g = 105, considered as 100%). While, the treatment of Pb (100 ppm) was lower caused lower g value than control sample (g = 52.3 considered 49.8%) this combined with the appearance of six characteristic fine peaks of Mn. For this reason, the free radical peaks did not appear clearly in those two treatments. Heavy metals can influence the physical and chemical processes in plants by directly inducing ROS production (Fenton reaction), by blocking functional groups of proteins and glutathione and by displacing essential metals like zinc or selenium from proteins and zinc-finger motifs of transcription factors40. One possible mechanism by which excess heavy metals may damage plant tissues is the stimulation of free radical production, by imposing oxidative stress41.
| Table 1: | Lead accumulation and its effect on micronutrient contents (ppm) in leaves and roots of radish plants under Pb stress after 40 days growth |
Mean values are presented ±SE (n = 3). Values with different letters in the vertical column differ significantly at p<0.05 | |
| Table 2: | Enzymes activity (U mg–1 protein) in leaves and roots of Pb treated radish plants after 40 days growth period |
Mean values are presented ±SE (n = 3). Values with different letters in the vertical column differ significantly at p<0.05 | |
Reactive Oxygen Species (ROS) such as singlet oxygen (1O2), superoxide radical (O2˙¯), hydrogen peroxide (H2O2) and hydroxyl radical (OH˙) are formed in metabolic processes occurring in plants, but when they are created on non-controlled levels they can lead to disturbances and injury in cell metabolism or even to cell death42,43. The higher Pb concentration in the soil environment results in an enhanced Pb uptake by roots and leaves. In particular, the level of antioxidant enzyme is a good indicator for the impacts of pollutants like heavy metals. The heavy metal damage is an important factor in many pathological and toxicological processes44. The presence of toxic metal in the cell led to formation of free radical species, which cause severe damage to different cell organelles and biomolecules45,46. On contrary in the highest treatment of Pb (500 ppm) the spectrum area was higher than control sample (g = 134.9, considered 128.5%). The intensity which related to free radical formation may be correspond to the stable free radicals came from the reaction of superoxide free radical with one or more of the constituents of the root cells such as DNA, lipids and proteins. It was found that two g-values were consistent with quinone radicals, although the exact origin of these radicals was not determined. Atherton et al.47 suggested that precursors of these radicals might be either quinone involved in electron transport pathways, simple phenolic secondary metabolites or more complex polyphenols. The present results are in agreement with Rucinsk et al.48 who found that Pb treatment in lupine roots induced ESR signal for paramagnetic radical, which derived from quinine.
Enzyme specific activities: A significant inhibition in acid phosphatase (AP) activity was observed in seedlings of both leaves and roots of radish with low Pb treatments (Table 2). The results appeared that with Pb treatments (25, 50, 100 and 150 ppm) a clearly reduction in AP activity in leaves (17, 17.7, 23 and 12%), respectively, in comparison with control treatments. Additionally, the concentration (25 and 50 ppm) of Pb caused also reduction of acid phosphatase activity in roots (7 and 15%), respectively, compared with control plants. Under (250 and 500 ppm) Pb treatment about 4 and 13% increase in AP activity was observed in leaves of radish plants as compared with control. Furthermore, AP activity significantly sharply increased at (8, 26.2, 50.2 and 91%) in roots with the highest levels of Pb (100, 150, 250 and 500 ppm) as compared with control. The change of enzyme activity, however, cannot support the presumption that AP is directly involved in Pb detoxification, although it might help the plant to resist the nutrient inadequacy that often accompanies heavy metal stress49. In addition, the increases in AP activity with Pb treatments might be due to the decline of phosphate (Pi) level in the cell of Pi starvation and that intracellular and extracellular AP are integral components of plants response to Pi deficiency50. Abiotic environmental stress such as salinity, water stress and heavy metals lead to an alteration of gene expression in plants, which in turn leads to alteration in its metabolism and development51. However, over expression of AP, accelerating the degradative processes in cellular metabolism, are non-specific or exclusive responses to heavy metal toxicity52. Thus, some investigators have suggested that AP activity increases under the stress of high levels of heavy metals and is one possible process of detoxification and resistance53.
Esterase (EST) specific activity show different pattern in leaves and roots of radish plant under Pb treatments as presented in Table 2. The results of Pb treatments (25, 50, 100, 150 ppm) appeared that a significant increase of EST activity (59.4, 79.3, 26.5 and 21.6%) in leaves compared to control sample. While in high dose of Pb (250 and 500 ppm) showed a noticed inhibition in EST activity (6 and 36.3%), respectively, in comparison with control sample. Furthermore, Pb treatments in root, showed a variable significant induction of EST activity (28.6, 46.6, 76.8, 134, 80 and 42.4%) as compared to control of radish plants. These results are in agreement with Amal et al.54 who found the trend of EST activity in leaves and roots of radish under Cd stress. The increase of EST activity in leaves and roots of radish plants in low doses of Pb treatments may be due to its role in detoxification mechanisms for heavy metals resistance in the medium or due to the de novo protein synthesis under conditions of metal stress55. In addition, the increased activity of enzyme after treatment of plants with heavy metals can be interpreted as an indication of damage to membranes, which leads to liberation of enzymes from cell structures56. While, the reduction in enzyme activity in the high level of Pb may be due to the interaction of both metals with functional sulphydryl (SH)-groups of the enzyme57.
Polyphenol oxidase (PPO) is very important enzyme for plant respiration. The PPO is a terminal oxidase which can directly pass electrons to O2 when the intermediate products of plant respiration are oxidized. It could catalyze the oxidization of such a group of compounds as phenol to quinone. The PPO has some relationship with the synthesis of cell compounds containing phenol groups such as lignin58. The activity of polyphenol oxidase enzyme was increased by increasing Pb treatments in the growth media and reached its maximum activity at 500 ppm as shown in Table 2. The results showed that a marked significant increase in PPO specific activity with Pb treatments in roots about 10 fold (5.21 and 52.4) and followed by leaves about 7.6 fold (3.35-26.1) (U mg–1 protein) compared to control. The PPO specific activity were enhanced by increasing Pb concentration and reached its maximum increase with 500 ppm even in roots 52.4 and leaves 26.1 as noticed in Table 2. It was suggested that activity of PPO enhanced under stress conditions caused by the presence of toxic heavy metals is responsible for binding and detoxification of heavy metals in Nymphet epidermal glands. In Arabidopsis thaliana the responses to Pb toxicity and to bacterial infection are similar and include an increase in peroxidase activity with the accumulation of polyphenol deposits59. Increases in phenol concentration in response to heavy metal stress have also been noted in several plants such as tobacco, maize and pine60-62. Saffar63 reported that in Arabidopsis thaliana PPO activity might be the result from prolonged heavy metal stress.
Isoenzymes detection: Isoenzyme profiles of acid phosphatase (AP) in leaves and roots at 40 day-old grown under control and Pb treated radish plants are shown in Fig. 2. It is evident from the zymogram that, an obvious decrease in leaves AP band under Pb treatments (25, 50, 100 and 150 ppm) were observed, while at highest Pb treatments (250 and 500 ppm) the intensity and mobility of this band was enhanced comparing with control band. Moreover, the results of roots treated with Pb appeared that, a marked increase in the intensity of the basic three-isoenzyme bands of acid phosphatase with Pb treatments. In addition, two new bands were appeared at high treatments of Pb (100, 150, 250 and 500 ppm) with increasing in their intensity compared with control sample. According to Mukherji and Gupta64, the increased level of enzyme activity can be attributed to an accelerated synthesis of protein. The stimulation of enzyme by metal ions is assumed to be an indirect effect. However, this stimulation is known to be caused by a metal induced de novo synthesis of the enzyme protein65. Generally, increased the activity of hydrolytic enzymes (ribonuclease, deoxyribonuclease and acid phosphatase) after treatment of plants with heavy metals reflect a general senescence response induced by heavy metal ions66. These results in agreement with many researchers who reported that heavy metals modified the pattern of AP isoenzymes in many plants such as Glycine max, Zea mays and lupin67,68.
Esterase (EST) isoenzyme polymorphism was photographed and presented in Fig. 3. Zones Est1 and Est2 at leaves of radish showed a decrease in their intensity at high doses of Pb. While zones Est3 and Est4 were represent appearance of new isoenzyme bands and disappearance of others at Pb exposed sample compared with unexposed ones. Moreover, no changes were seen at the pattern zones of Est1 and Est2 in root exposed to Pb compared with control. Otherwise, zones Est3 and Est4 showed different changes under Pb stress. New isoenzyme bands appeared at strong intensity and others at weak intensity in the exposed samples compared with unexposed sample.
| Fig. 2(a-b): | Acid phosphatase isoenzyme patterns of radish tissues (a) Leaves and (b) Roots at harvest time (40 days growth) in plants grown in Pb containing media, lanes: 1: Pb 0.0 ppm, 2: Pb 25 ppm, 3: Pb 50 ppm, 4: Pb 100 ppm, 5: Pb 150 ppm, 6: Pb 250 ppm, 7: Pb 500 ppm |
| Fig. 3(a-b): | Esterase isoenzyme patterns of radish tissues (a) Leaves and (b) Roots at harvest time (40 days growth) in plants grown in Pb containing media, lanes: 1: Pb 0.0 ppm, 2: Pb 25 ppm, 3: Pb 50 ppm, 4: Pb 100 ppm, 5: Pb 150 ppm, 6: 250 ppm, 7: Pb 500 ppm |
Therefore, the appearance of new bands or disappearance of others at zones Est3 and Est4 in the metal exposed radish samples could be due to adaptation of the leaves and roots to the metal exposed environment69. In addition, the reduction of iso-esterase bands intensity or the disappearance of the fast bands may be due to heavy metals acts at the nucleic acid level in which transcriptional, post-transcriptional, translational or other inhibition processes may be involved. Evidence has been found of inhibition of RNA synthesis and the inhibition of DNA polymerase I and II activity has been confirmed69. The isoenzyme patterns of EST are particularly affected by metals. Finally, these results are agreement with previous studies who noted that, Treatment of Triticum aestivum, Silene vulgaris and Raphanus sativus with heavy metals (Zn, Cu, Pb, Cd and Ni) concentrations of verifying toxicity resulted in differenced in the number of iso-esterase bands and in quantitative changes of band activity54,70.
| Fig. 4(a-b): | Polyphenol oxidase isoenzyme patterns of radish tissues (a) Leaves and (b) Roots at harvest time (40 days growth) in plants grown in Pb containing media, lanes: 1: Pb 0.0 ppm, 2: Pb 25 ppm, 3: Pb 50 ppm, 4: Pb 100 ppm, 5: Pb 150 ppm, 6: Pb 250 ppm, 7: Pb 500 ppm |
The results of PPO profile in Pb-treated radish plants appeared that, a marked induction at the intensity of two isoenzyme bands of PPO with Pb treatments (Fig. 4). The fastest migrating isoenzyme of PPO was almost undetectable in the control, but was clearly visible in Pb-treated leaves. The mobility of this fast band was increased at the high treatments of Pb (150, 250 and 500 ppm) compared with control. In addition, the root extracts used for PPO staining following native PAGE showed a dramatic increase at the intensity of three PPO isoenzyme bands under Pb treatments. However, the induction of PPO activity may be due to its role in phenolic compound synthesis which playing an important role in detoxification of heavy metals in plants71. Detoxification of heavy metals in plants was achieved by the formation different complexes and amino acids and phenolic compounds have all been implicated in the chelation and precipitation of heavy metals72.
CONCLUSION
Based on the present study, it can be concluded that Pb causes oxidative stress as evidenced by significant increase in enzyme activity. Generally, Fe, Zn, Mn and Cu declined in leaves compared to the roots. Moreover, the data demonstrated that Pb induced changes in some enzymes activity such as acid phosphatase (AP), esterase (EST) and polyphenol oxidase (PPO) in leaves and roots of radish plant.
The Pb stress also induced several changes in AP, EST and PPO isoenzyme profiles. It remains to be seen whether these increases may be due to induced gene transcriptional and de novo synthesis of protein or are due to posttranslational modification of existing protein. Finally, the results of Electron Spin Resonance (ESR) showed a clear disorder in the Pb treated plant. The detection of such parameters is useful, early and sensitive indicators for prediction of heavy metal toxicity in plant.
SIGNIFICANCE STATEMENT
| • | Lead concentrations in roots and leaves increased with increasing Pb concentration |
| • | Micronutrients decline in leaves compared with increasing Pb concentration |
| • | The ESR show decrease in free radical in roots, followed by increase with increasing Pb |
| • | The Pb induces changes in enzymes activity leaves and roots of radish plant |
| • | Results of isoenzymes were consistent with changes of enzyme activities assayed |
ACKNOWLEDGMENTS
Authors would like to thank the management of the Faculty of Agriculture, Cairo University and National Research Center for ongoing cooperation to support research for providing funds and facilities necessary to achieve the desired goals of research.