Co-exposure Effects of Selenium and Mercury on Phaseolus vulgaris Excised Leaves Segment by Enhancing the NR, Anti-oxidative Enzyme Activity and Detoxification Mechanisms

Agricultural useful soils in many parts of India and the world are slightly contaminated by presence of heavy metal toxicity. Few as Cd, Cu, Zn, Ni, Co, Cr, Pb, and As. This because long-term use of phosphatic fertilizers, sewage sludge application, dust from smelters, industrial waste and bad watering practices in agricultural lands [13]. Toxics Link in India has been involved both at the global and the national level in working on the issues of mercury (Hg). Hg distribution in the environment has been a focus of scientific attention because of the potential health risks posed by Hg exposure [4].


Introduction
Agricultural useful soils in many parts of India and the world are slightly contaminated by presence of heavy metal toxicity. Few as Cd, Cu, Zn, Ni, Co, Cr, Pb, and As. This because long-term use of phosphatic fertilizers, sewage sludge application, dust from smelters, industrial waste and bad watering practices in agricultural lands [1][2][3]. Toxics Link in India has been involved both at the global and the national level in working on the issues of mercury (Hg). Hg distribution in the environment has been a focus of scientific attention because of the potential health risks posed by Hg exposure [4]. Selenium (Se) is an essential micronutrient and has important benefits for animal and human nutrition, but in trace amount after that limit it causes toxicity in human and plants [5,6]. The presumed protective effect of Se against cadmium and mercury toxicity is through the diversion in their binding from low molecular weight proteins to higher molecular weight ones [7]. The Se distribution pattern was found to be unaffected by the presence of Hg, but the amount of Se assimilated was found to be higher in plants co-exposed to Hg [8].
However, until now the study of interaction of selenium and mercury in phaseolus vulgaris plants have not yet been reported. In this study, pot culture method under lab condition was used to study the interaction between (rajmah) selenium and mercury, which was of practical significance.

Materials and Method Plant growth and metal treatment
Seeds of phaseolus vulgaris were rinsed in running water for 2 min. Seeds were surface sterilized with 0.1% HgCl 2 for 30 sec. and then washed with three times autoclaved double distill sterilized water. Sterilized seeds were sown on plastic pot containing 1 kg acid washed sand and 20 seeds per pot. Pots were placed in continuous light 30 wm -2 intensity supplied by fluorescent tubes at 26 ± 2ºC for 7-8 days. Half strength Hoagland solution without nitrogen was used for watering. Mercury Chloride (HgCl 2 ) and Sodium Selenite (Na 2 SeO 3 ) was used as test chemical. Different concentrations of the compounds were prepared (namely 0, 0.001, 0.01, 0.1, and 1 mM) using half strength Hoagland solution as solvent.
Metal treatment: Treatment with metal at two levels in order to correlate uptake, accumulation and comparison of the same with the following treatment schedules. a) Seeds were treated with Hg metals for 2 hrs and 4 hrs followed by thorough wash and subsequently planted on acid washed sand contained in plastic pots. b) Different concentration of metal treated acid washed sand for 24 hrs contained in plastic pots.
Leaves floating: For Hg treated soil (for 24 hrs) and seed treated (two and four hour) in continuous light leaf were floated on 1/4 th strength Hoagland solution. The young two cotyledons leaves were co treated with selenium (c (without treatment, 0.001, 0.01, 0.1, 1mM) different concentration to see effect on different enzymes and correlate with detoxification mechanism. Incubation in continuous light inside "Indosan growth chamber" BOD. The leaf is then used in estimation of nitrate reductase in vivo and endogenous nitrate pool, peroxidase and catalase assay.
Nitrate reductase assay: The activity of nitrate reductase was estimated by in vivo nr by Srivastava [9] method with slight modification. Briefly About 0.25 g of leaf material were incubated with 10 ml of incuba tion medium consisting of 0.1 M sodium phosphate buffer (pH 7.2), 0.2 M KNO 3 , and 25% iso-propanol in dark vial of 20 ml capacity. The whole set was incubated in dark for 30 min at 30ºC. Nitrite released in the incubation mixture due to enzyme activity was measured by colour development by the formation of diazo compound with sulfanilamide and nitrate coupled with NED to give a red dye. The absorbance was read at 540 nm after 20 min by using UVspectrophotometer.
Endogenous nitrate pool in the leaf segments were estimated according to Aslam [10]. 20 ml incubation buffer containing 0.1 M phosphate buffer and 25% isopropanol. Incubated for one hour at 30ºC. It was mixed with sulfanilamide and NED. After 20-25 min the reading is taken at 540 nm.
Antioxidant enzyme: Peroxidase was estimated according to Maehly [11]. The leaf tissue was extracted in Phosphate buffer (pH 6.8) at 0-4°C. The reaction mixtures contained distill water, 0.1 M buffer, 0.22% Guaiacol, 3% Hydrogen peroxide and enzyme. POD catalyses the transformation of guaiacol to tetraguaiacol (brown product). The oxidation of guaiacol was measured by the increase in absorbance at 436 nm for 1 min.
Activities of cata lase (EC 1.11.1.6) was assayed in fresh leaf tissue extracts by using a modifica tion of the method of Zhou, 2001 andZhang, 1990 as mentioned in Cui and Wang, 2006 [12-14] with slight modification. Briefly, the samples were prepared for catalase analysis by homogenization of fresh tis sue with a mortar and pestle in a buffer solution containing 0.2M buffer (pH 7.8). After the homogenate was centrifuged at 10,000 rpm for 20 min at 4°C, the supernatant was used immediately to determined catalase activity by monitoring the disappearance of H 2 O 2 by measuring the decrease in absorbance at 240 nm of a reaction mixture containing 0.1M H 2 O 2 , 0.2M buffer solution (pH 7.8), deionized water and enzyme extract. Catalase activity was expressed as μmol of H 2 O 2 decomposed per min per gram of fresh weight (μ mol/min/g FW).

Statistical Analysis
Each experiment was repeated at least thrice and data presented are the average value and standard deviation value of findings. Statistical data collected from one-way ANOVA test software.

Sand treated
In vivo NR and endogenous NR: During in vivo, Supply of Hg in sand 1mM shows significant (<0.001) inhibition than followed by 0.01, 0.001 mM as compared to control. Whereas when Se supplied with Hg (co-exposure) it enhanced activity with increase in concentration. As compared to control maximum enhanced activity in 1mM with 194 % time was observed (Table 1).
During endogenous NR, Supply of mercury shows significantly decrease in activity at 0.1 mM followed by 1 and 0.001 mM conc. Whereas in compared to Se treatment with Hg maximum activity was found in 0.001mM concentration with 212 % times then followed by 0.01, 0.1 and 1 mM (Table 1).
Antioxidant enzymes: During peroxidase, a concentration dependent activity of peroxidase was observed from 0.001 to 0.1 mM of Hg. The activity slightly inhibited in 0.01 and 0.1 mM in sand treated with Hg. This enzyme activity enhanced in presence of selenium and 0.1 mM concentration with 221% times followed by 1 mM with 169% maximum activity was observed (Table 1).
During catalase, Supply of Hg in sand treatment shows more inhibition in 0.01 mM than followed by 0.1 in compare to control. Se with mercury shows maximum inhibition in 0.01 mM with 159 % times and minimum in 1 mM concentration as compared to control (Table 1).

Seed treatment
In vivo NR and endogenous NR: In two hour 1 mM concentration shows significant inhibition (<0.001) than followed by 0.01 and 0.1 mM whereas in four hour 0.01 mM concentration shows more significantly enzyme inhibition. When Se supplied with mercury, activity enhanced with increase in concentration (   (Table 3).
Hg inhibited endo nr activity in seed treated two hour at 0.1 mM concentration whereas in four hour 0.001 mM concentration significantly (Tables 2 and 3). When Se supplied with mercury, enhanced activity in 0.001 then 0.1mM concentration was observed during two hour seed treatment in comparison to this four hour seed treatment shows maximum activity in 0.001 mM than 0.1 and 0.01 mM concentration (Tables 2 and 3).

Antioxidant enzymes:
Peroxidase activity when Hg treatment supplied to seed with two hour only in 1mM conc shows significantly increase in activity whereas in four hour 71 times more fold inhibition in 0.1mM followed by 0.001mM conc. or least inhibition in 0.01 mM conc. When Hg was co exposure supplied with selenium, peroxidase activity at 0.001 mM and 0.1mM shows more inhibition and 100 times more enhanced activity than control in both two hour and four hour (Tables 2 and 3).
During catalase activity, when only Hg treatment was given during seed treatment with two hour only in 1mM conc shows significantly decrease in activity whereas in four hour 93 times more fold inhibition in 0.01mM followed by 0.001mM conc. or least inhibition in 0.1 mM conc. When co exposure with Se mercury treatment supplied the activity enhanced and shows more detoxification. During two hour 0.1 mM shows more inhibition than 0.01mM, 0.001mM and 1 mM with compare to control. During four hour, 0.01 mM and 0.1mM shows more inhibition (Tables 2 and 3).

Discussion and Conclusion
Obtained from the present study showed reduced enzyme activity under Hg stress which might be associated with inhibited contribution of essential component required for the particular enzyme activity of plant. Similar research also done earlier for identify mercury toxicity on plants or ecosystem. The growth pattern of phaseolus seedlings in the presence of Hg was different from pea and spear mint [15], ryegrass [16,17], tomato [18], Sesbania grandiflora [19], Mentha arvensis [20].
The activities of anti-oxidative enzymes in the seedlings of Phaseolus aureus found that effects of Hg and cadmium (Cd) had little primary damaging effect on membranes [21]. Antioxidant effect of HgCl 2 was in selected plant Clitoria ternatea L. It increases H 2 O 2 content and the antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were observed in HgCl 2 treated plants when compared with control [22].
Hg causes phyto toxicity and oxidative stress in wheat (Triticum aestivum L.) plants [23]. Plants treated with higher concentration of Hg were subjected to comparatively greater oxidative damage and demonstrated that the antioxidative components were not able to remove the stress due to higher concentration of Hg and thus might affect the productivity in plants. Hg also showed inhibition property towards physiological parameters such as chlorophyll, protein, nitrate, and endogenous pool. Higher concentrations of HgCl 2 were found to be more toxic [24].
The selenium content and species of both plant and animal foodstuffs depend on environmental conditions, in particular, the quantity and species of selenium to which the animal/plant is exposed. Selenomethionine is predominant in cereals, and selenium concentrations vary from 0.01 to 0.55 lg/g fresh weight [25], whereas in other plant foods the content is generally lower. Se existence supports the expression of the selenoprotein, which have important antioxidant and detoxification function [26]. These forms of selenium can combine with Hg 2+ to form insoluble Hg-Se complex in rice root surrounding environment or root surface [27].
Co-exposure of Se with Hg enhanced NR enzyme activity and improved antioxidant mechanisms seedlings which could be attributed to their synergistic effect and the role of Se to regulate the plant growth and detoxifying mercury toxicity.
The interaction between mercury (Hg (II)) with selenium Se (VI) and Se (IV) in Tomato (Lycopersicum esculentum) experimented in Sand and soil culture resulted in Decreases Hg uptake [28]. Another study shows that Hg (II) and Se (VI) and Se (IV) in Radish plants (Raphanus sativus) performed in Pot culture decreases Hg uptake and possibly forms Hg-Se insoluble complex in soil [29]. Mercury Hg (II) Se (IV) in Glycine max (soybean) experimented with Soil and a 50:50 mix of soil and ProMix resulted forms a high molecular weight entity containing Se and Hg in plants [8].
Se may play an important role in limiting the bioaccessibility, absorption, and translocation/bioaccumulation of mercury in the aerial rice plant, which may be related to the formation of an Hg-Se insoluble complex in the rhizospheres and/or roots [30].
Se antagonistic study with other heavy metal also studied such as arsenic, cadmium etc. Arsenic interaction with Se in Hydroponic culture and found that decreases As uptake and lipid peroxidation; enhances levels of antioxidants, chlorophyll, MTs, thiols and GST; increases cellular viability, and rebuilds membranes. Enhances As concentration, Se induces more production of thiols and GSH to counterbalance the negative effects of increased As and inhibits lipid peroxidation in Pteris vittata L [31,32]. In Garlic (Allium sativum) found decreases Cd uptake and stimulates growth of root and seedlings. Another study in Rape (Brassica napus L., Polish genotype cv. Bojan) and wheat (Triticum aestivum, Polish genotype cv. Kamila and a Finnish genotype cv. Manu) results shows Promotes growth, reduces lipid peroxidation, enhances membrane stability, and counterbalances the Cd-induced changes in nutrients [33,34] (Figures 1-3  The robust data presented in the above work shows that co-exposure of Se is more effective in the alleviation of Hg stress. Improved Hg tolerance in the presence of Se involves physiological, and biochemical interaction in a synergistic or additive way. Therefore, the present study gives a new strategy to minimize the Hg toxicity in crop plants, especially in rajmah (phaseolus vulgaris) using beneficial application of Se as protective mechanism against mercury toxicity.