Exogenous hemin improves Cd2+ tolerance and remediation potential in Vigna radiata by intensifying the HO-1 mediated antioxidant defence system

The present study evaluated the effects of exogenous hemin on cadmium toxicity in terms of metal accretion and stress resilience in Vigna radiata L. (Wilczek). One-week-old seedlings were treated with CdCl2 (50 μM) alone and in combination with hemin (0.5 mM) in half-strength Hoagland medium for 96 h. The optimum concentrations of Cd and hemin were determined on the basis of haem oxygenase-1 activity. The results demonstrated that under Cd stress, plants accumulated a considerable amount of metal in their tissues, and the accumulation was higher in roots than in leaves, which significantly reduced the plant biomass and chlorophyll content by increasing the oxidative stress (MDA and H2O2 content). However, hemin supplementation under Cd,-stress improved plant growth by enhancing the harvestable biomass and photosynthetic pigments, increasing antioxidant activities (SOD, APX, POD, HO-1 and proline), lowering oxidative damage and increasing Cd tolerance in plants. Furthermore, the application of hemin enhances the removal efficiency of Cd in V. radiata by increasing the uptake of Cd via roots and its translocation from roots to foliar tissues. Thus, the study suggests that hemin has the potential to improve the stress tolerance and phytoremediation ability of heavy metal-tolerant plants so that they can be used instead of hyperaccumulators for remediation of Cd-contaminated environments.


Results
Impact of Cd and hemin on crop morphology. The length and biomass of V. radiata seedlings were studied to evaluate the adverse impact of cadmium on crops and the role of hemin against Cd toxicity. In the current work, cadmium negatively affected plant morphology. Compared with the control, the plant biomass (fresh and dry weight) in the Cd-treated plants decreased by 33.33% and 42.86%, respectively, whereas the dose of hemin, under Cd stress conditions (T3), recovered the biomass by 1.17-(fresh weight) and 1.13 (dry weight)fold more than that under the Cd treatment (T2) ( Table 1). Similar trends were observed for plant height and the tolerance index. A 22.86% increase in root length and 24.14% increase in shoot length was noted when seedlings of V. radiata were exposed to the combined treatment of hemin + Cd compared with the Cd treatment alone (Fig. 1). The tolerance index decreased by 33.3% under Cd stress, which improved by 17.2% under an exogenous supply of hemin. However, the improvements in biomass and height of the plants in the combined treatment were lower than those of untreated seedlings (Table 1).
Chlorophyll contents. The level of photosynthetic pigments (chlorophyll a, chlorophyll b and total chlorophyll) decreased markedly under cadmium stress. In comparison to the control, decreases of 18.8% (chlorophyll a), 32.3% (chlorophyll b) and 25.7% (total chlorophyll) were recorded when crop seedlings were subjected to cadmium stress. The exogenous application of hemin increased the chlorophyll content under both control and stress conditions. Under control conditions, the hemin treatment (T1) increased the photosynthetic pigments by 1.03 (Chl a), 1.29 (Chl b) and 1.16 (total Chl) times that of the control. Moreover, the cotreatment of 50 µM Cd and 0.5 mM hemin elevated the chlorophyll contents by 1.4 (Chl a), 1.68 (Chl b) and 1.52 fold in comparison with the respective Cd-only treatments (Table 1). Table 1. Effect of an exogenous hemin (0.5 mM) on biomass, height, tolerance index, leaf water content and chlorophyll (Chl a, Chl b and Total Chl) content in seedlings of V. radiata treated with CdCl 2 (50 µM) for a period of 96 h. Values are mean ± SE (n = 3) from three replicates for each treatments. Values marked with different letters shows significant differences (p ≤ 0.05) within treatment. Here CK, T1, T2 and T3 indicate control, 0.5 mM hemin, 50 μM CdCl 2 and 50 μM CdCl 2 + 0.5 mM hemin respectively.

Treatments
Fresh weight (g) Dry weight (g)

Tolerance index (%)
Leaf water content (%) Proline content. To overcome the harmful effects of the cadmium treatment, plants accumulated a considerable amount of proline as a compatible osmolyte in their tissues. In the present study, under Cd stress, proline accumulation was approximately twice and 1.07 times in leaves and roots, respectively, that of control plants. However, the accretion of proline was further found to be increased by the application of exogenous hemin. In contrast to individual cadmium treatments, 0.5 mM hemin supplementation improved the proline content by 36% (leaves) and 40% (roots) in the Cd-treated seedlings of V. radiata (Fig. 3E).
Activity of antioxidant enzymes. SOD catalysis increased by 20% and 28.6% in leaves and roots, respectively, after CdCl 2 treatment in comparison to the control levels. Supplementation of 0.5 mM hemin with CdCl 2 significantly improved SOD activity, which was 1.17 (leaves) and 1.22 (roots) times higher than that under Cd treatment alone (Fig. 3D). Conversely, catalase activity in seedlings increased progressively upon Cd treatment, showing levels 2.71 and 3.49 times higher than those of the control. However, a sudden drop of 37.9% and 14.3% was recorded with the addition of hemin to cadmium-affected crops in comparison to the levels of plants under stress conditions only (Fig. 3B).  www.nature.com/scientificreports/ Peroxidase (POD) activity increased gradually by 24% (leaves) and 30% (roots) in seedlings of V. radiata treated with CdCl 2 stress. However, the addition of 0.5 mM hemin resulted in a subsequent enhancement in enzyme catalysis compared to that in plants under CdCl 2 stress alone (Fig. 3A). Similar trends of results were obtained for ascorbate peroxidase (APX). An increase in APX catalysis was recorded in both parts, with levels 1.45-(leaves) and 1.2-(roots) fold higher than those of the control upon exposure to Cd at a 50 μM concentration. Cd treatment with hemin further enhanced APX activity by 21% and 48% in roots and leaves, respectively (Fig. 3C).
The impact of various treatments on HO-1 catalysis is depicted in Fig. 3F. A considerable rise in HO-1 activity was observed upon treatment of V. radiata seedlings with CdCl 2 . However, the application of exogenous 0.5 mM hemin in the Cd treatment group restored HO-1 activity by 15.7% and 9.1% in leaves and roots, respectively, compared with Cd treatment alone (Fig. 3F).
Effect of exogenous hemin on the uptake, accumulation and translocation of Cd 2+ . Vigna radiata seedlings treated with 50 µM CdCl 2 showed higher accumulation of Cd 2+ in roots than leaves, as depicted in Table 2. In terms of phytoremediation, the addition of 0.5 mM hemin progressively increased the intracellular cadmium content by 40% and 17% in leaves and roots, respectively, in comparison to those of the seedlings subjected to CdCl 2 treatment only ( Table 2). www.nature.com/scientificreports/ Cadmium uptake from the liquid medium via roots and its translocation to the foliar tissue affected the BCF and TF. For the seedlings of V. radiata subjected to Cd stress, hemin increased the uptake and translocation of cadmium in the plants. Supplementation of Cd-treated plants with 0.5 mM hemin improved the BCF by 2.0-and 1.6-fold in leaves and roots, respectively. A similar pattern of results was obtained for the TF, and the transfer of Cd ions from roots to aerial parts increased by 18.8% upon the addition of hemin to the stressed plants ( Table 2).

Principal component analysis (PCA).
Principal component analysis was performed to determine the correlation between different evaluated parameters (morphological and physiological) in response to various treatments and to understand their importance in enhancing Cd tolerance in V. radiata seedlings supplemented with a low dose (0.5 mM) of hemin. The results showed that all the parameters (growth, total chlorophyll, tolerance index, stress and antioxidants) were grouped into two components (PC 1 and PC 2) and accounted for 97.5% of the total variance (Fig. 4). The first principal component showed 80.15% variance and correlated with the biomass (Fwt: fresh weight, Dwt: dry weight), plant height (SL: shoot length, RL: root length), leaf water content (LWC), total chlorophyll (Total Chl), tolerance index (TI) and antioxidants (SOD, POD, APX, proline), including HO-1. The second principal component contributed to 17.35% of the variance and was grouped with stress parameters (LPX and H 2 O 2 ), the LWC and antioxidants (SOD, POD, APX, proline, HO-1, CAT) (Fig. 4).

Discussion
As a serious environmental pollutant, cadmium causes severe damage to plants by negatively influencing its morphology and physiology 2,5,17 . The outcome of the work demonstrated that metal stress hinders overall crop development by reducing plant biomass and chlorophyll contents. However, hemin supplementation markedly alleviated the adverse impact of cadmium by improving the growth and physiology of the plants. This is in accordance with earlier reports on hemin-induced alleviation of the inhibitory impact of cadmium in Medicago sativa L. 11 and Brassica chinensis L. 5 . The morphology of root tissues is an imperative marker to assess the toxic effects of Cd 2+ as roots are the initial tissues that are associated with metal ions 17 . In this study, root length was significantly affected by Cd stress, which disturbs the uptake of water and nutrients via roots and reduces crop growth and biomass 5,18,19 . Thus, root length affects the uptake and translocation of Cd by plants. Increases in root length and the number of root hairs were observed with hemin supplementation, which increases the absorption Table 2. Intracellular Cd content, Biological concentration Factor (BCF) and Translocation Factor (TF) in seedlings of V. radiata exposed to different treatments for a period of 96 h.  www.nature.com/scientificreports/ of water and nutrients and ultimately enhances plant biomass. This might be because exogenous hemin advances auxin-stimulated lateral root growth, which increases root numbers by stimulating cell division 5,20 . Hence, hemin favours the uptake of nutrients and metal ions from the liquid medium 21,22 .
In addition to enhanced root growth, the impact of hemin on crop improvement was mainly reflected in the alterations in photosynthetic pigments, as photosynthesis is the basis of plant growth and development. In our study, the chlorophyll content noticeably declined upon exposure of crop seedlings to Cd stress, which is probably due to damage to the photosynthetic apparatus, specifically photosystems I and II 5,23 . Cadmium blocks the photoactivation of photosystems by hindering electron transport in plastids 24 . Moreover, Cd inhibits the Calvin cycle (an important cycle of the dark reaction during photosynthesis) by affecting enzyme activities, which results in a lower photosynthetic rate 25 . However, hemin supply progressively improved the chlorophyll content in the stressed seedlings. The role of hemin in improving the chlorophyll concentration might be correlated with augmented haem oxygenase-1 activity. The results of this study are comparable with prior research results that demonstrated the role of haem oxygenase as an essential element in chlorophyll biosynthesis [26][27][28][29] .
The cadmium concentration in liquid medium changes the osmotic balance of plant cells by hindering water uptake through roots and reducing the leaf water content (LWC) 2 . Conversely, our study showed no noticeable reduction in the leaf water content under Cd stress, which might have been due to the progressive increase in the proline content in V. radatia seedlings. Proline is an effective quencher of ROS and works as a metal chelator in stress environments 30,31 . The addition of hemin to Cd-stressed plants further improved the proline content, which restored the leaf water content in plants. The improvement in the LWC due to the increased proline content was probably due to the antioxidant nature of proline, which alters plant metabolic activities to improve stress tolerance 31,32 . A simultaneous increase in the proline content was reported earlier in different plant species subjected to Cd stress 2,33-35 .
The toxic effects of cadmium in the present work were indicated by the overproduction of H 2 O 2 and increased accumulation of MDA. The MDA content, a product of lipid peroxidation, is considered a signal of membrane destruction 27 . The study outcomes demonstrate an increase in toxicity in seedlings of V. radiata subjected to cadmium stress. Parallel effects of Cd in other plant species were also reported in earlier studies 2,5,33,34 . However, addition of hemin to metal-treated crops reduced the detrimental effect of cadmium in the seedlings and enhanced their physiology. These study results were in accordance with those of Zhu et al. 5 , who determined the mitigation of cadmium stress in Brassica chinensis L. with an application of exogenous hemin to the treated crop. The shielding impact of hemin might be associated with the increased activities of antioxidants, which were probably mediated by haem oxygenase-1 11 37 . These antioxidant enzymes show regular activity under normal conditions, but their catalytic reaction is magnified under stress 17 . In our study, the activity of all antioxidants, including that of SOD, CAT, POD and APX, increased under Cd stress, which shows the clear response of V. radiata seedlings to metal stress and is likely one of the reasons for their survival along with Cd accumulation. Similar trends in antioxidant activity were recorded in different plant species exposed to Cd stress 2,9,17,33,34 .
However, exogenous application of 0.5 mM hemin further augmented the catalysis of APX, SOD and POD, which decreased the ROS concentration in V. radiata seedlings. These findings are comparable with those of Zhu et al. 5 , which verified the positive effect of hemin on antioxidant enzyme activity in seedlings of Brassica chinensis L. treated with Cd stress. Similarly, Chen et al. 38 reported that supplementation with 1 μM and 5 μM hemin enhanced SOD and APX activity in Oryza sativa L. seedlings exposed to Zn (1 μM), Pb and Cr (5 μM)treated hydroponic medium. The affirmative impact of hemin on antioxidant activities might be associated with the role of haem oxygenase-1. Hemin is a potent stimulator of HO-1 39 , which oxidizes haem to biliverdin (BV), Fe 2+ and carbon monoxide by employing NADH as a reducing equivalent 40 . Haem oxygenase-1 maintains the normal physiology of plants under stress environment by regulating antioxidant activities which neutralizes the negative effect of metal stress 17,41 . In recent findings, supplementation of 0.5 mM hemin to the Cd stress medium up-regulates the activity of haem oxygenase-1 enzyme to many folds (1.35 folds higher than control) which induces the activities of APX, SOD, POD and proline. The stimulated activity of antioxidants (APX, SOD, POD and proline) by HO-1 was clearly apparent in Fig. 4 which shows a linear co-relation with HO-1. The highest correlation of HO-1 was recorded with SOD and APX (Fig. 4). Additionally, CAT, POD and APX are haem enzymes with Fe in their structures, so Fe deficiency (due to competition between Cd and bivalent ions) is probably a limiting factor for their activities under Cd stress 3 . Hemin application in the Cd-treated medium elevates the activity of these antioxidants by increasing accessibility to iron (formed as a by-product during haem catalysis by HO-1) 17 . The study is supported by numerous reports that validate the shielding action of haem oxygenase towards cadmium treatment in different plant genotypes 17,[41][42][43] . Furthermore, the PCA results revealed that augmented antioxidant activity improved the harvestable biomass and chlorophyll content of V. radiata under the combined treatment, and these parameters were negatively correlated with the H 2 O 2 content and membrane damage (LPX) (Fig. 4).Thus our findings display the exact mechanism of hemin to tolerate Cd 2+ by intensifying the activities of antioxidants which is mediated by hemin induced upregulated activity of HO-1.
To understand the uptake and translocation behaviour of metals in plants, it is important to select a suitable agent for improving the phytoremediation and stress tolerance of plants. Hemin, as a biostimulator, is known to enhance stress resilience in crops 5,38 . Similar to the results from other prior studies, the intracellular cadmium concentration in our study was far higher in roots than in leaves 3,17,33,34 . However, the intracellular Cd concentration in both tissues, viz. leaves and roots, was much lower than the critical concentration reported from Cd hyperaccumulators, which is 100 mg kg −1 dry weight of tissue 3 . Moreover, the BCF of leaves and roots as well as www.nature.com/scientificreports/ the TF were also less than 1. Thus, in our study, Vigna radiata did not display the characteristics of a cadmium hyperaccumulator according to the standard described by Baker and Brooks 44 but was considered a cadmium eliminator that efficiently removes Cd from the contaminated environment.
In the current investigation, BCF and TF declined upon exposure of V. radiata seedlings to cadmium, as revealed by Mahmud et al. 2 and Nabei and Amooaghaie 3 in Brassica juncea L. and Catharanthus roseus (L.) G. Don; treated with Cd concentrations. This decrease may be due to the reduction in biomass and chlorophyll content of seedlings. Moreover, the decline can also be attributed to the saturation of metal uptake and rootto-leaf translocation 45 . Interestingly, exogenous application of hemin at a lower dose (0.5 mM) enhanced metal accretion in V. radiata tissues, which confirmed the efficiency of hemin for phytoremediation. Correspondingly, the uptake and transfer of metal from roots to leaves was noticeably amplified upon supplementation of hemin to Cd-stressed medium ( Table 2). The effective promotion of the BCF and TF with the application of hemin was due to many reasons. The first probable reason is that hemin promotes auxin (specifically IAA)-induced lateral root development, which has already been reported in several studies conducted on different plant species [46][47][48][49] . Increased production of endogenous auxin stimulates the activity of membrane H + ATPases that alter the transport of cation transporters 50 and advances the absorption of Cd by V. radiata. A similar increase in the efficient absorption of Cd and U ions by an exogenous treatment of IAA was reported by Chen et al. 9 in B. juncea. Moreover, auxin increases the solubility and bioavailability of Cd in the growing medium by decreasing the pH of the medium, which enhances metal absorption via roots and transfer to aerial tissues 9,26 . Second, hemin supplementation of Cd-stressed plants triggers a haem oxygenase-1 mediated antioxidant defence mechanism that improves the endogenous concentration of Fe 2+ and CO (by-products of the HO-1 catalytic reaction) in the liquid medium. Improved Fe 2+ and CO concentrations increase the uptake and accumulation of Cd while decreasing micronutrient uptake in plant tissues 51 . This might be understood on the basis of competition between Cd and micronutrients, particularly bivalent ions (Fe, Mg, Zn, Cu and Ca), for the transport channels that are involved in the translocation of these ions 3 .
Additionally, in the present study, hemin increased the tolerance index along with Cd accumulation in plant tissues, which suggests that the defensive role of hemin is not associated with the inhibition of uptake and transfer of metals but is possibly involved in activating the endogenous mechanism of cadmium detoxification in V. radiata. This assumption was subsequently verified by the finding that the application of hemin increased Cd translocation to the foliar parts, and despite the higher Cd concentration in the aerial tissues, the growth and physiology of the plant improved. This might be due to the release of some strong ligands by hemin to the Hoagland medium, which counterbalanced the Cd ion concentration in the plants. Moreover, hemin may also act as a metal chelator and efficiently translocate Cd over long distances through the xylem to avoid the high toxicity of Cd ions in the edible parts of plants, similar to other biostimulators 3 . Our results are in contrast with previous reports on Chinese cabbage 5 , rice 38 and alfalfa 11 , where hemin application inhibited metal uptake and accumulation in plants to mitigate the noxious effects of cadmium ions. These discrepancies in the results are probably because the impact of hemin cotreatment on heavy metal accumulation depends on the type of plant species, concentration of the metal and hemin, exposure time and experimental conditions. Additionally, hemin might also increase metal accumulation in tolerant plants while decreasing metal accumulation in vulnerable species 7,52 which is why diverse effects were observed based on the physiological nature of the plants.

Conclusion
The present investigation explores the role of hemin application in the advancement of Cd stress tolerance and remediation efficiency using Vigna radiata. Cadmium adversely affects plant growth by reducing the harvestable biomass and chlorophyll content and increasing oxidative stress (H 2 O 2 and MDA content). However, exogenous supplementation of hemin in liquid medium that also contains Cd mediates the initiation of tolerance mechanisms in plants through upregulation of antioxidant activities. Moreover, this study presented strong evidence that hemin supports the removal efficiency of V. radiata by (1) improving the plant biomass and chlorophyll content and (2) enhancing Cd uptake and its translocation from roots to foliar tissues. Thus, our data concluded that hemin, as a biostimulator, has the potential to improve the phytoremediation efficiency of heavy metal tolerant plants so that they can be used as an alternative to hyperaccumulators to remediate Cd contamination. Future studies in the natural environment need to be carried out to further verify the significance of hemin for cadmium tolerance and remediation.

Materials and methods
Processing of experimental material and stress treatments. Vigna radiata var. PDM 54 seeds were collected from the National Bureau of Plant Genetic Resources, Jodhpur, India and disinfected with mercuric chloride (0.1% w/v) for one minute followed by rinsing with autoclaved deionized water (4-5 times) to remove the remaining traces of HgCl 2 . Disinfected seeds were germinated in sterile conditions in petri-plates with blotting paper soaked with distilled water at room temperature in the dark in a seed germinator. At the two-leaf stage, the germinated seedlings were transferred to half-strength Hoagland medium (pH 6.8-6.9) and placed under thermostatically controlled conditions (50% relative humidity and 25 ± 2 °C temperature). The liquid medium was replaced with fresh medium every other day and aerated twice daily to avoid nutrient and oxygen deficiency in the seedlings.
After 1 week, seedlings successfully adapted to hydroponic culture were treated with exogenous hemin at concentrations ranging from 0.1 to 20 mM (0.1, 0.5, 1.0, 5.0, 10, 20 mM) for 96 h [53][54][55][56][57] . Liquid medium without treatment was considered a control and utilized to evaluate the effect of hemin on haem oxygenase-1 catalytic activity. Maximum haem oxygenase-1 (HO-1) catalysis was observed under the 0.5 mM treatment (Fig. 5E) www.nature.com/scientificreports/ HO-1 in mitigating Cd stress in V. radiata seedlings 17 , we selected a 50 µM CdCl 2 concentration for the present study because at this treatment level, the activities of all the antioxidants, including haem oxygenase-1, were maximal (Fig. 5A,B), which resulted in the lowering of oxidative damage at this concentration (Fig. 5C,D). Thus, the seedlings of V. radiata used in the present study were subjected to the following treatments:  www.nature.com/scientificreports/ Morphological parameters. To study morphological parameters, freshly harvested crop seedlings were first washed with distilled water. Crop morphology was examined with regard to seedling height (root and shoot length), biomass (fresh and dry weight), a tolerance index (TI) and the leaf water content (LWC). The height of the seedlings was individually computed in centimetres. To calculate the dry weight, fresh seedlings were desiccated overnight at 65 ˚C in an oven, and the weight of dehydrated seedlings was measured. The TI was measured according to the Wilkins 58 method and presented as percent tolerance. The LWC was calculated from seedling biomass by utilizing the equation (fresh weight − dry weight/fresh weight) × 100 59  Estimation of proline content. The proline content was determined by the procedure of Bates et al. 63 .
Freshly harvested tissues were extracted in sulphosalicylic acid (3% w/v) and subsequently centrifuged for 20 min at 3000×g. The reaction mixture (equivalent volume of acid ninhydrin + supernatant + glacial acetic acid) was incubated for an hour at 60 °C in a water bath and transferred to an ice bath to terminate the reaction. The proline concentration (µg g −1 fresh weight of tissue) was estimated by documenting the optical density of an organic chromophore at 520 nm after adding toluene to the terminated reaction mixture 63 .
Extraction and assay of antioxidant enzymes. Freshly harvested crop seedlings were pulverized in NaPO 4 buffer (50 mM, pH 7.0) in cold conditions. The homogenate was centrifuged for 20 min at 10,000×g at 4 °C. The procured supernatant was utilized for further analysis. Superoxide dismutase (SOD) catalysis was assayed by the Beuchamp and Fridovich 64 , protocol. The catalytic reaction of SOD was determined by documenting the optical density of the assay compound [NaPO 4 buffer (50 mM, pH 7) + methionine (13 mM) + nitroblue tetrazolium (NBT) (75 µM) + riboflavin (2 mM) + EDTA (0.1 mM) + enzyme extract] at 560 nm after 30 min of incubation under bright light, which indicates the capability of an enzyme to hinder the photolytic reduction of NBT (with a proportionality constant of 100 mM −1 cm −1 ).
The catalytic reaction of the catalase (CAT) enzyme was measured via the Aebi 65 , procedure. The decomposition rate of H 2 O 2 (mM H 2 O 2 degraded min −1 g −1 fresh weight of tissue) was evaluated by the decline in optical density of the assay mixture [NaPO 4 buffer (50 mM, pH 7) + H 2 O 2 (9 mM) + enzyme extract] at 240 nm by utilizing 0.039 mM -1 cm -1 as the molar absorption coefficient 65 .
The catalysis of ascorbate peroxidase (APX) was determined via Chen and Asada 66 . The oxidation rate of ascorbate (mM ascorbate oxidized min −1 g −1 fresh weight of tissue) was computed by the decline in optical density of an assay compound [NaPO 4 buffer (50 mM, pH 7) + H 2 O 2 (10% v/v) + enzyme extract + ascorbate (0.6 mM)] at 290 nm by employing 2.8 mM −1 cm −1 as a proportionality constant 66 .
The peroxidase (POD) catalysis was measured by using the method of Putter 67 . The rate of formation of tetraguaiacol (mM min −1 g −1 fresh weight of tissue) was calculated by the rise in optical density of an assay mixture [NaPO 4 buffer (50 mM, pH 7) + guaiacol (20 mM) + H 2 O 2 (3.7 mM) + enzyme extract] at 436 nm by employing 26.6 mM -1 cm -1 as a proportionality constant 67 .
The catalysis of the haem oxygenase-1 (HO-1) enzyme was evaluated according to the Balestrasse et al. 68  www.nature.com/scientificreports/ acid mixture containing HNO 3 (70% v/v) + H 2 O 2 (30% v/v) + deionised water in the proportion of 1:1:3. The colourless remains of the mixture were diluted in HNO 3 (2% v/v) and employed for estimation of the Cd content 69 .

Measurement of biological concentration factor (BCF) and translocation factor (TF). The bio-
logical concentration factor reveals the ability of corresponding plant species to uptake the particular metal ion into tissues to its proportion in the related surroundings and was calculated by the formula 17 : The translocation factor demonstrates the transfer ability of metal ion in particular plant 70 . The formula for estimating TF is: Statistical analysis. For graphical representation of data Sigma plot version 12.0 (Chicago, IL, USA) software (http://www.sigma plot.co.uk/produ cts/sigma plot/produ pdate s/prod-updat es5.php) was used. Data were statistically examined by one way ANOVA and the correlation between different evaluated parameters were analyzed by principal component analysis using SPSS 16 version software. The data were considered as average (± standard error) of individual replicas (n = 3) of every test conducted separately. To check the reproducibility of results, three independent biological replicates were studied following completely randomized design (CRD). The significant variations among treated and untreated seedlings were illustrated at 0.05% significant level by applying Duncan's Multiple Range test (DMRT) 17,59 . . TF = Metal µg g −1 DW (leaf) Metal µg g −1 DW (root) .