Iron–Lysine Mediated Alleviation of Chromium Toxicity in Spinach ( Spinacia oleracea L.) Plants in Relation to Morpho-Physiological Traits and Iron Uptake When Irrigated with Tannery Wastewater

: Chromium (Cr) is among the most widespread toxic trace elements found in agricultural soils due to various anthropogenic activities. However, the role of micronutrient-amino chelates on reducing Cr toxicity in crop plants was recently introduced. In the current experiment, the exogenous application of micronutrients [iron (Fe)] chelated with amino acid [lysine (lys)] was examined, using an in vivo approach that involved plant growth and biomass, photosynthetic pigments and gaseous exchange parameters, oxidative stress indicators and antioxidant response. The uptake and accumulation of Fe and Cr were determined under di ﬀ erent levels of tannery wastewater (33, 66, 100%) used along with the exogenous supplementation of Fe-lys (5 mM) to Spinacia oleracea plants. Results revealed that tannery wastewater in the soil decreased plant growth and growth-related attributes, photosynthetic apparatus and Fe contents in di ﬀ erent parts of the plants. In contrast, the addition of di ﬀ erent levels of tannery wastewater to the soil signiﬁcantly increased the contents of malondialdehyde (MDA), hydrogen peroxide (H 2 O 2 ) and electrolyte leakage (EL), which induced oxidative damage in the roots and leaves of S . oleracea plants. However, S . oleracea application of Fe-lys. In conclusion, the application of Fe-lys is an innovative approach to mitigate Cr stress in spinach plants, which not only increased plant growth and biomass but also decreased the Cr contents in di ﬀ erent plant organs.

Recently, various techniques and methodologies have been developed to reduce the heavy metal stress in plants [37][38][39][40]. In the past few years, a new method has been adopted to emphasize the use of amino acids as chelates to reduce toxic heavy metals [14,26,41]. Souri [42] has previously investigated the role of iron (Fe) chelated fertilizer with the amino acid lysine in improving crop growth and yield. Because Fe is an essential micronutrient for proper plant growth and development, it plays a crucial role in plant metabolism in enzymatic and metabolic processes [1,43,44]. Conversely, Fe deficiency significantly alters the morpho-physiological characteristics of plants and results in stunned plant growth and biomass [45]. Nevertheless, the application of Fe complex with lysine (lys) greatly reduces the toxicity induced by heavy metals stress. Fe complex with lys significantly reduced the Cd stress by improving rice growth, photosynthesis and the antioxidant system [45]. Large amounts of heavy metals in plant organs seriously affected the quality of their vegetative characters. In order to overcome this drawback, the current study examined the exogenous impact of Fe-lys on plant growth and biomass by decreasing the consumption of Cr in plant organs grown in soil irrigated with wastewater tanning.

Collection and Analysis of Wastewater and Soil
Samples of soil used to perform the current experimental study were collected from the Department of Botany, University of Punjab Lahore, Pakistan (31.4015 • N, 74.3070 • E), from an average depth of 0-15 cm. The 2 mm sieve was used to separate mud and debris from the dried soil intentionally. A comprehensive analysis of organic soil content was performed following the reported procedures [46]. For determining the precise size of the soil elements, a hydrometer was used [47]. Likewise, electrical conductivity (EC), sodium adsorption ratio (SAR) and soil ions were also carefully measured following the reported method [48]. Soil sampling was carried out with the help of ammonium bicarbonate diethylenetriamine penta acetic acid (AB-DTPA) solution for the sufficient estimation of extractable trace components [49,50]. The physicochemical characteristics of the soil under study are given in Table 1. The tannery wastewater used for the pot experiment was collected from the tannery industries based in Kasur, Punjab, Pakistan. The physicochemical properties of the tannery wastewater used in the present study were estimated according to the set protocols [51]. Comprehensive details of major characteristics of the tannery wastewater used in this experiment are presented in Table 2. The same soil with the tannery wastewater was used in our previous study, by Zaheer et al. [5].

Pot Experiment and Treatments
This study was conducted in a botanical garden under a glasshouse environment, at the Department of Botany of the University of Punjab Lahore 54,000, Pakistan. Healthy and mature seeds of spinach (Spinacia oleracea L.) were collected from Ayyub Agriculture Research Institute Faisalabad, Pakistan. S. oleracea seeds have been carefully sowed in experimental pots filled with approximately 5 kg of soil after diligent washing with H 2 O 2 (10%) (to avoid fungal or bacterial infection) and rising with deionized water. Five seeds were sowed in each pot. After thinning, three seedlings remained in each pot. After 2 weeks of seed germination, foliar application of Fe-lys (5 mg·L −1 ), along with various levels of tannery wastewater (33, 66 and 100%), were gently applied. A hand sprayer was used for the exogenous supplementation of Fe and Fe-lys to the S. oleracea plants. A total volume of 2 L of Fe-lys and 1 L of Fe-lys was used in the whole experiment in each treatment, and every treatment was replicated five times, with three plants in each pot. Under the control conditions of the glasshouse, tannery wastewater was added in the pots, and all pots were given 0, 33, 66 and 100% wastewater on the basis of the treatments, when needed with other intercultural operations such as weeding. Moreover, with the addition of tannery wastewater (at least) once a week, we also sprayed Zn-lys regularly, on the basis of demand, on whole plant parts. In order to maintain an optimum amount of micronutrients in plant organs, specific amounts of fertilizers in the form of phosphate and potassium sulphate were also applied, as previously described [45]. The pots used in this study (under the glasshouse environment) were rotated regularly in order to avoid environmental effects on the plants.

Plant Harvesting
Plants of S. oleracea were carefully rooted-up after 30 days of experimental treatment (precisely after 60 days of germination) and washed gently with the help of distilled water to eliminate the aerial dust and deposition. All the harvested plants were divided into two parts, i.e., roots and shoots to study different biological traits. Plant height was measured straightway after the harvesting using a measuring scale, and the number of leaves per plant was also counted. The harvested plant samples were attentively washed with the help of de-ionized water, followed by oven-drying at 70 • C for three days, and were ground in a stainless steel mill and passed through a 0.1 mm nylon sieve for further investigation.

Determination of Photosynthetic Pigments and Gaseous Exchange Parameters
A certain weight of plant leaf samples was crushed and kept in the tubes containing 85% acetone (v/v). The tubes were placed in the dark for the extraction of pigments for 24 h. The tubes were then centrifuged at 4000× g at 4 • C for 10 min. The supernatant was used to measure absorbance at 470, 647 and 664.5 nm by using a spectrophotometer (Halo DB-20/ DB-20S, UK). Chlorophyll contents were measured as recommended by Lichtenthaler [53]. Net photosynthesis, stomatal conductance, Sustainability 2020, 12, 6690 5 of 22 transpiration rate and water use efficiency were measured from three different plants in each treatment group. The measurements were conducted between 9 am and 11 am on days with clear sky with a portable IRGA (Infra-Red Gas Analyzer, Hoddesdon, UK).
2.5. Determination of Malondialdehyde (MDA), Hydogen Peroxide (H 2 O 2 ) and Electrolyte Leakage (EL) The degree of lipid peroxidation was evaluated as malondialdehyde (MDA) content. Briefly, 0.1 g of frozen leaves were ground at 4 • C in a mortar with 25 mL of 50 mM phosphate buffer solution (pH 7.8) containing 1% polyethene pyrrole. The homogenate was centrifuged at 10,000× g at 4 • C for 15 min. The mixtures were heated at 100 • C for 15-30 min and then quickly cooled in an ice bath. The absorbance of the supernatant was recorded using a spectrophotometer (xMark™ microplate absorbance spectrophotometer; Bio-Rad, United States) at wavelengths of 532, 600 and 450 nm. Lipid peroxidation was expressed as l mol g −1 using the following formula: 6.45 (A532−A600)−0.56 A450. Lipid peroxidation was measured using a method previously published by Heath and Packer [54].
To estimate the H 2 O 2 content of plant tissues (root and leaf), 3 mL of sample extract was mixed with 1 mL of 0.1% titanium sulfate in 20% (v/v) H 2 SO 4 and centrifuged at 6000 g for 15 min. The yellow color intensity was evaluated at 410 nm. The H 2 O 2 level was computed by an extinction coefficient of 0.28 mmol −1 cm −1 .
The stress-induced electrolyte leakage (EL) of uppermost stretched leaves was determined by Dionisio-Sese and Tobita's [55] method. The leaves were cut into minor slices (5 mm length) and placed in test tubes containing 8 mL distilled water. These tubes were incubated and transferred into a water bath for 2 h prior to measuring the initial electrical conductivity (EC 1 ). The samples were autoclaved at 121 • C for 20 min, and then cooled down to 25 • C before measuring the final electrical conductivity (EC 2 ). Electrolyte leakage was measured using a pH/conductivity meter (model 720, INCO-LAB Company, Kuwait) and calculated as: To evaluate enzyme activities, fresh leaves (0.5 g) were homogenized in liquid nitrogen and 5 mL of 50 mmol sodium phosphate buffer (pH 7.0) including 0.5 mmol ethylenediaminetetraacetic acid (EDTA) and 0.15 mol NaCl. The homogenate was centrifuged at 12,000× g for 10 min at 4 • C, and the supernatant was used for the measurement of SOD and POD activities. SOD activity was assayed in a 3 mL reaction mixture containing 50 mM sodium phosphate buffer (pH 7), 56 mM nitro blue tetrazolium, 1.17 mM riboflavin, 10 mM methionine and 100 µL enzyme extract. Finally, the sample was measured using a spectrophotometer (xMark™ microplate absorbance spectrophotometer; Bio-Rad). Enzyme activity was measured using a method by Chen and Pan [56] and expressed as U g −1 FW. POD activity in the leaves was estimated using the method of Sakharov and Ardila [57] using guaiacol as the substrate. A reaction mixture (3 mL) containing 0.05 mL of enzyme extract, 2.75 mL of 50 mM phosphate buffer (pH 7.0), 0.1 mL of 1% H 2 O 2 and 0.1 mL of 4% guaiacol solution was prepared. Increases in the absorbance at 470 nm because of guaiacol oxidation were recorded for 2 min. One unit of enzyme activity was defined as the amount of the enzyme.
Catalase activity was analyzed according to Aebi [58]. The assay mixture (3.0 mL) was comprised of 100 µL enzyme extract, 100 µL H 2 O 2 (300 mM) and 2.8 mL of 50 mM phosphate buffer with 2 mM ETDA (pH 7.0). The CAT activity was measured from the decline in absorbance at 240 nm as a result of H 2 O 2 loss (ε = 39.4 mM −1 cm −1 ).

Determination of Iron (Fe) and Cromium (Cr) Contents from the Plants
The digestion of plant samples was carried out through the di-acid (HNO 3 -HClO 4 ) method. Dry samples (0.5 g) of both roots and leaves were added to a flask containing 10 mL of HNO3-HClO4 (3:1, v:v), and the mixture was kept overnight. After that, 5 mL HNO 3 was added, and the samples were completely digested by placing them on a hot plate as described by Rehman et al. [60]. The concentration of Fe and Cr in the roots and leaves of the plants was measured using an atomic absorption spectrophotometer.

Statistical Analysis
The normality of data was analyzed using IBM SPSS software (Version 21.0. Armonk, NY, USA: IBM Corp) through a multivariate post hoc test, followed by a Duncan's test in order to determine the interaction among significant values. One-way analysis of variance (ANOVA) was used to assess the significance of the variations of Cr among the different plant parts, followed by highest significant deviation (HSD) tests. Where significant, Tukey's HSD post hoc test was used to compare the multiple comparisons of means. The analysis showed that the data in this study were almost normally distributed. Thus, the mean difference between the treatments was deemed significant at p ≤ 0.05 between the treatments. The graphical presentation was carried out using SigmaPlot 12 (Systat Software Inc., San Jose, CA 95110, USA). The Pearson correlation coefficients and heatmap between the measured variables of S. oleracea were also calculated. The plots of principal component analysis on rapeseed parameters were carried out using the Rstudio software (4.3.1) (Rstudio PBC, Boston, MA, USA).

Effect of Foliar Application of Fe-lys on Plant Growth and Biomass under Different Levels of Tannery Wastewater
Alterations in different morphological traits in S. oleracea plants were observed when grown in tannery wastewater. Data regarding different morphological parameters of S. oleracea plants are presented in Figures 1 and 2. According to the results, the toxic contents of Cr in the wastewater caused a significant decrease in all the morphological traits of S. oleracea plants studied in this study (Figures 1 and 2). Compared to the control, a maximum decrease (significantly, p < 0.05) in plant height, number of leaves, leaf area, root length, root fresh weight, root dry weight, leaf fresh weight and leaf dry weight by 52.8, 47.5, 79.7, 48.2, 56.7, 55.0, 67.7 and 68.8%, respectively, was recorded in the plants grown in 100% addition of tannery wastewater without the application of Fe-lys. Consequently, the exogenous supplementation of Fe-lys is helpful to the plants, increasing all the morphological parameters both in a tannery wastewater environment alone and treated with wastewater in the soil. At all levels of tannery wastewater (33%, 66% and 100%), the application of Fe-lys improved all morphological traits, while at 100% tannery wastewater treatment the above mentioned morphological traits increased by 18.1, 34.1, 26.3, 28.0, 15.0, 18.0, 23.2 and 12.7%, respectively, when the exogenous foliar application of Fe-lys was applied. Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed, and mean differences were tested by highest significant deviation (HSD) (P < 0.05). Different lowercase letters on the error bars indicate a significant difference between the treatments. Relative radiance of plastic filter used: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater). Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed, and mean differences were tested by highest significant deviation (HSD) (p < 0.05). Different lowercase letters on the error bars indicate a significant difference between the treatments. Relative radiance of plastic filter used: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater). Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). Oneway ANOVA was performed, and mean differences were tested by HSD (P < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Relative radiance of plastic filter used: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

Effect of Foliar Application of Fe-lys on Chlorophyll Contents and Gaseous Exchange Attributes under Different Levels of Tannery Wastewater
Photosynthetic pigments, along with different gaseous exchange parameters, were significantly decreased in S. oleracea plants when growing in soil having a large amount of tannery wastewater. The increasing level of tannery wastewater (33, 66 and 100%) in soil reduced the photosynthetic pigments and gaseous exchange parameters of S. oleracea plants when compared to control treatment. The data regarding these parameters are presented in Figure 3. Figure 3 shows that the maximum contents of chl, carot, Tr, Gs, Pn and Wi in the plants grown under 100% addition of tannery wastewater in the soil decreased (significantly, P < 0.05) by 43.1, 28.4, 35.7, 30.1, 71.1 and 41.8%, respectively, compared to the control treatment. Although the exogenous supplementation of Fe-lys increased (significantly, P < 0.05) chl, carot, Tr, Gs, Pn and Wi by 16.4, 22.5, 4.5, 34.5, 6.9 and 89.0%, respectively, in the plants grown under 100% addition of tannery wastewater in the soil without supplementation of Fe-lys, compared to the plants grown under 100% addition of tannery wastewater in the soil with the supplementation of Fe-lys. Moreover, it was also noticed that the Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed, and mean differences were tested by HSD (p < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Relative radiance of plastic filter used: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

Effect of Foliar Application of Fe-lys on Chlorophyll Contents and Gaseous Exchange Attributes under Different Levels of Tannery Wastewater
Photosynthetic pigments, along with different gaseous exchange parameters, were significantly decreased in S. oleracea plants when growing in soil having a large amount of tannery wastewater. The increasing level of tannery wastewater (33, 66 and 100%) in soil reduced the photosynthetic pigments and gaseous exchange parameters of S. oleracea plants when compared to control treatment. The data regarding these parameters are presented in Figure 3. Figure 3 shows that the maximum contents of chl, carot, Tr, Gs, Pn and Wi in the plants grown under 100% addition of tannery wastewater in the soil decreased (significantly, p < 0.05) by 43.1, 28.4, 35.7, 30.1, 71.1 and 41.8%, respectively, compared to the control treatment. Although the exogenous supplementation of Fe-lys increased (significantly, p < 0.05) chl, carot, Tr, Gs, Pn and Wi by 16.4, 22.5, 4.5, 34.5, 6.9 and 89.0%, respectively, in the plants grown under 100% addition of tannery wastewater in the soil without supplementation of Fe-lys, compared to the plants grown under 100% addition of tannery wastewater in the soil with the supplementation of Fe-lys. Moreover, it was also noticed that the application of Fe-lys also increased the photosynthetic pigments and gaseous exchange parameters without the addition of different levels of tannery wastewater in the soil.
Sustainability 2020, 12, x FOR PEER REVIEW 9 of 23 application of Fe-lys also increased the photosynthetic pigments and gaseous exchange parameters without the addition of different levels of tannery wastewater in the soil.  Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed, and mean differences were tested by HSD (p < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Relative radiance of plastic filter used: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

Effect of Foliar Application of Fe-lys on Oxidative Stress and Antioxidant Response under Different Levels of Tannery Wastewater
The increased contents of MDA, H 2 O 2 and EL (%) indicate that the high contents of metal in the soil used caused oxidative damage in S. oleracea plants. Similarly, the activities of various antioxidative enzymes also increased with the addition of tannery wastewater in the soil, when compared to the control treatment. The data regarding oxidative stress and antioxidant response in the roots and leaves of S. oleracea plants are presented in Figures 4-6, respectively. Compared to the control treatment, maximum contents of MDA, H 2 O 2 initiation and EL (%) were shown in the plants grown in 100% addition of tannery wastewater in the soil, which increased 3.9, 1.8 and 3.6 times, respectively, in the roots and 2.6, 3.4 and 3.2 times, respectively, in the leaves. However, the exogenous application of      Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed, and mean differences were tested by HSD (p < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Relative radiance of plastic filter used: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).
in the roots, under the application of iron-lysine to S. oleracea plants. Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed, and mean differences were tested by HSD (P < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Relative radiance of plastic filter used: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater). Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed, and mean differences were tested by HSD (P < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Relative radiance of plastic filter used: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

Effect of Foliar Application of Fe-lys on the Uptake and Accumulation of Cr and Fe under Different Levels of Tannery Wastewater
The results of the Cr and Fe uptake in the roots and shoots of S. oleracea plants were also determined in this study (Figure 7). The data suggested that increasing levels of wastewater in the soil caused a significant increase in Cr uptake in all organs of the plants. At the same time, a further addition of wastewater caused a significant decrease in Fe contents in all organs of the S. oleracea plants (Figure 7). According to the results, maximum contents of Cr were observed in the plants grown in 100% addition of tannery wastewater in the soil, which were 118 mg kg −1 in the roots and 33.5 mg kg −1 in the shoots of S. oleracea plants. In contrast, Fe content was found to be in its maximum level in the control treatment (201 mg kg −1 in roots and 132 mg kg −1 in the shoots). At the same time, a further addition of wastewater in the soil significantly decreased Fe contents in all organs of S. oleracea plants. The exogenous application of Fe-lys decreased Cr contents and increased Fe contents in all the treatments of wastewater compared to the treatments without application of Fe-lys and grown in tannery wastewater. However, we also noticed that the application of Fe-lys decreased Cr contents and increased Fe contents in the control treatment or the plants grown without addition of tannery wastewater in the soil. Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed, and mean differences were tested by HSD (p < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Relative radiance of plastic filter used: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

Effect of Foliar Application of Fe-lys on the Uptake and Accumulation of Cr and Fe under Different Levels of Tannery Wastewater
The results of the Cr and Fe uptake in the roots and shoots of S. oleracea plants were also determined in this study (Figure 7). The data suggested that increasing levels of wastewater in the soil caused a significant increase in Cr uptake in all organs of the plants. At the same time, a further addition of wastewater caused a significant decrease in Fe contents in all organs of the S. oleracea plants (Figure 7). According to the results, maximum contents of Cr were observed in the plants grown in 100% addition of tannery wastewater in the soil, which were 118 mg kg −1 in the roots and 33.5 mg kg −1 in the shoots of S. oleracea plants. In contrast, Fe content was found to be in its maximum level in the control treatment (201 mg kg −1 in roots and 132 mg kg −1 in the shoots). At the same time, a further addition of wastewater in the soil significantly decreased Fe contents in all organs of S. oleracea plants. The exogenous application of Fe-lys decreased Cr contents and increased Fe contents in all the treatments of wastewater compared to the treatments without application of Fe-lys and grown in tannery wastewater. However, we also noticed that the application of Fe-lys decreased Cr contents and increased Fe contents in the control treatment or the plants grown without addition of tannery wastewater in the soil. Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed, and mean differences were tested by HSD (P < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Relative radiance of plastic filter used: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

Relationship between Morpho-Physiological Attributes and Cr Uptake in Different Parts of the Plants
The Pearson correlation analysis was carried out to quantify the relationship between Cr uptake/accumulation and different morphological and physiological attributes of S. oleracea plants (Figure 8). Cr concentration in the roots was positively correlated with Cr concentration in the shoots, malondialdehyde contents in the roots and leaves, hydrogen peroxide contents in the roots and leaves and electrolyte leakage in the roots and leaves of S. oleracea plants while negatively correlated with plant height, fresh biomass of roots and leaves, dry biomass of roots and leaves, root length, number of leaves, leaf area, total chlorophyll contents, carotenoid contents, transpiration rate, stomatal conductance, net photosynthesis, water use efficiency, superoxide dismutase activity in the roots and leaves, peroxidase activity in the roots and leaves, catalase activity in the roots and leaves, ascorbate peroxidase activity in the roots and leaves and iron contents in the roots and shoots of S. oleracea. Similarly, Cr concentration in the shoots was positively correlated with Cr concentration in the roots, oxidative stress indicators while negative correlated with all other morpho physiological traits and Fe uptake. This correlation reflected the close connection between Cr uptake and growth in S. oleracea. Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed, and mean differences were tested by HSD (p < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Relative radiance of plastic filter used: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

Relationship between Morpho-Physiological Attributes and Cr Uptake in Different Parts of the Plants
The Pearson correlation analysis was carried out to quantify the relationship between Cr uptake/accumulation and different morphological and physiological attributes of S. oleracea plants (Figure 8). Cr concentration in the roots was positively correlated with Cr concentration in the shoots, malondialdehyde contents in the roots and leaves, hydrogen peroxide contents in the roots and leaves and electrolyte leakage in the roots and leaves of S. oleracea plants while negatively correlated with plant height, fresh biomass of roots and leaves, dry biomass of roots and leaves, root length, number of leaves, leaf area, total chlorophyll contents, carotenoid contents, transpiration rate, stomatal conductance, net photosynthesis, water use efficiency, superoxide dismutase activity in the roots and leaves, peroxidase activity in the roots and leaves, catalase activity in the roots and leaves, ascorbate peroxidase activity in the roots and leaves and iron contents in the roots and shoots of S. oleracea.
Similarly, Cr concentration in the shoots was positively correlated with Cr concentration in the roots, oxidative stress indicators while negative correlated with all other morpho physiological traits and Fe uptake. This correlation reflected the close connection between Cr uptake and growth in S. oleracea.

Principal Component Analysis
The score and loading plots of principal component analysis (PCA) to evaluate the effects of various levels of levels of tannery wastewater with the foliar application of Fe lys on some important studied attributes of S. oleracea plants are given in Figure 9. Among all the principal components, first two components i.e. PC1 (Dim 1) and PC2 (Dim 2) exhibited maximum contribution and accounted for 94.8% of the total variance in the database (Figure 9). Of which, PC1 contributed 74.2%, while PC2 contributed 20.6%, accordingly. All of the 8 treatments were distributed successfully by first two principal components. This distribution of treatments gave a clear indication that increasing level of tannery wastewater had significant effects on studied attributes of S. oleracea s compared to control. The tannery wastewater treatments were displaced control (1), indicating that higher Cr concentration imposed hazardous impacts on growth and ecophysiology of S. oleracea plants. The

Principal Component Analysis
The score and loading plots of principal component analysis (PCA) to evaluate the effects of various levels of levels of tannery wastewater with the foliar application of Fe lys on some important studied attributes of S. oleracea plants are given in Figure 9. Among all the principal components, first two components i.e. PC1 (Dim 1) and PC2 (Dim 2) exhibited maximum contribution and accounted for 94.8% of the total variance in the database (Figure 9). Of which, PC1 contributed 74.2%, while PC2 contributed 20.6%, accordingly. All of the 8 treatments were distributed successfully by first two principal components. This distribution of treatments gave a clear indication that increasing level of tannery wastewater had significant effects on studied attributes of S. oleracea s compared to control. The tannery wastewater treatments were displaced control (1), indicating that higher Cr concentration imposed hazardous impacts on growth and ecophysiology of S. oleracea plants. The first group of variables with which PC1 is positively correlated include CAT, EL and Cr contents in the leaves. While, a significant negative correlation of PC1 variables was found with the variables aligned with SOD activity in the roots, Fe contents in the leaves, plant height, leaf area and carotenoid contents in the leaves.

Discussion
High levels of heavy metals in arable land, due to the existence of industrial effluents and industrial waste, pose a serious ecological threat to plants having various important antioxidants [61][62][63][64][65]. The addition of wastewater seriously affects plant growth and biomass [1,6], due to the toxic effects of heavy metals in the soil [66][67][68]. In the current study, increasing levels of wastewater (33, 66 and 100%) in soil displayed inhibitory effects on all morphological traits of the S. oleracea plants, by declining different parameters of plant growth and biomass when compared to the control treatment (Figures 2 and 3). We also elucidated that the application of Fe-lys increased plant growth and biomass even in the toxic levels of Cr in soil (Figures 2 and 3). Several studies showed that the addition of wastewater in the soil directly influenced the plants' composition and caused their significant reduction (P < 0.05) [11,34]. This is because Cr is the most toxic heavy metal, causes a delay in seed germination [25] and ultimately decreases plant growth and biomass [16,21,27]. However, the application of Fe-lys ameliorates Cr toxicity in the S. oleracea plants by Fe fortification. A similar trend was recorded by Bashir et al. [45], who studied Oryza sative growth under toxic levels of Cd and reported that the foliar spray of Fe-lys ameliorates Cd toxicity by decreasing its contents and increasing plant growth and biomass, compared to plants grown in the absence of Fe-lys. In the present study, we also noticed that the application of Fe-lys increased plant growth and biomass in the plants which are not grown in the tannery wastewater soil (Figures 2 and 3). This is because Fe-

Discussion
High levels of heavy metals in arable land, due to the existence of industrial effluents and industrial waste, pose a serious ecological threat to plants having various important antioxidants [61][62][63][64][65]. The addition of wastewater seriously affects plant growth and biomass [1,6], due to the toxic effects of heavy metals in the soil [66][67][68]. In the current study, increasing levels of wastewater (33, 66 and 100%) in soil displayed inhibitory effects on all morphological traits of the S. oleracea plants, by declining different parameters of plant growth and biomass when compared to the control treatment (Figures 2  and 3). We also elucidated that the application of Fe-lys increased plant growth and biomass even in the toxic levels of Cr in soil (Figures 2 and 3). Several studies showed that the addition of wastewater in the soil directly influenced the plants' composition and caused their significant reduction (p < 0.05) [11,34]. This is because Cr is the most toxic heavy metal, causes a delay in seed germination [25] and ultimately decreases plant growth and biomass [16,21,27]. However, the application of Fe-lys ameliorates Cr toxicity in the S. oleracea plants by Fe fortification. A similar trend was recorded by Bashir et al. [45], who studied Oryza sative growth under toxic levels of Cd and reported that the foliar spray of Fe-lys ameliorates Cd toxicity by decreasing its contents and increasing plant growth and biomass, compared to plants grown in the absence of Fe-lys. In the present study, we also noticed that the application of Fe-lys increased plant growth and biomass in the plants which are not grown in the tannery wastewater soil (Figures 2 and 3). This is because Fe-lys enhances protein properties (metabolic function and stock of amino acid function in plants), increases the photosynthetic processes for creating a healthy plant and provides a substantial growth in a short time in the stress condition or even in the plants grown in a normal soil [45].
Photosynthetic traits are important biological parameters when studying environmental stress response in plants [65,[69][70][71]. In the current study, photosynthetic pigments and gaseous exchange traits were affected significantly (p < 0.05) by the addition of different levels of tannery wastewater (33, 66 and 100%) in the soil (Figure 3), while under toxic concentrations of Cr in soil, the photosynthetic machinery was improved by the exogenous application of Fe-lys to Cr-stressed plants (Figure 3). The decrease in chlorophyll contents and gaseous exchange traits under Cr stress is the common response in plants, when they are subjected to the Cr-stressed environment [5,20,72]. Several experiments showed that the photosynthetic machinery was severely affected under a toxic concentration of Cr in soil [17,21,30]. Micronutrients chelated with amino acids increased photosynthetic pigments, compared to plants grown without the application of micronutrient chelation with amino acids [42,73]. In a Cr stress environment, the increase in photosynthetic pigments and gaseous exchange traits might be due to the increase in nutrient uptake and decrease in Cr contents by the plants [74,75].
In addition to the decrease in plant growth and composition, redox imbalance is another toxic effect of Cr in the plants [11,21,26]. Previously, the phytotoxicity of Cr caused oxidative stress in the plants through the generation of extra reactive oxygen species (ROS) [24,72]. However, plants have well-organized enzymatic and non-enzymatic antioxidants which scavenge ROS production [16,76,77]. Toxic levels of Cr in the soil led to oxidative damage to the membranous bounded organelles in Oryza sative [27], Brassica napus [78] and Helianthus annuus [25]. The increasing contents of MDA, initiation of H 2 O 2 and EL (%) suggested that the phytotoxicity of Cr may damage cellular organelles and alter the function of the plasma membrane, which ultimately decreases plant growth and biomass, as shown in Helianthus annuus by Farid et al. [79]. Cr toxicity also induced oxidative damage by increasing the contents of MDA, H 2 O 2 and EL (%) in the roots and leaves of S. oleracea plants (Figure 4), and increased the activities of various antioxidants studied by increasing the addition of tannery wastewater in the soil (Figures 5 and 6). Increasing the activities of various antioxidants suggests that the plant has a better defense system, which can scavenge ROS generation [80][81][82]. Furthermore, foliar applications of Fe-lys to the Cr-stressed plants further enhanced the activities of various antioxidants studied in this experiment, compared to plants grown in the absence of Fe-lys ( Figures 5 and 6). Moreover, lower oxidative stress indicators in the plants treated with Fe-lys might be due to the decreased Cr contents and increased Fe contents in the different plant organs (Figure 7). It is well known that the exogenous supplementation of Fe-lys decreases oxidative stress in plants [74,75]. Previously, the foliar spray of Fe-lys decreased the oxidative stress in Oryza sative by increasing the activities of various antioxidants, compared to plants grown in the absence of Fe-lys [45].
In the current study, the increasing levels of wastewater (33, 66 and 100%) in the soil increased Cr contents and decreased Fe contents in all organs of S. oleracea plants (Figure 7). However, the exogenous supplementation of Fe-lys decreased Cr contents and increased the essential micronutrient (Fe) in all organs of S. oleracea plants (Figure 7). In many previous studies, it was also observed that the increasing levels of Cr in the soil caused a significant (p < 0.05) increase in Cr contents in all organs of the plants [5,11,19,21]. The decrease in essential nutrients such as Fe might be linked with the decrease in the nutrient uptake by the plants, which might be due to excess contents of Cr in the soil [18,19,24,41]. It is also well documented that Cr is extremely toxic for plants and disturbs metabolic processes, which ultimately decreases the quantity of essential nutrients in plant organs and thus affects plant growth and yield [16,72,83]. Previously, Zaheer et al. [5] also reported the same trend, showing that various levels of Cr in the soil also enhance Cr contents in different plant parts and reduce essential nutrients due to excess contents of Cr in plant tissues. However, the availability and toxicity of Cr can be overcome by the exogenous application of micronutrients chelated with amino acids [42,75]. The present study also demonstrated that the exogenous supplementation of Fe-lys decreased Cr contents and increased Fe contents in various parts of S. oleracea plants, compared to plants grown in the absence of Fe-lys (Figure 7). The reduced Cr concentration in all organs of S. oleracea plants might be due to the higher Fe contents in the plants, as Cr and Fe showed antagonistic effects reported previously by Danish et al. [29]. However, the reduction in Cr contents might also be due to the increase in Fe contents in different organs of the plants (Figure 7). Moreover, under flooded conditions, Fe 2+ is the main form of Fe in the soil, which may compete with Cr at the root surface during plant uptake [11]. Bashir et al. [45] reported that foliar spray of Fe-lys increased the Fe contents and decreased the heavy metal (Cd) contents in all parts of rice (Oryza sative) plants, compared to plants grown in the absence of Fe-lys. It was also reported that both Fe and Cr compete in an environment with an excess of Cr, while the external application of Fe-lys helps the plants to uptake large amounts of Fe and decrease the uptake of Cr by the plant organs [45]. Taken together, the application of Fe-lys helps the plants to maintain their growth and composition and also promotes the uptake of Fe contents and decrease Cr contents in plants grown in an environment with an excess of Cr.

Conclusions
The irrigation of green leafy vegetable crops, such as S. oleracea, with tannery wastewater is dangerous because the latter may contain toxic pollutants (i.e., Cr) which significantly affect the plants' growth and yield. The phytotoxicity of Cr also decreases plant photosynthetic pigments and induces oxidative damage due to the overproduction of ROS in internal tissues. Moreover, a toxic level of Cr in the soil decreases the essential nutrients (i.e., Fe) in the plant organs, which are essential for the normal growth and development of the plant's body. The phytotoxicity of Cr can be overcome by the exogenous application of Fe-lys, which increases not only the morphological traits but also the activities of various antioxidants, which helps in mitigating oxidative stress in the plants. Moreover, the supplementation of Fe-lys increases the amino acids in the plant organs which restrict the movement of toxic heavy metals and reduce their availability for the plant. Hence, the application of Fe-lys under a heavy metal stress environment is a safer option to get the maximum yield from green leafy vegetable crops, to fulfill the market demand for the vegetables.