Introduction

Heavy metal contaminants in the environment are the most significant abiotic stress which constrains the productivity and quality of crop plants. The availability and the toxicity of heavy metals in plants depend on the complex rhizospheric reaction between the soil, plants, and microbes. Among an array of heavy metals, cadmium and nickel are the most phytotoxic redox inactive metals which are released into the arable soil from farming and industrial processes and deteriorate the development process of plants by incorporating into the food chain leading to its biomagnifications (Gill and Tuteja 2011; Gill et al. 2011, 2012). Cadmium, one of the chief agents of the abiotic stress (Ona et al. 2006), is toxic to plants at almost all concentrations. The permissible limit of Cd in agricultural soil is 100 mg kg−1 of soil (Mwegoha and Kihampa 2010). Cd is a needless element for plants which is easily absorbed by the roots and transported to the aerial plant parts, thus entering into the food chain causing health problems in biological organisms. Its increasing concentration in soil leads to decline plant growth by affecting nutrient uptake, water uptake, loss of photosynthesis activity, degradation of chlorophyll activity, loss in enzymatic activity, and high production of reactive oxygen species (ROS) leading to oxidative stress (Gill and Tuteja 2011). Thus, due to elevated toxicity and high solubility in water, cadmium (Cd) is a significant environmental pollutant (Daud et al. 2009).

Nickel is the other essential transition metal import into the surroundings due to smelting wastes, extensive mining, and continuous use of pesticides and phosphate fertilizers. Ni is an essential element for plants as it is a component of a number of enzymes, such as glyoxalases (family I), peptide deformylases, methyl-CoM reductase, ureases, superoxide dismutases, and hydrogenases (Ermler et al. 1998; Kupper and Kroneck 2007) which plays an important role in various metabolic processes including ureolysis, hydrogen metabolism, methane biogenesis, and acidogenesis (Maier et al. 1993; Collard et al. 1994; Ragsdale 1998; Mulrooney and Hausinger 2003). But in greater concentration, it leads to disparity in nutrient uptake which results in interruption of important biological processes in plants leading to leaf chlorosis and necrosis, decline growth responses, and failure of cell membrane function (Ahmad et al. 2008; Siddiqui et al. 2011). Recent reports on Ni toxicity reveal that Ni induced lipid peroxidation and declines water uptake in plants which extensively hamper the membrane functionality. Enormously high concentrations of Ni in soil destroy the farmland and make it unsuitable for vegetation (Duarte et al. 2007). Thus, different heavy metals exert different toxic effects in plants (Sethy and Ghosh 2013).

Heavy metal-stimulated destruction in plants consist of a series of reactions, the major step of which includes linking of metal to the cysteine residue of proteins or enzymes and the production of ROS (Mehrag 1994; Zhang et al. 2004). In addition, heavy metals also promote the generation of ROS and lipid peroxidation which enhances the antioxidant defense system in plants (Sharma and Dietz 2009). Plant protection system to ROS comprises free oxygen radical detoxifying enzymes like catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), guaicol peroxidase (GPX), and glutathione reductase (GR) as well as supplementary antioxidant molecules namely reduced glutathione (GSH), carotenoids, proline, thiols, and other compounds able of quenching ROS (Liu et al. 2009; Demirevska-Kepova et al. 2004). All the above antioxidant enzymes and molecules have biological role under non-stressed environment, but their activity or quantity enhanced during oxidative stress. Induction of the antioxidant actions of enzymes via oxidative stress represents the response of a plant towards unfavorable conditions (Foyer et al. 1994) which vary with plant species, tissues analyzed, and the kind, intensity, and duration of the stress treatment (Schutzendubel and Polle 2002). ROS detoxification process begins with the activity of SOD, the first metalloenzyme which converts O2 − radicals to H2O2 and O2 (Alscher et al. 2002). Accumulation of H2O2 is prohibited inside the cell by CAT activity or through the ascorbate–glutathione cycle where ascorbate peroxidase reduces it to H2O so that the deposition of O2 − and H2O2 is effectively prevented (Sharma and Dietz 2009). Recently, Cui et al. (2013) reported that hydrogen-rich water (HRW) has the ability to improve the effects due to cadmium-induced oxidative stress in Medicago sativa, which is consistent with a significant enhancement of the ratio of reduced/oxidized (homo) glutathione ((h) GSH).

Recent advances achieved in elucidating the role of heme oxygenase a novel antioxidant enzyme in plant cell defense against oxidative stress. Heme oxygenases are the universal and highly active family of enzymes which catalyzes the oxidative degradation of Fe(III) protoporhyrin IΧ (heme) to biliverdin, Fe(II), and CO in the presence of reducing equivalents (Shekhawat and Verma 2010). The enzyme was originally identified in the animal system where its role is in the bilirubin production in rat liver (Tenhunen et al. 1968). Now, the genes encoding HOs have been isolated from a wide variety of living system including animals, algae, cyanobacteria, cryptophyta, higher plants, and pathogenic bacteria. In photosynthetic organism, HO gene was first identified in red algae (Cyanidium caldarium) (Shekhawat and Verma 2010). Further studies on HO suggested that the enzyme exists in three isoforms: an inducible heme oxygenase 1 (HO 1) and constitutive heme oxygenases HO 2 and HO 3 (Maines 1988). Until now, gene encoding HO 1 has been isolated and strongly characterized as a defending element in cellular protection against oxidative stress in animal system (Tomaro and Batlle 2002; Otterbein et al. 2003). Conversely, fewer reports discussed the functions of HO 1 in quenching ROS in plants. HO 1 role in providing protection against the oxidative stress induced by cadmium has been well reported in Glycine max (Balestrasse et al. 2005, 2008; Shekhawat et al. 2011). The HO 1 role in soybean plants was also confirmed by pretreatment of ascorbic acid prior to UV B treatment where HO 1 expression is a dosage-dependent phenomenon (Yannarelli et al. 2006; Santa-Cruz et al. 2017). Other reports on HO 1 depicted its role in abiotic stress including wheat, M. sativa, and Chinese cabbage (Cui et al. 2011; Fu et al. 2011; Xu et al. 2011). Additionally, protection exerted by Jasmonic acid against Cd stress in soybean roots has also been reported (Noriega et al. 2012). Recent reports about heme-heme oxygenase 1 (heme-HO 1), a novel antioxidant system, which results in the regulation of ammonium tolerance in rice HO 1 (OsSE5), were investigated. Besides cellular defense mechanism, several other roles of HO 1 involvement have also been reported in plants, viz. in biosynthetic pathways leading to phytochrome chromophore synthesis (Terry and Kendrick 1999; Davis et al. 2001; Emborg et al. 2006; Linley et al. 2006) and chlorophyll synthesis (Terry et al. 2001; Li et al. 2014; Zhu et al. 2017), developmental role in lateral and adventitious root development (Xu et al. 2011; Cao et al. 2011; Li et al. 2015), and stomatal movement (Cao et al. 2007).

On the basis of the recent information on HO 1 concerning its antioxidant role, the present study is performed to evaluate the regulatory changes of the various physiological processes that occur due to the exposure of cadmium and nickel in the seedlings of Vigna radiata and to investigate the HO 1 gene expression in the seedlings subjected to metal toxicity. The purpose of the research is to ensure the probable functions of HO 1 in cellular protection against Cd- and Ni-induced oxidative stress. Therefore, the current investigation is mainly based on the significance of the heme oxygenase 1 in plant response to metal exposure. The investigation may enhance our understanding to interpret the upregulation as well as downregulation activity of heme oxygenase with other enzymatic and non-enzymatic parameters. Additionally, the current study will be important as it discusses the leading role of HO 1 in providing defense against metal-induced oxidative stress which has been previously unexplored in V. radiata, a heavy metal-tolerant species. The crop is a rich source of protein (18–36%) and iron (Bains et al. 2003) and easily survives in the stress conditions which is the precondition of the Thar desert.

Materials and methods

Plant material and establishment of hydroponic culture

Seeds of V. radiata var. PDM 54 were collected from NBPGR, Jodhpur, surface sterilized with 0.1% mercuric chloride for 1–2 min to avoid fungal contamination, and washed thoroughly four to five times with autoclaved distilled water to remove the remaining traces of mercuric chloride in the seeds. Sterilized seeds were sprouted in an autoclaved glass petri dish (10 cm) having autoclaved blotting paper imbibed with sterile deionized water (10–15 ml) at 25 °C in a BOD incubator (vaiometra) under dark conditions. Germinated seeds were shifted to light in thermo statically culture room sustained at 25 ± 2 °C and 50% relative humidity. After a week growth in petri dishes under the above mentioned condition, the hydroponic cultures were established by transferring the uniformly germinated seedlings in plastic pots (12 × 12 cm) containing 1000 ml of Hoagland nutrient solution (pH 6.8 to 6.9, Elico LI 120 pH meter). Ten seedlings were transferred per pot. These pots were transferred to the thermostatically controlled culture room maintained at 25 ± 2 °C and 50% relative humidity (Fig. 1). The hydroponic solution was aerated twice a day with glass rod to supply adequate oxygen as well as to avoid precipitation of salts. On every third day, the Hoagland medium was changed to evade nutrient deficiencies to seedlings.

Fig. 1
figure 1

Represents the schematic procedure of the successful establishment of hydroponic culture. Seeds of V. radiata (PDM 54) (A), sterilized seeds of V. radiata germinated in a petri dish under controlled conditions (25 ± 2 °C) (B), germinated seedlings (C), randomly selected seedlings of V. radiata at cotyledonary stage for the establishment of hydroponic culture (D), and successful establishment of hydroponic culture (E)

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Heavy metal treatment in growth medium

The two to four leaf stage (1-week-old) adapted seedlings were further utilized for practical work. The seedlings were subjected to cadmium and nickel stress at different concentrations ranging from 10 to 100 μM. A control having only Hoagland nutrient solution was used to compare the effect of heavy metals on crop plant. After 96 h of treatment, the stressed plant harvested to study the various physiological parameters.

Plant growth parameters

Growth parameters were measured in terms of fresh weight, dry weight, root length, and shoot length of seedlings. For growth parameter studies, seedlings were harvested and washed with deionized water. To determine the dry weight, the fresh tissue was kept in a hot air oven (Nsico Hicon Oven) at 65 °C for drying and the weight (Adair Dutt 125 A SCS) of dried tissue was calculated.

Tolerance index of plant was determined from fresh weight (FW) by using the formula (FW treated / FW control) × 100 and characterized in percent tolerance (Wilkins 1978).

Estimation of chlorophyll content and CSI

Young leaves of V. radiata seedlings were assembled and processed for resolving chlorophyll pigments in which 0.1 g of fresh leaf tissues was homogenized in 80% chilled acetone in cold conditions; homogenate was centrifuged (Sigma 3K15) at 10,000×g for 15 min at 4 °C. After centrifugation, the absorbance of the supernatant was recorded at 645 and 663 nm, respectively. The chlorophyll a, chlorophyll b, and total chlorophyll content were determined by Arnon (1949) method. Chlorophyll stability index (CSI) was calculated by the formula (amount of chlorophyll under stress condition / amount of chlorophyll in control condition) × 100.

Estimation of total protein content

Protein was estimated by using the method of Lowry et al. (1951). One milliliter of the extracted sample was taken which was homogenized in 50 mM phosphate buffer (pH 7.0). 4.5 ml of reagent I [48 ml of 2% Na2CO3 (sodium carbonate in 0.1 N NaOH), 1 ml of 0.5% CuSO4·5H2O (copper sulfate), and 1 ml of 1% sodium potassium tartarate], was added. A measure of 0.5 ml of Folin phenol reagent was added after 10-min incubation. Thirty-minute incubation produces a blue color complex in the mixture. Absorbance was taken at 660 nm against a blank without sample (using Elico double beam SL 210 UV VIS spectrophotometer). Protein content was calculated from a standard curve prepared by using bovine serum albumin (BSA) as a standard.

Lipid peroxidation

Lipid peroxidation was predicted by computing the production of malondialdehyde (MDA) content with 2-thiobarbituric acid (TBA) according to De Vos et al. (1989). Plant tissue (0.5 g) was crushed in 10 ml of 0.25% TBA in 10% trichloroacetic acid. The extract was heated at 95 °C for half an hour in a water bath and then brings it to the room temperature by immediately cooling it on ice. Centrifuge it at 10,000×g for 15 min, the absorbance of supernatant was recorded at 532 and 600 nm. The MDA content (nmol cm−1 g−1 fresh weight of tissue) was calculated through Beer Lambert’s law by applying extinction coefficient of 155 mM−1 cm−1. The specific absorbance was determined by subtracting the absorbance at 600 nm from absorbance at 532 nm.

Determination of H2O2 content

H2O2 production in seedlings of plant was determined spectrophotometrically by the method described by Alexieva et al. (2001). For H2O2 determination, 0.5 g fresh plant tissue was homogenized in 5 ml of 0.1% (w/v) TCA in an ice bath. Homogenate was centrifuged at 12,000×g for 15 min. The reaction mixture consists of 0.5 ml of the supernatant, 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0), and 1 ml of 1 M KI. Reaction was carried out for 1 h in darkness, and absorbance was measured at 390 nm (using Elico double beam SL 210 UV VIS spectrophotometer). Amount of H2O2 was calculated by using the extinction coefficient of 0.28 μM−1 cm−1.

Non-enzymatic parameters

Proline estimation

Proline estimation was performed by the procedure given by Bates et al. (1973). Plant material (500 mg) was homogenized in 5 ml of freshly prepared 3% sulfosalicylic acid. Centrifuge it at 3000×g for 20 min, 2 ml of supernatant was reacted with 2 ml of acid ninhydrin (1.25 g ninhydrin in 30 ml glacial acetic acid and 20 ml 6 M phosphoric acid) and 2 ml of glacial acetic acid. Incubate it at 100 °C for 60 min in a water bath and immediately keep it on ice to terminate the reaction. To this, 4 ml of toluene was introduced and then spin for 1 min. Absorbance of chromophore was recorded at 520 nm. The amount of proline was calculated by using a standard curve made by L-proline. Proline content was expressed in micrograms per gram fresh weight of tissue.

Estimation of thiol content

The thiol content (reduced GSH and oxidized GSSG) was estimated following the method of Anderson (1985). For total glutathione determination, 0.5 g of fresh plant tissue was homogenized in 3 ml of 5% (w/v) sulfosalicylic acid under cold conditions. The homogenate was centrifuged at 1000×g for 10 min. To 0.5 ml of supernatant, 0.5 ml of reaction mixture [100 mM potassium phosphate buffer (pH 7.0) and 3 mM Na2EDTA] and 50 μl of 0.15% DTNB (in absolute methanol) were added. After 5 min, the absorbance was recorded at 412 nm using Elico double beam SL 210 UV VIS spectrophotometer. GSSG was assayed by the same method in the presence of 2-vinylpyridine. Standard curves with known concentrations of GSH and GSSG were used. The content of GSH was calculated from the difference between total GSH and GSSG.

For non-protein thiol (NPT) determination, 0.5 g plant tissue was homogenized in 5 ml of 5% (w/v) TCA. Incubate it on ice for 10 min, and further centrifuge it at 8000×g for 10 min. One milliliter of supernatant was reacted with 3 ml of 0.2 M Tris–HCl (pH 8.5) and 0.1 ml of 10 mM DTNB (in absolute methanol). Incubate it at room temperature for 2 min, and absorbance was calculated at 412 nm (Sedlak and Lindsay 1968). Non-protein thiol content was calculated in millimolar per gram fresh weight of tissue by using the extinction coefficient 13.6 mM−1 cm−1.

Enzymatic parameters

For extorting antioxidative enzyme, 0.5 g of plant tissue was crushed in 5.0 ml of 50 mM phosphate buffer, pH 7.0. The extract was centrifuged at 5000×g for 20 min at 4 °C. The supernatant was preserved at − 20 °C for the antioxidative enzyme assay.

CAT (EC 1.11.1.6) activity was measured by the method of Aebi (1974). The assay system comprises of 50 mM phosphate buffer (pH 7.0), 9 mM H2O2, and a suitable aliquot of enzyme in the final volume of 3 ml. Decrease in the absorbance was taken at 240 nm. The molar extinction coefficient of H2O2 at 240 nm was taken as 0.039 mM−1 cm−1.

Activity of APX (EC 1.11.1.11) is the rate of H2O2-dependent oxidation of ascorbic acid. Its activity was estimated by the procedure given by Chen and Asada (1989). Ascorbic acid oxidation rate was determined by recording the decrease in absorbance at 290 nm for 3 min (extinction coefficient 2.8 mM−1 cm−1).

Guaicol peroxidase (GPX) (EC 1.11.1.7) was calculated by the procedure of Putter (1974). The assay mixture comprises of 50 mM phosphate buffer (pH 7.0), 20 mM guaicol, 3.7 mM H2O2, and a suitable aliquot of enzyme in the final volume 3 ml. Increase in the absorbance was recorded at 436 nm (extinction coefficient 26.6 mM−1 cm−1).

Heme oxygenase assay

HO (EC 1.14.99.3) was determined by the procedure given by Balestrasse et al. (2005). Plant sample (0.3 g) was crushed in 4 ml of extraction buffer (ice-cold solution of 0.25 M sucrose solution containing 1 mM PMSF, 0.2 mM EDTA, and 50 mM potassium phosphate buffer (7.4)). Mixture was centrifuged at 20,000×g for 20 min, and supernatant was used for estimating HO activity. The reaction mixture consists of 50 mM potassium phosphate buffer (pH 7.4), 60 nM NADPH, 250 μl HO extract (0.5 mg of protein), and 200 nM hemin up to a final volume of 500 μl. Samples were incubated at 37 °C for 1 h, and absorbance was recorded at 650 nm. Activity of HO was estimated by calculating the production of biliverdin (extinction coefficient 6.25 μM−1 cm−1).

Intracellular metal (Cd and Ni) content determination

Uptake of intracellular metal ion was estimated by the procedure given by Bates et al. (1982). Plant tissues were oven dried at 80 °C (Nsico Hicon Oven) overnight, and dry weight was calculated. One hundred milligrams of dried plant sample was digested in 5 ml of digestion mixture containing HNO3 (70%) + H2O2 (30%) + deionized water in 1:1:3 ratio until the solution becomes colorless. Residual solution was dissolved in 2% (v/v) nitric acid to a final volume of 5 ml, and Cd and Ni concentration was estimated by atomic absorption spectrophotometer.

The bioconcentration factor (BCF) is used to reflect the ability of the corresponding plant species to take up a particular metal into root/leaf with respect to its concentration in the associated environment. It is the ratio of root/leaf metal to whole metal concentration present in the nutrient solution and was calculated according to the formula given by Zayed et al. (1998). The translocation factor (TF) shows the metal translocation properties of corresponding metal species (Stoltz and Greger 2002). The TF were calculated by the formula TF = [metal μg g−1 DW]leaf / [metal μg g−1 DW]root × 100.

Statistical analysis

Data were statistically analyzed in experimental observations using Sigma 12.0 version software, and the results were expressed as mean (±standard error) of three independent replicates of each independent experiment.

Results

Growth parameters of two to four leaf stage seedlings of V. radiata were analyzed by measuring fresh weight, dry weight, shoot length, and root length. As shown in Table 1, increasing concentration of cadmium progressively decreases the fresh weight and dry weight up to 33.86 and 55.32% with respect to control. The effect of cadmium on fresh and dry weight was noticed maximum at 50 μM concentration after 96 h of treatment. Root length on exposure to cadmium decreases progressively with increasing concentration of CdCl2, whereas no significant effect of cadmium was observed on shoot length after fourth day of treatment (Fig. 2a) (Table 1). Similar results of fresh weight and dry weight were observed on treating the seedlings with NiSO4 (Table 2). Shoot length of V. radiata seedlings initially increases and then decreases progressively on increasing the concentration of nickel (Fig. 2b). The maximum shoot length was observed at 20 μM which was 1.8-fold with respect to control. Effect of nickel on root length was noticed at initial concentration of NiSO4, 20 μM at which root length was 1.62-fold in comparison to control (Fig. 2b) (Table 2).

Fig. 2
figure 2

a Morphological alteration in root and shoot length in Vigna radiata seedlings after treated it with different concentrations of CdCl2 (10–100 μM) for a period of 96 h. b Morphological alteration in root and shoot length in V. radiata seedlings after treatment for a period of 96 h with different concentrations of NiSO4 (10–100 μM)

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Table 1 Effect of cadmium on fresh weight, dry weight, shoot length, and root length of seedlings of V. radiata
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Table 2 Effect of nickel on fresh weight, dry weight, shoot length, and root length of seedlings of V. radiata
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Photosynthetic pigments increase progressively with increasing concentration of cadmium up to 30 μM; at 50 μM, a significant decrease was observed in Chl a, Chl b, and total chlorophyll in the case of leaves (Table 3). Whereas in the case of shoots, different results were observed; at initial concentration of cadmium up to 30 μM, a significant decline in photosynthetic pigment was observed, but on further increasing the concentration up to 100 μM, a gradual increase in chlorophyll content was observed. Percentage decrease at 30 μM concentration was more in Chl a in comparison to Chl b in shoots (Table 3). Stability index of chlorophyll was also calculated, and differing results were obtained in the case of leaves and shoots. It was observed that at 50 μM, 66.05% increase and 9.8% decrease in leaves and shoots, respectively, in CSI were noticed as compared to control after 96 h of Cd treatment (Table 3). The effect of nickel on photosynthetic pigments was also determined. Chlorophyll content decreases gradually with increasing concentration of nickel which results in chlorosis of the leaves. At 60 μM concentration, approximately 25.19% inhibition in total chlorophyll pigment was noticed in leaves (Table 4). In shoots, a noticeable increase was observed in Chl a and total chlorophyll content. At 60 μM Ni, 25% decline in chlorophyll stability index of leaves was observed after 96 h (Table 4).

Table 3 Chlorophyll a, chlorophyll b, and total chlorophyll content in seedlings of Vigna radiata exposed to different concentrations of cadmium chloride after 96 h
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Table 4 Chlorophyll a, chlorophyll b, and total chlorophyll content in seedlings of Vigna radiata exposed to different concentrations of nickel after 96 h
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Protein content in seedlings of V. radiata treated with Cd increases at initial concentration, but a significant increase was observed at 50 μM concentration of CdCl2 (Fig. 3a). At 50 μM Cd treatment, 117.6, 174, and 87.58% increase in protein content was observed in leaves, shoots, and roots, respectively. However, after 50 μM concentration, a slight decline in protein quantity was noticed in leaves, but the recorded concentrations were notably elevated with respect to control. Except 50 μM concentration, no significant change was observed in protein content in the case of shoots and roots (Fig. 3a). The protein content increases progressively with increase in Ni concentrations. At 60 μM Ni treatment, 59.2 and 49.7% increase in protein content was recorded in leaves and roots, respectively. No significant effect of Ni was noticed in the protein content of shoots (Fig. 3b).

Fig. 3
figure 3

a NPT and total protein content in seedlings of Vigna radiata. Seedlings were treated with 10, 30, 50, 70, and 100 μM cadmium for a period of 96 h. Vertical bars represent SE (n = 3). b NPT and total protein content in seedlings of V. radiata. Seedlings were treated with 10, 20, 40, 60, 80, and 100 μM nickel for a period of 96 h. Vertical bars represent SE (n = 3)

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The responses of the MDA content to the applied concentrations of cadmium and nickel are depicted in Fig. 4a, b. A substantial elevation in MDA amount in V. radiata seedlings treated with different cadmium concentrations was observed after 96 h of treatment. Plant tissue treated with 50 μM Cd shows 161.49% increase in MDA content with respect to control in leaves. Whereas, MDA content at 50 μM Cd was 4.02 times in comparison to control in root tissues. However, after 50 μM Cd concentration, a slight decrease in MDA content (at 70 μM Cd) was recorded (Fig. 4a). Considerable increase in the MDA content was also depicted with increasing concentration of Ni in Fig. 4b. At 60 μM Ni treatment, 150.48, 122.2, and 67.49% increase in MDA content in comparison to control was observed in leaves, shoots, and roots, respectively. But, after 60 μM concentration, a minute decline in MDA content was noticed (at 80 μM Ni) which again rises at higher concentration (100 μM Ni treatment). To understand the time-dependent effect of Cd and Ni on MDA content, a study was made after 0.5, 6, 15, 24, 48, 72, and 96 h for which 50 μM Cd and 60 μM Ni concentrations were selected, as maximum effects were observed at these concentrations. As shown in Fig. 4c at 50 μM Cd and 60 μM Ni, the MDA content increases significantly till 24 h and then decreases. MDA content at 50 μM Cd and 60 μM Ni at 24 h was 1.79 and 1.72-fold higher in comparison to initial time interval in leaf tissue. Maximum increase in MDA content in leaves at 50 μM Cd was observed after 96 h of treatment which is 3.2 times than MDA content at 0.5 h. The linear increase in MDA content after individual treatment of 50 μM Cd and 60 μM Ni was also observed in roots, but no significant change in MDA content was noticed in shoots. The increased accumulation of lipid peroxides in both Cd and Ni is an indication of enhanced production of reactive oxygen species like H2O2. H2O2 showed a significant increase in leaves of Cd-treated seedlings (Fig. 5a). The level of H2O2 was 1.69 times (at 10 μM Cd) in comparison to control over a period of 96 h, and thereafter, no significant change was observed at high Cd treatment. Effect of Ni on H2O2 content was also noticed in leaves of V. radiata. H2O2 content initially increases with increase in Ni concentration. At the highest Ni treatment, the H2O2 content was found to increase. At 10 μM Ni, the H2O2 content was increased by 1.24-fold as compared to control (Fig. 5b). H2O2 content was also studied with respect to the time duration of Cd and Ni exposure. The studies were conducted after 0.5, 6, 15, 24, 48, 72, and 96 h. Treatment with 50 μM Cd induced a rapid increase in H2O2 content at initial hours. The threshold level of H2O2 was observed at 6 h which is 1.29-fold higher than the H2O2 content at 0.5 h (Fig. 5c). Similar results were obtained with 60 μM Ni. The peak value of H2O2 with 60 μM Ni was noticed at 6 h which is 1.15-fold higher than the H2O2 content at initial hours (Fig. 5c).

Fig. 4
figure 4

a Proline and MDA content in seedlings of Vigna radiata. Seedlings were treated with 10, 30, 50, 70, and 100 μM cadmium for a period of 4 days. Vertical bars represent SE (n = 3). b Proline and MDA content in V. radiata seedlings. Seedlings were treated with 10, 20, 40, 60, 80, and 100 μM nickel for a period of 4 days. Vertical bars represent SE (n = 3). c The time duration of MDA content at 50 μM cadmium and 60 μM nickel at equal time interval for a period of 96 h. Values are mean ± SE (n = 3)

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Fig. 5
figure 5

a H2O2 content and APX activity in seedlings of Vigna radiata. Seedlings were treated with equimolar concentration of cadmium chloride for a period of 96 h. Vertical bars represent SE (n = 3). b H2O2 content and APX activity in seedlings of V. radiata. Seedlings were treated with 10, 20, 40, 60, 80, and 100 μM concentration of nickel sulfate for a period of 96 h. Vertical bars represent SE (n = 3). c The effect of time on H2O2 content at 50 μM cadmium and 60 μM nickel at 0.5, 6, 15, 24, 48, 72, and 96 h. Values are mean ± SE (n = 3)

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Proline content in leaf tissue of V. radiata on exposure to CdCl2 increased significantly with increasing concentration of Cd (Fig. 4a). At 70 μM Cd treatment, proline content was found to be maximum which was 2.79-fold with respect to control in leaves. Whereas in root tissues, 166.5% increase in proline content was found at initial Cd concentration (10 μM Cd). In shoots, no significant change was observed in proline content on treating the seedlings with Cd (Fig. 4a). However, the effect of Ni on proline content was only observed in leaves in which the significant increase was observed at 60 μM Ni concentration which was 6.15-fold in comparison to control (Fig. 4b).

V. radiata seedlings on exposure to cadmium show progressive increase in glutathione content with increasing concentration of CdCl2. The oxidized and reduced glutathione (GSSG and GSH) content increases with elevated CdCl2 concentration till 50 μM and then decreases at higher Cd treatment (Table 5). At 50 μM Cd, the GSSG content in leaves, shoots, and roots was 1.13, 1.17, and 1.27-fold with respect to control, while percent increase in GSH content at this concentration was 28, 19.68, and 5.27% than control in all the three tissues, viz. leaves, shoots, and roots (Table 5). The GSH/GSSG ratio after treated V. radiata seedlings with CdCl2 for a period of 96 h progressively increases with rise in Cd concentration in leaves and shoots. However, in the case of roots, the GSH/GSSG ratio decreases significantly till 50 μM Cd treatment. After 50 μM, the GSH/GSSG ratio was found to increase in root tissue (Table 5). Moreover, non-protein thiol content was observed maximum at 50 μM Cd which is 41.4% in comparison to control in leaves. Whereas, no significant change was observed in shoot and root tissues (Fig. 3a). The effect of Ni on glutathione and non-protein thiol content was also recorded. The GSSG content initially increases at initial Ni concentration till 40 μM and then decreases with further increase in concentration in leaf tissue. While in shoot and root tissues, the GSSG content declines significantly with elevated NiSO4 concentration. Similarly, GSH content decreases progressively with increase in Ni concentration in shoot and root tissues. But no significant effect of Ni on GSH content in leaf tissue was noticed (Table 6). Furthermore, the GSH/GSSG ratio in seedlings of V. radiata after NiSO4 treatment decreases with rise in metal concentration till 60 μM treatment and then increases with further increase in metal concentration in leaf tissue. While in shoots and roots, the GSH/GSSG ratio decreases progressively with increase in NiSO4 concentration (Table 6). Non-protein thiol content initially decreases and then increases with increasing concentration of Ni. At 60 μM Ni, non-protein thiol was found to be maximum which was 54.8% with respect to control in leaves. No considerable change was observed in non-protein thiol in both shoot and root tissues, respectively (Fig. 3b).

Table 5 Oxidized glutathione (GSSG) and reduced glutathione (GSH) content and GSH/GSSG ratio in seedlings of Vigna radiata exposed to different concentrations of cadmium after 96 h
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Table 6 Oxidized glutathione (GSSG) and reduced glutathione (GSH) content and GSH/GSSG ratio in seedlings of Vigna radiata exposed to different concentrations of nickel after 96 h
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Changes in the activity of antioxidant enzymes were found in the two to four leaf stage seedlings of V. radiata treated with CdCl2 and NiSO4. GPX activity increased significantly after Cd exposure up to 50 μM concentration in leaves and shoots which was 2.46 and 3.11-fold with respect to control at 50 μM (Fig. 6a). In roots, GPX activity after Cd treatment was found maximum at 10 μM concentrations in which 102.3% increase in GPX activity was recorded with respect to control (Fig. 6a). Whereas, GPX activity in the NiSO4-treated seedlings of V. radiata increases significantly with increase in concentration. GPX activity in roots at 10 μM concentration was found to be maximum which was 65.49% in comparison to control (Fig. 6b). Catalase activity on Cd exposure was found to increase with increase in Cd concentration in leaves, and at 50 μM Cd treatment, the catalase activity was found to be 48.03% with respect to control. After 50 μM concentration, the activity of catalase was found to be almost constant in leaves. No significant change was observed in catalase activity in roots and shoots on exposure to Cd metal (Fig. 6a). Effect of Ni on catalase activity was observed in leaves of V. radiata which was 36.5% with respect to control at 60 μM concentration (Fig. 6b). An increase of 36.3 and 10.23% in the activity of ascorbate peroxidase was observed in roots and leaves, respectively, on exposure to Cd metal at 50 μM (Fig. 5a). No significant change was observed in shoots after Cd treatment (Fig. 5a). An increase of 194.7% in APX activity was observed in roots with respect to control at 60 μM Ni treatments. No significant change was observed in APX activity in shoots and leaves on exposure to Ni (Fig. 5b).

Fig. 6
figure 6

a Catalase and guaicol peroxidase activity in Vigna radiata seedlings. Seedlings were treated with 10, 30, 50, 70, and 100 μM cadmium for a period of 4 days. Vertical bars represent SE (n = 3). b Catalase and guaicol peroxidase activity in V. radiata seedlings. Seedlings were treated with 10, 20, 40, 60, 80, and 100 μM cadmium for a period of 4 days. Vertical bars represent SE (n = 3). c The effect of time on guaicol peroxidase activity in V. radiata seedlings at 50 μM cadmium and 60 μM nickel at equal time interval for a period of 96 h. Values are mean ± SE (n = 3). d The effect of time on catalase activity in V. radiata seedlings at 50 μM cadmium and 60 μM nickel at equal time interval for a period of 96 h. Values are mean ± SE (n = 3)

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Antioxidant enzyme activity was also calculated in a time-dependent manner which is based on the duration of the interaction of Cd and Ni with the seedlings. The studies were conducted after 0.5, 6, 15, 24, 48, 72, and 96 h on 50 μM CdCl2 and 60 μM NiSO4. GPX activity in V. radiata seedlings was noticed from the beginning of the 50 μM Cd and 60 μM Ni treatment. The increase in GPX activity at 50 μM Cd lasted for 96 h, but the linear increase was found to be after 48 h, and the maximum activity was noticed at 72 h in leaves, shoots, and roots which was 2.02, 1.43, and 2.08 times higher than the activity observed just after the treatment (Fig. 6c). Time-dependent effect of 60 μM Ni treatment on GPX activity was found to increase till 72 h in leaves and shoots which was 2.03 and 1.78 times higher than the GPX activity at 0.5 h, but in roots, the GPX activity was found to decrease after 6 h up till 48 h, and then, a sudden increase was observed at 72 h at which the activity was 1.60 times higher than the activity at 0.5 h (Fig. 6c). Treatment with 50 μM Cd induced a rapid increase in catalase activity. The peak level of catalase was observed at 24 h which were 2.02, 2.64, and 2.35 times higher than the study conducted at 0.5 h of treatment in leaves, shoots, and roots, respectively (Fig. 6d). Catalase activity after exposing the seedlings of V. radiata with 60 μM Ni linearly increases with increase in time intervals. The threshold catalase activity was observed at 24 h which were 1.24, 2.69, and 4.71-fold higher than the activity observed just after the treatment in leaves, shoots, and roots (Fig. 6d).

Heme oxygenase activity was higher in leaf tissue in comparison to shoots and roots on treating the seedlings with CdCl2. In leaf tissue, HO significantly increased up to 50 μM CdCl2, and after that, constant HO activity was observed. At 50 μM CdCl2 concentration, a 38.8% increase in HO activity was observed in leaf tissue. HO activity in roots was found maximum at 100 μM CdCl2, whereas no significant change was observed in HO activity in shoots (Fig. 7a). Effect of Ni on heme oxygenase activity was found to be higher in leaves at 60 μM concentration which was 43.67% in comparison to control. No significant change was observed in shoot and root tissues on treating the seedlings with NiSO4 (Fig. 7b). Time-dependent measurements of HO content which depend on the duration of Cd and Ni exposure were examined. The study was conducted after 0.5, 6, 15, 24, 48, 72, and 96 h. At 50 μM Cd treatment, a rapid increase in HO activity with increase in time exposure was observed. The peak value of HO activity was observed at 24 h which is 2.12, 2.08, and 2.74 times higher than the HO activity just after the treatment (Fig. 7c). Similar results were obtained with 60 μM Ni. The HO activity observed at 24 h with 60 μM Ni was 1.18, 3.18, and 7.17 times higher than the activity of HO at 0.5 h (Fig. 7c).

Fig. 7
figure 7

a Heme oxygenase 1 activity in seedlings of Vigna radiata. Seedlings were treated with 10, 30, 50, 70, and 100 μM cadmium for a period of 96 h. Vertical bars represent SE (n = 3). b Heme oxygenase 1 activity in seedlings of V. radiata. Seedlings were treated with 10, 20, 40, 60, 80, and 100 μM nickel for a period of 96 h. Vertical bars represent SE (n = 3). c Effect of time on HO 1 activity in V. radiata seedlings at 50 μM cadmium and 60 μM nickel at equal time interval for a period of 96 h. Values are mean ± SE (n = 3)

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The intracellular Cd and Ni content in leaves and roots of V. radiata seedlings was investigated (Table 7). Intracellular Cd accumulation decreases with increase in Cd content till 50 μM Cd and further increases at higher concentration in leaf tissue. The highest Cd accumulation was noticed at 10 μM Cd (9.86 times) and minimum at 50 μM Cd (1.45 times). However, in roots, the intracellular Cd content increases, with rise in Cd concentration. The maximum Cd accumulation was at 70 μM Cd which is 54.71-fold, and minimum accumulation was at 10 μM Cd that is 9.38 times as compared to control (Table 7). Intracellular Ni content in leaf and root tissues of V. radiata seedlings increases with elevated concentration of Ni till 50 μM and then decreases. The intracellular Ni content gradually decreased from 53.7-fold (at 50 μM Ni) to 45.3-fold (at 60 μM Ni) in leaf tissue over a period of 4 days (96 h). Ni accumulation in roots was greatest at 50 μM Ni which is 57.03 times and lowest at 40 μM Ni concentration which is 14.49 times in comparison to control (Table 7) .

Table 7 Intracellular metal ion content in root and leaf tissues of V. radiata seedlings exposed to different concentrations of Cd and Ni after 96 h
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Besides, intracellular metal accumulation BCF has been suggested as the other parameter for recognizing the metal extraction and accumulation ability of V. radiata. The BCF in different tissues of V. radiata seedlings was evaluated for Cd and Ni after 96 h of treatment. BCF decreases progressively with increasing concentration of Cd in the nutrient solution up to 50 μM and then increases in the case of leaves. But the increase in BCF at 70 μM Cd was 92.7% less than 10 μM Cd. The BCF of Cd in root is significantly higher at low Cd concentrations (at 10 μM Cd) (Table 7). Similar pattern was obtained for BCF in Ni which was ranged from 0.3309 to 0.12013 (from 10 to 70 μM) in leaves and 0.7553 to 0.2171 (from 10 to 70 μM) in roots.

The TF signifies the internal metal transportation. In the current study, the TF of Cd decreases at initial concentration till 50 μM CdCl2 and then increases with further increase in concentration. The maximum TF was observed at 10 μM Cd which was 31.4 (Table 7). However, similar pattern of TF was observed in the case of NiSO4. The peak value of TF was observed at 70 μM Ni which was 55.33 (Table 7).

Discussion

In plants, heavy metals (Cd and Ni) in a micromolar concentration promote toxicity by generating ROS and lipid peroxidation (Hsu and Kao 2004; Shekhawat et al. 2008) which results in the reduction of photosynthetic rate, growth inhibition, and disturbances in antioxidant defense system (Sandalio et al. 2001). The produced ROS (H2O2, OH− radicals) acts as a messenger for the induction of multiple stress-responsive genes responsible for the heavy metal acclimatization and repair. Hence, the aim of the present study is to investigate the possible role of HO 1 in providing cellular defense to the hydroponically grown V. radiata seedlings which is subjected to different Cd- and Ni-induced oxidative stress. The study is conducted by examining the different morphological parameters and non-enzymatic as well as enzymatic parameters associated to the antioxidant defense mechanism. The research is significant as it is helpful in selecting an appropriate crop species to study the leading role of HO 1 in antioxidant defense mechanism and to produce a metal-tolerant crop plant in the near future.

Plant primarily responds to metal toxicity by inhibiting its overall development which is in terms of decline in fresh weight and dry weight, shoot length reduction, and root length reduction. The present study on two to four leaf stage V. radiata seedlings treated with varying concentrations of Cd and Ni illustrates a regular reduction in the overall development. This was in agreement with other studies on inhibitory effect of Cd using different plant species (Verma et al. 2008). The inhibitory effect on the growth of seedlings by Cd has earlier been reported by Ferreira et al. 2002 and by Ni has recently been reported by Hu et al. (2015).

In the present study, reduction in chlorophyll contents (Chl a, Chl b, and total Chl) with increasing concentration of Cd and Ni was observed (Tables 3 and 4). Heavy metal causes strong oxidation of chlorophyll machinery, hence reduces the size and density of chloroplast (Sidlecka and Baszynsky 1993). Decrease quantity of photosynthetic pigments in leaves subjected to Cd stress was also discussed in Phragmites australis (Pietrini et al. 2003). Protein content initially increased in the leaves with increasing concentration of Cd and Ni in the nutrient media and decreased at higher concentration (Fig. 7a, b). Elevated protein content at lower metal concentration is recognized in stimulation of stress proteins (Sanita di Toppi and Gabbrielli 1999). Production of ROS like H2O2 and other species has been straightly interrelated with protein destruction (Romero-Puertas et al. 2002). Thiol content (glutathione and NPT) is associated to the sequestration of heavy metals which maintain the level of ROS and hence considered as an essential element of cellular antioxidant defense system (Noctor and Foyer 1998). The higher thiol content (GSSG, GSH, and NPT) in foliar tissue exposed to sublethal metal concentration (50 μM Cd and 60 μM Ni) was interpreted as an acclimation which strengthens the defense system (Tausz et al. 2004).

Accumulation of proline has been found to increase with increasing concentration of Cd and Ni. Proline deposition in the current study has been comparable as reported earlier with the study conducted on Cd (Hare and Cress 1997; Shekhawat et al. 2009). Higher proline concentration has been related with the enhanced tolerance against metal-stimulated oxidative stress (Siripornadulsil et al. 2002). Resistance towards oxidative stress was projected to relate with ROS detoxifying capability of proline (Tripathi et al. 2004, 2013).

The MDA content level, one of the major TBA reactive metabolites, increased with increase in Cd and Ni concentration in leaf tissue. The similar pattern of results was reported by Shekhawat et al. (2009) and Verma et al. (2013). Increase in MDA content with time (Fig. 4c) recommended that the MDA content in leaves was associated to the exposure period of heavy metal. Heavy metal enhances the production of ROS which results in the elimination of hydrogen from unsaturated fatty acid, and thus forms various lipid radical and reactive aldehydes which is the basis of cell membrane distraction (Reinheckel et al. 1998). Further lipid peroxidation depends on the activity of lipoxygenase which oxidizes lipid to produce lipid hydroperoxides (Arvind and Prasad 2003) that was also demonstrated under heavy metal stress in Phaseolus vulgaris (Somashekaraiah et al. 1992) and Arabidopsis thaliana (Skórzynska-Polit et al. 2006). In plant cells, H2O2 is produced in a wide range of biotic and abiotic stresses (Laloi et al. 2004). In the current investigation, we observed that increasing concentration of Cd and Ni accumulated more amount of H2O2 in the leaf tissue of V. radiata seedlings. The accumulation of H2O2 in Brassica juncea in the presence of Cd has been well reported by Verma et al. (2013) and Verma et al. (2008). Increase in H2O2 contents at high concentration of heavy metal may be due to the toxic effects of metals that cause metabolic imbalance and finally lead to the cell death (Verma et al. 2013). Because Cd-stimulated cell death was correlated to the H2O2, burst has been studied by Schutzendubel et al. (2001) in Pinus sylvestris.

The present investigation shows the outcome of HO 1 on the metabolic process of Vigna under Cd and Ni stress. Our data signify that plants produce elevated level of HO 1 in leaves in comparison to roots with increasing concentration of both Cd and Ni. The increase in HO 1 level depends on the concentration and duration of metal (Fig. 7a, b). The previous studies demonstrated the role of HO 1 in providing defense against the reactive oxygen species due to the oxidative stress generated by the cadmium-treated soybean plants (Balestrasse et al. 2005, 2008; Yannarelli et al. 2006). Accumulation of HO under stress indicates that heavy metal provokes the antioxidant defense mechanism which might be arbitrated by HO. In support to this, numerous reports have revealed the defensive role of HO against abiotic stress which is strongly associated to the HO-arbitrated reduction of ROS in plants (Yannarelli et al. 2006; Noriega et al. 2012; Xie et al. 2015). High dosage as well as exposure time of metal did not induce endogenous HO 1 production which might be due to the cell metabolism destruction. However, lower concentration of metal in the hydroponic nutrient medium elevates the HO 1 activity as a result of metal-induced oxidative stress.

Results reveal that seedlings treated with sublethal metal concentration (50 μM Cd and 60 μM Ni) exhibit visible changes in antioxidant defense machinery in comparison to control. To evaluate that metal toxicity reduction may be credited to antioxidant properties of HO 1, the antioxidant enzyme activities like CAT, APX, and GPX were studied. The convincing outline was observed for these antioxidant enzymes where the HO 1 activity is increased in seedlings of V. radiata which were induced by metal stress (in both Cd and Ni stress). In the current study at sublethal concentration of the studied metal (50 μM Cd and 60 μM Ni), the highest activity of antioxidant enzymes along with HO 1 was found in different parts of the plant. In higher plants, indication has been provide that surplus metal in the nutrient medium stimulates GPX, APX, and CAT activity has been provided (Shekhawat et al. 2009). However, the activities of all these antioxidant enzymes were lowest at the highest metal concentration (100 μM) probably due to deactivation. The prohibition of enzyme activities at greater concentrations of stress might be attributed to ROS-stimulated alteration such as transductional alterations, DNA damage, improved vulnerability, and protein disintegration to proteolysis (Smirnoff 1998).

ROS (H2O2) level increases with increasing concentration of metal and time exposure that is at 10 μM CdCl2 and 10 μM NiSO4 as well as after 6 h of metal exposure (50 μM Cd and 60 μM Ni), but a decrease was observed at sublethal metal concentrations and on increasing exposure time. Reduction in H2O2 intensity might be due to scavenging through APX since at these concentrations of metal, APX activity was highest in root tissue. Similarly in B. juncea, Shekhawat et al. (2009) reported a rise in APX activity with declining H2O2 content. Histochemical analysis conducted by Zilli et al. (2009) of H2O2 in soybean leaves under salinity stress demonstrated a decrease in H2O2 content at 100 mM concentration, although the activity of other antioxidant enzymes including HO 1 was highest at this concentration. Time-dependent increase in antioxidant enzyme activity including HO 1 (Figs. 6c, d and 7c) suggests that accumulation of antioxidant enzymes was apparently related to the plant metal exposure period. At 24 h, the highest activity of antioxidant enzymes including HO 1 was observed. At this time period, H2O2 content was found to be lowest. The low activity of antioxidant enzymes along with increase in H2O2 content at high metal concentration (100 μM) may be due to the participation of H2O2 in the signal transduction needed for the action of antioxidant enzymes to prevail over the effects of stress (Vranova et al. 2002; Zilli et al. 2009). Due to decrease in antioxidant enzyme activity at high metal concentration, H2O2 accumulation increases in all plant parts.

In the recent study, leaf tissue elucidates enhanced ROS scavenging and avoidance of membrane damage than root tissue which is confirmed from the AAS study on different plant parts of V. radiata seedlings. The current investigation reveals that at sublethal concentrations of heavy metals (50 μM Cd and 60 μM Ni), the intracellular accumulation of Cd and Ni in leaf tissue decreases 85.26% (from 10 to 50 μM) and 29.26% (from 40 to 60 μM) in Cd and Ni, respectively. While at the same concentrations, intracellular heavy metal (Cd and Ni) accumulation is higher in roots in comparison to leaves. This indicates greater level of HO 1 in leaf tissue (Fig. 7a, b). High level of HO 1 in leaf tissue is based on the fact that heme oxygenase activity is localized in plastids (Cornejo et al. 1998; Muramoto et al. 1999). HO 1 localized in plastid is highly inducible by H2O2 (Chen et al. 2009) which suggests that HO 1 might be using H2O2 as a substrate along with CAT and APX-mediating complex signal transduction network. Higher HO 1 activity in leaves in comparison to roots may be due to the fact that plants are adapted to cope up with metal stress through various physiological adaptations including changes in membrane permeability which exclude excess of metal, ability to sequester excess ions in cellular compartments including vacuole, ability to transport ions in the older leaves that undergo senescence, and operate in order to reduce the metal concentration or to alleviate the deleterious effects of metal (Dixit et al. 2014). Due to high mobility, Cd and Ni are readily uptaken by plants. Majority of the metal ions (Cd and Ni) are retained in the root tissues, and minute quantity is translocated to foliar tissues (Cataldo et al. 1983). Compared to the control, V. radiata seedlings exposed to metal stress illustrate high intracellular metal content in root tissues. Decline in BCF and TF with elevated Cd and Ni concentration indicates the defensive role of HO 1. Nahar et al. (2016) demonstrated similar results while studying the exogenous effect of spermine in mung bean exposed to Cd-induced oxidative stress.

In conclusion, the consequences of the study signify that heavy metal (Cd and Ni) are the basis of oxidative stress in V. radiata ensuing in overall growth inhibition. HO 1 plays an imperative role in series of reactions dependable for metal tolerance by regulating antioxidant stimulation or diminution. Thus, based on previous study, HO 1 works within an assembly of antioxidant enzymes in which some enzymes upregulate while other downregulate with HO 1 that generate the defense mechanism for plant’s existence. Recent outcomes specify an elementary plant reaction through antioxidant enzymes (APX, CAT, and GPX) that is widespread to the majority of stress response cycle and a particular response of HO 1 that was earlier not known in V. radiata. As these antioxidant enzymes including HO 1 became tolerant, the H2O2 level was plagued. Activation of HO 1 illustrates that HO 1 has a chief function in the defense system towards metal stress. The study enhances our understanding of the intricacy of the defense group against metal stress which will be useful in developing metal-tolerant varieties for future research.