Combined application of selected heavy metals and EDTA reduced the growth of Petunia hybrida L.

Up till now, despite of well-developed ornamental market, very little information is available on Petunia hybrida L. tolerance against heavy metals (HMs), which can contribute in both beautification of urban dwellings, as well as potential in phytoremediation. Therefore, hydroponic study was conducted to check the effects of Cd, Cr, Cu, Ni and Pb individually (50 and 100 μM) and with co-application of EDTA (2.5 mM) in Hoagland’s nutrient solution. Results indicated higher uptake of Cd, Cr, Ni and Pb in above ground parts, and Cu in roots, further the co-application of EDTA enhanced HMs uptake in P. hybrida L. This uptake accompanied changes in biochemical stress indicators, included significantly higher MDA, H2O2 contents and electrolyte leakage with reduced chlorophyll a, chlorophyll b, total chlorophyll and carotenoid content. Upon exposure to HMs increased antioxidant enzyme activities (CAT, POX, GST, APX, and SOD) were noted. Though selected HMs can be removed by using P. hybrida L., the findings of current study indicated that the direct exposure of P. hybrida L. to Cd, Cr, Cu, Ni and Pb damaged the plant’s aesthetics, and to use P. hybrida L. for beautification of urban landscape or phytoremediation, appropriate soil modification should be included.

www.nature.com/scientificreports www.nature.com/scientificreports/ fresh and dried weight were found negatively affected, at the p = 0.05 where n = 18, with reference to Pb and Ni. (Supplementary Table S1). The R 2 value for each of the selected metal uptake model was higher than 0.9, indicating the good fit of the model (Fig. 1). In case of Cd, and Cu the CPC was highest for the root FW (23, and 19%, respectively), shoot length in Cr (19%), while for Ni and Pb the highest CPC was noted for shoot dried wright (18, and 25%, respectively). stress injury. Effects of selected HMs application on chlorophyll content (chlorophyll per g −1 of FW), including chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (Chl T) and carotenoid (Car) content were presented in Fig. 2. Abrupt decline in Chl a (74.5%), Chl b (79%) and Chl T (76.8%) were noted for Cd treatments, and significantly highest in all cases of 100 µM, when compared with control. For Car, statistically significant decline was noted for Cd treatment at 50 and 100 µM, and with Cu at 100 µM + EDTA (~67%), in comparison to control. The addition of EDTA significantly improved plant growth, in the case of Cd. While, no variation for Cr and Ni was noted. Significantly highest lipid peroxidation (µM of MDA g −1 of FW), EL, and H 2 O 2 were noted for 100 µM Cd treatment with EDTA (Fig. 3). In case of Cr and Pb, highest MDA contents were noted at 100 µM metal concentration (0.013 and 0.011, respectively), while for Cd, Cu and Ni, 100 µM metal treatment with EDTA resulted in highest MDA contents (0.025, 0.018 and 0.012 µM g −1 FW, respectively). The highest percentages for EL were noted for Cd, Cr, Cu and Ni, at 100 µM metal treatments with EDTA, with the following trend, Cd (97.7%) > Ni (86.7%) > Cr (74.7%) > Cu (74%). In case of Pb statistically significantly higher EL was recorded for 100 µM Pb concentration (72%), as compared to control. The H 2 O 2 contents for Cd and Cu were highest, and for Cd and Pb, were also higher with 100 µM metal treatments with EDTA, while in case of Ni at 100 µM metal treatments, in both conditions with and without EDTA, were resulted significantly highest H 2 O 2 content. Supplementary Table S2, represented the coefficient correlation between selected HMs and stress indicator. MDA content, EL and H 2 O 2 contents were found strongly positively correlated, while were found negatively correlated for Chl a, Chl b, Chl T and Car with metal uptake. The coefficient correlations were high to moderate between each case. MLR equations and cumulative percentage contributions were presented for metal uptake and studied stress indicators (Fig. 4). MDA contents was found to have highest percentage contribution (39,37,33, and 27%, respectively) for Ni, Cd, Pb and Cr. While, for Cu the H 2 O 2 content resulted in highest CPC (39%). The high MLR coefficients (R 2 ) were observed for each metal ranging from 0.873 to 0.925. www.nature.com/scientificreports www.nature.com/scientificreports/ Antioxidant enzyme activity. The activities of CAT, POX, GST, APX, and SOD content in leaves of P. hybrida exposed to Cd, Cr, Cu, Ni and Pb with or without EDTA were presented in Table 2. Enzyme activities (including CAT, POX, GST, APX and SOD) increased with increasing the concentrations of selected HMs from 0 to 50 and 100 µM. The addition of EDTA with HMs, resulted in statistically significant amplification in all enzyme activities of P. hybrida, except in the case of POX activity, where highest value was noted for Cr 100 µM (0.88 U g −1 of FW), and reduction in POX activity was noted with the addition of EDTA along with Cr at 50 and 100 µM (0.   mM EDTA) by using the same control (CB) and control + EDTA (CE). Data are in means (n = 3 ± SD). Alphabets represent statistical differences, number followed by alphabets in CB and CE represents difference with metals (1 = Cd, 2 = Cr, 3 = Cu, 4 = Ni, and 5 = Pb), significantly highest mean was "a" in each series of bars followed by later alphabets for lower means. *Represents significantly highest reduction.
www.nature.com/scientificreports www.nature.com/scientificreports/  Figure 5 represented the cumulative percentage contribution and MLR equation for each selected metal and studied enzyme activities. In case of Cr, Cu, Ni and Pb significantly higher MLR coefficients (R 2 ) were observed. While, with Cd the model's R 2 value was found to be moderate. Highest CPC noted for uptake of Cd was with SOD (38%), for Cr with CAT (30%), Cu and Ni for APX (70 and 44%, respectively), and for Pb with CAT and POX (27, and 27%, respectively).
Heavy metal uptake. Heavy metal uptake by Petunia hybrida L. was dose dependent, as increase in metal concentration resulted in increased HM content with in the plant tissues (Fig. 6). The use of EDTA also exacerbated the metal content uptake. However, the pattern of compartmentalization, i.e. metal content in each of the plant compartment (root, shoot and leaf), varied. In case of Cd, the distribution of metal was found comparatively uniform, but with the addition of EDTA higher Cd content was noted in leaf, followed by shoot and root. = 50 µM + 2.5 mM EDTA, 100 + EDTA = 100 µM + 2.5 mM EDTA) by using the same control (CB) and control + EDTA (CE). Data are in means (n = 3 ± SD). Alphabets represent statistical differences, number followed by alphabets in CB and CE represents difference with metals (1 = Cd, 2 = Cr, 3 = Cu, 4 = Ni, and 5 = Pb), significantly highest mean was "a" in each series of bars followed by later alphabets for lower means. *Represents significantly highest reduction.
www.nature.com/scientificreports www.nature.com/scientificreports/ Leaf contained highest metal content, when plants were treated with Cr, followed by a dose dependent increase in shoot, while in root the content of Cr was comparatively uniform with no statistical difference. Application of EDTA with Cr resulted in highest content of Cr with in the leaf, followed by root and then shoot. Copper content was found highest in root in all the treatments, interestingly with co-application of EDTA, the shoot metal content increased significantly. With Ni and Pb, the metal uptake was found to be highest in leaf and is influenced by both the dose of application and addition of EDTA with metal treatments.

Discussion
Use of ornamental plant is expected to grow with the passage of time, not just because they can improve the aesthetics of the urban dwellings and medicinal advantages, but also in the field of phytoremediation, as this discipline is an ever evolving method and much attention was not given on the ornamental plants 9,10,13 . Therefore, it will be a good approach to investigate commonly used ornamental plants. This will not only help to identify the potential for any possible use in phytoremediation technology, but will also in the scaling the tolerance limit against HMs, assistance to maintain the vibrant aesthetics of these plants when used in urban surroundings (like roads, gardens, and walking lanes) under the expected stress of environmental contaminants spread due to anthropogenic activities (vehicular exhaust, irrigation with contaminated water, soil erosion, and even dust scattering due to pedestrian). This study was designed to understand the tolerance level and metal uptake pattern of Petunia hybrida L., when exposed to cadmium, chromium, copper, nickel and lead, with and without addition of EDTA, and to best of our knowledge no such competitive study was done. The use of EDTA for ameliorated extraction of heavy metals was well documented in already published data with other plants like Brassica napus 4,12 , Brassica rapa 14 , Phaseolus vulgaris and Zea mays 15 , and Helianthus annuus 13,16 . The effect of addition of EDTA was also found to vary (positive or negative) among different plants 4,13,14 . Effects of HMs and EDTA exposure on plant physiological status. In the present study, it was observed that addition of EDTA enhanced the HMs uptake, but enhanced metal uptake resulted in reduced plant growth. The studied physiological parameters of Petunia hybrida L. were negatively influenced by the increasing the concentration of selected HMs in the hydroponic media (Table 1 and Supplementary Table S1). The impact was further escalated, when the HMs were provided with co-application of EDTA. The negative impact of HMs was dose dependent, while for EDTA the negative impact was due to increased toxic HMs uptake and the hindrance in essential metal uptake as EDTA bind non-specially with metals, resulting in chelation of metals required by plant along with the toxic HMs 16 . Further the chelator mediated toxicity also reduced the plant biomass, limiting the metal uptake 17 , as with low biomass limited HM is expected to be translocate. It should further be noted that a higher HM concentration in plant tissue by the addition of metal chelating agent did not necessarily mean a better removal efficiency, as the change in biomass and other physiological parameter www.nature.com/scientificreports www.nature.com/scientificreports/ are the determining factors 16 . This can be better understood by identifying the contribution of each parameter towards HM uptake. Cumulative percentage contribution of each of the physiological parameters against total metal uptake ( Fig. 1) was performed and checked how metal uptake is affected by reduction or increase in each of studied parameter. For Cd, and Cu the highest CPC was for the root FW (23, and 19%, respectively), indicating that root fresh weight was significantly reduced by the uptake of these metals (Supplementary Table S1). Root fresh weight and Cu and Cd uptake were negatively co-related. Shoot length the physiological parameter is most affected. Ivanov, et al. 18 performed root toxicity assay with Zea mays L. and proposed that metal toxicity was declined as follow Cu ≈ Ti > Ag > Cd > Hg > Co > Zn > Pb, and the results were in accordance to our Petunia hybrida L. HMs exposure experimentations. When Petunia hybrida L. plants were exposed to Cr, Ni and Pb highest CPC (19,18, and 25%, respectively), was noted with shoot length for Cr and shoot dried weights for Ni and Pb, suggesting that negative influence on shoot occurred upon exposure of these HMs, and this was also evident by seeing the coefficient co-relation (Supplementary Table S1). plant biochemical and enzymatic status with reference to HMs and eDtA stress. Upon exposure to HMs, plants are expected to undergo stressed condition, contributing to metal uptake that leads to generation of reactive oxygen species (ROS), reduced plant turgidity, lipid peroxidation and decreased chlorophyll content 19 . Multiple toxic effects occur upon exposure to HMs, primarily due to formation of ROS, inhibiting cellular processes at different levels. The instability of ROS contributes; (a) damage cellular components, and (b) an important tributary for the induction of defence system 20 . In the present study reduced (negative correlation) in Chl a, Chl b, Chl T, and Car, while increased (positive correlation) MDA, H 2 O 2 contents and EL were recorded upon the stress induced by each selected HM at 50 and 100 µM (Figs 2 and 3, Supplementary Table S2). The addition of EDTA resulted in improvement of plant pigments in case of Cd, reduction with Cr, Cu and Ni, while no significant changes in Pb. The MDA content represents damage due to lipid peroxidation, electrolyte leakage is a good indicator to check the integrity of plant cell membrane, while higher H 2 O 2 content shows the production of ROS 12,21 . Upon exposure to HMs, and successive increase in the exposure concentrations led to increment in EL, MDA, and H 2 O 2 content, while the addition of EDTA did not showed any positive or negative impact on these parameters, except for Cd, where significantly higher EL, MDA, and H 2 O 2 contents were noted. Hence, it can be said that the HM induced stress to Petunia hybrida L. was not reduced upon addition of EDTA, despite it was known for the metal chelating activities 4,12,17 . The effects on plant that occur due to EDTA facilitated HMs uptake vary broadly based on HM, and the plant effected 4,12 . The lipid peroxidation and oxidative damage due to metal uptake is detrimental, and in this work MDA content (for Cd, Cu, Ni and Pb) and H 2 O 2 (for Cu) Catalase + Guaiacol peroxidase + Glutathione S-transferase + Ascorbate peroxidase + Superoxide dismutase + Control a 0.53 ± 0.06 c12345 0.14 ± 0.02 c15,d234 0.33 ± 0.01 c125, d3, e4 0.03 ± 0.00 c14,d235  Table 2. Effect on enzyme activities of P. hybrida L. upon exposure of HMs and EDTA. In each column of metal treatment, statistical comparison was done with the same control and control + EDTA. Data was presented in enzyme unit per mg of fresh weight. Data are in means (n = 3 ± SD). a Alphabets followed by number (1 = Cd, 2 = Cr, 3 = Cu, 4 = Ni, and 5 = Pb) represents statistical differences of control to each of the HMs. Significantly highest mean was "a" column wise followed by later alphabets for lower means. Similar small letter in same column within same metal treatment of experiment are non-significant. *Represents statistically highest mean among all metal treatments and controls. + All enzyme activities are expressed in U g −1 FW of plant, while for GST is in μM min −1 g −1 FW of plant.
www.nature.com/scientificreports www.nature.com/scientificreports/ were found to have highest CPC, for metal uptake model. It is inferred that upon exposure to HMs, the growth of Petunia hybrida L. plant was decrease due to ROS formation and lipid damage due to peroxidation. If there are no other options for the irrigation of Petunia hybrida L. except with HMs contaminated water, appropriate measures should be taken. These options include soil conditioners (individually or in combination) like, biochar, biosolids, compost, organic acids, and plant growth promoting bacterial amendments, as they have proven to reduce the toxic impacts of HMs on plants, which were not found achievable by using synthetic chelator like EDTA 17,22,23 , and in the current investigation as well.
The stress due to oxidative damage, induced by HMs uptake, is tackled plants with the help of defence mechanisms against oxidants. One of these mechanisms, is the use of antioxidant enzyme arsenal, including CAT, POX, GST, APX, and SOD regulating the concentration of stressors, cellular superoxide (O 2− ) and hydrogen peroxide (H 2 O 2 ), which upon uptake of heavy metals limit the production of OH radicals 4 . Different heavy metal effect the plants by versatile ways. As discussed earlier, some directly influence production of ROS, while others interfere with antioxidant defence mechanism 24 , hence a different enzymatic profile against each metal should be expected. In current study, exposure of Petunia hybrida L. to the selected HMs resulted in significantly higher antioxidant enzyme activity, which was enhanced with the increasing concentrations of HMs, and co-addition of EDTA along with HMs (Table 2). Similar findings were reported by other studies 4,12 . Moderate to high positive correlations were noted between all enzyme activities against selected HMs, except for APX with Cd (Supplementary Table S3). The increase in enzyme activates indicated stress induced by the HMs, as there were no significant variations in enzyme activities among the controls (with and without EDTA) 17 . Specific responses of anti-oxidant enzymes against a particular HM play an important role in metal toxicity using cellular defence strategy 25,26 . In this study, similar pattern was observed (Fig. 5) that the enzymatic profiles of P. hybrida L. for each of the HMs were different. A different mix of antioxidant enzyme CPC was noted for each HM, except in case of Cu, where APX activity was found having a very high CPC (70%). In another study, Wang, et al. 27 found the same pattern of enzyme activity. They suggested that higher accumulation of HM (Cd) in non-accumulator ornamental plants, including African marigold (Tagetes erecta), scarlet sage (Salvia splendens) and sweet hibiscus (Abelmoschus manihot), resulted in such an effect.
Heavy metal uptake and compartmentalization by P. hybrida L. The HM uptake was found to be dose dependent, and further enhanced by the addition of EDTA in the studied three compartments of P. hybrida L., comparable results were noted by Chen and Cutright 3 and Kanwal, et al. 4 , while the HMs compartmentalization with in P. hybrida L. was found to vary for each of HMs (Fig. 6). With Cr, Ni, and Pb, significantly higher compartmentalization occurred in P. hybrida L. leaf. Similar outcome was achieved with Cd, but the distribution of Cd was comparatively homogenous than the other three. This represented that translocate HMs to above www.nature.com/scientificreports www.nature.com/scientificreports/ ground parts P. hybrida L. can lead to reduced plant vigour and aesthetics, as observed in this work that enhanced HM uptake occurred with facilitation of EDTA but with negative impacts on plant growth. For Cu, significantly higher levels were noted in roots, when P. hybrida L. was exposed to Cu stress, which increased with increasing the Cu in root and concentration in the external solution. Upon addition of EDTA the levels of Cu were increased in roots as well as in stem. This was due to formation of stable Cu-EDTA complex that are transported into shoot, and higher concentration end up there as compared to the treatment with no EDTA addition and Cu stress 28 . Other explanation to this variable metal compartmentalization was due to differential expression of proteins responsible for metal transporting with in different part of plant. These protein families included copper transporters (CTR or COPT) 29 , Cation Diffusion Facilitator (CDF) 30 , ZRT/IRT-like Protein (ZIP) 31 , Cation Exchanger (CAX) 32 , Natural Resistance-Associated Macrophage Protein (NRAMP) 30 , and Heavy Metal ATPase (HMA) 33 .

Conclusion
The hydroponics system can provide an exact picture of the plant tolerance level for heavy metals due to non-interference of soil properties, micro/macro organisms and presence of other contaminating agents. We conclude that P. hybrida L. accumulated elevated levels of Cu, Cd, Ni and Pb in above ground parts, but this led to reduction in plant's aesthetics, and reduced plant vigour. P. hybrida L. reduced Cu mobility to aerial plant parts and maintained higher concentration in the roots. The enzyme activates were increased, and this effect was dose dependent of HM concentrations while physiological and other biochemical parameters were influenced negatively by HMs uptake. The presence of chelating agent i.e. EDTA showed deleterious effects on plant health due www.nature.com/scientificreports www.nature.com/scientificreports/ to increased metal uptake, leading to physiological and biochemical stress on P. hybrida. To use P. hybrida L. in phytoremediation, the phytoextraction is not a good option. The use of P. hybrida L. with an appropriate combination of soil conditioners and plant growth promoting bacteria for other phytoremediation techniques, like phytostabilization is an unexplored area of research and may be feasible. Further, it is also important to investigate other ornamental plants, to know their metals/contaminants tolerance potential. Flowering plant belonging to Catharanthus, Celosia, Cosmos, Dahlia, Mirabilis, and Nicotiana genus are the good candidates for investigation due to wide cultivation, established agronomic practices, and higher biomass.  SO 4 ). Pots were kept in dark till the seeds started to germinate, at 30 ± 1 °C in the day time and 24 ± 1 °C at night with a photoperiod of 16:8 (day:night), similar condition were maintained throughout the experiment. Upon seed germination, seedlings were transferred to wire house and after two weeks, equal sized uniform seedlings were carefully wrapped with filter wool between root and shoot, were held on thermophore sheet and were transferred to plastic containers containing full strength Hoagland's nutrient solution (pH 5.6), lined with polythene sheet. The plants were acclimatized for 2 weeks, along with refreshing of Hoagland's nutrient media and polythene sheet lining weekly, to prevent media contamination and depletion of nutrients.

Materials and
After the acclimation, Petunia hybrida L. plants were treated for three weeks with HMs and EDTA as T1: Blank Control, T2: EDTA control (2.5 mM), T3: HM (50 μM), T4: HM (100 μM), T5: HM (50 μM) + EDTA (2.5 mM), and T6: HM (100 μM) + EDTA (2.5 mM). It is to be noted that all treatments were conducted in biological triplicates, T3, T4, T5, and T6 were different for each HM. Salts used for making representative HMs stocks were, cadmium chloride dihydrate (CdCl 2 .2H 2 O) for Cd, chromium nitrate (Cr(NO 3 ) 2 ) for Cr, copper sulphate pentahydrate (CuSO 4 .5H 2 O) for Cu, nickel chloride (NiCl 2 ) for Ni, and lead nitrate (Pb(NO 3 ) 2 ) for Pb. After 3 weeks, plants were harvested, roots surface were washed with distilled water, and were used for plant physiological and metal uptake analysis. Samples for biochemical analysis were stored at −80 °C (for enzyme activities only) to avoid any disturbance in the activity. Each analysis was performed in biological triplicates. plant physiological parameters. Plant physiological parameters, including plant height, root length, total number of leaf per plant, fresh and dried weight (FW and DW) of root, shoot and leaves were recorded using standard method as done by Arshad, et al. 34 and Habiba, et al. 12 . Leaf area was calculated using ImageJ software 35 . Samples were dried at 60 °C till the constant weight and dried samples were used for acid digestion and then for atomic absorption spectrometry (AAS), with the help of Perkin Elmer, AAS-700, analysis for metal comparison in different plant compartments. plant biochemical characters. Among plant biochemical characters of plant, stress injury and antioxidant enzyme activities were noted. For stress injury chlorophyll a, b, total chlorophyll and carotenoid content were quantified by using method described by Arnon 36 and values were expressed in mg of chlorophyll g −1 of FW. Lipid peroxidation was noted in terms of malondialdehyde (MDA) by method adopted by Venkatachalam, et al. 37 , and was expressed in µM of MDA g −1 of FW. Electrolyte leakage (EL) was determined by the method as describe by Nishiyama, et al. 38 and was expressed in percentage. H 2 O 2 contents were determined according to Habiba, et al. 12 . Absorbance was taken at 410 nm and extinction coefficient of 0.28 µM −1 cm −1 was used for calculating H 2 O 2 contents. The values were expressed in µM of H 2 O 2 g −1 of FW. Quantification of plant enzyme and H 2 O 2 content was done by preparation of plant extract. It was prepared using 100 mg of leaf 's tissue, homogenised with the help of pre-chilled mortar and pestle in 1 ml of potassium phosphate buffer (50 mM, pH 7.4 containing 0.5 mM EDTA). Resulted extracts were collected in 2 ml tube and were centrifuged at 10000 g for 15 min at 4 °C. After centrifugation, supernatant was carefully collected in 1.5 ml tube and was used and stored at 4 °C to prevent the deterioration 37 . Superoxide dismutase (SOD) activity was assayed by measuring its ability to inhibit the photochemical reduction of NBT using the method of Dhindsa, et al. 39 , by noting the absorbance at 560 nm of the reaction mixture. One unit of SOD activity was determined as the quantity of enzyme that induced 50% prohibition of photochemical reduction of the NBT. Catalase (CAT) activity was assayed by measuring the rate of disappearance of H 2 O 2 in reaction mixture using the method of Maehly 40 . The decrease in H 2 O 2 was followed as a decline in absorbance at 240 nm after 1 min (ε = 39.4 mM −1 cm −1 ). Ascorbate peroxidase (APX) activity was determined according to the method of Chen and Asada 41 , with minor modification. The oxidation of ascorbate was followed by the decrease in the absorbance at 240 nm (ε = 2.8 mM −1 cm −1 ). Guaiacol peroxidase (POX) activity was determined according to Upadhyaya,et al. 42 , and the activity was computed using the extinction coefficient of 26.6 mM −1 cm −1 . Glutation-s-Transferase (GST) activity was assayed spectrophotometrically by measuring change of A 340 43 . Reactions were initiated by the addition of 1-chloro-2,4-dinitrobenzene (CDNB), and A 340 was monitored for 120 s in model of time-driver and values were computed using extinction coefficient of CDNB-glutathione conjugate (ε = 9.6 mM −1 cm −1 ). Values are expressed in Units g −1 of FW of sample for all enzyme activities, except for GST which was expressed in μM min −1 g −1 of FW. statistical analysis. One-way ANVOA between individual metal treatment and among all metals was performed using SPSS, followed by Duncan's multiple range test on each studied parameter. To find the correlation between metal uptake and studied parameters, Pearson's correlation analysis was conducted, after testing the www.nature.com/scientificreports www.nature.com/scientificreports/ normality of data using the Shapiro-Wilk normality test. Further to find the relation of studied parameters with cumulative metal uptake stepwise multiple linear regressions (MLR) was employed. Data was min-max normalized prior to MLR, for the prevention of large numeric ranges dominating those with small numeric range, to reduce the potential bias into the data values exactly. In this normalization method, the recorded case value is subtracted with the minimum value of recorded in that case from each value of the attribute and followed by dividing the difference by the range of the studied case.
Where, Z is the normalized observed value of x, min and max are the minimum and maximum values in x given its range. The normalized values lay in the range [0, 1]. The advantage of this normalization is that it preserves all relationships of the data. Using MLR, multivariate model was constructed for metal uptake, the dependent variable Y, based on consciously selected studied variables (X). MLR coefficient (R 2 ) showing highest value is ideal for the best equation. Based on these assumptions we can say: Where, Y is the metal uptake (dependent variable), X 1 , X 2 , …, X n are descriptive studied variables for plant physiology, enzyme activity, biochemical characteristic, and induced stress (independent variables), b 0 is the constant, where the regression line intercepts the Y axis; b ith (1 ≤ i ≤ nth) is the standard partial regression coefficient, representing the amount, the response variable Y changes when the descriptive studied variables changes 1 unit. This represents a model of the system under study, which can be used to investigate which variables influence its response and at what extent, and/or to predict the value of one variable when the others are known. The coefficient of regression (R 2 ), had reliable competence between the predicted and measured values, and a higher R 2 , more than 0.75, is considered a good indicator of good fit model for stepwise MLR for cumulative metal uptake and studied parameters. Cumulative percentage contributions (CPC) of studied parameter with cumulative metal uptake for each metal was calculated using following equation; where, B i = MLR coefficient for specific parameter and ∑ B i = sum MLR coefficient of all parameters 42,43 .