Biochemical Changes under Chromium Stress on Germinating Seedlings of Vigna rad i

Hexavalant chromium is considered the most toxic form because of its high solubility in water. Cr is known to induce production of elevated concentration of reactive oxygen species (ROS) resulted in macromolecule damage. Plants are having unique mechanisms to overcome ROS induced damage by accumulation of proline, ascorbate and glutathione and increasing the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and ascorbate peroxidaes (APX), peroxidise (POX). In the present investigation effects of chromium on seed germination of Mung bean (Vigna radiata ‛Gujarat Mung-4’) were studied. Seeds were treated with different Cr concentrations (50, 100, 150 and 200 μM) for seven days. On 7 day root and shoot length was measured and activities of antioxidant enzyme SOD, APX, POX, CAT and GR were checked along with protein, proline and lipid peroxidation. It was observed that there is gradual decrease in shoot and root length with respect to the increase in Cr concentration. Level of lipid peroxidation significantly increased along with proline and antioxidant enzyme activity at higher Cr concentration. Lipid peroxidation is an indication of membrane damage due to elevated production of reactive oxygen species (ROS). To combat oxidative damage by ROS antioxidant enzyme activity increased significantly, which indicates that antioxidant enzymes (SOD, CAT, APX and GR) play a crucial role during Cr stress during germination of V. radiata.


Introduction
Agricultural land contamination with heavy metals is a serious problem, which has gathered considerable public attention in recent decades.Some metals are required for plant growth and development but some of them are very toxic at elevated concentration.Sources of heavy metal contamination include anthropogenic activities, like use of pesticides, agriculture waste and industrial waste.Hexavalant chromium is considered the most toxic form because of its high solubility in water, which usually occurs amalgamated with oxygen as chromate or dichromate (Samantaray et al., 1998, Caldelas et al., 2012;Hayat et al., 2012).Cr is known to induce production of elevated concentration of ROS resulted in macromolecule damage.Plants have evolved mechanisms to overcome ROS induced damage by accumulation of proline, ascorbate and glutathione and increasing the activities of SOD, CAT, GR, POX and APX (Samantaray et al., 1998;Nataraj et al., 2009, Thounaojam et al., 2012;Tripathi et al. 2012;).Antioxidants are the compounds, which act as osmotic buffers; beside this, they scavenge ROS (Nataraj and Paramar, 2008, Nataraj and Roshan, 2008, Nataraj et al., 2009).Soluble protein content of germinating seedling is an important indicator of metabolic changes under various stress conditions and predicts physiological status of seedlings (Nataraj andRoshan, 2008, Martins et al., 2013).Mung bean is an important staple food of the world population having high nutritional quality among pulses.
The present investigation was undertaken to test the effects of chromium on growth, induction of oxidative stress and antioxidative responses in Mung bean cultivar ‛Gujarat Mung-4.'

Plant material, growth conditions and treatment
The present study was carried out on Mung (Vigna radiata) 'Gujarat Mung-4', which was collected from research centre of Junagadh Agriculture University (JAU), Junagadh.The seeds were surface sterilized with 0.1% HgCl2 (w/v) for one minute.Solutions of different heavy metal concentrations (50, 100, 150, 200 µM) were prepared, using K2Cr2O7 and distilled water.Six seeds were placed on Petri plates over Whatman no -1 filter paper and treated with 5 ml solution of different chromium (Cr) concentrations.For every treatment three replicates, each with six seeds, were maintained.The fresh solution was added after 48 hours to the above described Petri plates.Seven day old seedlings were selected for bio-chemical analysis.

Total soluble protein, proline and lipid peroxidation
Protein content was determined as described by Lowry et al. (1951).Proline content was estimated following the procedure of Bates et al. (1973).The extent of lipid peroxidation in terms of malondialdehyde (MDA) formation was measured according to the method of Esterbauer and Cheeseman (1990).

Determination of enzymatic antioxidants
After 7 days of treatment, 0.50 g fresh seedlings were homogenized with 8 ml 50 mM phosphate buffer solution (pH 7.8) in an ice bath and then centrifuged at 10,000 x g for 15 min at 4 ○ C. The supernatant was designated as crude enzyme extract and stored at 4 ○ C for further enzyme assay.
Peroxidase (EC 1.11.1.7)Peroxidase content was determined using the method of Chance and Maehly (1955).The amount of purpurogalline formed was determined by reading the absorbance at 420 nm against a blank prepared by adding the extract after the addition of 1 ml 2.5N H2SO4.
Statistical analysis: Presented data represent the mean values with standard deviation (S.D.).All results were subjected to one-way analysis of variance (ANOVA) using the statistical software package (Graph pad PRISM 3.0).The significance of differences between exposed and control plants was tested by Tukey's multiple comparison tests.The difference was considered significant at p levels lower than 0.05 (p < 0.05).

Results and discussions
The effect of Cr on shoot length and root length is presented in Fig. 1 and Fig. 2. Shoot and root length significantly decreased with respect to increase in Cr concentration.Similar responses of inhibitory activity of Cr on root and shoot length was reported in rice (Tripathi et al., 2012).This reduction could be due to the accumulation of high concentration of Cr in roots and/or a nonexistence of any defined Cr translocation mechanism, thereby enhancing the Cr sequestration in the tissue thus, inhibiting root development (Diwan et al., 2010;Lu et al., 2004).
As shown in Fig. 3, total soluble protein content increased with the increasing Cr +6 concentrations in the treatment.At lower concentration 50 µM of Cr +6 exposures a statistically non-significant increase of total soluble protein was observed.A significant increase in protein content found in seeds treated with 150 µM and 200 µM Cr +6 (p<0.001).Similar observations were reported in Brassica juncea under cadmium stress (Seth et al., 2008, Szollosi et al. 2009).in 100 µM to (p<0.001) 200 µM Cr +6 treated seeds.Proline accumulation in response to heavy metal stress was reported in several plants (Nataraj and Paramar, 2008;Nataraj et al. 2009, Yilmaz andParlak, 2011).Lipid peroxidation is measured in terms of MDA content.MDA content increased in all the Cr treated germinating seeds (Fig. 4.).A non-significant increase was found during the 50 µM treatment, while a significant increase (p<0.001) in MDA content was found at 150 and 200 µM Cr +6 .As MDA content is a product of lipid peroxidation, the elevated concentration of MDA content clearly reflects membrane damage due peroxidation of membrane's lipid content in the presence of ROS.MDA level is regarded as a biochemical indicator for injury mediated by ROS (Ozdener et al., 2011).Our results indicate that excessive heavy metal increases oxidative stress as it is evident from increased lipid peroxidation.It is in accordance with Ozdener et al. (2011) andTripathi et al. (2012).SOD, CAT, GR, POX and APX are important components in preventing oxidative stress in plants by scavenging free radicals and peroxides with the elevation of their activities when exposed to heavy metal stress (Shanker et al., 2005).Antioxidant enzyme activities after exposure to Cr +6 are shown in Fig. 6 to 10.In the present investigation, it was found that SOD, GR and POX activity increased significantly at (p<0.001) 100,150 and 200 µM, whereas CAT and APX activity significantly increased (p<0.001) at 50 µM in comparison with 200 µM in V. radiata 'Gujarat Mung-4'.Enhanced activities of these enzymes suggest the active involvement of SOD in the removal of superoxide radical and H2O2 by CAT and GR.Similar reports were presented earlier due to metal treated germinating seeds of raya and fenugreek (Nataraj and Parmar, 2008;Nataraj and Roshan, 2008;Nataraj et al., 2009).