Introduction

The persistent nature of xenobiotic compounds, particularly explosives, pyrotechnic compositions, and propellants, in the environment has become an international alarm, explicitly concerning military base closures and their associated policies (Rocheleau et al. 2010). The major sources of explosive compounds in areas of military activities are partially detonated or damaged munitions. These munitions are highly mobile in nature and interfere with the surrounding flora and fauna (Pichtel 2012; Shukla et al. 2023). The nitroaromatic recalcitrant explosive compound 2,4-dinitrotoluene (DNT) is a common soil contaminant at munitions manufacturing sites and army installations and is enumerated among one of the priority pollutants by the US Environmental Protection Agency (Lent et al. 2012; Chatterjee et al. 2017; Heisnam et al. 2023). The DNT is also used in dye processes, smokeless gun powders, and the manufacturing of polyurethanes (Rocheleau et al. 2010; Podlipna et al. 2015; Okozide et al. 2020). Moreover, the most commonly used explosive material 2,4,6-trinitotoulene (TNT), is also known to exhibit the potential synthesis of other nitroaromatic explosive compounds including DNT (Su et al. 2021). Structurally, DNT possesses an aromatic ring with two nitro groups, making it relatively resistant towards environmental degradation. Thus, the fate of these explosives and their by-products is an issue of great concern due to their low sensitivity towards natural degradation processes like biodegradation, hydrolysis, and volatilization (Chandra et al. 2021; Anjum and Quraishi 2022).

The primary route of the interaction of these compounds with the plants is by the bulk flow of water in the xylem due to evapotranspiration (Mahish et al. 2019). Su and Liang (2011) revealed in their experiment that DNT transportation occurs mainly via the symplastic pathway. In a study, the concentration of DNT in seedlings of Solanum lycopersicum and Triticum aestivum were positively correlated with its concentration in the external solution (Su and Liang 2011). Plant uptake of DNT and its isomers has been revealed to have inhibitory effects on plant development (Kiiskila et al. 2015). Seeds act as the primary dissemination unit and comprise the complete genetic makeup of the species. However, under stressful environmental conditions, the performance is likely to be affected by various physiological changes such as increased mean time of germination, decreased germinability, in conjunction with abnormal seedlings (Yadu et al. 2017; Kaur and Keshavkant 2021). Contaminants are mechanically absorbed by seeds as they imbibe water to initiate germination (Rajjou et al. 2012), while both juvenile and adult life stages pull contaminants from the soil matrix along with water moving in the soil-plant-atmosphere continuum. Krishnan et al. (2000) and Best et al. (2007) reported successful germination in Bromus inermis, Andropogon geraldii, Bouteloua gracilis, and Panicum virgatum in the presence of TNT but showed a significant reduction in plant health at similar concentrations with an increase in age.

These compounds can root damage to the plants at a molecular level (gene expression, regulation, and signal transduction) (Adomako-Bonsu et al. 2024). Certain actions like disruption of cellular metabolic balance and numerous secondary signaling molecules inducing overproduction of reactive oxygen species (ROS) [superoxide radical (O₂˙ˉ), hydrogen peroxide (H₂O₂), and hydroxyl radical (˙OH)], are leading to physiological changes in plants to a great extent (Choudhary et al. 2014; Adomako-Bonsu et al. 2024). Plants possess natural/ inherent mechanisms to scavenge the ROS, which is a complex antioxidative defence system comprising both enzymatic [superoxide dismutase (SOD), catalase (CAT), guaicol peroxidase (POX), ascorbate peroxidase (APX) and glutathione reductase] and non-enzymatic (ascorbic acid, α-tocopherol, flavonoids, glutathione, and proline) components (Sharma et al. 2012; Mansoor et al. 2023). These are accountable for stabilization of structures of proteins, protection of membranes, and activities of essential metabolic enzymes (Gadi et al. 2012).

Vigna radiata L. (V. radiata) is one of the most prominent pulse crops with highly nutritious edible seeds, beneficial for healing numerous ailments. It is an excellent source of vitamins, proteins, dietary fibers, and notable amounts of bioactive compounds, including peptides, polysaccharides, and poly-phenols, hence an excellent food crop for fostering wellness (Hou et al. 2019). Chhattisgarh is an important contributor of V. radiata in India with an average productivity of 456 kg ha^-1 (Parihar et al. 2018). Vigna radiata is rich in various essential amino acids like leucine, isoleucine, valine, etc. (Mubarak 2005). Seeds of it comprise various antioxidants which neutralize harmful molecules and reduce the risk of chronic inflammation, heart diseases and certain cancers too (Khansari et al. 2009). These are very good source of potassium, magnesium, and fiber, thus reducing the risk of high blood pressure (Ganeshan and Xu 2017). Moreover, due to dense forest, many areas of Chhattisgarh fell prey to Naxalism and were affected by landmines. Explosive materials used in landmines are hazardous, non-biodegradable, toxic chemicals that can severely affect the health of plants.

The present study aimed to monitor the amendments in physiological, biochemical, and molecular parameters under DNT-induced toxicities on V.  radiata seeds. No studies have been performed to unravel the impact of DNT and its metabolites on V. radiata. The proposed study will help to reveal the critical effect of DNT on seed germination and growth, alongside an assessment of alterations in levels of ROS, metabolism of lipids and antioxidants, and also to observe variations in RNA content and RNA profile during stress conditions.

Materials and Methods

Seed Collection and Preparation of Treatment Solutions

Vigna radiata L. seeds were acquired from the local market of Raipur, India. The chemical used in this study i.e. 2,4-dinitrotoluene (DNT), was purchased from M/s HiMedia Lab Pvt. Ltd., Mumbai. Due to the less solubility of DNT in the aqueous environment, a small amount of acetone was used for initial solubilization, which was further used as a stock solution for the preparation of various concentrations of DNT (0, 0.05, 0.15, 0.3%) solution. Petri plates were then covered with the appropriate amount of DNT and left overnight to evaporate the acetone at room temperature.

Growth Conditions

Seeds of V. radiata were surface sterilized with 0.1% (v/v) sodium hypochlorite for 5 min followed by thorough washing (3-4 times) with MilliQ water (MW, Millipore, Gradient A-10, USA). Afterwards, seeds were allowed to imbibe for 2h in MW (Yadu et al. 2017). The imbibed seeds (10 each) were then allowed to germinate over the two layers of filter paper towels kept on pre-coated (MW, acetone, 0.05, 0.15, and 0.3% DNT) Petri plates. All the plates were incubated in dark at 25±2ºC for 6 days, and seeds were irrigated with a fixed volume of MW after every 24 hrs. Percentage germination (%) was recorded daily for each of the treatments.

Radicle Length and Biomass

The lengths of radicles (n=10) were carefully calculated in five replicates with the help of a scale (cm), to record differences in their lengths (Yadu et al. 2018). The biomass [fresh mass (FM) and dry mass (DM)] of radicles from each treatment was measured by weighing freshly harvested and after 48 hrs of oven drying at 60 °C (Sartorius, Sweden) and expressed in milligrams (mg) (Yadu et al. 2017).

Electrolyte Leakage

Electrolyte leakage (EL) of the radicles was assessed by following the method of Blum and Ebercon (1981). Briefly, 0.2g of excised radicles was plunged in 20ml of deionized water and incubated for 24 h in a shaker (50rpm) at ambient conditions. Then, their electrical conductance was calculated using conductivity meter (Eutech™ Expert CTS, Thermo Scientific, USA) and expressed as mS g^-1 FM.

Estimation of Reactive Oxygen Species

Histochemical Detection

Histochemical detection of O₂^·− and H₂O₂ was done by the method of Georgiou et al. (2005) and Hempel et al. (1999), respectively. Radicles from each treatment were dipped in 50 mM potassium phosphate buffer (pH 6.8), abiding dihydro ethidium (10μM) and 2′,7′-dichlorofluorescin diacetate (50μM), individually, for detection of precise location of O₂^·− and H₂O₂, respectively, and kept for 30 min in the dark. After that, cross-sections of radicles were mounted over a glass slide and imaged under fluorescence microscope [Confocal microscope DM 2000 LED (Leica, Germany)].

Quantitative Estimation

The biochemical estimation of O₂^·− was done following the method of Sangeetha et al. (1990). Five radicles (0.2 g) were homogenized with 2 ml of 0.2M sodium phosphate buffer (pH 7.2) having 0.001M diethyl dithiocarbamate (DDC) and centrifuged (14,000 rpm) for 15 min at 4ᵒC. Afterwards, 200μL of supernatant was added to 1.8 ml of sodium phosphate buffer (0.2 M, pH 7.2). The absorbance of the medium was noted immediately after addition of nitroblue tetrazolium (NBT, 100 μL, 2.5×10-4 M), and after 6 min, respectively, at 540 nm using UV-spectrophotometer (Lambda-25, Perkin Elmer USA) and expressed as μmol gˉˡ FM.

The method of Velikova et al. (2000) has been followed to assay H2O2 content. Briefly, 0.2 g of radicles was weighed and homogenized with trichloroacetic acid [2 ml, 0.1% (w/v)] followed by centrifuged at 12,000 rpm for 15 min. Then, an equal volume of sodium phosphate buffer (10 mM, pH 7.0) and 1 M potassium iodide was added with 200 μL of supernatant, and the absorbance of the sample was recorded at 390 nm. Concentration of H₂O₂ was calculated using the extinction coefficient of 0.28 μM-ˡ cm-ˡ and expressed as μmol g-ˡ FM.

Determination of malondialdehyde

To monitor malondialdehyde (MDA), a lipid peroxidized product, weighed amounts (0.1 g) of liquid nitrogen crushed fine tissue powders were extracted with 20% (w/v) TCA consisting 0.5% (w/v) 2-thiobarbituric acid (TBA). The homogenates were boiled for 30 min at 100ᵒC, cooled and centrifuged (11,000 rpm, 10 min). Absorbance (at 532 nm) of the supernatant was recorded, and the level of MDA was derived following the extinction coefficient 157 mmol^-1 cm^-1 (Hodges et al. 1999) and data expressed as nmol g^-1 FM.

Extraction of protein and enzymes, and their estimations

0.2 g of radicles were homogenized with 2ml of sodium phosphate buffer (10mM, pH 7.2), containing 1 M ethylenediaminetetraacetic acid, 2 mM dithiothreitol, 0.2% (w/v) triton X-100 and 1 mM phenylmethylsulfonyl fluoride (Zivy et al. 1983), followed by centrifugation at 14,000 rpm for 20 min at 4ᵒC. The obtained supernatant was collected and stored for the determination of total protein and enzymes. Total protein content was assayed following the protocol of Bradford (1976) and expressed as mg g^-1 FM.

The SOD (EC 1.15.1.1) activity was estimated by following the protocol of Marklund and Marklund (1974). The inhibition % of pyrogallol auto-oxidation, source of O₂˙ˉ by enzyme, was spectrophotometrically measured at 420 nm and expressed as Units of SOD min^-1 g^-1 FM.

Analysis of CAT (EC 1.11.1.6) activity was carried out by method of Chance and Maehly (1955). The activity was determined by decomposition of H₂O₂ at an absorbance of 240 nm. The enzyme activity was computed using the extinction coefficient of 39.4 mM^-1 cm^-1 and expressed as μmol min^-1 g^-1 FM. The method of Nakano and Asada (1981) has been followed for the estimation of APX (EC 1.11.1.11) activity. The ascorbate oxidation rate of extracted enzyme was spectrophotometrically analyzed at 290 nm and enzymatic activity was estimated by extinction coefficient of 2.8 mol^-1 cm^-1 and expressed as μmol min^-1 g^-1 FM.

Gene Expression Analysis

Total RNA Extraction

Total RNA was extracted following the hot-phenol method of Verwoerd et al. (1989). Both concentration and quality of extracted RNA was estimated by Nanodrop spectrophotometer (ND1000, Thermo Scientific, USA). Further, RNA integrity was confirmed by counting two intact bands (28s and 18s) of rRNA in the intensity ratio of 2:1 in agarose gel (1.5%, w/v) electrophoresis. After that, this RNA was employed as template to synthesize cDNA following the Superscript VILO kit (Invitrogen, USA).

Reverse Transcriptase (RT)‑PCR

The RT-PCR was executed in a reaction volume of 10 μL with the aid of precise gene-specific primers (Table 1). The β-actin, a housekeeping gene, was employed for the template normalization. Both forward and reverse primers were designed following a web-based primer designing tool, Primer-3 (https://frodo.wi.mit.edu) and synthesized from M/s Eurofins Genomics India Pvt. Ltd., Bengaluru, India (Nair and Chung, 2015). The 10 μL of reaction volume comprising 1 μL 10X buffer, 0.5 μL (25 mM) magnesium chloride, 1 μL (2.5 mM) dNTP mix, 1 μL (10 nM) forward primer, 1 μL (10 nM) reverse primer, 0.2 μL Taq DNA polymerase, 1 μL cDNA and 4.3 μL nuclease-free water was used in the process of PCR reaction. The PCR reaction was carried out following cycle conditions: an initial denaturation (96ᵒC) for 60 s, thereafter 35 cycles (at 95ᵒC) for 15 s, 52-60ᵒC for 45 s, 72ᵒC for 45 s and final extension for 5 min at 72ᵒC. The PCR products were separated on 1.5% (w/v) agarose gel by electrophoresis with constant voltage of 50 V for 40 min and gel was analyzed under a Gel-Doc (BioRad, USA).

Statistical Analysis

All the data were analyzed using one-way ANOVA, followed by Least Significance Difference (LSD) for mean separation, analyzed by Duncan’s multiple range tests (DMRT), at P < 0.05 using SPSS software (version 16.0), to observe the detrimental effect of DNT on seeds of V. radiata. Data depicted are mean ± standard error (SE) of five sampling replicates. Stained radicles were assessed by fluorescence microscopy for the presence and intensity of dye. Gene expressions were analyzed and inferred by comparing the fluorescence intensities of the bands under Gel-Doc (BioRad, USA) system.

Table 1: List of RT-PCR primers

APX: Ascorbate peroxidase; CAT: Catalase; SOD: Superoxide dismutase

Result and Discussion

Physiological impacts

Growth Assessments

Growth and development are necessary for the continuing survival and multiplication of any plant species. Physiological factors such as %G, radicle length, FM, DM and EL of seeds/seedlings reflect the actual growth and developmental status of plants. Germination studies on V. radiata seeds after DNT treatment (0, 0.05, 0.15, and 0.3%) for 6 days outlined no effect on %G. Rather, 100% germination was attained by all the treated samples, which might be owing to the seed coat of V. radiata being impregnable, thus restraining the absorption/ perforation of DNT. However, a significant decline was detected in radicle length i.e. 2.6-10.2 folds with an increase in the concentration of DNT (0.05, 0.15, and 0.3%) (Fig. 1). Additionally, many morpho-physiological alterations were noticed in the radicles such as abnormal growth and browning of root tips, which were more prominent with inclining concentrations of DNT. Further decline in the length of radicles was observed with an increase in concentrations of DNT. Acetone was used for dissolution of DNT, due to its low solubility in aqueous medium. Thus, the impact of acetone alone was also observed, but a non-significant alteration in radicle length was recorded on the acetone-exposed seeds.

Fig.1: Alteration in the length of the radicles of Vigna radiata treated with different concentrations of 2,4-dinitrotoluene (DNT). Each bar represents mean±SE of three replicates. Data points showing the same letter are non-significant at p < 0.05 level.

Earlier studies have demonstrated that nitroaromatics have an inhibitory effect on plant growth and development. Nitroaromatics like TNT and its by-products are largely bound in below-ground tissues, resulting in altered morpho-physiological responses (Via et al. 2016). In the year 1996, Peterson et al. reported that along with germination, the emergence of radicle from the seed might cause exposure to DNT, thereby absorbing it into the cells. These reductions are possibly due to the augmented permeability of cell membranes; thus, loss of constituents and improper nutrient uptake, which are the basic requirements of any tissue or organ to grow and develop optimally (Yadu et al. 2018). In addition, arsenic (As) exposure leads to reduced rate of cell elongation that can also contribute to the inhibition of RL. Diminution in biomass accumulation was the result of water loss and also a decrease in water uptake by the root cells during As-stress (Chandrakar et al. 2016). Additionally, Rocheleau et al. (2010) further studied the toxicity effect of 2,4-DNT on Echinochloa crus-galli, Lolium perenne, and Medicago sativa. The outcome of this study was reflected in the alteration of shoot mass with varying soil-organic matter. A comparison in the growth patterns of crop plants (Sinapis alba, Helianthus annus, Linum usitatissimum, and Cannabis sativa) grown on soils contaminated with DNT has been performed. The growth stimulations of plants were noted at 0.252 mg g^-1 DNT, whereas the lethal concentration (LC50) of 1 mg g^-1 inhibited plant growth. Consequently, this study reported that the toxicity of DNT was comparatively higher than the parental compound (Podlipna et al. 2015). Furthermore, Arabidopsis thaliana seedlings were tested with different isomers of DNT viz., 2,3-DNT, 2,4-DNT, 2,6-DNT, 3,4-DNT, and aminonitrotoluene. The isomers of DNT showed varying toxicity levels, while 2,6-DNT was the most toxic among all tested DNT isomers. At higher concentrations, incomplete degradation of the reduced products was observed. Reduced metabolites of DNT were retrieved from the body of aphids feeding on Arabidopsis thaliana, which points towards the potential distribution in the environment via the food chain (Nisar et al. 2018). The phytotoxicity of toluene nitroderivative- 2,4-DNT was analyzed, where the LC50 of 2,4-DNT in Triticum aestivum, Sinapis alba, Lactuca saliva, and Lens culinaris were evidenced at 0.049 mg g^-1, 0.12 mg g^-1, 0.08 mg g^-1, and 0.17 mg g^-1, respectively, therefore, reported that 2,4-DNT is phytotoxic to these four plants (Picka and Friedl 2004).

Fig. 2:     Biomass (Fresh mass and Dry mass) of Vigna radiata radicles in response to increasing concentrations of 2,4-dinitrotoluene (DNT). Each bar represents mean±SE of three replicates. Data points showing the same letter are non-significant at p < 0.05 level.

Furthermore, treatments of DNT significantly decline the FM and DM of radicles. Different concentrations (0.05, 0.15, and 0.3%) of DNT significantly affected the biomass [FM and DM (Fig. 2)] of V. radiata radicles. A prominent (3.6 fold) reduction in the FM of tissues was observed after treatment with 0.05% DNT, which further increased to 4.8 and 10.5 folds with 0.15 and 0.3% DNT, respectively, compared to control (Fig. 2). Likewise, 0.05% DNT treated radicles showed a 2.9 fold decrease in the DM, whereas 0.15% and 0.3% DNT depicted 12.4 and 12.9 fold reductions respectively, when compared to control (Fig. 2). However, a non-significant (p < 0.05) alteration in biomass was recorded with acetone-treated radicles compared to the control. Doherty et al. (2019) reported the inhibitory effects of DNT on different physiological parameters including radical emergence, FM and DM of Calamagrostis Canadensis, Oxytropis campestris, Polemonium pulcherrium, and Picea glauca. 2,4-DNT stress tolerance capacity of plants is strongly correlated to temperature. The plants adapted to cold conditions were seen to be more tolerant of DNT under low temperature conditions. Yoon et al. (2006) researched the toxic effects of 2,6-DNT on Arabidopsis thaliana and reported its inhibitory effects on DM and root elongation of the plant. At higher concentrations of 2,6-DNT, biomass and root growth were severely inhibited.

Electrolyte leakage

The leakage of electrolyte is a key marker of membrane permeability (Yadu et al. 2017). Lowest and non-significant difference in EL was observed in the radicles of MW and acetone-treated ones (Fig. 3). On the other hand, treatment of radicles with different concentrations of DNT depicted an inclined level of EL. The leakage of electrolytes increased by 2, 2.5, and 4.3 folds from the radicles treated with 0.05%, 0.15%, and 0.3% DNT, respectively, when compared with the control (Fig. 3). Similarly, an augmented level of EL has been reported by Hniličková et al. (2019) in Lactuca sativa, Tetragonia tetragonoide, and Portulaca oleraceaunder under salt stress. Likewise, in the tissues of Phaseolus aureus, enhanced releases of electrolytes mediated cellular damage were observed under arsenic stress (Singh et al. 2007). Change in membrane integrity under DNT toxicity assayed in terms of EL, was significantly increased in V. radiata radicles. This increment might be correlated with several factors such as overproduction of ROS, peroxidation of membrane acyl lipids, water use efficiency, and leakage of cellular constituents (Chandrakar et al. 2016). Plant responses to stress (salinity, drought, heavy metals, pathogen attack, etc.) are accompanied by EL with increased membrane permeability. Accumulating evidence shows that ROS are ‘partners’ of potassium (K⁺) leakage in plant stress response, leading to loss of electrolytes associated with programmed cell death (Demidchik et al. 2014).

Fig. 3: Change in the rate of electrolyte leakage from Vigna radiata radicles treated with different concentrations of 2,4-dinitrotoluene (DNT). Each bar represents mean±SE of three replicates. Data points showing the same letter are non-significant at p<0.05 level.

Biochemical and molecular alterations

Reactive oxygen species

The present work revealed that production of O₂˙ˉ was visualized under a fluorescence microscope, which revealed that fluorescence intensity unambiguously increases upon DNT exposure (Fig. 4a). The radicles exposed to DNT showed extensive staining with higher intensity than those subjected to acetone and control. The highest production of O₂˙ˉ was observed with 0.3% DNT, while their levels were comparatively lower in the control and acetone-treated radicles, suggesting that the DNT provokes the O₂˙ˉ accumulation. Furthermore, the spectrophotometric estimation of O₂˙ˉ has been done in the radicles subjected to MW, acetone, and DNT (0.05%, 0.15%, and 0.3%). Around 1.57-fold rise in O₂˙ˉ content in 0.05% DNT treated radicles was observed, which further showed manifold increase in 0.15% (2.57 fold) and 0.3% DNT (3.42 fold) treated radicles, respectively (Fig. 4b) when compared with control.

Fig. 4a:   Histochemical localization of superoxide radicals in Vigna radiata radicles in response to different concentrations of 2,4-dinitrotoluene (DNT) (A;control, B;acetone, C;0.05% DNT, D;0.15% DNT, E;0.3% DNT).

Fig. 4b:   Amount of superoxide radicals in the radicles of Vigna radiata treated with different concentrations of 2,4-dinitrotoluene (DNT). Each bar represents mean ± SE of three replicates. Data points showing the same letter are non-significant at p < 0.05 level.

Likewise, fluorescence microscopy has been done to visualize the production of H₂O₂. An augmented level of intensity was observed with increasing concentration of DNT. Maximum production of H₂O₂ was recorded with 0.3% DNT, suggesting the inceptive role of DNT for H₂O₂ accumulation (Fig. 5a). Intensity of fluorescence emission explicitly increased upon treatment of DNT compared to control and acetone. In addition to that, a significant (1.47 fold) rise in H₂O₂ content was spectrometrically detected in 0.05% DNT applied tissues, which further rose to 1.93 and 2.39-fold in response to 0.15% and 0.3% DNT, respectively (Fig. 5b) compare to control.

Fig. 5a: Histochemical localization of hydrogen peroxide in Vigna radiata radicles in response to different concentrations of 2,4-dinitrotoluene (DNT) (A;control, B;acetone, C;0.05% DNT, D;0.15% DNT, E;0.3% DNT).

To validate the analytics, staining of DNT stressed radicles was visualized with NBT and DAB, which confirmed the upgradation in ROS level. With simultaneous rise in DNT level, the coloration propagated all through the radicle, implying the tip of the radicle being the primary place of ROS generation (Rughani et al. 2016). Similar findings under Cd and Ni stress, respectively, have been documented for Pisum sativum (Tomero-Puertas et al. 2004) and Pennisetum typhoideum radicles (Rughani et al. 2016). In the transgenic Nicotiana tabacum plants, Kayihan et al. (2021) investigated overexpression of a cold-adaptive nitroreductase gene, which exhibited an enhanced rate of DNT detoxification at low temperature. After an exposure to DNT, both transgenic and wild-type plants exhibited an increase in H₂O₂ level at 15ᵒC, while at 4ᵒC the wild-type plant did not demonstrate any significant change, but the transgenic plant had a decrease in H₂O₂ content.

Fig. 5b: Level of hydrogen peroxide in Vigna radiata radicles subjected to different concentrations of 2,4-dinitrotoluene (DNT). Each bar represents mean ± SE of three replicates. Data points showing the same letter are non-significant at p < 0.05 level.

Malondialdehyde

In stressed plants, subsequent deterioration of the membrane and cellular dysfunction leads to the production of MDA, and thereby its content reflects the degree of damage. Lipid catabolism is chiefly regulated by the lipoxygenase enzyme, thus activating a cascade of reactions. Membrane peroxidation reduces fluidity, causing substances to leak through the membrane. Pisum sativum treated with Cd and Ni had significantly higher MDA levels, indicating lipid peroxidation (El-Amier  et al. 2019). Similar observation was recorded in arsenic-subjected Cajanus cajan by Yadu et al. (2018). A considerably higher amount of MDA was determined in the radicles treated with different concentrations of DNT. The peroxidized products measured in the 0.05%, 0.15%, and 0.3% DNT treated radicles showed 2.28, 3.45, and 5.45 folds increments as compared to the control, respectively (Fig. 6). Increased vulnerability of ROS to attack membrane lipids with the release of cytotoxic product (MDA) may be closely linked with lipid peroxidation. A positive correlation was found between ROS (O₂˙ˉand H₂O₂) and MDA accumulation. Enhanced ROS accumulation along with the escalation in MDA and EL was observed in Oryza sativa under 2,4-dichlorophenoxyacetic acid stress (Islam et al. 2016).

Fig. 6: Effects of different concentrations of 2,4-dinitrotoluene (DNT) on malondialdehyde content of Vigna radiata. Each bar represents mean±SE of three replicates. Data points showing the same letter are non-significant at p < 0.05 level.

Till date, studies suggested that augmented ROS accumulation is one of the key factors for the induced cellular-mediated toxicity under herbicide or saline stress conditions (Parihar et al. 2018). These herbicides tend to bind with phospholipids and alter the physical interactions, thereby increasing the peroxidation of lipids (Bukowska et al. 2006). The present study revealed no significant change in ROS content of 0.5% DNT subjected radicles, revealing possible involvement of the exposed tissue in confronting DNT stress.

Protein content

Figure 7 represents the amount of protein in V. radiata after treated with DNT (0.05%, 0.15%, and 0.3%). A significant increase in the level of protein was recorded after treated with 0.05% DNT (1.14), while 1.35 (0.15%) and 1.81 (0.3%, p<0.05) fold reduction was noted with increasing concentrations of DNT when compared to control.

Fig. 7: Alteration in the content of protein in Vigna radiata radicles treated with various concentrations of 2,4-dinitrotoluene (DNT). Each bar represents mean±SE of three replicates. Data points showing the same letter are non-significant at p < 0.05 level.

Activities and gene expressions of antioxidants

Plants are equipped with an antioxidant defense system to counterbalance the effect of oxidants so as to maintain the redox homeostasis (Chandrakar et al. 2016). Researchers have reported that differential activities of antioxidant enzymes vary according to the type of stress, exposure time, and life stage of the plant (Hasanuzzaman et al. 2020). Antioxidants are constitutively present in plants, and their higher concentration plays a major role in deactivation and detoxification of long-lived oxy-products such as O₂˙ˉand H₂O₂ (Gechev et al. 2002).

Fig. 8a: Variation in the activity of superoxide dismutase, catalase and ascorbate peroxidase in Vigna radiata radicles treated with various concentrations of 2,4-dinitrotoluene (DNT). Each bar represents mean ± SE of three replicates. Data points showing the same letter are non-significant at p < 0.05 level.

The tolerance mechanisms induced by the spontaneous functioning of various antioxidant enzymes help in slowing down the oxidation of biomolecules and block the process of oxidative chain reactions. This role of antioxidant enzymes enables the plant to survive in stressful conditions, which is reached when plants accumulate excessive concentrated metals in their organs. It can be inferred from the reported studies that antioxidant enzymes protect the plants to some extent against oxidative damage (Jamal-Abad et al. 2007, Gechev et al. 2002).

Fig. 8b: Gene expression analysis of superoxide dismutase, catalase and ascorbate peroxidase in Vigna radiata radicles treated with different concentrations of 2,4-dinitrotoluene (DNT) (where, A;control, B;acetone, C;0.05% DNT, D;0.15% DNT, E;0.3% DNT).

Antioxidants like SOD, APX, and CAT work together to scavenge ROS and keep the body in a homeostasis state (Cho and Seo, 2005). Alterations in the activities of enzymatic antioxidants (SOD, CAT, and APX) were observed in response to 0.05%, 0.15%, and 0.3% DNT in the radicles of V. radiata. An upsurge in activities of SOD, CAT, and APX (Fig. 8a) were detected in tissues subjected to a mild concentration of DNT (0.05%). However, after the exposure of 0.15% and 0.3% DNT, the activity of SOD decreased by 1.21 and 1.55 folds in comparison with the control (Fig. 8a).

A non-significant change in the activity of CAT in MW and acetone-treated radicles, while a significant reduction in its activity was observed with higher concentrations of DNT in the radicles of V. radiata. Further, a 1.25-fold rise in the CAT activity was observed in 0.05% compared to the control. In contrast to that, a drop in CAT was measured in 0.15% (1.51 fold) and 0.3% (2.04 fold) DNT-treated radicles than control (Fig. 8a). Similarly, the inclined activity of APX was observed in the radicles treated with 0.05% (1.67 fold) DNT only, but declined after 0.15% (1.18 fold) and 0.3% (1.28 fold) DNT treated ones (Fig. 8a).

The RT-PCR analysis has been carried out for the gene expression studies, in which β-actin was used as a housekeeping gene. This study revealed that the genes of SOD, CAT, and APX (Fig. 8b) exhibited significant alterations or up-/down-regulation in agreement with their spectrophotometric data.

Treatment of DNT not only affected physiological and biochemical attributes but also shook the defence system. It significantly disturbed the antioxidants of V. radiata by altering the expressions of genes of SOD, CAT, and APX (Figs. 8a and 8b). The radicles treated with DNT showed lesser activities of antioxidants when compared to the tissues treated with DNT and proline (Figs. 8a and 8b). Many researchers reported that the differential activities of antioxidants vary with respect to exposure time, type of stress, and status of the plant life cycle (Hasanuzzaman et al., 2020). It is evident that plants can counterbalance the harmful effects of ROS by taking the help of antioxidants (Keshavkant and Naithani, 2001; Chandrakar et al., 2016). However external application of DNT to V. radiata damaged the radicles by significantly enhancing ROS production compared to that determined in the control tissues. Activities of antioxidants were observed to be higher in the radicles treated with DNT than in the MW-grown control. On the other hand, in response to the application of proline, activities of SOD, CAT, and APX (Fig. 8a) were significantly improved, hence neutralizing the effect of O₂˙ˉand H₂O₂, to some extent, thereby reduced oxidative stress and maintained cellular integrity. In a study by Hoque et al. (2007), it was observed that the activities of antioxidative enzymes (SOD, CAT, and APX) were significantly increased after exogenous supplementation of proline to Nicotiana tabacum suspension cultures subjected to salinity stress. Similarly, the application of proline to the roots of Arabidopsis thaliana considerably reduced the ROS, indicating free radical scavenging potential of proline (Cuin and Shabala, 2007).

In this study, activity of SOD in the radicles changed significantly with DNT concentration. This can be supported by the data of gel doc visualization. SOD is attributed as the first line of defense, directly establishing its action on O₂˙ˉ. The upsurge of SOD might be closely linked with increased ROS, thus up-regulating the expression of the gene through de novo synthesis of the enzyme or by stimulation of the existent enzyme pool assay (Jiang and Yang 2009). Increased SOD activity in Abelmoschus esculentus and Zea mays was investigated under mercury and zinc stress, respectively (Liu et al. 2025; Hameed et al. 2017). Likewise, increased activities of SOD in arsenic subjected V. radiata and V. mungo advocate their protective functions against toxicity (Singh et al. 2007; Srivastava and Sharma 2013). Song et al. (2007) and Yin et al. (2008) reported growth inhibition as the principal discernible symptom in response to herbicides. Chlorotoluron-treated seedlings of Triticum aestivum relatively showed an upsurge in ROS, SOD, and APX levels accompanied by a remarkable peroxidation of lipids, thus damaging the plant cell (Song et al. 2007). Alterations in the activities of enzymatic antioxidants (SOD, CAT, and APX) were observed in response to DNT exposure on radicles of V. radiata. Lowest SOD activity (0.08unit min^-1g^-1 FM) was observed in control, which showed non-significant alterations after acetone and 0.05% DNT exposure. Further increase in DNT concentration significantly enhanced (1060.8%) the SOD activity. Enhanced SOD activities with DNT accumulation witness their protective role under stress condition.

CAT activity was stimulated at a low level of concentration, although inhibited at higher concentrations of DNT. Depletion of CAT activity in response to DNT toxicity perhaps can be related to elevated O₂˙ˉand H₂O₂, thus impairing the activity of tetrameric heme-containing enzyme, CAT. Furthermore, increased APX activity was observed and studied using spectrophotometry and gel staining in comparison to the control. APX is considered a more potent antioxidant enzyme, thus accountable for the regulation of ROS. A considerable increment is most likely due to the synergetic working of SOD and APX. Increased SOD works in complementation to APX, thus higher SOD expression resulted in ROS scavenging because of the presence of high levels of APX (Jiang and Yang 2009). Similar trend in CAT and APX activity has been noted in Triticum aestivum under prometryne stress by Jiang and Yang in 2009. Enhanced APX activity in Zea mays under cadmium (Jamal-Abad  et al. 2007) and Abelmoschus esculentus exposed to mercury stress (Hameed et al. 2017) has been reported. Studies have shown that increased levels of these scavenging enzymes may potentially help in restricting the photo damage to plants (Hodges et al. 1999). This is the first time reported that changes in antioxidant enzymes such as SOD, CAT, and APX have been quantified in any plant species exposed to DNT. On the contrary, increased (243.73%) CAT activity was recorded in radicles of V. radiata with 0.05% DNT compared to control. Moreover, subsequent decline (31.86 and 81.27%) in CAT activities was observed with further increase in DNT concentrations (0.15 and 0.3%). Additionally, an irregular trend was marked for APX enzymatic assay in radicles of V. radiata. 0.05% concentration of DNT boosts the APX activity by 477.70% compared to control. Moreover, a further increase in concentration up to 0.15% considerably reduced (68.90%) the APX activity. Maximum APX activity was recorded with 0.3% DNT, which was 45.57% higher than 0.05% DNT treated radicles.

Gene expression studies have been done by RT PCR, using β-actin as house-keeping gene. β-actin showed similar intensity of bands with control and all the treatments. Moreover, the enzymatic antioxidants (SOD, CAT, and APX) revealed similar up-regulations and down-regulations as observed in spectrophotometric data of control, acetone, and DNT (0.05, 0.15, and 0.3%) treated radicles of V. radiata.

Conclusion

Different concentrations [0.05%, 0.15% and 0.3%, w/v] of DNT caused several deleterious alterations in the growth parameters of Vigna radiata. All the assessed parameters [%G, FW, DW, EL, ROS level and MDA content] were found to be sensitive towards different concentrations of DNT, and significantly affected by DNT exposure. To deal with the DNT-induced oxidative stress, V. radiata seedlings activated a variety of anti-oxidative enzymes such as SOD, CAT, and APX. These enzymatic antioxidants have been known to combat the excessively generated ROS. Upon exposure to the highest concentration of DNT [0.3%], activities of these antioxidant enzymes [except for CAT] were observed to be enhanced. Although at lower concentrations, reduced activities of these enzymes were observed. Prolonged exposure to DNT affects the overall growth and development of plants, right from radicle emergence to expression of genes. Toxicity of DNT at the molecular level was evaluated by RT-PCR analysis, which showed several alterations in the intensities and appearance of bands. The results of this study revealed the phytotoxicity of DNT at physiological, biochemical, and molecular levels. However, the mechanism[s] involved in the DNT mediated damages will need to be studied further. Additionally, the determination of suitable molecule[s] to ameliorate the DNT-induced injuries in growing seedlings of V. radiata is yet to be done.

Acknowledgement

I sincerely thank my research supervisor, Dr. Keshavkant Sahu, Professor and Head, Department of Biotechnology, Pt. Ravishankar Shukla University, Raipur for his invaluable guidance, constant support, and expert supervision throughout this research work. The authors would also like to thank University Grants Commission, New Delhi, and Council of Scientific and Industrial Research, New Delhi, for awarding fellowship to Anita Bhoi under Research Fellowship [F.No. 16-6(DEC. 2018)/2019(NET/CSIR), dated July 24, 2019].

Conflict of interest Author declares that there is no conflict of interest.

Funding information not applicable.

Ethical approval not applicable.

References

Adomako-Bonsu AG, Jacobsen J, Maser E (2024) Metabolic activation of 2, 4, 6-trinitrotoluene; a case for ROS-induced cell damage. Redox Biology. 72:103082. Doi:10.1016/j.redox.2024.103082.

Anjum A, Quraishi A (2022) Investigation of biochemical changes in the leaves of Curcuma caesia Roxb. under sucrose-induced osmotic stress environment. NewBioWorld A Journal of Alumni Association of Biotechnology,4(1):1-6. 

Blum A, Ebercon A (1981) Cell membrane stability as a measure of drought and heat tolerance in wheat. Crop Science. doi:10.2135/cropsci1981.0011183X00210001- 0013x.

Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. doi:10.1006/abio.1976.9999.

Bukowska B (2006) Toxicity of 2, 4-Dichlorophenoxyacetic Acid--Molecular Mechanisms. Polish Journal of Environmental Studies. 15(3).

Chance B, Maehly AC (1955) Assay of catalases and peroxidases. Methods in Enzymology. doi:10.1016/S0076-6879(55)02300-8.

Chandra J, Xalxo R, Pandey N, Keshavkant S (2021) Biodegradation of explosives by transgenic plants. Handbook of Bioremediation. Academic Press.

Chandrakar V, Naithani SC, Keshavkant S (2016) Arsenic-induced metabolic disturbances and their mitigation mechanisms in crop plants: A review. Biologia. doi:10.1515/biolog-2016-0052.

Chatterjee S, Deb U, Datta S, Walther C, Gupta DK (2017) Common explosives (TNT, RDX, HMX) and their fate in the environment: Emphasizing bioremediation. Chemosphere. doi:10.1016/j.chemosphere.2017.06.008.

Cho UH, Seo NH (2005) Oxidative stress in Arabidopsis thaliana exposed to cadmium is due to hydrogen peroxide accumulation. Plant Science. 168(1):113-20. doi:10.1016/j.plantsci.2004.07.021.

Choudhary KK, Agrawal SB (2014) Cultivar specificity of tropical mung bean (Vigna radiata L.) to elevated ultraviolet-B: Changes in antioxidative defense system, nitrogen metabolism and accumulation of jasmonic and salicylic acids. Environmental and Experimental Botany. 99:122-32. Doi:10.1016/j.envexpbot.2013.11.006.

Cuin TA, Shabala S (2007) Compatible solutes reduce ROS-induced potassium efflux in Arabidopsis roots. Plant, Cell and Environment. doi:10.1111/j.1365-3040.2007.01674.x.

Demidchik V, Straltsova D, Medvedev SS, Pozhvanov GA, Sokolik A, Yurin V (2014) Stress-induced electrolyte leakage. Journal of Experimental Botany. doi:10.1093/jxb/eru004.

Doherty SJ, Messan KS, Busby RR, Barbato RA (2019) Ecotoxicity of 2,4-dinitrotoluene to cold tolerant plant species in a sub-Arctic soil. International Journal of Phytoremediation. Doi:10.1080/15226514.2019.15837- 20.

El-Amier Y, Elhindi K, El-Hendawy S, Al-Rashed S, Abd-ElGawad A (2019) Antioxidant system and biomolecules alteration in Pisum sativum under heavy metal stress and possible alleviation by 5-aminolevulinic acid. Molecules. 24(22):4194. Doi:10.3390/molecules24224194

Gadi BR, Pooja V, Ram A (2012) Influence of NaF on seed germination, membrane stability and biochemical contents in Vigna seedlings. Journal of Chemical and Biological Physical Sciences.

Ganesan K, Xu B (2017) Polyphenol-rich lentils and their health promoting effects. International journal of molecular sciences. 18(11):2390. Doi:10.3390/ijms18112390

Gechev TS, Gadjev I, Van Breusegem F, Inzé D, Dukiandjiev S, Toneva V, Minkov I (2002) Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes. Cellular and Molecular Life Sciences CMLS. 59(4):708-14. Doi: 10.1007/s00018-002-8459-x

Georgiou CD, Papapostolou I, Patsoukis N, Tsegenidis T, Sideris T (2005) An ultrasensitive fluorescent assay for in vivo quantification of superoxide radical. Analytical Biochemistry. doi:10.1016/j.ab.2005.09.013.

Hameed A, Qadri TN, Mahmooduzzafar TO, Ozturk M, Altay V, Ahmad P (2017) Physio-biochemical and nutritional responses of Abelmoschus esculentus (L.) Moench (Okra) under mercury contamination. Fresenius Environmental Bulletin. 26(10):5814-23.

Hasanuzzaman M, Bhuyan MHB, Zulfiqar F, Raza A, Mohsin SM, Mahmud JA, Fujita M, Fotopoulos V (2020) Reactive oxygen species and antioxidant defense in plants under abiotic stress. Antioxidants. doi:10.3390/antiox9080681.

Heisnam D, Shankar S, Chandra D, Goel D, Mishra A, Kushwaha M (2023) Explosive contamination in soil: sources, environmental concerns, and phytoremediation. InXenobiotics in Urban Ecosystems: Sources, Distribution and Health Impacts 2023 Nov 1 (pp. 241-260). Cham: Springer International Publishing.

Hniličková H, Hnilička F, Orsák M, Hejnák V (2019) Effect of salt stress on growth and electrolyte leakage. Plant, Soil and Environment. doi:10.17221/620/2018-PSE.

Hodges DM, DeLong JM, Forney CF, Prange RK (1999) Improving TBARS assay for lipid peroxidation. Planta. doi:10.1007/s004250050524.

Hoque MA, Banu MN, Okuma E, Amako K, Nakamura Y, Shimoishi Y (2007) Exogenous proline and glycinebetaine increase NaCl-induced ascorbate–glutathione cycle enzyme activities, and proline improves salt tolerance more than glycinebetaine in tobacco Bright Yellow-2 suspension-cultured cells. Journal of Plant Physiology. https://doi.org/10.1016/j.jplph.2006.10.004.

Hou D, Yousaf L, Xue Y, Hu J, Wu J, Hu X, Feng N, Shen Q (2019) Mung bean (Vigna radiata L.): Bioactive polyphenols, polysaccharides, peptides, and health benefits. Nutrients. 11(6):1238. Doi:10.3390/nu11061238.

Islam F, Ali B, Wang J, Farooq MA, Gill RA, Ali S, Wang D, Zhou W (2016) Combined herbicide and saline stress differentially modulates hormonal regulation and antioxidant defense system in Oryza sativa cultivars. Plant Physiology and Biochemistry. 107:82-95. Doi:10.1016/j.plaphy.2016.05.027.

Jamal-Abad AZ, Rahmatzadeh S (2007) Cadmium toxicity in maize seedlings: Changes in antioxidant enzyme activities and root growth. Pakistan Journal of Biological Sciences. 10(1):127-31.

Jiang L, Yang H (2009) Prometryne-induced oxidative stress and impact on antioxidant enzymes in wheat. Ecotoxicology and Environmental Safety. 72(6):1687-93. Doi:10.1016/j.ecoenv.2009.04.025.

Kayihan DS, Kayıhan C, Özden Çiftçi Y (2021) Transgenic tobacco plants overexpressing a cold-adaptive nitroreductase gene exhibited enhanced 2, 4-dinitrotoluene detoxification rate at low temperature. International journal of phytoremediation. 23(1):1-9. Doi:10.1080/15226514.2020.1786795.

Keshavkant S, Naithani SC (2001) Chilling-induced oxidative stress in sal seedlings. Acta Physiologiae Plantarum. doi:10.1007/s11738-001-0056-3.

Khansari N, Shakiba Y, Mahmoudi M (2009) Chronic inflammation and oxidative stress as a major cause of age-related diseases and cancer. Recent patents on inflammation & allergy drug discovery. 3(1):73-80. Doi:10.2174/187221309787158371.

Kiiskila JD, Das P, Sarkar D, Datta R (2015) Phytoremediation of explosive-contaminated soils. Current Pollution Reports. 1(1):23-34. Doi:10.1007/s40726-015-0003-3.

Krishnan G, Horst GL, Shea PJ (2000) Differential Tolerance of Cool-and Warm-Season Grasses to TNT-Contaminated Soil. International Journal of Phytoremediation. 2(4):369-82. doi:10.1080/15226510008500045.

Lent EM, Crouse LC, Quinn Jr MJ, Wallace SM (2012) Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays. Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 742(1-2):54-60. doi:10.1016/j.mrgentox.2011.11.013.

Liu X, Lan S, Sha J, Zhang Z, Chang Y, Chen Y, Yang S (2025) Transcriptome Analysis Reveals the Molecular Mechanisms Through Which Exogenous Methyl Jasmonate Enhances Mercury Stress Tolerance in Maize Seedlings. Agronomy. 15(6):1369. doi:10.3390/agronomy15061369.

Mansoor S, Ali A, Kour N, Bornhorst J, AlHarbi K, Rinklebe J, Abd El Moneim D, Ahmad P, Chung YS (2023) Heavy metal induced oxidative stress mitigation and ROS scavenging in plants. Plants. 12(16):3003. doi:10.3390/plants12163003.

Marklund S, Marklund G (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. European journal of biochemistry. 47(3):469-74.

Pramod Kumar Mahish, KL Tiwari and SK Jadhav (2019) Lead Toxicity, Measurement and Control. NewBioWorld A Journal of Alumni Association of Biotechnology,1(2):1-8.

Mubarak AE (2005) Nutritional composition and antinutritional factors of mung bean seeds (Phaseolus aureus) as affected by some home traditional processes. Food chemistry. 89(4):489-95. Doi:10.1016/j.foodchem.2004.01.007.

Nair PM, Chung IM (2015) Changes in the growth, redox status and expression of oxidative stress related genes in chickpea (Cicer arietinum L.) in response to copper oxide nanoparticle exposure. Journal of Plant Growth Regulation. 34(2):350-61. Doi:10.1007/s00344-014-9468-3.

Nakano Y, Asada K (1981) Hydrogen peroxide scavenging by ascorbate peroxidase. Plant and Cell Physiology. doi:10.1093/oxfordjournals.pcp.a076232.

Nisar N, Cheema KJ, Powell G, Bennett M, Chaudhary SU, Qadri R, Yang Y, Azam M, Rossiter JT (2018) Reduced metabolites of nitroaromatics are distributed in the environment via the food chain. Journal of hazardous materials. 355:170-9. doi:10.1016/j.jhazmat.2018.05.028.

Okozide OE, Adebusoye SA, Obayori OS (2020) Aerobic degradation of 2, 4‐dinitrotoluene: Effect of raw organic wastes and nitrogen fortification. Journal of Environmental Quality. doi:10.1002/jeq2.20088.

Parihar RA, Agrawal P, Sharma DJ, Minz MG (2018) Character association and path analysis studies on seed yield and its yield attributing traits in mungbean (Vigna radiata (L.) Wilczek). Journal of Pharmacognosy and Phytochemistry. 7(1):2148-50.

Peterson MM, Horst GL, Shea PJ, Comfort SD, Peterson RK (1996) TNT and 4-amino-2, 6-dinitrotoluene influence on germination and early seedling development of tall fescue. Environmental Pollution. 93(1):57-62. doi:10.1016/0269-7491(96)00016-4.

Pichtel J (2012) Distribution and fate of military explosives and propellants in soil: a review. Applied and Environmental Soil Science. 2012(1):617236. doi:10.1155/2012/617236.

Picka K, Friedl Z (2004) Phytotoxicity of some toluene nitroderivatives and products of their reduction. Fresenius Environmental Bulletin. 13(8):789-94.

Pilon-Smits EA (2014) Selenium in plants. InProgress in Botany: Vol. 76, 2014 Aug 6 (pp. 93-107). Cham: Springer International Publishing.

Podlipná R, Pospíšilová B, Vaněk T (2015) Biodegradation of 2, 4-dinitrotoluene by different plant species. Ecotoxicology and environmental safety. 112:54-9. Doi:10.1016/j.ecoenv.2014.07.026

Rajjou L, Duval M, Gallardo K, Catusse J, Bally J, Job C, Job D (2012) Seed germination and vigor. Annual review of plant biology. 63:507-33. Doi:10.1146/annurev-arplant-042811-105550.

Rasleen Kaur and S. Keshavkant (2021) Enzyme Based Biosensor for Onsite Detection of Chromium. NewBioWorld A Journal of Alumni Association of Biotechnology,3(2):1-7.

Rocheleau S, Kuperman RG, Simini M, Hawari J, Checkai RT, Thiboutot S, Ampleman G, Sunahara GI (2010) Toxicity of 2, 4-dinitrotoluene to terrestrial plants in natural soils. Science of the total environment. 408(16):3193-9. Doi:10.1016/j.scitotenv.2010.04.010.

Rughani G, Chandra J, Chandrakar V, Keshavkant S (2016) Production and in-situ localization of ROS in Pennisetum typhoideum indulged with heavy metal stress. CSVTU Int J Biotechnol Bioinf Biomed. 1(1):8-13.

Sangeetha P, Das UN, Koratkar R, Suryaprabha P (1990) Increase in free radical generation and lipid peroxidation following chemotherapy in patients with cancer. Free Radical Biology and Medicine. 8(1):15-9. Doi:10.1016/0891-5849(90)90139-A.

Sharma P, Jha AB, Dubey RS, Pessarakli M (2012) Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. Journal of botany. 2012(1):217037. Doi:10.1155/2012/217037.

Shukla AK, Sisodiya DS, Savita, Chattopadhyay A, Bhattacharya A (2023) To Quench or Not: Extending a β-Carboline Fluorophore for TNT Detection in Aqueous Media via Simultaneous ESPT Destabilization and AIE. The Journal of Physical Chemistry B. 127(46):10025-34. Doi:10.1021/acs.jpcb.3c05936.

Singh HP, Batish DR, Kohli RK, Arora K (2007) Arsenic-induced root growth inhibition in mung bean (Phaseolus aureus Roxb.) is due to oxidative stress resulting from enhanced lipid peroxidation. Plant Growth Regulation. 53(1):65-73. Doi:10.1007/s10725-007-9205-z.

Song NH, Le Yin X, Chen GF, Yang H (2007) Biological responses of wheat (Triticum aestivum) plants to the herbicide chlorotoluron in soils. Chemosphere. 68:1779-87. Doi:10.1016/j.chemosphere.2007.03.023.

Srivastava MP, Sharma YK (2017) Enhancement of morphological and physiological parameters in Vigna radiata (l.) Wilczek treated with Zinc and Trichoderma sp. Advance Pharmaceutical Journal. 2(6): 235-242

Su K, Wu Z, Liu Y, Jiang S, Ma D, Wang Y, Fu C (2021) Highly efficient detoxification of dinitrotoluene by transgenic switchgrass overexpressing bacterial nitroreductase. Plant, Cell & Environment. 44(9):3173-83. Doi:10.1111/pce.14099.

Su YH, Liang YC (2011) Transport via xylem of atrazine, 2, 4-dinitrotoluene, and 1, 2, 3-trichlorobenzene in tomato and wheat seedlings. Pesticide biochemistry and physiology. 100:284-8. Doi:10.1016/j.pestbp.2011.05.001

Tomero-Puertas MC, Rodiguez-Serrano M, Corpas FJ, Gomez M, Delrio LA, Dalio San LM (2004) Cadmium induced subcellular accumulation of superoxide and hydrogen peroxide in pea leaves. Plant Cell Environ, 27(9), 1122-1134.

Velikova V, Yordanov I, Edreva A (2000) Oxidative stress and antioxidant systems in acid rain-treated plants. Plant Science. doi:10.1016/S0168-9452(99)00197-1.

Verwoerd TC, Dekker BM, Hoekema A (1989) Rapid isolation of plant RNAs. Nucleic Acids Research. doi:10.1093/nar/17.6.2362.

Via SM, Zinnert JC (2016) Impacts of explosive compounds on vegetation: A need for community scale investigations. Environmental Pollution. 208:495-505. Doi:10.1016/j.envpol.2015.10.020

Yadu B, Chandrakar V, Meena RK, Keshavkant S (2017) Glycine betaine reduces oxidative injury under fluoride stress. South African Journal of Botany. doi:10.1016/j.sajb.2017.03.023.

Yin XL, Jiang L, Song NH, Yang H (2008) Toxic reactivity of wheat (Triticum aestivum) plants to herbicide isoproturon. Journal of agricultural and food chemistry. 56(12):4825-31. Doi:10.1021/jf800795v.

Yoon JM, Oliver DJ, Shanks JV (2006) Phytotransformation of 2,4-dinitrotoluene in Arabidopsis thaliana. Biotechnology Progress. doi:10.1021/bp0601443.

Zivy M, Thiellement H, De Vienne D, Hofmann JP (1983) Nuclear and cytoplasmic genome expression in wheat. Theoretical and Applied Genetics. doi:10.1007/BF00281838.