The results obtained showed that A.tricolor has Al-tolerant mechanism. Al accumulation in the roots as a result of metal stress appeared to play an important role in the acclimation of the genotype to Al stress, suggesting that they could be used as physiological markers during the screening for Al-tolerance. Total protein, proline, ascorbate and scavenging enzymes responded distinctly to AlCl3 stress, suggesting divergent of response mechanisms in the species. Genetic evaluation of genotypes based on Al tolerance indices could be exploited in the breeding of Al tolerant genotypes.
We are extremely grateful to Vision Group on Science and Technology (VGST) Govt. of Karnataka for funding (VGST/P-15/K-FIST Level- I/2010-11/724) the project. Our sincere thanks go to Dr. V.Kumar, Davangere University for his guidance.
Pollution of the biosphere with toxic metals has accelerated dramatically since the beginning of the industrial revolution1 . Not only the humankind, but also plants and other organisms are affected by metal pollution. Trace metals such as Aluminum, Zinc, Arsenic, Cadmium, Manganese, Nickel, and Selenium etc., have been considered to be the major environmental pollutants and their phyto - toxicity is well established2 . Aluminum (Al) is the most abundant metal in the world and the third most common element in the earth crust. It is the primary limiting factor in crop productivity particularly in acid soils in the tropic and sub tropics3 . Evidences have demonstrated that the root apex is the primary site of Alinduced root inhibition4 . Al can interact with root cell walls, disrupts plasma membrane transport system and interacts with calmudulin and therefore plant signaling system5 . Al stress induced genes are identified and characterized from wheat, tobacco and Arabidopsis6,7,8 . A common aspect of all environmental stresses is the enhanced production of reactive oxygen species (ROS) within several subcellular compartments of the plant cell9 . ROS, if not detoxified cause serious damage to proteins, lipids and nucleic acids10. To minimize the harmful effects of ROS, plants have evolved an effective defense system. This includes both enzymatic antioxidants and non-enzymatic antioxidants. Among the antioxidant defense system, ascorbate (AsA), glutathione (GSH) and related enzymes (such as ascorbate peroxidase, glutathione reductase and superoxide dismutase) play a pivotal role in scavenging of ROS from plant cells11 . Amaranthus tricolor L belongs to amaranthaceae family, commonly known as ?Dantu sappu? (in Southern part of India) is a widely cultivated plant in the world, particularly in tropical Asia12. It is one of the major leafy vegetable available in hot summer. It is an upright branched annual herb, which grows about 0.45m ? 1m12. The seeds are small and brown. Mature leaves contain red-violet pigments, Betacyanin, Amaranthin and Isoamaranthin13. The plant leaves contains 38.3% dry matter as protein, 0.25% fats and 6.6% carbohydrates, along with other minerals and vitamins14. The role of Amaranthus as an under exploited plant with promising economic value has been recognized by the National Academy of Sciences12. A wide range of environmental stresses, such as extreme temperatures, drought, salinity, UV radiation and metals are potentially harmful to plant growth15. Hence, the present study aimed to investigate the toxicity of Al on growth, antioxidant profile of Amaranthus tricolor.
2.1 Seed germination and seedling growth Seeds of Amaranthus tricolor L were purchased from the local market, Bangalore city, Karnataka, India and verified by Dr. Shiddamallayya N, National Ayurveda Dietetics Research Institute, Bangalore (Voucher no. Drug Authentication/SMPU/ NADRI/BNG/2013-14/418). The healthy and uniform sized seeds were separated. The seeds were washed and surface sterilized with 1.0 % mercuric chloride. The seedlings were watered with Hoagland nutrient solution16 for 14 days, and then stress treatments were started. 2.2 Stress treatments Hoagland nutrient solution (1X) was supplemented with AlCl3 solution at different concentrations of 20 ?M, 60 ?M, and 100 ?M (pH-5.5). The control seedlings were maintained by watering normal Hoagland nutrient solution. Stress treatments were continued for, 3, 6, 9, and 12 days. 2.3 Assay of the Plant growth parameters 2.3.1 Root, shoot length and dry weight Root and shoot lengths were measured in the intact seedlings with the help of a scale and thread. The measurements were determined for 5 seedlings from each treatment and controls, then average values were calculated. Dry weight of detached shoots and roots of both control and Al stressed plants were determined separately. The samples were oven-dried at 80oC for 15 min, then vacuum-dried at 40 oC to constant weight and the dry weights (DW) were measured. 2.3.2 Estimation of Total Protein Two fifty (250) mg of root and shoot extract was homogenized separately in phosphate buffer (1.5ml) under ice cold conditions. The homogenate was centrifuged and the supernatant was estimated for total protein content spectrophotometrically. The concentration of the protein in supernatant was calculated using the formula as per the method of Layne, E. 195717 and UV spectrophotometrically. Here the O.D of the protein solution was read at 280 nm and 260 nm for possible nucleic acid contamination using the phosphate buffer (20mM) as the blank. Then the following formula was used to estimate the protein concentration of the enzyme sample. 2.3.3 Estimation of Proline Proline was estimated by Bates method18spectrophotometrically. The amount of proline in the test sample was calculated from the standard curve constructed by appropriate concentration of proline. Express the proline content on fresh-weight-basis as follows: 2.3.4 Ascorbate assay Ascorbate was determined at 525nm spectrophotometrically according to the modified procedure of Law et al19. Fresh root and shoot samples (500 mg) of both control and Al treated were separately homogenized into 3.0 ml of 5% metaphosphoric acid and centrifuged at 22,000?g for 15 min at 25 oC, using cooling centrifuge (REMI, India; Model C-24 BL). Supernatant was saved and used for the estimation of ascorbate. The supernatant was initially treated with dithiothreitol (for reducing dehydroascorbate to ASC). The supernatant (0.2 ml) was added to 0.5 ml of 150 mM phosphate buffer (pH 7.4) containing 5mM EDTA and 0.1 ml of 0.5% (w/v) Nethylmaleimide. After adding 0.4 ml of 10% (w/v) trichloroacetic acid, 0.4 ml of 44% (v/v) orthophosphoric acid, 0.4 ml of 4% (w/v) 2,2?-bipyridyl in 70% (v/v) ethanol and 0.2 ml of 3% (w/v) ferric chloride, the mixture was incubated at 40 oC for 40 min. The absorption of the color developed was measured at 525 nm using a UV-Visible spectrophotometer (Systronics -117). Total ascorbate was calculated using a standard curve for pure ascorbate. 2.4 Antioxidant Assay 2.4.1 Glutathion assay Extraction and estimation of total glutathione in both control and stressed tissues was carried out according to Griffith20 at 412 nm spectrophotometrically using 5,5?-dithiobis-2-nitro benzoic acid (DTNB) reagent. The concentration of the glutathione was calculated by using a standard calibration curve constructed by appropriate concentrations of glutathione (reduced; GSH) standard. 2.4.2 Catalase assay Catalase was estimated in the tissue extract by the method of Aebi21 using a UV visible spectrophotometer. The amount of H2O2 reduced was determined by using molar extinction coefficient of H2O2. The activity was expressed in terms of ?mol of H2O2 reduced min-1g -1 tissue fresh weight at 25 ? 2?C. 2.4.3 Superoxide dismutase (SOD) assay Superoxide dismutase activity was assayed by using the photochemical NBT method22. The homogenate was filtered through four layers of muslin cloth and centrifuged at 10,000 rpm for 20 min at 4oC, using cooling centrifuge and the supernatants were used for protein concentration determination and enzyme assays. Fresh roots and shoots (500 mg) of both control and aluminum stressed were separately homogenized into 4 ml extraction buffer consisting of 100 mM potassium phosphate buffer (pH 7.0) containing 1% PVP. The assay mixture in 3 ml contained 50 mM phosphate buffer, pH 7.8, 9.9 mM L-methionine, 57 mM NBT, 0.025% (w/v) Triton X-100, and 0.0044% (w/v) riboflavin. The contents were mixed rapidly and kept below 30 cm of light (at the light intensity of 300 mmol -1 m-2 s-1) for 10 minutes along with enzyme control lacking extract. The photoreduction of NBT (formation of purple formazan) was measured at 560 nm using a UV-Visible spectrophotometer. An inhibition curve was made against different volumes of extract. One unit of SOD is defined as being present in the volume of extract that causes inhibition of the photo-reduction of NBT by 50%, that was calculated with the help of following formula. 1.00000000000000000000000000000 SOD (units) = Vol. of extract (ml) required to cause 50% of NBT inhibition X 10-3 2.5 Estimation of Al accumulation in plant root and shoot Plant samples were gently ground using electrical grinder. Three gm of plant sample was digested with 20ml of HNO3:HClO4 ( 2:1). The mixture was heated in a beaker until dissolved and then cooled. The digested plant samples were then re-dissolved in 10% HClO4 and filtered through Whatman no. 40 filter papers, and the volume was adjusted to 50ml with 10% HClO4 in polyethylene volumetric flask. Reagent blanks for plant filters were also prepared by carrying out the whole extraction procedure, but without samples23. The Al in plant samples were estimated using flame atomic absorption spectroscopy method (GBC AVANTA Model 324). 2.6 Statistical analysis Statistical analysis of data was conducted using one-way Analysis of Variance (ANOVA) using SPSS 17.0 software. Values in the figures indicate the mean values?SD based on independent three determinations (n = 3). Least Significant mmoles per g tissue = mg proline/mL x mL toluene x 5 molecular weight of proline g sample Bhanuprakash et al Int. J. Fundamental Applied Sci. Vol. 2, No. 4 (2013) 64-68 66 Difference (LSD) test was used to assess the differences between control and different treatments; p<0.05 was considered statistically significant.
3.1 Plant growth The growth parameters were used as useful bioindicators of Al toxicity in Amaranthus seedlings. These parameters are expressed as root and shoot length and dry weight. A gradual reduction in growth parameters were observed with increased Al3+ concentration (Fig. Ia-c). Al3+ exhibited injurious effects followed by the death of Amaranthus seedlings when added at the highest (100 ?M) level. Inhibition of growth and reduction of biomass production are general responses of some plants to metal toxicity and are often a reliable indication of plant's sensitivity to their stresses24 . Our result also suggested that higher concentration of Al3+ (60- 100) ?M, exerts stress effect on Amaranthus and inhibits all the growth parameters. The total protein contents both in root and shoot were increased especially under (20-40) ?M concentration of Al3+ upon 3-6 days treatment (Fig Id, e). However with higher concentration of Al3+ and longer period of incubation the total protein content decreased. The decrease in protein content may be due to the enhanced of protein degradation as a result of increased protease activity under Al3+ stress condition25 . 3.1.1 Total Proline Content The accumulation of proline has been considered as a result of metal stress in plant26. Schat H et al, (1997)suggested that the increased level of proline enhanced the plant?s tolerance level through mechanisms like osmoregulation, stabilization of protein etc27. In the present study the total proline content (Fig. II a, b) has increased as the concentration of Al3+ was increased both in root and shoot samples. This is an indication of metal stress and plant?s tolerance level. The elevated level of proline could be either due to de novo synthesis or decreased degradation or both as suggested by Kasai Y et al (1998)28 . 3.1.2 Ascorbate Estimation Ascorbate is an essential compound in plant tissues, reacts rapidly with superoxide and singlet oxygen (chemically), and hydrogen peroxide (enzymatically). Ascorbate content in the roots and shoots of Amaranthus tricolor exhibited a significant increase at metal stress (40-100 ?M) on 6th day as compared to their respective controls 3.2 Antioxidant assay 3.2.1 Glutathion Assay GSH plays several roles in cell metabolism such as redox state regulation, oxidative stress control, and defense against heavy metals29. Induction of GSH level is an important protective mechanism to minimize oxidative damage in plants exposed to metals. A marked increase in GSH content of the amaranthus root samples was observed under Al stress (60?M) on 6th day when compared to the controls (Fig. IV). This result is in agreement with other plants exposed to metals30 .3.2.2 Catalase and SOD Estimation The SOD converts superoxide radical into hydrogen peroxide and molecular oxygen, whereas the catalase converts hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide are converted to the harmless product water. Induction and activation of superoxide dismutase (SOD) and catalase are some of the major metal detoxification mechanisms in plants. Gwozdz et al. (1997) found that at lower heavy metal concentrations, activity of antioxidant enzymes increased, whereas at higher concentrations, the SOD activity did not increase further and catalase activity decreased31. Our result showed an initial increase, and subsequent decrease in SOD level (Table I), which actually supports Gwozdz et al?s (1997), finding. Peixoto P H P et al (1999) suggested that catalase enzyme activity decreased after Al treatment both in roots and shoots of the plant32. Similar results have been obtained by Richards et al. (1998) who has also observed a reduction in specific mRNA for these catalases in Al3+ treated plants8 .The present study shows a decrease in catalase activity (Table I) upon (100 ?M) Al3+ treatment in Amaranthus which supports the previous findings. 3.3 Al Accumulation The toxic effects of Al are primarily root related. The present study confirmed the primary site of Al accumulation in Amaranthus tricolor is in the root (Table II). A higher amount of Al accumulation was observed upon 9-12 days incubation with (20-60) ?M Al3+. Ciamporova M (2002) suggested that longer Al treatment is required to reduce cell division or to interfere with nucleic acids in the root apex33. Further investigation is required to elucidate the genomic interference of Al at root apex in Amaranthus tricolor.
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