Title: The Cumulative Dose of AMF and GB is More Effective in The Amelioration of Cr(VI) Toxicity in Sorghum (Sorghum Bicolor L.) Than Individually

Industrial and anthropogenic activities are the major source of heavy metal toxicants in agricultural soils. Among, heavy metal toxicants, hexavalent chromium is the most toxic toxicant that negatively affects plant’s metabolic activities and yield. It reduces the plant growth and development by inuencing the antioxidant defence system’s activities. In the present experiment, two different soil applied dozes of GB viz. 50 and 100mM, and AMF, both individually and in combination were tested for their capability to ameliorate Cr toxicity in sorghum. The promotive behaviour of these treatments for antioxidant defence system was analysed at vegetative (35 DAS) and grain lling stage (95 DAS) in three varieties of sorghum viz. SSG 59-3, HJ 513 (multi-cut) and HJ 541 (single-cut) under 2 and 4 ppm Cr toxicity. At the same time resultant effects of this behaviour on Cr accumulation, grain yield and indices of oxidative stress was also studied. In this experiment antioxidant defence system includes enzymes viz. SOD, APX, CAT, GR, POD and metabolites viz. glutathione, ascorbate, proline, β-carotene and indices of oxidative stress includes parameters viz. PPO, H 2 O 2 and MDA. The results delineated that Cr accumulation and indices of oxidative stress were increased with increasing concentration of Cr stress in all the varieties at both growth stages. Chromium stress at high concentration (4 ppm), decreased the grain yield (71.69 %) as compared with control. Due to 4 ppm Cr stress, PPO activity, MDA and H 2 O 2 accumulation increased signicantly (72.29 %, 73.15 %, 79 % respectively, at 35 DAS and 70.36 %, 74.78 %, 79.83 % respectively, at 95 DAS). GB and AMF individually increased antioxidant activity but in combination, further signicantly increased antioxidant defence system’s activity which in turn decreased indices of oxidative stress and reduced the Cr toxicity and increased grain yield of sorghum in all varieties at both the growth stages. However, treatment of 100mM GB with AMF was observed most signicant in decreasing oxidative stress and improved antioxidant system’s activities and grain yield as compared with all other treatments at both growth stages in all the varieties. SSG 59-3 cultivar showed lowest chromium content (1.60 and 8.61 ppm), indices of oxidative stress and highest antioxidant system’s activity as compared to HJ 513 followed by HJ 541 variety, at 35 and 95 DAS respectively. Thus, among the varieties SSG 59-3 was found most tolerant as compared to HJ 513 followed by HJ 541 variety. These ndings suggest that both GB and AMF, either individually or combined can play a positive role to reduce oxidative stress and increased yield attributes under Cr toxicity in sorghum.


Introduction
Sorghum crop [Sorghum bicolor (L.) Moench] a member of family Poaceae, is grown worldwide in over 41.14 million hectares of area and accounts for production of 58.72 million metric tons of grains with an average yield of 1.43 metric tons per hectare [1]. India ranks second in terms of area under sorghum cultivation. In India, Sorghum is cultivated in over 5.00 million hectares and accounts for production of 4.50 million metric tons of grains with an average yield of 0.90 metric tons per hectares [2]. Sorghum is one of the ve top cereal crops and is consumed after rice, wheat, maize and barley [3]. It is an important kharif season crop which is directly or indirectly utilized for nourishment of humans, animals [4]. Sorghum is a C4 plant that is highly e cient in converting solar energy to chemical energy, and also in under stressful conditions. In some plants, the natural accumulation of GB, is not enough to protect them from abiotic stresses. Under such conditions, exogenous application of GB may help to reduce the adverse effects of various environmental stresses [27]. GB is environmentally safe, nontoxic, and watersoluble [28]. There is strong evidence that GB plays an important role in plants against tolerance to abiotic stresses [29]. Previous studies on amelioration of heavy metal toxicity using GB in plants, suggested that 50 and 100 mM concentration was effective in the amelioration of heavy metal toxicity [30]. Furthermore, Arbuscular mycorrhizal fungi are recognized as biological agents that potentially increase the tolerance of plants to heavy metal toxicity [31]. The reduction of growth due to chromium interference with nutritional elements uptake can be improved through mycorrhizal inoculation. Karagiannidis and Hadjisavva-Zinoviadi, [32] showed that arbuscular mycorrhizal fungi (AMF) can enhance yield by simultaneously reducing the chromium content in crop plants. However, no one have reported about amelioration of hexavalent Cr toxicity by using combined dose of GB and AMF in sorghum. In this research, we tested the hypothesis that whether the combination of GB and AMF ameliorates Cr toxic effects and improves the yield in sorghum. Outcomes of our research would possibly depict a potential way to prevent Cr toxicity in sorghum.

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 lling (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 (HgCl 2 ) solution for 10 minutes, followed by 5 times washing with distilled water. The plants were raised in earthen pots lined with polyethylene bags lled 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).

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. 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. Signi cant (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 ( and available potassium was extracted by using neutral normal ammonium acetate and the content was determined by aspirating the extract into ame 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 ask 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 ask. 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 lling stage in sorghum plants.
Extract preparation for the estimation of enzymatic antioxidants: The complete extraction procedure was carried out below 4 0 C. 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 H 2 O 2 and molecular oxygen. It is a key antioxidant in aerobic cells and establishes the rst 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 ribo avin, 3.0 mM EDTA and enzyme extract. Ribo avin was added at the end. The tubes were shaked properly and placed 30 cm below light source consisting of three 20 W-uorescent 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 nonirradiated 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 de ned 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 H 2 O 2 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 H 2 O 2 . 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 coe cient (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 × × с
Where, A is the amount of light absorbed by the sample at a given wavelength, ε is the molar extinction coe cient, 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 detoxi es hydrogen peroxide without overwhelming cellular reducing equivalents and provides cell with energy e cient 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 modi ed 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 coe cient (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× × с
Where, A is the amount of light absorbed by the sample at a given wavelength, ε is the molar extinction coe cient, 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-speci c in nature. It utilize different compounds as substrates to metabolize H 2 O 2 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 lling 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. 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 ltered through Whatman No. 2 lter paper. Two mL of ltrate 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 modi ed procedure of Oser, [80].
Extract preparation: One g of the plant tissue was homogenised in 6 ml of ice-cold 0.8 N HClO 4 and centrifuged at 4 0 C, 10000 rpm for 30 minutes. The supernatant was collected and neutralized with 5M K 2 CO 3 . It was centrifuged again at same conditions (4 0 C 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 nal 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% H 2 SO 4 was added to the tubes at 4 o C 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. βcarotene 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 watersaturated 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 ltration through Whatman lter paper No. 1. The total volume of ltrate was made up to 100 ml. The absorbance (A) of the clear ltrate 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 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-speci c absorption at 600 nm were subtracted from it and the concentration of MDA was calculated by using the molar extinction coe cient 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 signi cant at ≤ 0.05.

Results
The present investigation was carried out on three varieties of sorghum viz. varieties for enzymatic activities. Among the varieties, SSG 59-3 variety showed highest activity of these antioxidative enzymes, followed by HJ 513 and lowest in HJ 541 variety (Fig 4).
The antioxidative defence systems include both enzymatic and non-enzymatic antioxidant components. Apart from enzymatic, non-enzymatic antioxidants such as Glutathione (GSH and GSSG), Ascorbate (AsA), Proline and β-carotene, are crucial for plant defence against oxidative stress. They play a key role as antioxidant buffers. Glutathione reductase is responsible for maintaining the supply of reduced glutathione. It is one of the most abundant reducing thiols in majority of cells. GSH plays a key role in the cellular control of ROS. The major role of APX is detoxifying hydrogen peroxide in plant cells via, ascorbate-glutathione cycle, in which, ascorbate acts as a speci c electron donor for APX enzymes in catalyzing the conversion of H 2 O 2 into H 2 O.
To determine the ameliorative effect of GB and AMF against hexavalent Cr in sorghum, non-enzymatic antioxidant components were also analysed. Non-enzymatic antioxidant components, namely total glutathione, reduced glutathione (GSH), oxidized glutathione (GSSG), ascorbate, proline and β-carotene were studied. Among them, except β-carotene all other metabolites, increased signi cantly with increasing concentration of Cr stress at both the growth stages, in all the varieties (Figs 5 and 6). The βcarotene content decreased signi cantly with increasing concentration of Cr (VI), at both the growth stages in all the varieties (Fig 6). All other properties observed were similar to other antioxidative metabolites contents. Along with β-carotene and except GSSG, further increase in the content of these metabolites was observed on exogenous application of GB and AMF, either individually or in combination, at both the growth stages in all the varieties. In contrast, GSSG content decreased on GB and AMF application, either individually or in combination, at both the growth stages in all the varieties (Fig 5). Effect of GB and AMF treatments on grain yield (100 grains weight) in sorghum under chromium toxicity There was a progressive decrease in grain yield with increasing concentration of Cr (VI), at both the growth stages, in all the varieties (Fig 7). Increase in grain yield was observed on exogenous application of GB and AMF, either individually or in combination, at both the growth stages in all the varieties.
Maximum increase was observed in plants provided with the combination of 100 mM GB and AMF, at both the growth stages in all the varieties.

Discussion
Chromium toxicity in cultivable lands has become a serious problem all over the world [33]. It reduces growth and yield of the sorghum crop [34]. There are many reports on Cr (VI) toxicity causing hazardous effects in plants. However, reports on amelioration of chromium toxicity by using GB and AMF together are scanty in literature. In this study, the ameliorative effect of exogenously applied GB and AMF (individually and in combination) against Cr (VI) toxicity was investigated on antioxidative defence system in sorghum. During present research, increasing levels of Cr treatments was resulted in increased Cr content in sorghum. It seems that after application of only 2 and 4 ppm of Cr, the Cr content in roots, stem and leaves increased many folds i.e. more than the highest treatment of 4 ppm. The reason behind this might be the lower weight of dried sorghum plant as compared to weight of soil (5 kg pot -1 ) because the concentration of matter changes with respect to weight of medium, when it is expressed in terms of weight. It increases as weight of medium decreases. Similar reports have been reported earlier also [35,36]. The Cr content was higher in roots followed by stem and leaves indicated that sorghum plants might have abundant resistance against Cr stress as reported by other researcher in chickpea [37]. Reduction in Cr content of plant samples might be due to GB and AMF, either individually or in combination maintains cell membranes integrity and protects cells from damages which in turn limits the entry of Cr in to the cell. The reduction in Cr absorption by plants on GB application might also be due to shielding nature of GB towards cell membranes that reduces chromium movement to cells [38,39].
Similar results have been reported for Pb and Cd contents in mung bean [40], rice [41] and wheat [42].
Karagiannidis and Hadjisavva, [32] reported that AMF inoculation increased nutrient uptake and supresses Cr, Mn, Fe, Co, Ni, and Pb absorption in duram wheat. It suggested, other possibility in reduction of Cr absorption with AMF and GB application might be the competition between nutrients and Cr for entry in to the cells. Many reports on heavy metal resistant microorganisms have indicated exceptional ability of AMF to promote the growth of host plant under stressful conditions [31,43]. Moreover, AMF also has been recognized as a potential biological agent that increases the tolerance capacity of host plant under heavy metal stress.
It was noticed that Cr enhanced ROS generation such as H 2 O 2 and hydroxyl compounds which in turn increases MDA level and PPO activity. It was reported earlier that Cr is non-essential for plants and generates toxic stress by causing reduction of molecular oxygen and producing intermediate products called ROS such as superoxide radicals, hydroxyl radicals and H 2 O 2 . Interestingly, generation of ROS is the rst line of defence reaction exhibited by any plant cell in response to stress. They further induce the synthesis of other biomolecules (metabolites) and activation of enzymes of various pathways as a defence mechanism. The level of these compounds signi es the extent of stress and are known as indices of oxidative stress. Membrane lipids and proteins are more liable to be attacked by ROS making them reliable indicators of oxidative stress in plants.
In present study activities of antioxidant enzymes and metabolites were increased with increasing levels of Cr treatments (Figs 4-6). But this increase was not su cient in scavenging the ROS generated under Cr stress as was evident from increased H 2 O 2 , MDA and PPO activities at same treatments of Cr. Further, exogenous application of AMF and GB both individually or in combination enhanced antioxidant enzymes and metabolites activities at same Cr treatments in Sorghum and alleviates chromium induced toxicity as was evident from reduced H 2 O 2 , MDA and PPO activities on GB and AMF application (Fig 3).
The reason behind promotive role of GB and AMF towards antioxidants activities might be the inhibition of Cr absorption and increased nutrient absorption as studied by Jabeen et al. [44] in mung bean under Cr toxicity. Moreover, GB itself acts as compatible solutes and AMF helps in accumulation of them that functions as osmoprotectants and counteracts the oxidative stress by elevating the levels of antioxidant enzymes and metabolites [45]. Hisyam et al. [46] have also reported increased antioxidant system activities on exogenous GB application to counteract the stress caused by water defciency in rice plants.
Wang et al. [47] were also of similar view that GB acts as osmoprotectant, which in turn protects the plant cells from osmotic stresses and resulted in decreased PPO activity, while working on GB accumulation in wheat. Raza et al. [48] and Gill et al. [15] also got similar reports on exogenous GB application in wheat and brassica under Cr toxicity. These reports are supportive of the ndings of present investigation.
In the present experiment loss of grain yield on Cr application was noticed that might be due to excessive production of ROS which is toxic to plants and cause oxidative damage to cellular constituents that resulted in loss of growth and yield as reported by Khaliq et al. [49], who studied the effect of Cd toxicity in duram wheat. The other reason might be increased PPO activity which causes oxidation of polyphenols that reduces the chances of plants growth and reduces the yield under stressful conditions [50,51]. Apart from that H 2 O 2 is also very toxic compound and a higher content of it, produces injuries through lipid peroxidation in plant cells which in turn increases MDA content in plants that might also be the cause for reduced yield during stressed conditions [52,53]. The decrease in grain yield under Cr toxicity may also be due to increased Cr absorption with increasing Cr stress in plant that caused damaging of roots, chlorosis, necrosis, loss of mineral nutrition, and loss of water balance, ultimately resulted in to reduced yield of plants as also suggested by Ali et al. [54] in barley, Gill et al. [55] in oilseed rape cultivars under Cr toxicity and Kanwal et al. [56] in wheat under lead toxicity. The reduction in yield under Cr (VI) toxicity has also been reported widely in literature [30,57,58,59].
The results of present investigation revealed an increased yield on GB and AMF application both individually or in combination (Fig 7). This increase in grain yield might be resulted due to reduction in Cr uptake on GB and AMF application which in turn decreased stress level by maintaining proper stomatal conductance, chloroplast ultrastructure, RuBisCo activity, photosynthetic capacity and proper nutrient uptake [60]. Glycine betaine increased the antioxidant systems activity which in turn prevents plants from oxidative damages caused by ROS generated due to stressed conditions that might resulted in enhanced grain yield [61-63] demonstrated similar effects on AMF inoculation in sun ower under Cd toxicity, as were observed during present investigation. Similarly, GB application increased the growth and yield in rice plants under Cd toxicity [41]. Bharwana et al. [64] also obtained similar results that foliar application of GB increased the yield of cotton plants grown under lead (Pb) toxicity. However, the mechanism(s) involved in enhancement of growth and yield of the plant by GB and AMF application are still not clear. In present experiment, variety SSG 59-3 showed highest grain yield as compared to HJ 513 and HJ 541 (Fig   7). This might be ascribed to highest level of antioxidant enzymes and metabolites activities (Figs 4-6), and lowest level of Cr accumulation and indices of oxidative stress parameters (Figs 2 and 3) in SSG 59-3 variety followed by HJ 513 and lowest in HJ 541.
To sum up, our ndings revealed that Cr stresses signi cantly reduced the grain yield, antioxidant enzymes and metabolites activities. Indices of oxidative stress parameters were dominant due to Cr toxicity. However, the exogenous application of GB and AMF both individually and in combination signi cantly enhanced the grain yield and reduced the indices of oxidative stress parameters by improving antioxidant enzymes and metabolites activities under Cr stresses. The GB and AMF application also reduced the Cr accumulation and transport. No reports are available about the mechanism of GB and AMF combination in sorghum under Cr stress. Hence, further studies are needed at eld level in order to see the role of GB and AMF combinations and its mechanism towards various plant species under heavy metal stresses.

Conclusions
To conclude, Cr (VI) toxicity (2 & 4 ppm) produced biochemical changes in sorghum (Sorghum bicolor L.) plants resulted in increased ROS levels in all the varieties at both vegetative and grain lling stage. The deleterious effects increased with the increasing concentration of Cr. This may be due to increased Cr uptake which resulted in increased indices of oxidative stress. Through, the components of the antioxidant defence system increased under Cr toxicity. However it seems that it was not su cient to combat the toxicity stress. As revealed by high level of indices of oxidative stress parameters of the plant.
Exogenous application of GB and AMF, however improved the stress tolerance due to further increase in enzymes and metabolites of antioxidant defence system and reduction in indices of oxidative stress. The treatment of GB at both 50 and 100 mM level, applied in soil, signi cantly ameliorated Cr toxicity. However, AMF (10 g) concomitantly with GB, at both 50 & 100 mM level, further ameliorated the effects of Cr toxicity in sorghum plants at both growth stages (35 & 95 DAS). But the AMF application with GB at 100 mM level was found more bene cial at both growth stages. The combination of GB (100 mM) alongwith AMF (10 g) was observed most effective and best concentration among all the treatments, for the amelioration of Cr toxicity in sorghum plants at both growth stages. However, the effects were found more prominent at 35 DAS than 95 DAS. Based on results obtained in present investigation, the variety SSG 59-3 was observed to be more tolerant to Cr toxicity followed by HJ 513 and HJ 541. Further studies in eld conditions are necessary to con rm the mechanisms and ndings of this experiment.

Declarations Data availability
All data generated or analysed during this study are included in this article le.

Figure 1
Chromium toxicity reduces Sorghum plants growth and yield as compared to control plants.