Amelioration of Chromium VI Toxicity in Sorghum (Sorghum bicolor L.) using Glycine Betaine

The main objective of the present research work was to study the effect of Cr toxicity and its amelioration by glycine betaine (GB) in sorghum (HJ 541 and SSG 59-3). Chromium (Cr VI), 2 and 4 ppm led to a significant reduction in plant height, root length, chlorophyll content, antioxidant enzymes viz. catalase, peroxidase, ascorbate peroxidase, glutathione reductase, polyphenol oxidase, and superoxide dismutase; and metabolites viz. ascorbate, proline, and glutathione. The results of the present study supported the findings that the application of GB can minimize or reduce the toxic effects caused by Cr VI which reaches the plants via soil, water, and air pollution. It is concluded that GB at both 50, as well as 100 mM concentrations, successfully ameliorated Cr VI (up to 4 ppm) toxicity and its application may be recommended for crops affected by Cr VI toxicity to get better growth and yield.

species in response to different environmental stresses. GB is expected to contribute to enhancing the HM stress tolerance in plants. The tolerant or sensitive species may be differentiated depending on the accumulated amount of GB during heavy metal chromium stress. GB is non-toxic, soluble in water 13 and one of the best-studied compatible solutes 14 . It is a quaternary ammonium compound that is found in bacteria, marine invertebrates, hemophilic archaebacteria, plants and mammals 15,16 . It gets accumulated to significant levels in salt-tolerant plants and halotolerant cyanobacteria 17,18 . The GB level varies significantly among different plant species and organs. Low levels of GB are found to be in the plants of distant species (taxonomically). However, when plants are subjected to abiotic stresses 19 , large amounts of GB accumulation has been reported. However, there are some plant species which do not produce GB under normal or stressful conditions 19 .
The available literature indicated that GB plays an important role in the amelioration of heavy metal Cr (VI) toxicity by increasing the activity of the antioxidative enzymes of the plant. Keeping the above view, the present study was planned to examine the effect of Cr (VI) toxicity and GB application on different morphophysiological & biochemical parameters in sorghum plants. Two different sorghum cultivars were selected for this experimental study, on the basis that SSG 59-3 is a multicut 20 while HJ 541 is a single cut cultivar. Moreover, SSG 59-3 is sweeter than HJ 541. They are widely grown in Haryana region for the nourishment of animals and industrial purposes. They are the only source of forage in dryland during the summer season. Both the cultivars differ from each other in quality parameters. However, there are no reports about the sensitivity of two cultivars against Cr (VI) stress.

Results
GB ameliorated the toxic effect of Cr (VI) stress on plant growth, chlorophyll content, antioxidative enzymes, and metabolites reflecting a significant increase in their amounts. The physical appearance of sorghum plants differ significantly in control plants, GB treated plants and Cr (VI) stressed plants. The plants with Cr (VI) treatment alone were shorter than those grown with GB (50 & 100 mM) treatments. The results of the present study are as follows.

Effect of exogenous GB on Cr (VI) induced suppression in Morphophysiological parameters.
Chromium VI toxicity reduced the plant growth and development with increasing Cr concentrations (0-4 ppm). The effect of Cr (VI) toxicity on growth and development of sorghum was evaluated by six characters, i.e. fresh weight, dry weight, root length, shoot length, chlorophyll content and grain yield. The effect of exogenously supplied GB on growth characters of Sorghum plants under Cr (VI) stress is shown in Figs 1 and 2. The results obtained show that the growth characters of sorghum plants significantly decreased under chromium stress in comparison with control plants.
Chlorophyll content. There was a decrease of 0.77% and 6.71% at 35 DAS (Fig. 1) (Figs 1 and 2). The increase in root length was 22.86% and 47.50% in HJ 541 and 29.94% and 45.07% in SSG 59-3  Grain yield. It was observed that there was 24.95% and 31.64% decrease in grain yield of HJ 541 under 2 and 4 ppm Cr, respectively. In SSG 59-3, the decrease of grain yield was 15.51% and 29.76% under 2 and 4 ppm Cr, respectively; (Fig. 2). The exogenous application of GB (50 and 100 mM) significantly increased grain yield in both the varieties (Fig. 2). The increase in grain yield was 17.23% and 36.99% in HJ 541 and 10.81% and 15.15% in SSG 59-3 plants grown under 2 and 4 ppm Cr, respectively; by 50 mM GB treatment. The yield was increased further by 15.49% and 31.13% in HJ 541 and 2.04% and 4.42% in SSG 59-3 plants grown under 2 and 4 ppm Cr, respectively; on 100 mM GB application.
It owes, therefore, be concluded that both 50 and 100 mM concentrations of GB significantly improved the growth characters against chromium toxicity in sorghum. But, the values of all the remaining enzyme activities were more in SSG 59-3 variety compared to HJ 541, which indicated that the former can tolerate the toxic stress, especially chromium toxicity, more strongly.
Effect on antioxidative metabolites level. The results (Figs 3 and 4) indicated the same pattern for antioxidative metabolites as for antioxidative enzymes during Cr (VI) stress at both the stages in both varieties. The content of all metabolites viz. ascorbate, glutathione, and proline increased with increasing concentrations of Cr VI and was highest at 4 ppm. However, the treatment of GB at 50 mM further increased the content of by 12.38% for ascorbate, 9.41% for glutathione and 4.36% for proline, significantly at 35 DAS in HJ 541 plants grown under 2 ppm Cr (Fig. 3). At 4 ppm Cr, the increase was 12.46% for ascorbate, 6.48% for glutathione and 3.63% for proline at 35 DAS in HJ 541 plants. The increase in antioxidative metabolites was highest 15.25% for ascorbate, 7.21%   www.nature.com/scientificreports www.nature.com/scientificreports/ observed that the content of all these parameters increased (50-60%) with increasing concentration of Cr (VI) as compared to control, at both stages in both varieties (Tables 1 and 2), respectively. But the treatment of GB at 50 and 100 mM concentrations decreased the content (23-33%) of all these parameters at both Cr, levels (2 & 4 ppm) in both varieties at both stages. The decrease was more at 100 mM treatment of GB under 2 ppm Cr stress as compared to 4 ppm of Cr toxic stress and 50 mM GB treatment in both varieties at both stages. It was observed that GB 50 and 100 mM caused a significant reduction in ADF, NDF, cellulose, hemicellulose, lignin, pectin, and silica content at both toxic levels of Cr in both the varieties at both the stages.
The content of ADF, NDF, cellulose, hemicellulose, lignin, pectin, and silica also increased (40-45%) significantly along with growth stages of the plant in both varieties. But, the increase in quality parameters was more in HJ 541 compared to SSG 59-3. Moreover, the later was found to be more tolerant towards Cr toxicity in comparison to the former at both stages. The high rate of decrease in the content of these parameters was observed at 100 mM concentration of GB at both stages in both varieties.

Discussion
Chromium toxicity has become a serious problem in agricultural soil all over the world and requires an immediate solution 21,22 . Chromium (VI) pollution has produced many negative effects on plant's and animal's health 23 . High concentrations of Cr (VI) inhibit seed germination and plant growth by affecting many biochemical and physiological processes such as protein synthesis, photosynthesis, enzymatic and non-enzymatic antioxidative defense system (viz. catalase, peroxidase, superoxide dismutase, ascorbate peroxidase, glutathione reductase, polyphenol oxidase, and metabolites glutathione, proline, and ascorbate) 24,25 . Chromium (VI) toxicity also affects the quality and resistance capacity of plants 26,27 . In the present research, efforts have been made to study the  [30][31][32] . It might be due to the change in EC, pH and OC properties (Table 3) of the soil on Cr (VI) application. It is well reported by Gomes et al 33 . during his study on the absorption of Cr, Cd, Cu, Ni, Zn and Pb by the plants. Soil properties (pH, EC, OC) has a significant effect on the sorption of HM in soils 34 . A low pH value leads to a reduction in sorption which consequently enhances the bioavailability or mobility of HM 35 . The presence of organic matter in the soil has a major influence on the nature of trace metals like Cr. Organic matter possess negatively charged surfaces which play a significant role in cation exchange capacity in the soil 36 . It causes more availability of positively charged metals like Cr to plant roots and results in increased Cr level in Cr (VI) treated plants. GB application in soil decreased the Cr accumulation and total Cr uptake by sorghum plants compared to respective Cr (VI) treatment alone. The reduction in uptake of heavy metal like Cd and Pb by plant roots because of GB application was also reported earlier in mung bean, rice, and cotton crops [37][38][39] . It might be due to the shielding nature of GB that inhibits the entry of Cr (VI) in the cytoplasm via cell membrane or the other way of competition between Cr (VI) with other nutrients' uptake by the plant 40 .

Exogenous GB offsets Cr (VI) induced inhibition in Morphophysiological Parameters.
The results of the present study have shown (Figs 1 and 2) that 4 ppm chromium greatly reduced the chlorophyll content in sorghum plants. But, the application of GB (50 and 100 mM) significantly increased (25-27%) chlorophyll content. The maximum increase was observed in 100 mM treatment of GB in sorghum plants. GB application clearly affected the photosynthetic pigments and improved it, by increasing the plant performance like nutrient uptake and antioxidative defense system. Similar results were observed by Bharwana et al. 39 (Figs 1 and 2). Similar observations have been made by Ali et al. 41 . The reason might be chelating nature of GB for Cr which blocks the movement of Cr from soil to plant and in plant parts. It reduces the Cr stress level in plants which in turn increased plant growth. The increased plant growth by GB, under Cr (VI) stress, might be due to the better development in nutrient uptake and gas exchange attributes of plants on GB application, as reported by Iqbal et al. 42 and Shahbaz et al. 43 in case of wheat under abiotic drought stress conditions. Moreover, GB may protect CO 2 fixing enzymes like RuBisCo and RuBisCo activase under abiotic stress, and thus, leading to an improvement in plant growth 44 .

Exogenous GB counteracts Cr-VI induced alterations in the Antioxidative defense system.
Plants are able to protect themselves from the harmful effects of heavy metal stress by reducing reactive oxygen species (ROS) accumulation using enzymes, such as ascorbate peroxidase, catalase, superoxide dismutase, polyphenol oxidase, peroxidase, glutathione reductase and metabolites like glutathione, proline, and ascorbate 45,46 . The results of the present investigation (Figs 2-6) showed that GB application (50 & 100 mM) increased the activities of antioxidant enzymes and metabolite in Sorghum plants grown under chromium stress. Glycine betaine treatment significantly increased (25-28%) peroxidase and catalase enzymes activities compared to control as well as Cr (VI) treated plants. Reports suggested that ascorbate and proline may consume the ROS generated in plants due to stress conditions 47,48 . Proline, a basic amino acid, is found in high percentage in protein. Free proline plays a crucial role in plants during stress. Though the molecular mechanism has not yet been recognized regarding the increased level of proline, one of the hypotheses refers to the breakdown of protein into amino acids followed by conversion to proline for storage. Many researchers have reported a several-fold increase in the proline content under physiological and pathological stress conditions. Increased levels of glutathione, proline, and ascorbate with increasing concentration of GB under different treatments of Cr (VI) have also been observed in the present study (Figs 5 and 6) suggesting the protective role of GB against HM stress. Similar observations were also reported by Arafa et al. 49 in sorghum plants under saline stress and Ali et al. 23 in wheat under Cr stress. The GB treatments were found to be effective in the amelioration of Cr (VI) toxicity as evident from the better growth of sorghum plants (Figs 1 and 2) and reduction of Cr (VI) level in roots (Figs 5 and 6). Similar results were also obtained in case of rice and mung bean plants under Cadmium (Cd) stress 37,38 and cotton under lead (Pb) stress with the exogenous application of GB 39 . Cha-um et al. 50 reported the similar results in the activities of antioxidative enzymes by GB under drought stress. Park et al. 51 reported that in tomato under chilling stress, the expression of catalase synthesis initiating genes was enhanced by GB application. The reason behind, the increase in the enzymatic activities after GB treatment might be due to the decrease in Cr uptake or reduction in electrolyte leakage 52 . The action of the antioxidative defense system (enzymatic and non-enzymatic) may protect the plant cells from oxidative damage by quenching or converting the ROS into harmless forms 53 .
In the present study, the activity of all antioxidative enzymes increased by the application of GB during the Cr stress. Similar observations were also reported by Gill et al. 53 in their study on Brassica napus under Cr stress. The results of the present study also revealed an increase in antioxidative enzymes and metabolites activity in plants under Cr VI treatment alone (Figs 3 and 4). But, the plant growth was less in these plants (under 2 & 4 ppm of Cr treatment alone) as compared to control and plants provided with GB (Figs 1 and 2), which indicates that the increment in antioxidative enzyme activities under Cr treatment alone was not enough to support the plant growth and development compared to both control as well as GB treated plants. Moreover, continuous Cr stress leads to a reduction in the capacity of the antioxidative defense mechanism of sorghum plants against Cr stress. The decrease in activities of the antioxidative  www.nature.com/scientificreports www.nature.com/scientificreports/ defense system in Cr treated plants causes' reduction in the efficiency of antioxidants to consume ROS that increased the chances of ROS accumulation in the plant cell, which ultimately causes plant death. But the soil application of GB further increased the activity and efficiency of the antioxidative defense system in those plants (under 2 & 4 ppm of Cr treatment alone), which leads to the decreased stressed condition due to excess ROS, and increased growth and quality parameters by the reduction in Cr accumulation. It suggested the amelioration property of GB relating to Cr toxicity. These results were in accordance with Raza et al. 54 and Molla et al. 55 who studied the physiology of wheat and lentil under drought stress. They reported that the GB application mitigated the adverse effects of drought and improved the plant's tolerance capacity to stress. Likewise, Einset et al. 56 reported that GB might activate the expression of genes responsible for ROS scavenging enzyme synthesis, which may protect the photosynthetic apparatus of plants under stressful conditions. In the present study, increase in plant height, root length, chlorophyll content, and quality parameters might be due to the GB induced decrease in Cr uptake in plants and increase in activity of antioxidative enzymes as well as metabolites. GB increases or favors the growth of mycorrhizal fungi around plant roots. Mycorrhizal fungi reduce the HMs uptake by plant roots either by chelating HMs or storing more HMs in their vacuoles. exogenous GB mitigates cr (Vi) induced damage to forage Quality of sorghum. Forage quality parameters like ADF, NDF, cellulose, hemicellulose, lignin, pectin, and silica are the measures, which reveal the digestibility, i.e. how easily or in how much amount will an animal digest the feed. In other words, these parameters are used to determine the nutrition value of a particular crop for animal feed. The results of the present study (Tables 1 and 2) showed that chromium toxicity reduced the quality of sorghum by increasing the amount of ADF, NDF, cellulose, hemicellulose, lignin, pectin, and silica, but GB application increased the quality of sorghum by reducing the Cr absorption in sorghum roots (Figs 5 and 6) along with other processes like increased activity of antioxidative enzymes (Figs 3 and 4) and metabolites (Figs 5 and 6). Daud et al. 57 observed that the plant cells under stressful conditions induce more lignification, silicification, ADF, NDF synthesis, to make the cell-wall stronger and thicker against osmotic burst. This favors the survival of plants by protecting the cells from osmotic stress caused by heavy metal. These results (Tables 1 and 2) were also in accordance with results obtained by Daud et al. 57 in cluster beans. Thus, due to the decrease in Cr (VI) absorption by sorghum plants, on GB application the toxic stress was reduced in sorghum plants, which in turn induces plant cell to bring normal synthesis of lignification, silicification, and structural carbohydrates in the cell-wall that leads to enhanced quality of sorghum digestibility by the animals. This might be the reason for increased forage quality of sorghum on the GB application. Available reports in literature on Cr toxicity and tolerance reported that GB helps in chelation of heavy metals in the cellular vacuoles and causes the blockage of heavy metal movement or transportation 44 . This might be the reason behind the Cr VI toxicity tolerance and amelioration of toxic effects caused in sorghum by GB application which were recorded during this experimental study.

conclusion
From the results of the present investigation, it may be concluded that Cr (VI) is a non-essential element for plants and toxic heavy metal for sorghum that affects the plant morpho-physiological, biochemical quality at the molecular level. Application of exogenous GB has been found to inhibit Cr (VI) uptake by sorghum plants which might be due to GB induced chelation of heavy metal in cellular vacuoles. Thus, GB causes blockage of heavy metal Cr (VI) movement. This might be the reason behind the ameliorative effect of GB in sorghum also. Hence, application of exogenous GB may be used in the improvement of quality and yield of sorghum in Cr affected areas.
Antioxidative system. The complete extraction procedure for both, the enzymes as well as metabolites was carried out, at 0-4 °C. Two gm of fresh and cleaned leaf tissue were homogenized in 10 ml of 0.1 M potassium phosphate buffer (pH 7.0) by using a previously chilled mortar and pestle. The homogenate was centrifuged at 10,000 rpm for 15 minutes. The supernatant labeled as crude extract was collected and used at the same time for measurement activity of all the enzymes as well as estimation of metabolites. The same crude extract was used for total soluble protein estimation.
Superoxide dismutase (EC 1.15.1.1). Superoxide dismutase was assayed by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium by adopting the method of Giannopolities and Ries 63 . One enzyme unit is defined as the amount of enzyme, which could cause 50% inhibition of the photochemical reaction.
Catalase. Catalase activity was determined by the procedure of Sinha 64 . One enzyme unit is defined as the amount of enzyme, which catalyzed the oxidation reaction of 1 µmole H 2 O 2 minute −1 under assay conditions. Peroxidase (EC 1.11.1.7). The enzyme activity was estimated by the method of Shannon et al. 65 . One unit of peroxidase is defined as the amount of enzyme required to cause a change in 0.1 O.D. minute −1 under assay conditions.
Ascorbate peroxidase (EC 1.11.1.11). The enzyme activity was determined by following the oxidation of ascorbic acid 66 . One enzyme unit is defined as the amount of enzyme required to oxidize 1 nmol of ascorbic acid minute −1 at 290 nm.
Glutathione reductase (EC 1. 6.4.2). Method of Halliwell and Foyer 67 was followed to measure the enzyme activity. One enzyme unit is defined as the amount of enzyme required to oxidize 1.0 nmol of NADPH oxidized minute −1 .
Polyphenol oxidase (E.C. 1.10.3.1). Polyphenol oxidase activity was assayed by using the modified method of Taneja and Sachar 68 . One unit of enzyme activity is expressed as a change in 0.01 absorbance minute −1 mg −1 protein.
Ascorbate content. 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 69 , which was originally developed by Roe 70 . The amount of ascorbate was determined by using a reference curve of ascorbate and expressed as µmoles gm −1 fresh weight.
Proline content. The estimation of the proline content in plants was examined by implementing the method of Bates et al. 61 . The amount of proline content present in the samples was determined from the standard curve of proline and has been expressed as µmoles gm −1 fresh weight.
Glutathione content. It is a major water-soluble antioxidant involved in maintaining the low redox potential and a highly reduced intracellular environment. It is also involved in scavenging of reactive oxygen species. Level of glutathione was estimated by using the method of Smith 71 . Glutathione content was calculated from a standard curve of GSH and is expressed as µmoles gm −1 fresh weight. Statistical analysis. All the results were analyzed by following a three-factorial (the First factor included varieties "2 varieties as HJ 541 and SSG 59-3", second included chromium levels as control, 2 and 4 ppm, and the third factor included glycine betaine administration at control, 50 & 100 mM) analysis of variance (ANOVA) by using IBM SPSS Statistics 23 software along with post hoc Tukey test. On the basis of CD values obtained after this analysis for each parameter at both 35 and 95 DAS, differences between the treatment doses were evaluated 72 . Based on the ANOVA test, the interactions were found to be significant.

Data availability
All data generated or analyzed during this study are included in this article file.