Interactive role of zinc and iron lysine on Spinacia oleracea L. growth, photosynthesis and antioxidant capacity irrigated with tannery wastewater

Abstract

Abstract

Untreated wastewater contains toxic amounts of heavy metals such as chromium (Cr), which poses a serious threat to the growth and physiology of plants when used in irrigation. Though, Cr is among the most widespread toxic trace elements found in agricultural soils due to various anthropogenic activities. To explore the interactive effects of micronutrients with amino acid chelators [iron-lysine (Fe-lys) and zinc-lysine (Zn-lys)], pot experiments were conducted in a controlled environment, using spinach (Spinacia oleracea L.) plant irrigated with tannery wastewater. S. oleracea was treated without Fe and Zn-lys (0 mg/L Zn-lys and 0 mg/L Fe-lys) and also treated with various combinations of (interactive application) Fe and Zn-lys (10 mg/L Zn-lys and 5 mg/L Fe-lys), when cultivated at different levels [0 (control) 33, 66 and 100%) of tannery wastewater in the soil having a toxic level of Cr in it. According to the results, we have found that, high concentration of Cr in the soil significantly (P < 0.05) reduced 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 (E), stomatal conductance (gs), net photosynthesis (PN), and water use efficiency (WUE) and the contents of Zn and Fe in the plant organs without foliar application of Zn and Fe-lys. Moreover, phytotoxicity of Cr increased malondialdehyde (MDA) contents in the plant organs (roots and leaves), which induced oxidative damage in S. oleracea manifested by the contents of hydrogen peroxide (H₂O₂) and membrane leakage. The negative effects of Cr toxicity could be overturned by Zn and Fe-lys application, which significantly (P < 0.05) increase plant growth, biomass, chlorophyll content, and gaseous exchange attributes by reducing oxidative stress (H₂O₂, MDA, EL) and increasing the activities of various antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD) catalase (CAT) and ascorbate peroxidase (APX). Furthermore, the supplementation of Zn and Fe-lys increased the contents of essential nutrients (Fe and Zn) and decreased the content of Cr in all plant parts compared to the plants cultivated in tannery wastewater without application of Fe-lys. Taken together, foliar supplementation of Zn and Fe-lys alleviates Cr toxicity in S. oleracea by increased morpho-physiological attributes of the plants, decreased Cr contents and increased micronutrients uptake by the soil, and can be an effective in heavy metal toxicity remedial approach for other crops.

Graphic abstract

Introduction

Heavy metals are significant environmental pollutants, and their toxicity is a problem of increasing significance for ecological, evolutionary, nutritional and environmental reasons (Ashraf et al. 2017; Nagajyoti et al. 2010). Heavy metals are largely found in dispersed form in rock formations. Industrialization and urbanization have increased the anthropogenic contribution of heavy metals in biosphere. They also have largest availability in soil and aquatic ecosystems and to a relatively smaller proportion in atmosphere as particulate or vapours (Kamran et al. 2020; Rehman et al. 2019b). Moreover, heavy metal toxicity in plants varies with plant species, specific metal, concentration, chemical form and soil composition and pH, as many heavy metals are considered to be essential for plant growth (Saleem et al. 2020a). Triggered by exorbitantly increasing population, rapid industrialization and urbanization posed the menace of air water and soil pollution which is a potential threat to mankind. Among industries, tanneries played a leading role in polluting the soil and water bodies. The wastewater discharged from the tanning industry contains numerous pollutants including heavy metals (Riaz et al. 2019; Zaheer et al. 2020a). While wastewater use can carry essential nutrients and other elements that the plants need to grow and develop normally, and also reduce the use of important fertilizers such as nitrogen (N), phosphorus (P) and potassium (K) (Hussain et al. 2018; Rizwan et al. 2017). Although the use of wastewater for crop irrigation has fewer advantages, it is very harmful and dangerous to use for vegetative crop cultivation, as it may contain harmful organic or inorganic pollutants, which may reduce crop yields and productivity (Maqbool et al. 2018). Additionally, wastewater irrigation can also carry some toxic heavy metals such as chromium (Cr) in the soil, which is highly toxic to normal plant growth and development and major risk for human health (Gill et al. 2015; Hussain et al. 2018). Tannery wastewater can easily contaminate the food chain with different toxic metals particularly with Cr. More than 5.0 mg L^–1 concentration of Cr in growing medium induces toxic effects in plants (Kumar et al. 2016; Shahid et al. 2017). However, the toxicity was dependent upon the Cr species, nature of soil, plant type and cultivar. It has been reported that Cr stress imposed severe toxic effects on plants (Zaheer et al. 2020b). Excess Cr in plants may cause toxic effects in plants and reduce the growth, photosynthesis, mineral nutrients, and quality of the crops (Ranieri et al. 2020; Zaheer et al. 2020a). Combustion of oil, coal and waste from chemical, metallurgy and tannery industrial effluents adds 2000–5000 mg Cr L^–1 in contrast to the acceptable limit of 2 mg Cr L^–1 which degrade the soil through excessive uptake of Cr (Gill et al. 2015; Shahid et al. 2017).

Higher Cr levels in plants cause ultra-structural alteration (Madhu and Sadagopan 2020), oxidative stress in plants and increased electrolyte leakage (EL), malondialdehyde (MDA) concentrations, whereas induced alterations in antioxidant enzyme activities such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and ascorbate peroxidase (APX) (Imran et al. 2019; Rehman et al. 2019a; Saleem et al. 2020f). The overproduction of ROS is generally considered the primary response of plants to heavy metal stress (Parveen et al. 2020; Saleem et al. 2020c). These ROS include: hydrogen peroxide (H₂O₂), singlet oxygen (½O₂), superoxide anion (O₂^•–), hydroxyl (HO^•), alkoxyl (RO^•), peroxyl (RO₂^•), and organic hydroperoxide (ROOH). These ROS are naturally produced in plants as by-products of numerous normal aerobic biochemical reactions taking place in various plant organelles such as peroxisomes, mitochondria and chloroplasts (Saleem et al. 2020b; Zaheer et al. 2020c). Different environmental stresses, such as heavy metal stress, drought or salinity can disrupt a delicate balance in ROS production and ROS scavenging (Kamran et al. 2019; Maqbool et al. 2018; Zaheer et al. 2019). Under normal/natural conditions, ROS are involved in various essential metabolisms of plants such as regulation of stomatal conductance, signal transduction for programmed cell death, alleviation of seed dormancy, senescence, growth regulation, fruit ripening and initiation of defense metabolism under stress (Danish et al. 2019a; Tang et al. 2019). In order to control the deleterious effect of ROS-induced oxidative stress, plants have evolved a well-developed and complex ROS scavenging enzyme mechanism comprising of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) (Mohamed et al. 2020; Saleem et al. 2020g; Zaheer et al. 2020b). Previously, increasing the activities of various antioxidant enzymes were found in Zea mays (Danish et al. 2019a), Helianthus annuus (Qadir et al. 2020), and Lemna minor (Varga et al. 2013) when grown in the toxic level of Cr in the soil. Moreover, spinach (Spinacia oleracea L.) also can tolerate heavy metal stress, due to its strong antioxidant defence system and various other physiological mechanisms (Maqbool et al. 2018; Zaheer et al. 2019; Zubair et al. 2019). S. oleracea is a green leafy flowering plant, belongs to the family Amaranthaceae (commonly known as Amaranth family), and an edible vegetable crop. It is an annual crop, grown mostly in the world's temperate region and native to central and western Asia (Danish et al. 2019b). Since S. oleracea has the ability to cultivate various stresses to the environment, due to its specific biological and physiological processes, it can also withstand Cr stress (Maqbool et al. 2018; Zaheer et al. 2020c; Zubair et al. 2019). We have also demonstrated previously that S. oleracea can tolerate Cr stress when irrigated with various levels of tannery wastewater in the soil (Zaheer et al. 2019).

With the advancement of scientific knowledge, many new techniques are introduced in the field which helps a plant tolerate under conditions of abiotic stress. Amino acids are organic compounds and protein building blocks, plays a crucial role in many processes and mitigates the impact on a plant from environmental stress conditions (Rehman et al. 2020; Saleem et al. 2020d). On the other hand, iron (Fe) and zinc (Zn) are essential micronutrients and their deficiency can reduce the growth, yield, and nutritional quality of agricultural crops worldwide. Recently, researchers have introduced a new method for using lysine [lys (amino acid)] chelated zinc and iron, which helps in a plant's stress condition. In many previous studies, micronutrients such as Zn and Fe chelated with lys have been reported to relieve heavy metal stress by increasing plant growth and biomass (Rafie et al. 2017; Saifullah et al. 2016; Souri 2016). Furthermore, application of Zn and Fe chelated with lys also boosted the activities of antioxidative enzymes and increased the chlorophyll pigments in the plant. This is because of Zn and Fe are an essential micronutrient for the plants and perform a very vital role in different metabolic processes of plant like photosynthesis, respiration and DNA synthesis and also act as a cofactor for many enzymes involved in oxygen and electron transfer. Application of these micronutrients greatly reduced the toxicity induced by the toxic heavy metals. Exogenous supplementation of Fe-lys increased plant growth and biomass in Oryza sativa (Bashir et al. 2018) and S. oleracea (Zaheer et al. 2019) while application of Zn-lys reported to increase plant growth and biomass in Triticum aestivum (Rizwan et al. 2017), Oryza sativa (Hussain et al. 2018) under excess concentration of heavy metals in the soil. Although there are many previous studies on Fe and Zn-lys on different crops under different heavy metal stresses (Bashir et al. 2018; Hussain et al. 2018; Rizwan et al. 2017; Saifullah et al. 2016; Souri 2016; Zaheer et al. 2019, 2020a, b), but there are very few studies on the combination of Fe and Zn-lys on any crop. Thus, in this study, we demonstrated the effect of the concentrations of Fe and Zn-lys on S. oleracea, when grown at different levels of tannery wastewater in the soil. The results from the present study will add to our knowledge about (i) the role of micronutrients chelated with amino acids on plant growth and biomass, (ii) photosynthetic pigments and gaseous exchange attributes, (iii) oxidative stress and antioxidant response and (iv) uptake and accumulation of Cr and micronutrients (Fe and Zn) in different parts of S. oleracea plant, when grown in different levels of tannery wastewater. However, it was also hypothesized that exogenous application of Fe and Zn-lys decreases the Cr concentration in S. oleracea plant by enhancing the contents of essential nutrients from the soil irrigated with tannery wastewater. In addition, it was also assumed that the application of amino acid chelated micronutrients often improves the plant's morphology and physiology by reducing the plant's oxidative stress. The current experiments will therefore offer a new insight into the interactive application of Fe and Zn-lys for Cr uptake/accumulation in various parts of S. oleracea and promises combined supplementation of the Fe and Zn-lys complex under heavy metal stress.

Materials and methods

Collection and analysis of wastewater and soil

Clay loam sand used in this analysis was collected from the botanical garden of the University of Punjab Lahore, Pakistan (31.4015^◦ N, 74.3070^◦ E) from an average depth of 0–20 cm. The soil was thoroughly sieved to 2 mm in order to completely remove the unwanted materials such as previous crop residues and debris. A comprehensive analysis of organic soil content was performed by the process described by Walkley and Black (1934). For the precise size of the soil elements, the hydrometer was used (Bouyoucos 1962). Likewise, electrical conductivity (EC) and sodium adsorption ratio (SAR) and soil ions were also carefully measured by the method of Page (1965). 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 (Amacher 1996; Soltanpour 1985). The physico-chemical characteristics of the soil under study are given in Table 1S. The tannery wastewater used for the pot experiment was collected from the tannery industries based in Kasur, Punjab, Pakistan. Physico-chemical properties of tannery wastewater used in the present study were estimated according to the set protocols of (Apha). Comprehensive details of major characteristics of tannery wastewater used in this experiment are presented in Table 2S. The same soil with the tannery wastewater was used in our previous study, by (Zaheer et al. 2019).

Table 1.

Physico-chemical properties of loam soil used in the pot experiments

Texture	Clay loam
Sand	62.6
Silt	11.9
Clay	23.4
pHs	7.1
ECe (dS m−1)	3.86
Cation exchange capacity (CEC) (cmol kg−1) 4.9	4.78
Soluble CO3−2 − (mmol L−1)	0.85
Soluble HCO3 − (mmol L−1)	3.45
Soluble Cl− (mmol L−1)	5.91
Soluble Ca2+  + Mg2+ (mmol L−1)	14.93
Organic matter (%)	0.52
Ni (mg kg−1)	0.21
Cu (mg kg−1)	0.39
Zn (mg kg−1)	0.64
Cr (mg kg−1)	0.10

Table 2.

Characteristics of tannery wastewater used for irrigation of the soil used in the pot experiments

Parameters	Values	Permissible limits**
EC (dS m−1)	1.41	< 1.5
SAR (mmol L−1)1/2	4.02	< 7.5
RSC (mmol c L−1)	2.24	< 2.0
Ni (mg L−1)	0.09	0.20
Cd (mg L−1)	0.04	0.01
Pb (mg L−1)	1.24	5.0
Co (mg L−1)	0.02	0.05
Cr (mg L−1)	4.03	0.10
Zn (mg L−1)	1.95	2.00

**Ayers and Westcot (1985)

Pot experiment

This study was conducted in botanical garden under glass house environment, of the Department of Botany, University of Punjab Lahore, Pakistan. Healthy and mature seeds of spinach (Spinacia oleracea L.) were collected from Ayyub Agriculture Research Institute Faisalabad, Pakistan. Previously, S. oleracea has been used in the many studies related with Cr-stressed soil (Danish et al. 2019b; Maqbool et al. 2018; Zaheer et al. 2019, 2020c). S. oleracea seeds have been carefully sowed in experimental pots filled with approximately 5 kg of soil after diligent washing with H₂O₂ (10%) (to avoid fungal or bacterial infection) and rinsing with deionized water. Later, five seeds were sowed in each pot. After thinning, three seedlings remained in each pot. After 2 weeks of seed germination, S. oleracea was treated with Fe and Zn-lys (10 mg/L Zn-lys and 5 mg/L Fe-lys), when cultivated at different levels [0 (control) 33, 66 and 100%) of tannery wastewater in the soil having toxic concentration of Cr in it. A hand sprayer was used for the exogenous supplementation of Fe and Zn–lys to the S. oleracea plants. A total volume of 2L of Zn-lys and 1L of Fe-lys used in the whole experiment to each treatment and every treatment has five replications per treatment with three plants in each pot. The present experimental work was conducted in the wire house Department of Botany, the University of the Punjab Lahore, Pakistan, in an open environment, protected from human and animal interactions. The rainfall was controlled, or plants were protected from rainfall by covering the whole wire house with a plastic sheet. In order to maintain optimum amount of micronutrients in plant organs, specific amount of fertilizers in the form of phosphate and potassium sulphate were also applied as described by Bashir et al. (2018). The pots used in this study, (under control condition), were rotated regularly in order to alleviate environmental stress conditions.

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. The sampled leaves were washed with distilled water, immediately placed in liquid nitrogen, and stored in a freezer at low temperature (80 °C) for further analysis. All the harvested plants were divided into two parts i.e. roots and shoots to study different biological traits. Three plants representing per treatment were selected randomly for morphological study. Plant height was measured straightway after the harvesting using measuring scale and number of leaves per plant were also counted after it. The number of leaves and leaf area was measured, and the fresh and dry biomass of the leaves were measured, and then the remaining were analysed for Cr and Zn content from the shoots, after mixing with stems and other shoot parts. All the plant samples (after harvest) were attentively washed with the help of de-ionized water and after that oven dried at 70 °C (for three days) and then grounded to very fine powder to complete further investigation. Roots were immersed in 20 mM Na₂EDTA for 15-20 min to remove Cr adhered to the surface of roots. Then, roots were washed thrice with distilled water and finally once with de-ionized water and dried for further analysis.

Determination of photosynthetic pigments and gaseous exchange parameters

A certain weight of plant leaf samples were 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 1987). Calculations were made by using the following formulas:

$$\text{Chlorophyll a}\left(\text{mg}/\text{g FW}\right)=0.{\text{999A}}_{\text{663}}-0.0{\text{989A}}_{\text{645}}$$

$$\text{Chlorophyll b}\left(\text{mg}/\text{g FW}\right)=0.{\text{328A}}_{\text{663}}+\text{1}.{\text{77A}}_{\text{645}}$$

where C_a is chlorophyll a content and C_b is chlorophyll b contents.

While at bright sunny day (9:00 am – 11:00 am) gas exchange parameters were measured using portable IRGA (Infra-Red Gas Analyzer, Hoddesdon, England) before harvesting the plants. For different gas exchange parameters such as transpiration rate (Tr), stomatal conductance (Gs), Net photosynthesis (Pn), and water use efficiency (Wi), three leaves were selected randomly from each treatment and from three different plants of a single treatment. And we selected three different leaves from each treatment which were alike (means no difference in age were observed).

Determination of malondialdehyde (MDA), hydogen peroxide (H₂O₂) 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 (1968).

To estimate the H₂O₂ 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) H₂SO₄ and centrifuged at 6000 g for 15 min. The yellow color intensity was evaluated at 410 nm. The H₂O₂ 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 (1998) 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 (EC₁). The samples were autoclaved at 121 °C for 20 min, and then cooled down to 25 °C before measuring the final electrical conductivity (EC₂). Electrolyte leakage was measured using a pH/conductivity meter (model 720, INCO-LAB Company, Kuwait) and calculated as:

$$\text{EL}=\left({\text{EC}}_1/{\text{EC}}_2\right)=\times 100$$

Determination of superoxidase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) activity

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 (1996) and expressed as U g⁻¹ FW.

POD activity in the leaves was estimated using the method of Sakharov and Ardila (1999) 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% H₂O₂ and 0.1 mL of 4% guaiacol solution was prepared. Increase 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 (1984). The assay mixture (3.0 mL) was comprised of 100 μL enzyme extract, 100 μL H₂O₂ (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 H₂O₂ loss (ε = 39.4 mM⁻¹ cm⁻¹).

Ascorbate peroxidase activity was measured according to Nakano and Asada (1981). The mixture containing 100 μL enzyme extract, 100 μL ascorbate (7.5 mM), 100 μL H₂O₂ (300 mM) and 2.7 mL 25 mM potassium phosphate buffer with 2 mM EDTA (pH 7.0) was used for measuring APX activity. The oxidation pattern of ascorbate was estimated from the variations in wavelength at 290 nm (ε = 2.8 mM⁻¹ cm⁻¹).

Determination of iron (Fe), Zinc (Zn) and cromium (Cr) contents from the plants

Plant samples were vigilantly digested via di-acid (HNO₃-HClO₄) technique. 0.5 g dry sample of roots and shoots of the plants were taken into the flask having 10 mL of HNO₃-HClO₄ (3:1, v:v), this collection was then retained overnight. Final digestion of these plants’ samples was completed after the addition of HNO₃ (5 mL) and then placed on the hot plate for complete digestion as described by Rehman et al. (2015). Atomic absorption spectrophotometer (AAS) was used to investigate the exact amount of Cr, Fe and Zn in shoots and roots of the plant.

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 the 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. The graphical presentation was carried out using GraphPad prism 8. The Pearson correlation coefficients and heatmap between the measured variables of S. oleracea were also calculated. The plots of principal component analysis on S. oleracea parameters were carried out using the Rstudio software (4.3.1).

Results

Effect of foliar application of Zn and Fe-lys on plant growth and biomass under different levels of tannery wastewater in the soil

In the present study, different growth parameters were measured under different levels of tannery wastewater (33, 66 and 100%) in the soil with or without application of different concentrations of Zn (10 mg/L) and Fe (5 mg/L) lys. The data regarding different morphological traits of S. oleracea grown under different levels of tannery wastewater with or without the application of Zn and Fe-lys are presented in Fig. 1. According to the given results, it has been observed that increasing levels of tannery wastewater in the soil decreased plant growth and biomass [significantly (P < 0.05)], when compared to the plants grown without irrigation with wastewater (Fig. 1). The maximum decrease in leaf fresh and dry weight, root fresh and dry weight, plant and root length, leaf area and number of leaves were observed in the plants grown in 100% addition of tannery wastewater in the soil, which were decreased by 68, 67, 51, 53, 61, 54, 80 and 45% respectively, compared to the plants grown without addition of tannery wastewater in the soil. Although, exogenous supplementation of Zn and Fe-lys increased plant growth and biomass, even grown in different levels of tannery wastewater in the soil (Fig. 1). Figure 1 also presented that at every level of tannery wastewater (33, 66 and 100%) in the soil, interactive application of Zn (10 mg/L) and Fe (5 mg/L) lys increased all growth and biomass-related parameters of S. oleracea studied in this experiment. The plants grown in 100% irrigation with tannery wastewater with the application of Zn and Fe-lys increased leaf fresh and dry weight, root fresh and dry weight, plant and root length, leaf area and number of leaves by 25, 26, 27, 31, 21, 30, 35 and 23% respectively, compared to the plants grown without application of Zn and Fe-lys, when irrigated with 100% tannery wastewater.

Fig. 1.

Effect of Zn and Fe-lys on leaf fresh and dry weight (a), roots fresh and dry weight (b), plant and root length (c), leaf area (d) and number of leaves (e) of S. oleracea, when grown in different levels of tannery wastewater in the soil. Bars sharing similar letter(s) within a column for each parameter do not differ significantly at P < 0.05. Data in the figures are means of three repeats (n = 3) of just one harvest of S. oleracea plants. Error bars represent standard deviation (SD) of three replicates. Different treatments used in this study are as follow: Ck (without irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T1 (without irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T2 (33% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T3 (33% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T4 (66% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T5 (66% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T6 (100% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys) and T7 (100% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys)

Effect of foliar application of Zn and Fe-lys on chlorophyll contents and gaseous exchange attributes under different levels of tannery wastewater in the soil

The contents of total chlorophyll and carotenoid along with different gaseous exchange parameters of S. oleracea grown under different levels of tannery wastewater with or without the application of Zn and Fe-lys are presented in Fig. 2. According to the results, addition of tannery wastewater in the soil, decreased significantly the contents of chlorophyll and carotenoid and gaseous exchange parameters of S. oleracea plants (Fig. 2) significantly (P < 0.05). According to the given results, the contents of total chlorophyll, carotenoid, transpiration rate, stomatal conductance, net photosynthesis and water use efficiency were decreased by 52, 13, 48, 47, 34 and 47% respectively, in the plants irrigated with 100% tannery wastewater compared to the plants grown without addition of tannery wastewater in the soil. However, the contents of chlorophyll and carotenoid and gaseous exchange parameters can be improved in S. oleracea plants by the application of Zn and Fe-lys, even in the plants grown in irrigated tannery wastewater in the soil (Fig. 2). The combined application of Zn and Fe-lys increased total chlorophyll, carotenoid, transpiration rate, stomatal conductance, net photosynthesis and water use efficiency by 28, 21, 29, 31, 27 and 33% in the plants grown 100% irrigated with tannery wastewater without the application of Zn and Fe-lys, compared to the plants grown in 100% irrigated with tannery wastewater with the application of Zn and Fe-lys.

Fig. 2.

Effect of Zn and Fe-lys on chlorophyll contents (a), carotenoid contents (b), transpiration rate (c), stomatal conductance (d), net photosynthesis (e) and water use efficiency (f) of S. oleracea, when grown in different levels of tannery wastewater in the soil. Bars sharing similar letter(s) within a column for each parameter do not differ significantly at P < 0.05. Data in the figures are means of three repeats (n = 3) of just one harvest of S. oleracea plants. Error bars represent standard deviation (SD) of three replicates. Different treatments used in this study are as follow: Ck (without irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T1 (without irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T2 (33% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T3 (33% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T4 (66% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T5 (66% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T6 (100% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys) and T7 (100% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys)

Effect of foliar application of Zn and Fe-lys on oxidative stress and antioxidant response under different levels of tannery wastewater in the soil

In the present study, the contents of malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and percentage of electrolyte leakage (EL) were also measured under different levels of tannery wastewater (33, 66 and 100%) in the soil with or without application of different concentrations of Zn (10 mg/L) and Fe (5 mg/L) lys. The results showed that, increasing concentration of MDA, H₂O₂ and EL (%) in S. oleracea plant under increasing levels of tannery wastewater (33, 66 and 100%) in the soil, showed that high concentrations of heavy metals (Cr) induced oxidative damaged in the roots and leaves of S. oleracea plant (Fig. 3). Compared to the plants grown in the control, the maximum increase of MDA, H₂O₂ and EL (%) by 39, 28 and 80% respectively, in the roots and also increased by 69, 64 and 67% respectively, in the leaves, in the plants grown in 100% irrigation with tannery wastewater in the soil. However, application of Zn and Fe-lys decreased oxidative stress in the roots and leaves of S. oleracea plant by decreasing the contents of MDA, H₂O₂ and EL (%), compared to the plants grown without application of Zn and Fe-lys, when irrigated with different levels of tannery wastewater in the soil (Fig. 3). Compared to the plants, grown in irrigation with 100% tannery wastewater, interactive application of Zn and Fe-lys decreased the contents of MDA, H₂O₂ and EL (%) by 22, 19 and 26% respectively, in the roots and also decreased by 20, 27 and 21% respectively, in the leaves of S. oleracea plants grown in irrigation with 100% tannery wastewater with the exogenous application of Zn and Fe-lys.

Fig. 3.

Effect of Zn and Fe-lys on MDA contents (a), H₂O₂ contents (b) and EL percentage (c) in the roots and leaves of S. oleracea, when grown in different levels of tannery wastewater in the soil. Bars sharing similar letter(s) within a column for each parameter do not differ significantly at P < 0.05. Data in the figures are means of three repeats (n = 3) of just one harvest of S. oleracea plants. Error bars represent standard deviation (SD) of three replicates. Different treatments used in this study are as follow: Ck (without irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T1 (without irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T2 (33% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T3 (33% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T4 (66% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T5 (66% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T6 (100% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys) and T7 (100% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys)

In the present study, irrigation with different levels of tannery wastewater (33, 66 and 100%) in the soil, increased the activities of various antioxidative enzymes such as superoxide dismutase (SOD), peroxidase (POD) catalase (CAT) and ascorbate peroxidase (APX), when compared with the plants grown without irrigation with tannery wastewater in the soil (Fig. 4). Although, the activities of various antioxidants (SOD, POD, CAT and APX) showed irrelevant behaviour i.e., increased initially up to the wastewater level of (33 and 66%), then decreased significantly (P < 0.05) in the plants grown in 100% tannery wastewater in the soil, compared to the plants grown in control (Fig. 4). Compared to the control (without irrigation with tannery wastewater in the soil), the maximum increase in the activities of SOD and POD by 39 and 47% respectively in the roots and also increased by 62 and 49% respectively in the leaves, in the plants grown in 33% irrigation with tannery wastewater in the soil. Similarly, the activities of CAT and APX were also increased by 206, 110% respectively in the roots and also increased by 146 and 79% in the leaves in the plants grown in 66% irrigation with tannery wastewater in the soil, compared to the control. However, combined application of Zn and Fe-lys increased the activities of various antioxidative enzymes studied in this experiment up to 66% of irrigation with tannery wastewater in the soil (Fig. 4). Compared to the control, the activities of SOD, POD, CAT and APX were increased by 46, 50, 53 and 47% in the roots, and also increased by 66, 45, 72 and 45% in the leaves, in the plants grown in 66% of tannery wastewater in the soil.

Fig. 4.

Effect of Zn and Fe-lys on the activities of SOD (a), POD (b), CAT (c) and APX (d) in the roots and leaves of S. oleracea, when grown in different levels of tannery wastewater in the soil. Bars sharing similar letter(s) within a column for each parameter do not differ significantly at P < 0.05. Data in the figures are means of three repeats (n = 3) of just one harvest of S. oleracea plants. Error bars represent standard deviation (SD) of three replicates. Different treatments used in this study are as follow: Ck (without irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T1 (without irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T2 (33% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T3 (33% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T4 (66% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T5 (66% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T6 (100% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys) and T7 (100% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys)

Effect of foliar application of Zn and Fe-lys on uptake and accumulation of Cr, Zn and Fe under different levels of tannery wastewater in the soil

In the present study, the contents of Cr, Zn and Fe from the roots and leaves of S. oleracea plants were also determined. The data regarding the contents of Cr, Zn and Fe of S. oleracea plants grown in different levels of tannery wastewater (33, 66 and 100%) in the soil with or without application of different concentrations of Zn (10 mg/L) and Fe (5 mg/L) lys are presented in Fig. 5. Figure 5 showed that the contents of Cr in the roots and leaves of S. oleracea plants were increased continuously with the increasing levels of tannery wastewater in the soil while the contents of Zn and Fe were decreased continuously in S. oleracea plants with the addition of tannery wastewater in the soil. According to the results, the maximum increase in Cr contents in the roots (128 mg kg^–1 DW) and leaves (34 mg kg^–1 DW) were observed in the plants which were irrigated with 100% tannery wastewater in the soil. However, the contents of Zn and Fe were decreased continuously with the addition of tannery wastewater in the soil and maximum decrease was observed in the roots (11 and 2 mg kg^–1 DW respectively) and leaves (2 and 14 mg kg^–1 DW respectively) in the plants grown in 100% irrigation with tannery wastewater in the soil, compared to the plants grown in control. However, combined application of Zn and Fe-lys decreased the Cr contents in the roots by 25% and in the leaves by 22%, while increased the contents of Zn and Fe by 29 and 36% respectively in the roots and also increased by 30 and 27% respectively in the plants grown in 100% irrigation with tannery wastewater with the combined application of Zn and Fe-lys, compared to those plants grown in 100% irrigation with tannery wastewater without the application of Zn and Fe-lys.

Fig. 5.

Effect of Zn and Fe-lys on the uptake of Cr (a), Zn (b) and Fe (c) contents in the roots and leaves of S. oleracea, when grown in different levels of tannery wastewater in the soil. Bars sharing similar letter(s) within a column for each parameter do not differ significantly at P < 0.05. Data in the figures are means of three repeats (n = 3) of just one harvest of S. oleracea plants. Error bars represent standard deviation (SD) of three replicates. Different treatments used in this study are as follow: Ck (without irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T1 (without irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T2 (33% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T3 (33% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T4 (66% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), T5 (66% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), T6 (100% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys) and T7 (100% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys)

Correlation between Cr uptake with different morphological and physiological attributes

The Pearson correlation analysis was carried out to quantify the relationship between Cr uptake/accumulation and different morphological and physiological attributes of S. oleracea (Fig. 6). Cr concentration in the roots was positively correlated with Cr concentration in the shoots, electrolyte leakage in roots, MDA contents in leaves, H₂O₂ initiation in leaves, MDA contents in roots, electrolyte leakage in leaves and H₂O₂ initiation in roots while negatively correlated with root dry weight, SOD activity in roots, Fe contents in roots, root fresh weight, Fe contents in leaves, leaf area, SOD activity in leaves, root length, Zn contents in leaves, POD activity in roots, plant height, stomatal conductance, Zn contents in roots, number of leaves, water use efficiency, leaves dry weight, transpiration rate, leaves fresh weight, POD activity in leaves, net photosynthesis, total chlorophyll contents, APX activity in leaves, APX activity in roots, carotenoid contents, CAT activity in leaves and CAT activity in roots. Similarly, Cr concentration in the shoots was also positively correlated with Cr concentration in the roots, electrolyte leakage in roots, MDA contents in leaves, H₂O₂ initiation in leaves, MDA contents in roots, electrolyte leakage in leaves and H₂O₂ initiation in roots while negatively correlated with root dry weight, SOD activity in roots, Fe contents in roots, root fresh weight, Fe contents in leaves, leaf area, SOD activity in leaves, root length, Zn contents in leaves, POD activity in roots, plant height, stomatal conductance, Zn contents in roots, number of leaves, water use efficiency, leaves dry weight, transpiration rate, leaves fresh weight, POD activity in leaves, net photosynthesis, total chlorophyll contents, APX activity in leaves, APX activity in roots, carotenoid contents, CAT activity in leaves and CAT activity in roots. This correlation reflected the close connection between Cr uptake and growth in S. oleracea.

Fig. 6.

Correlation between different biological attributes studied in this study. Different abbreviations used are as follow: CR-S (Cr contents in leaves), CR-R (Cr contents in roots), EL-R (electrolyte leakage in roots), MDA-L (MDA contents in leaves), H₂O₂-L (H₂O₂ initiation in leaves), MDA-R (MDA contents in roots), EL-L (electrolyte leakage in leaves), H₂O₂-R (H₂O₂ initiation in roots), RDW (root dry weight), SOD-R (SOD activity in roots), Fe-R (Fe contents in roots), RFW (root fresh weight), Fe-L (Fe contents in leaves), LA (leaf area), SOD-L (SOD activity in leaves), RL (root length), Zn-L (Zn contents in leaves), POD-R (POD activity in roots), PH (plant height), SC (stomatal conductance), Zn-R (Zn contents in roots), NOL (number of leaves), WUE (water use efficiency), LDW (leaves dry weight), TR (transpiration rate), LFW (leaves fresh weight), POD-L (POD activity in leaves), NP (net photosynthesis), TC (total chlorophyll contents), APX-L (APX activity in leaves), APX-R (APX activity in roots), Carot (carotenoid contents), CAT-L (CAT activity in leaves) and CAT-R (CAT activity in roots)

Principal component analysis

The scores and loading plots of Principal component analysis (PCA) to evaluate the effects of different levels of tannery wastewater with the foliar application of Zn and Fe-lys on some selected attributes of S. oleracea plants are presented in Fig. 7. Of all the main components, the first two components-Dim1 and Dim2-comprise more than 95% of the whole database and constitute the largest portion of all the components (Fig. 7a). Among this, Dim1 contributes 83%, and Dim2 contributes 12% of the whole dataset. According to the results, all the respective treatments were dispersed successfully in the whole dataset (Fig. 7a). The distribution of all the components in the dataset gives a clear indication that Cr toxicity in the soil significantly affected all the treatments studied with the interactive application of Zn and Fe-lys. Control (1) was most displaced from all other treatments of tannery wastewater in the soil, indicating that Cr toxicity in the soil significantly affected morpho-physiological traits of S. oleracea plants with the combined application of Zn and Fe-lys. However, Fig. 7b showed that MDA and Cr contents in the leaves were positively correlated in the dataset from all the variables. In contrast, SOD activity in the leaves, Zn and Fe contents in the leaves, plant height, total chlorophyll contents and net photosynthesis were negatively correlated in PCA (Fig. 7b).

Fig. 7.

Score (a) and loading plots (b) of principal component analysis (PCA) on different studied attributes of S. oleracea plants grown in different levels of tannery wastewater in the soil. Score plot represents separation of treatments: 1 (without irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), 2 (without irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), 3 (33% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), 4 (33% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), 5 (66% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys), 6 (66% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys), 7 (100% irrigation with wastewater + 0 mg/L Zn-lys + 0 mg/L Fe-lys) and 8 (100% irrigation with wastewater + 10 mg/L Zn-lys + 5 mg/L Fe-lys). The abbreviations are as follow: Fe–L (Fe contents in leaves), SOD-L (SOD activity in leaves), Zn-L (zinc contents in leaves), PH (plant height), NP (net photosynthesis), TC (total chlorophyll contents), MDA-L (MDA contents in leaves) and CR-R (Cr contents in roots)

Discussion

In the present study, the different morphological attributes in terms of plant growth and biomass decreased significantly (P < 0.05), with the increase of tannery wastewater in the soil (Fig. 1). This is because of the high contents of chromium (Cr) in the tannery wastewater in the soil, which decreased the plant growth and biomass (Fig. 1). The Cr toxicity decreased plant growth and biomass, which were reported previously in Brassica napus (Zaheer et al. 2020a), Helianthus annuus (Qadir et al. 2020) and Hibiscus cannabinus (Ding et al. 2016). The reduction in plant growth and biomass, under different levels of tannery wastewater (33, 66 and 100%) in the soil, might be due to less nutrients uptake by the plant, which was restricted due to high concentration of Cr in the soil (Danish et al. 2019a; Ding et al. 2016; Hussain et al. 2018; Maqbool et al. 2018). Under Cr stress, the reduction of plant growth and biomass, might be due to the alterations in the ultrastructure of mesophyll cell as suggested by Gill et al. (2015) in Brassica napus. In addition, Cr toxicity depends on plant growth and biomass, on the number of factors such as Cr concentration, time duration, plant species, and growth conditions (Gill et al. 2015; Madhu and Sadagopan 2020).

The decrease in chlorophyll content and gaseous exchange attributes in S. oleracea plants could be attributed to Cr toxicity in the present study (Fig. 2). Tannery wastewater has been reported to have reduced chlorophyll content and gaseous exchange which may be due to the Cr toxicity affecting photosynthetic apparatus of the plant by distributing its structure (Danish et al. 2019a). This could be due to the deterioration in chloroplast ultra-structure and disturbance in the electron transport chain, due to the toxic level of Cr in the soil tannery wastewater (Maqbool et al. 2018; Qadir et al. 2020). Prior to this, in many studies it has been reported that chlorophyll and gaseous exchange were reduced due to a high concentration of Cr in the soil (Zaheer et al. 2020a, 2020b).

Plants face severe oxidative damage in lipid bounded organelles due to the generation of extra reactive oxygen species (ROS) under high concentration of heavy metals in the soil (Kamran et al. 2020; Saleem et al. 2020e) and electrolyte leakage (Ghasemi et al. 2014; Parveen et al. 2020; Saleem et al. 2020g). Conditions of environmental stress (Cr stress) will disrupt and balance the equilibrium of ROS production and then remove it for healthy plant growth. Hence, ROS is accumulated in the plants under stress conditions, induces membrane lipid peroxidation, which disturebed the work of membrane-bounded structures (Hussain et al. 2018; Rafie et al. 2017; Zaheer et al. 2019). In previous studies, it was reported that toxic contents of Cr in the soil induces oxidative stress in Brassica napus (Zaheer et al. 2020a), S. oleracea (Maqbool et al. 2018), and Triticum aestivum (Datta et al. 2011) and increased contents of MDA, which is the indication of oxidative stress. Cr toxicity induces lipid peroxidation (Gill et al. 2015), could be supported by leakage of the cellular membrane (Rafie et al. 2017; Rizwan et al. 2017; Shahid et al. 2017). Our results reported that increasing tannery wastewater (Cr contents) in the soil, increased oxidative stress, and electrolyte leakage in S. oleracea plants (Fig. 3). The increase in oxidative stress, and electrolyte leakage in S. oleracea plants, which is attributable directly to the rising soil Cr content, induces physiological responses for plants (Datta et al. 2011; Maqbool et al. 2018; Zaheer et al. 2019).

ROS production in the cells/tissues of the plants is toxic, leading to the oxidative damage to various membrane-bound organelles. The plant, however, has a variety of antioxidants enzymes, which scavenge ROS production (Kamran et al. 2020; Saleem et al. 2020d). In the present study, antioxidant activities increased to a level under Cr stress but gradually decreased with the introduction of a large amount of tannery wastewater in the soil (Fig. 4). Increasing antioxidant activities have been found in Helianthus annuus (Qadir et al. 2020), Brassica napus (Zaheer et al. 2020a) and Oryza sativa (Hussain et al. 2018) under the toxic concentration of Cr in the soil. Increasing activity of antioxidant enzymes is seen as markers of increased generation and mitigation of ROS. Nonetheless, decreased activity of antioxidants under high Cr concentration in the soil may be due to the extreme Cr toxicity that denatures the enzymes (Maqbool et al. 2018; Zaheer et al. 2019).

Our results depicted that, increasing tannery wastewater (Cr contents) in the soil, caused a significant increase in Cr contents while decrease is reported in essential nutrients (Zn and Fe) in different parts (roots and leaves) of the S. oleracea plants (Fig. 5). Previously, it was observed that under the toxic levels of Cr in the soil, it causes a significant increase in Cr content in the different tissues of Brassica napus (Zaheer et al. 2020b), Zea mays (Danish et al. 2019a) and S. oleracea (Maqbool et al. 2018). Although, plants uptake low contents of Zn and Fe in their body parts, which might be due to the incapability of the roots to absorb these essential nutrients from the soil under Cr stress (Bashir et al. 2018; Rizwan et al. 2017). Furthermore, it was also reported that the plants which uptake a large amount of heavy metals (Cr contents) are unable to accumulate essential nutrients (Zn and Fe), which are important for their normal growth and development. This might be due to the defect in photosynthetic machinery and disrupture in ultra-structure of chloroplast (Bashir et al. 2018; Ghasemi et al. 2012, 2014). Similar findings were showed by Maqbool et al. (2018) when they observed that S. oleracea plants uptake a large amount of Cr contents when grown in the different levels of tannery wastewater. Although, in our previous study (Zaheer et al. 2019), we also noticed that, under the toxic concentration of Cr in the soil, S. oleracea is unable to accumulate a large amount of essential nutrients (Zn) when grown in the different levels of tannery wastewater in the soil.

The researchers used different strategies to mitigate Cr toxicity by alleviating oxidative stress and increasing crop yield and productivity (Bashir et al. 2018; Ghasemi et al. 2012, 2014; Hussain et al. 2018; Zaheer et al. 2019). Some the techniques are discussed in detail by Shahid et al. (2017). Amino acids are simple organic compounds, which constitutes proteins and it has been reported that amino acids chelated with micronutrients may alleviate abiotic stress in plants (Ghasemi et al. 2014; Rafie et al. 2017). Zn and Fe are the essential micronutrients for plants, however, and plants need to take them externally to sustain their body's normal growth and development (Rafie et al. 2017; Souri 2016; Zaheer et al. 2019). Recently, this technique has become interestingly popular in alleviating heavy metal stress in different plant species and used for enhancing plant growth and biomass (Bashir et al. 2018; Hussain et al. 2018; Rizwan et al. 2017; Saifullah et al. 2016; Zaheer et al. 2019). In the present study, higher plant growth and biomass under interactive application of Zn and Fe-lys under high concentration of Cr in the soil, might be due to the positive impacts of both amino acids (lys) and micronutrients (Zn and Fe) to the plants (Danish et al. 2019b; Souri 2016). Furthermore, amino acids (lys) also contain nitrogen (N) in their structure, which is also an essential component for plant growth. The plant can also uptake amino acids from the soil, which also plays a crucial role in the physiological mechanisms (such as photosynthesis) of the plants (Rafie et al. 2017; Teixeira et al. 2017). Previously, it was reported that the application of micronutrients (Zn and Fe) chelated with amino acids (lys), increased chlorophyll contents and improved photosynthetic machinery and water use efficiency in the plants (Hussain et al. 2018; Sadak and Abdelhamid 2015). The increase in photosynthetic pigments in S. oleracea plants under foliar application of Zn and Fe-lys might be attributed to the increase in plant nutrients uptake and/or decrease in Cr contents in various parts of the plants (Ghasemi et al. 2012, 2014). In our study, foliar application of Zn and Fe-lys decreased oxidative stress by increasing activities of various antioxidative enzymes of S. oleracea plants when grown under different levels of tannery wastewater in the soil (Figs. 3, 4). It is well-known that foliar application of Zn and Fe-lys decreased oxidative stress in the plants, when grown with or without abiotic stress conditions (Ghasemi et al. 2014; Rizwan et al. 2017). This is because amino acids has the efficiency for scavenging ROS production and decreasing oxidative stress in a number of plant species (Rafie et al. 2017; Shahid et al. 2017). Although Zn and Fe chelated with lys increased the activities of various antioxidant enzymes, plays a protective role by decreasing the contents of Cr in various parts of the plants (Ghasemi et al. 2012; Maqbool et al. 2018).

Uptake and accumulation of Cr in various parts of plants stimulated the use of amino acids (lys) chelated with micronutrients (Zn and Fe). Although, our findings suggested that, exogenous supplementation of Zn and Fe-lys significantly decreased Cr uptake/accumulation and enhanced the contents of Zn and Fe in different parts of the plants (Fig. 4). Exogenous supplementation of Zn-lys increased Zn contents in Triticum aestivum (Rizwan et al. 2017), Oryza sativa (Hussain et al. 2018) and S. oleracea (Zaheer et al. 2019) while application of Fe-lys increased Fe contents in Oryza sativa (Bashir et al. 2018) and Zea mays (Danish et al. 2019a). It has been reported that amino acids chelated with micronutrients enhanced plant growth and biomass by formation of complexes with different heavy metals which help them to uptake/move to various parts of the plants (Zaheer et al. 2019).

Conclusion

In the present study, we have observed that plant growth, biomass and gaseous exchange attributes of S. oleracea plants have been affected significantly due to the addition of different levels of tannery wastewater in the soil. The decrease in plant growth, biomass and gaseous exchange attributes of S. oleracea plants is due to the toxic concentration of Cr in the soil, which significantly decreased essential nutrients in the soil. Moreover, Cr toxicity also induces oxidative stress in the roots and leaves of S. oleracea plants by increasing lipid peroxidation and electrolyte leakage to the membrane bounded organelles. However, Cr toxicity was reportedly reduced by the interactive application of Zn and Fe-lys which significantly increased plant growth and biomass of S. oleracea plants when grown in different levels of tannery wastewater in the soil. In addition, the application of micronutrients chelated with amino acids scavenge ROS production and decreased oxidative stress in the plants by increasing the activities of various antioxidant enzymes. Although, further experiments are required to understand the mechanism of individual and combinatorial applications Zn and Fe-lys on various plants during field conditions.