Plant material selection
The present research was conducted in screen house of the department of biochemistry, college of basic sciences & humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana (India). Three varieties of sorghum (Sorghum bicolor L.) viz. HJ-541, HJ 513 and SSG 59-3 were procured from forage section of university. These varieties were selected because they are the only source of forage in dryland during the summer season and they are widely grown in Haryana region. Also, SSG 59-3 is sweeter than HJ 513 (multi-cut) variety and HJ 541 (single-cut) variety. Moreover, HJ 541 is suitable for both grain and fodder yield while HJ 513 is more suitable for grain yield. However, there are no reports about the sensitivity of these three cultivars for GB and AMF, under Cr (VI) toxicity. The toxic effects of hexavalent Cr, observed on sorghum plant growth along with possible reasons are depicted in Fig 1.
Experimental details and raising of the crop
Three varieties of sorghum at two growth stages viz. vegetative (35 DAS) and grain filling (95 DAS) stages were tested for amelioration of chromium toxicity (2 & 4 ppm) by exogenous application of GB (50 & 100 mM) and AMF in soil both individually and their combination, in completely randomized block design. The seeds of uniform size were selected and surface sterilized with 0.01 % mercuric chloride (HgCl2) solution for 10 minutes, followed by 5 times washing with distilled water. The plants were raised in earthen pots lined with polyethylene bags filled with 5 kg sandy loam, acid (5 % HCL) washed soil. The sterilised seeds were sown at 2 cm depth in the pots. Two weeks old seedlings of same size were transferred to other pots containing 5 kg soil. Soil properties are mentioned in Table 1. Separate pots were kept for control plants. Three replications were maintained for each treatment and control. All pots were irrigated with equal quantities of water and nutrient solution as per recommended package of practices (POP).
Table 1. Physicochemical properties of soil used during present experiment
Property
|
Value & unit
|
Evaluation
|
Texture
|
-
|
Sandy loam
|
Sand
|
71.70 %
|
-
|
Silt
|
18.96 %
|
-
|
Clay
|
9.34 %
|
-
|
pH
|
8.2
|
Basic
|
OC
|
0.32
|
Low
|
EC
|
0.17 DS meter-1
|
Normal
|
Nitrogen (N)
|
3 mg kg-1 soil
|
Low
|
Phosphorus (P)
|
8 mg kg-1 soil
|
Low
|
Potassium (K)
|
84 mg kg-1 soil
|
Normal
|
Zink (Zn)
|
0.61 mg kg-1 soil
|
Normal
|
Iron (Fe)
|
0.7 mg kg-1 soil
|
Low
|
Copper (Cu)
|
0.18 mg kg-1 soil
|
Normal
|
Manganese (Mn)
|
2.73 mg kg-1 soil
|
Normal
|
Chromium (Cr)
|
0.016 mg kg-1 soil
|
Low
|
Chemicals and reagents
The chemicals and reagents used during this research work were of high analytical grade. All the chemicals were procured from Sigma Chemicals Co. USA, Sisco Research Laboratories (SRL), Hi-Media and E. Merck Ltd.
Treatments and growth conditions
During present research, the treatments were provided on the basis of procedures followed in previous experiments [15]. The detailed composition of treatments used in this experiment is given in Table 2.
Table 2. Treatments details of AMF and GB provided in soil prior to plantation.
Treatment Name
|
Treatment Composition
|
C
|
Control
|
Control + AMF
|
T1
|
GB (50 mM)
|
GB (50 mM) + AMF
|
T2
|
GB (100 mM)
|
GB (100 mM) + AMF
|
T3
|
Cr (2 ppm)
|
Cr (2 ppm) + AMF
|
T4
|
Cr (2 ppm) + GB (50 mM)
|
Cr (2 ppm) + GB (50 mM) + AMF
|
T5
|
Cr (2 ppm) + GB (100 mM)
|
Cr (2 ppm) + GB (100 mM) + AMF
|
T6
|
Cr (4 ppm)
|
Cr (4 ppm) + AMF
|
T7
|
Cr (4 ppm) + GB (50 mM)
|
Cr (4 ppm) + GB (50 mM) + AMF
|
T8
|
Cr (4 ppm) + GB (100 mM)
|
Cr (4 ppm) + GB (100 mM) + AMF
|
Chromium stress treatments: Potassium dichromate salt (K2Cr2O7.7H2O) procured from Sigma Ltd. company, was used with distilled water to make two different levels of Cr stress solution (2 and 4 ppm). The soil in each pot was treated with 1 litre of respective, out of these two different levels of Cr stress solutions just after plantation of seedling. Level of respective stress was maintained by supplying respective Cr solution in the respective pots within the 7 days interval.
Glycine betaine treatments: Exogenously GB (50 and 100 mM) stalk solutions were prepared with distilled water and 1 litre of this from each was supplied in soil of respective pots just after plantation of seedling. The level of respective concentration of GB was maintained by supplying respective GB solution in the respective pots within a week interval.
Arbuscular mycorrhizal fungi (AMF) treatment: The AMF was supplied exogenously in soil before plantation of seedling. The treatment of AMF was provided by mixing 10 g of medium containing AMF in soil per pot. Generally, AMF can grow itself in the moist medium of soil and may increase their levels with time passes. So it was applied only once at the time of plantation of seedling in pots.
Plant sampling and analysis
The plant samples from control and each treatment, were collected at 35 and 95 DAS. A complete plant was collected in an ice cooled thermacol box. It was further divided in to leaf, shoot and root. Fresh leaves were used for the estimation of antioxidative enzymes, metabolites and indices of oxidative stress parameters. Shoot samples were hand homogenised and used immediately for the estimation of enzymes activity. Leaf, stem and root samples were dried in an oven for 72 h at 70 °C then Cr contents were estimated separately. The data was analysed by using a three-factorial, analysis of variance ANOVA, CRD design in SPSS software. Significant (P ≤ 0.05) differences between treatments were determined using critical difference.
Determination of soil properties
The soil was analysed for texture, pH, electrical conductivity, organic carbon, N, P, K, Fe, Mn, Cu, Zn and Cr (Table 2). Texture was determined by International Pipette method [65]. The pH of the soils was measured with glass electrode using soil suspension of 1:2 (soil: water) and electrical conductivity in supernatant as given in [66]. Organic carbon was determined by wet-oxidation method of Walkely and Black, [67]. Available nitrogen (N) was determined by alkaline permanganate method [68], available P content was determined by extracting the soil samples using 0.5M NaHCO3 and analysed by spectrophotometer [69] and available potassium was extracted by using neutral normal ammonium acetate and the content was determined by aspirating the extract into flame photometer. The available forms of Fe, Mn, Cu, Zn and Cr were extracted by DTPA at pH 7.3 and determined using atomic absorption spectrometer [70].
Determination of chromium contents
Chromium content was estimated in plant tissue (leaf, stem and roots) sample by using atomic absorption spectroscopy technique [70]. Five hundred mg tissue sample along with 20 ml digestion mixture (nitric acid and perchloric acid in 4:1 ratio, respectively) was digested overnight in a 100 ml conical flask at room temperature, followed by heating on an electric heater until a very small amount and colourless mixture (2-3 ml) was left in the flask. After cooling the total volume was made up to 25 ml with distilled water. The chromium content was determined in this digested mixture by calibration of standards of Cr (VI) in the form of potassium dichromate in the range 0 – 6 mg L-1 in water, and comparing with samples through atomic absorption spectroscopy (AAS). The results were expressed in ppm.
Determination of the enzymatic antioxidants
Following enzymatic antioxidants parameters were studied at vegetative and grain filling stage in sorghum plants.
Extract preparation for the estimation of enzymatic antioxidants: The complete extraction procedure was carried out below 40C. Two g of fresh and cleaned leaf tissue was homogenised in 10 ml of 0.1 M potassium phosphate buffer (pH-7.0) by using previously chilled mortar and pestle. The homogenate was centrifuged at 10,000 rpm for 15 minutes. The supernatant was collected as crude extract and stored in refrigerator for total soluble protein estimation. It was used for enzyme assay at same time.
Superoxide dismutase (SOD): The enzyme is a metalloprotein, which catalyses the dismutation of superoxide radical to H2O2 and molecular oxygen. It is a key antioxidant in aerobic cells and establishes the first line of defence against reactive oxygen species (ROS). Superoxide dismutase was assayed by measuring its ability to inhibit the photochemical reduction of nitro-blue tetrazolium (NBT) following the method of Beauchamp and Fridovich, [71]. The 3.0 ml reaction mixture contained 2.5 ml of 60 mM Tris-HCl (pH 7.8), 0.1 ml each of 420 mM L-methionine, 1.80 mM NBT, 90 µM riboflavin, 3.0 mM EDTA and enzyme extract. Riboflavin was added at the end. The tubes were shaked properly and placed 30 cm below light source consisting of three 20 W-fluorescent lamps (Phillips, India). The reaction was started by switching-on the light and terminated after 40 min of incubation by switching-off the light. After terminating the reaction, the tubes were covered with black cloth to protect them from light. A non-irradiated reaction mixture was kept that did not develop any colour and served as control. A separate blank was prepared for each sample, simultaneously by taking boiled enzyme extract. The reaction mixture without enzyme extract had developed maximum colour and its absorbance was decreased with the addition of enzyme. The amount of inhibition was used to quantify the enzyme. The absorbance were record at 560 nm. The Log A560 were plotted as a function of volume of enzyme extract used for reaction mixture. The volume of enzyme extract used in 50% inhibition of the photo-chemical reaction was considered as one enzyme unit. One enzyme unit was defined as the amount of enzyme required to inhibit the photo-reduction of one µmole of NBT. The enzyme activity was expressed in terms of unit g-1 fresh weight and were converted to unit mg-1 protein by estimating the total soluble proteins in the sample. The percent inhibition was calculated by following formula of Asada et al. [72].
V = Rate of assay reaction in absence of SOD.
v = Rate of assay reaction in presence of SOD.
Ascorbate peroxidase activity (APX): Ascorbate peroxidase is most widely distributed antioxidant enzyme. It reduces hydrogen peroxide to water using reduced ascorbate as the electron donor. It plays an important role in scavenging ROS than other antioxidative enzymes since ascorbate, in addition to reacting with H2O2 may react with superoxide, singlet oxygen and hydroxyl radical. Ascorbate peroxidase was assayed by the method of Nakano and Asada, [73]. Three ml reaction mixture contained 2.7 ml of 100 mM potassium phosphate buffer (pH 7.0), 0.1 ml L-ascorbate and 0.15 ml H2O2. The reaction was initiated by adding 50 µl of enzyme extract. Decrease in absorbance were recorded at 290 nm spectrophotometrically for 2 min against a suitable blank. A separate blank was prepared for each sample, simultaneously by taking boiled enzyme extract. The enzyme activity was calculated, using the molar extinction coefficient (Absorbance of one molar solution) of 2.8 mM-1 cm-1 for ascorbate in the standard equation for absorbance. One enzyme unit corresponds to the amount of enzyme required to oxidize one nmol of ascorbic acid min-1.
Standard equation for absorbance as A = ε × Ɩ × с
Where, A is the amount of light absorbed by the sample at a given wavelength, ε is the molar extinction coefficient, Ɩ is the distance that the light travels through the solution, and с is the concentration of the absorbing species.
Catalase activity (CAT): The enzyme catalase scavenges highly toxic hydrogen peroxide, produced in a number of reactions in the cell. Thus preventing metabolic machinery of the cell. It detoxifies hydrogen peroxide without overwhelming cellular reducing equivalents and provides cell with energy efficient mechanism to remove hydrogen peroxide. It exists profusely in plant tissues and its activity is connected with peroxisomes where, it removes hydrogen peroxide produced during photorespiration. The activity of enzyme was measured by slightly modified method of Sinha, [74]. The reaction mixture contained 0.55 ml of 0.1 M potassium phosphate buffer (pH 7.0), 0.4 ml of 0.2 M hydrogen peroxide and 50 µl of enzyme extract. It was mixed thoroughly and incubated for one minute at room temperature followed by addition of 3.0 ml dichromate reagent to it. A separate reaction was run for control, comprising 0.6 ml potassium phosphate buffer and 0.4 ml hydrogen peroxide (0.2 M), without enzyme extract. The tubes were kept in boiling water bath for 10 min. After cooling, the absorbance were recorded at 570 nm using a suitable blank containing boiled enzyme extract. The absorbance of sample were subtracted from that of control and the amount of hydrogen peroxide was calculated from standard curve. One enzyme unit correspond to the amount of enzyme required to breakdown one µmole of hydrogen peroxide min-1 or mg-1 protein.
Glutathione reductase activity (GR): Glutathione reductase catalyses the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) in a NADPH dependent reaction. Glutathione reductase was assayed using the procedure of Halliwell and Foyer, [75]. The assay mixture (3.0 ml) contained 2.5 ml of assay buffer buffer, 0.2 ml EDTA, 0.15 ml of 50 mM oxidized glutathione, 0.1 ml of 30 mM NADPH and 50 µl of enzyme extract. Assay reaction was initiated by adding NADPH at the end. Decrease in absorbance were recorded simultaneously, at 340 nm wavelength against a suitable blank containing boiled enzyme extract. Amount of NADPH oxidized were calculated by using an extinction coefficient (Absorbance of one molar solution) of 6.12 mM-1 cm-1 in the standard equation for absorbance. One unit activity of enzyme was correspond to the amount of enzyme required in the oxidation of one nmol of NADPH min-1.
Standard equation for absorbance as A = ε× Ɩ× с
Where, A is the amount of light absorbed by the sample at a given wavelength, ε is the molar extinction coefficient, Ɩ is the distance that the light travels through the solution, and с is the concentration of the absorbing species.
Peroxidase activity (POD): Peroxidase is non-specific in nature. It utilize different compounds as substrates to metabolize H2O2 preferably some phenolic compounds. During aging process, peroxidase catalyses cell wall softening reactions and plays an important role in response to environmental stresses. Peroxidase was assayed by the method of Shannon et al. [76]. Enzyme was assayed by putting 3.5 ml of assay buffer, 0.3 ml of o-dianisidine and 0.1 ml of diluted enzyme extract, in a cuvette of 5ml capacity. The solution was mixed well. The assay reaction was initiated by adding 0.1 ml of 0.2% hydrogen peroxide followed by recording the change in absorbance at 430 nm wavelength, simultaneously. A separate blank was prepared for each sample, simultaneously by taking boiled enzyme extract. The enzyme activity was expressed as change in 0.01 absorbance min-1 mg-1 protein.
Polyphenol oxidase (PPO): Polyphenol oxidase catalyses, o-hydroxylation of monophenols (phenol molecules with benzene ring containing, single hydroxyl substituent) to o-diphenols (phenols, with two hydroxyl substituents). They can further catalyse, the oxidation of o-diphenols to o-quinones. Polyphenol oxidase enzyme activity was assayed by the method of Taneja and Sachar, [77]. The assay mixture contained 1.8 ml of assay buffer, 2 ml catechol solution as substrate and 0.2 ml enzyme extract in glass test tubes. These test tubes were incubated at 37°C for 1 hour to take place the assay reaction followed by measuring absorbance at 430 nm on a UV-Vis spectrophotometer. A separate blank was prepared for each sample, simultaneously by taking boiled enzyme extract. The enzyme activity was expressed as change in 0.01 absorbance min-1 mg-1 protein.
Determination of the antioxidant metabolites
Following antioxidative metabolites were studied at vegetative and grain filling stage in sorghum plants under different treatments.
Glutathione: It is a low molecular weight thiol commonly found in both eukaryotic and prokaryotic cells. It is a most important water soluble antioxidant involved in preserving low redox potential and a highly reduced intracellular environment. It also take part in scavenging reactive oxygen species. The level of oxidized, reduced and total glutathione was estimated by the method of Smith, [78].
Extract preparation: One g of fresh leaf tissue was homogenised in 10 ml of 5% (w/v) sulphosalicylic acid using glass beads as abrasive, at 4ºC. Then, it was centrifuged at 30,000 x g for 20 min (4ºC) and the supernatant was collected for glutathione determination.
Assay: Total glutathione (GSH+GSSG), was determined by adding 0.1ml of 0.5 M potassium phosphate buffer (pH 7.5), 0.5 ml of 0.1 M sodium phosphate buffer (pH7.5) containing 5 mM EDTA, 0.1ml of 2mM NADPH, 0.1ml of glutathione reductase, 0.15 ml of 0.6 mM DTNB and 0.05 ml supernatant in a cuvette. The content was mixed thoroughly before the addition of supernatant, and the reaction was initiated by adding supernatant at the end of addition process. A separate blank tube was prepared by avoiding the addition of supernatant. The reduction rate of DTNB was monitored at 412 nm for 3 minutes. Total glutathione content was calculated from a standard curve of GSH (200-400 ng) plotted against the rate of increase of absorbance at 412 nm. Further, the oxidised glutathione (GSSG) content was determined by adding 1.5ml potassium phosphate buffer (0.5M, pH 7.5) and 0.2ml 4-vinylpyridine to 1ml supernatant in a test tube. The mixture was allowed to react for 1 hr to remove reduced glutathione (GSH). The GSSG content was measured using the same procedure as for total glutathione determination but with a GSSG standard curve (50-200 ng). Reduced glutathione (GSH) content was calculated by subtracting GSSG from the total glutathione content.
Proline: Proline is a basic amino acid found in high percentage in proteins. Free proline is said to play a role in plants under stress conditions. Though the molecular mechanism has not yet been established for the increased level of proline, one of the hypotheses refers to breakdown of protein into amino acids and conversion to proline for storage. Many workers have reported a several-fold increase in the proline content under physiological and pathological stress conditions. The proline content was estimated by the method of Bates et al. [79].
Extract preparation: One g of fresh leaves sample were homogenised in 10 ml of 3 % sulphosalicylic acid and centrifuged at 3000 rpm for 10 minutes. The supernatant was collected and used for proline estimation.
Assay: The extract was filtered through Whatman No. 2 filter paper. Two mL of filtrate along with 2mL of glacial acetic acid and 2mL acid ninhydrin were transferred in a test tube followed by heating in the boiling water bath for 1hr. The reaction was terminated by placing the tube in ice bath. Four mL toluene was added to the reaction mixture and stirred well for 20-30 sec. Toluene layer was separated and cooled to room temperature. The red coloured intensity of toluene was measure at 520 nm. Amount of proline present in the samples were determined from the standard curve (0.04 – 0.2 µg ml-1) of proline.
Where 115.5 is the molecular weight of proline.
Ascorbic acid: Ascorbic acid is an important antioxidant, when present in reduced form. It is widely distributed in fresh fruits like guava, mango, ber, papaya and leafy vegetables such as cabbage and spinach. Ascorbic acid was determined by the slightly modified procedure of Oser, [80].
Extract preparation: One g of the plant tissue was homogenised in 6 ml of ice-cold 0.8 N HClO4 and centrifuged at 40C, 10000 rpm for 30 minutes. The supernatant was collected and neutralized with 5M K2CO3. It was centrifuged again at same conditions (40C temperature, 10000 rpm for 30 minutes). Thus a clear supernatant was obtained, which were used for estimation of ascorbic acid content.
Assay: For estimation of total ascorbate, 1 ml extract was treated with equal volume (i.e. 1 ml) of 10% TCA. It was incubated in ice for 5 minutes. It was further mixed with 1 ml each of 5 M NaOH, 10 mM dithiothreitol (DTT) and 0.5% (w/v) N-ethyl maleimide (NEM) and 2 ml sodium phosphate buffer (pH7.4) in a final volume of 7 ml followed by 1 ml of 2% dinitrophenyl hydrazine and a drop of 10% thiourea, addition. Then the tubes were shaken vigorously and kept in boiling water bath for 15 minutes and cooled. After cooling 80% H2SO4 was added to the tubes at 4oC and vortexed. Then the absorbance were recorded at 530 nm against a suitable blank without the sample extract. The amount of ascorbate was determined by using a reference curve (0-100 nmoles) of ascorbate and expressed as µmoles g-1 fresh weight.
β-Carotene: It is a red-orange coloured pigment, found plentiful in cereals, vegetables, and fruits. β-carotene is a precursor of retinol (vitamin A). The absorption of β-carotene increases, if it is eaten with fats. The amount of β-carotene was determined by the method of AOAC, [81].
Assay: A homogeneous suspension was made by dispersing 10g of shoot sample in 50 ml of water-saturated n-butanol (The n-butanol and water were mixed in the ratio of 6:2 (v/v) and shacked vigorously. Then it was allowed to stand, till it separates into two phases. The upper clear layer was water saturated n-butanol). After vigorous shaking, it was allowed to stand overnight (16 hrs) at room temperature in dark. It was shacked again followed by filtration through Whatman filter paper No. 1. The total volume of filtrate was made up to 100 ml. The absorbance (A) of the clear filtrate was measured at 440 nm in Spectronic-20/spectrophotometer against a blank of saturated n-butanol. The amount of β-carotene were calculated from the following equation:
Detection of indices of oxidative stress
Following metabolites were studied as indices of oxidative stress at vegetative and grain filling stage in different treatments during the experimental analysis.
Hydrogen peroxide (H2O2): It is an important oxidant that initiates localized oxidative destruction which leads to disturbance of metabolic functions and damages of cellular integrity at site where it gathers. Hydrogen peroxide acts as a second messenger. It can diffuse across the cells and tissues; causes induction of proteins and genes, involved in stress defence system like CAT, APX. It is relatively stable.
Extraction: Two g tissue was macerated in 5ml of ice cold 0.01 M phosphate buffer (pH 7.0) and centrifuged at 8000 x g for 10 minutes. Supernatant was collected and used for the estimation of H2O2 content [75].
Assay: Fifty µl of extract were added to 1.95 ml of 0.01M potassium phosphate buffer (pH 7.0) and 2 ml of dichromate reagent to the mixture. It was kept in boiling water bath for 10 min and then cooled. After cooling, the absorbance were taken at 570 nm wavelength against a reagent blank without sample extract and the quantity of H2O2 were calculated from the standard calibration curve (10 to 160 µmole of H2O2).
Malondialdehyde (MDA): MDA content in plants reflects the extent of oxidative damage and hence membrane deterioration. It is produced during lipoxygenase (LOX) reaction.
Extraction: One g tissue were homogenized in 5 ml of TCA (0.1 % trichloroacetic acid; w/v) and centrifuged at 8000 x g for 15 min. The supernatant was used for MDA estimation by the method of Heath and Packer, [82].
Assay: The MDA estimation reaction was started by putting 1 ml of the supernatant, 4 ml of 20% TCA containing 0.5% 2-thiobarbituric acid (TBA). The content was heated in a boiling water bath at 95ºC for 30 minutes with constant stirring. Then it was cooled quickly in ice bath followed by centrifugation at 8000 x g for 10 min. The supernatant was decanted and the absorbance were recorded at 532 nm against distilled water as blank. The values for non-specific absorption at 600 nm were subtracted from it and the concentration of MDA was calculated by using the molar extinction coefficient at 155 mM-1 cm-1.
Grain yield determination
The grain yield was determined on 100 grains weight basis. One hundred grains from each replication were selected randomly and weighed, separately for each treatment, by using laboratory weighing balance. The average value of all replications was calculated and expressed as the yield in grams per 100 grains weight basis.
Statistical analysis
The present study was carried out in a completely randomized design (CRD) with three replications per treatment. All the results were analysed by using IBM SPSS Statistics 23 software for windows [83]. Comparison between different treatments was evaluated with a post hoc test followed by Tukey test. In the present study, the value for P was ascertained significant at ≤ 0.05.