Combined application of zinc and iron-lysine and its effects on morpho-physiological traits, antioxidant capacity and chromium uptake in rapeseed (Brassica napus L.).

Environmental contamination of chromium (Cr) has gained substantial consideration worldwide because of its high levels in the water and soil. A pot experiment using oil seed crop (rapeseed (Brassica napus L.)) grown under different levels of tannery wastewater (0, 33, 66 and 100%) in the soil using the foliar application of zinc (Zn) and iron (Fe)-lysine (lys) has been conducted. Results revealed that a considerable decline in the plant growth and biomass elevates with the addition of concentrations of tannery wastewater. Maximum decline in plant height, number of leaves, root length, fresh and dry biomass of root and leaves were recorded at the maximum level of tannery wastewater application (100%) compared to the plants grown without the addition of tannery wastewater (0%) in the soil. Similarly, contents of carotenoid and chlorophyll, gas exchange parameters and activities of various antioxidants (superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX)) were also reduced significantly (P < 0.05) with the increasing concentration of tannery wastewater (33, 66 and 100%) in the soil. In addition, a combined application of Zn and Fe-lys reduced the accumulation and uptake of toxic Cr, while boosting the uptake of essential micronutrients such as Zn and Fe in different tissues of the plants. Results concluded that exogenous application of micronutrients chelated with amino acid successfully mitigate Cr stress in B. napus. Under field conditions, supplementation with these micronutrient-chelated amino acids may be an effective method for alleviating metal stress in other essential seed crops.

1. Introduction

Contamination of soil with potentially toxic elements (PTEs) is a pressing and complex problem intensely felt worldwide, particularly in densely industrialized and populated areas [1-4]. Rapid growth of industries and manufacturing operations like smelting, melting, electroplating and mechanical set-ups leads to the extensive contamination of soil with contaminates like abovementioned PTEs [5-8]. However, anthropogenically origin sources related to the metal-enriched sewage sludges in agriculture, combustion, livestock manures, application of metal-based pesticides, electronics (manufacture, use, and disposal), volcanic eruptions, forest fires, industrial processes, municipal wastes, and agricultural activities [9, 10]. Extreme accumulation of PTEs in arable soil not only causes pollution of aquatic and terrestrial environments, but also escalates the likelihoods of human exposure with PTEs [3]. Copious public health distress rises if food products grown on such polluted soil come into national markets, which ultimately exposes of the wider population to PTEs contamination [11].

In Pakistan, aquifers have already been exhausted due to injudicious consumption of groundwater for irrigation purpose to produce crops and fodder [12-14]. So, there is a need to investigate some approaches to reduce the necessity of potable water in this sector. Reuse of wastewater as a renewable resource for irrigation can be a sustainable approach to mitigate the ever-increasing irrigation caused water scarcity around the globe [15, 16]. Nevertheless, depending upon the wastewater resources, it may contain non-biodegradable pollutants such as lead, cadmium, chromium, nickel, copper, zinc, mercury, and arsenic [17].

Prompt industrial and economic developments throughout the world has led to a range of environmental concerns including the contamination of soil with toxic PTEs [18, 19]. Rapid industrialization is causing water pollution as most of the wastewater is being dumped unprocessed back into the water system [20, 21]. Farmers are using wastewater for irrigation, especially in suburban areas to overcome the shortage of canal water [22-24]. Furthermore, wastewater also contains many nutrients which results in an increase in plant growth and productivity and may also reduce the need of excessive use of artificial fertilizers [25-27]. Besides having the several benefits of wastewater for agricultural crop production, it may contain many organic and inorganic contaminants which may deteriorate the quality of crops depending upon plant species and type of contaminant [19, 28]. Among different types of wastewater, tannery wastewater is being produced owing to the increase in the tanning industry in Pakistan and worldwide [3]. Tannery wastewater is used for the growth of crops, especially vegetables which may contaminate food with toxic metals, mainly chromium (Cr) [25, 26].

Cr is a potentially noxious metal and it does not have any vital function in metabolic activities of plants [29]. Industrial process (tanning effluents) along with natural and anthropogenic activities result in amplified accumulation of chromium in surrounding environments [18, 30]. Excess Cr in plants may cause toxic effects in plants and reduces growth, photosynthesis, mineral nutrients adsorption, and quality [31]. Combustion of oil, coal and waste from chemical, metallurgy and tannery industrial effluents adds 2,000–5,000 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 [32]. Higher Cr levels in plants cause ultra–structural alteration, oxidative stress in plants and increased electrolyte leakage (EL), malondialdehyde (MDA) concentrations, and induces alterations in antioxidant enzyme activities such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and ascorbate peroxidase (APX) [33-36]. Hence, it is required to safeguard plants from Cr toxicity to counter the phytotoxicity and oxidative stress triggered by the uptake of Cr in plants.

In the world, rapeseed (Brassica napus L.) is considered as essential oilseed crop and is regarded as the second major source of vegetable oil after soybean [37, 38]. Oil rapeseed (B. napus) has a tremendous capacity to combat the stress induced by the PTEs’ toxicity [39, 40]. Among different PTEs, contamination of Cr has shown noticeable influence on growth, ultra-structural, physicochemical and molecular profiling of the B. napus [41]. B. napus can tolerate various kinds of PTEs stresses, owing to its physiological and biological processes, and it also has the capacity to withstand chromium stress [15, 41].

To date, many scientific approaches have been employed to remediate the PTEs stress in plants, particularly by using exogenous applied amino acids. Amino acids play a very critical role in metal compartmentation, transport and tolerance in plants. Zinc (Zn) and iron (Fe) chelated fertilizers complexed with lysine (lys) as amino acid have been reported to improve the growth and yield of crops [42, 43]. Fruitful application of micronutrients expressively mitigates the toxic effects of PTEs stress in plants [44, 45]. Both Fe and Zn are important micronutrients as they play a very critical role in various metabolic processes in plants including photosynthesis, DNA synthesis and respiration [23]. Zn chelated with the amino acid lys effectively mitigates the Cr stress in Oryza sativa and S. oleracea and improved the growth of plant through Zn fortification [22, 46]. Similarly, a previous study reported that Fe-lys can be effectively used as operative amendment in the reduction of Cd stress in Oryza sativa [47]. However very little is known about the role of both Zn and Fe-lys regarding the mitigation of PTEs stress in pants.

There are various studies about the application of Zn and Fe-lys on different crops under range of PTEs stresses [11, 22, 46, 47], where there is no significant literature present on the combined application of Zn and Fe-lys on crops. This study aims to address this issue and to add knowledge about (i) the role of Zn and Fe–lys on plant growth, fresh weights, dry weights, chlorophyll contents, gas exchange characteristics, oxidative stress and antioxidant response and (ii) uptake and accumulation of Fe, Zn and Cr in different parts of B. napus when cultivated with wastewater rich in Cr concentration. To the best of our knowledge, this study is among the few studies which focuses on the metal tolerance and accumulation among oil seed crops in order to investigate their suitability for metal–contaminated sites. Findings from the present study will add to our understanding of the mechanism of Cr tolerance and accumulation in B. napus with the foliar application of Zn and Fe–lys.

2. Material and methods

2.1 Description of site and soil sampling

Soil surface samples (0-22cm) were carefully taken from the botanical garden, Department of Botany, University of Punjab Lahore, Pakistan (31.4015° N, 74.3070° E). Soil samples were crushed softly with hands, and all visible portions of dead and flourishing vegetation along with other dirt particles were detached at the site. The soil samples were air dried. Debris and mud particles were removed from the dried soil using a 2-mm sieve. All samples of soil were kept in plastic bags at were stored at room temperature.

2.2 Determination of soil and wastewater physico-chemical characteristics

Total organic content of the soil was estimated via subsequent titration and dichromate oxidation with ferrous ammonium sulfate according to the method describe by Walkley and Black [48]. Similarly, ion concentration in the soil, sodium adsorption ratio (SAR) and electrical conductivity (EC) were also carefully estimated with the method given by Page [49]. Soil sampling was performed with the assistance of ammonium bicarbonate diethylenetriamine penta acetic acid (AB-DTPA) solution for the appropriate measurement of extractable trace elements [50]. Physiochemical characteristics of the soil under study are given in S1 Table. Tannery wastewater used in this study was carefully collected from tannery industries located in Kasur, Punjab Pakistan. Physiochemical features of tannery wastewater used in the study were carefully estimated according to the set APHA protocols given for the estimation of water and wastewater. Comprehensive details of major characteristics of tannery wastewater used in this experiment are presented in S2 Table.

2.3 Experimental design

The present experimental work was conducted in the wire house Department of Botany, the University of the Punjab Lahore, Pakistan (31.4015° N, 74.3070° E), from an average depth of 0–15 cm, in an open environment, protected from human and animal interactions. The plants were protected from rainfall by covering the whole wire house with a plastic sheet. The pots used in this study were rotated regularly in order to avoid environmental effects on the plants and to avoid any effect of phototropism on the plants due to sunlight. Seeds of rapeseed (Brassica napus L.) (cv. Faisal canola) were collected from Ayub Agricultural Research Institute Faisalabad, Pakistan (31.4041°N, 73.0487°E). The seeds were surface sanitized with H2O2 solution and washed 5 times with distilled water before sowing in pots. B. napus seeds were uniformly sowed in plastic pots which was filled with 5 kg of soil. Experiments were conducted accordingly with completely randomized design (CRD) and with three replicates of each treatment. After a germination time of two weeks, B. napus seedling were treated with the foliar application of Zn and Fe-lys (Zn–lys 0, 10 mg/L and Fe–lys 0, 5 mg/L) along with treatment of tannery wastewater with different levels (0, 33, 66 and 100%). Irrigation with tannery wastewater was supplied once in a week. In addition, application of Zn and Fe-lys was also supplied once a week with a volume of one liter for each spray. We have used 4 L of Zn and Fe-lys in the whole experiment which was sprayed at 4 different intervals of the experiment. However, the controls grown without the application of Zn and Fe-lys was sprayed with the distilled water. In order to mitigate the crucial deficiency of macronutrients, potassium sulphate (SOP) and phosphate were carefully applied to plants as fertilizer by following the methodology described by Bashir et al. [47].

2.4 Treatments

Eight treatments regarding wastewater and Fe and Zn-lys applications was used for this study. A detailed description of these treatments are as follow; T1(control): tannery wastewater 0%, Zn-lys 0 and Fe-lys 0 mg/ L, T2: tannery wastewater 0%, Zn-lys 10 and Fe-lys 5 mg/ L, T3: tannery wastewater 33%, Zn-lys 0 and Fe-lys 0 mg/ L, T4: tannery wastewater 33%, Zn-lys 10 and Fe-lys 5 mg/ L, T5: tannery wastewater 66%, Zn-lys 0 and Fe-lys 0 mg/ L, T6: tannery wastewater 66%, Zn-lys 10 and Fe-lys 5 mg/ L, T7: tannery wastewater 100%, Zn-lys 0 and Fe-lys 0 mg/ L, T8: tannery wastewater 100%, Zn-lys 10 and Fe-lys 5mg/L.

2.5 Plant harvesting

60 days after sowing (DAS), plants were harvested and rinsed thoroughly with normal tap water to wash off the aerial deposition and then cautiously separated into roots and shoots. All plants were rooted up allowing to study different morpho-physiological traits in late December 2017. 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. Three plants per treatment were selected randomly for morphological studies. Several leaves were counted, and the plant height (cm) was recorded from the root neck to the upper most part of the shoot. The leaf area was measured by using a leaf area meter (LI-2000, LI-COR, USA). Leaves and roots fresh biomass was measured by weighing. Later, leaves and roots were dried in an oven at 105°C for 1 h, then at 70°C for 72 h allowing to determine the dry weight. Roots were immersed in 20 mM Na2EDTA for 15–20 min to remove Cr adhered to the surface of the roots. Then, roots were washed thrice with distilled water and finally once with de-ionized water and dried for further analysis.

2.6 Determination of chlorophyll contents and gas exchange parameters

For chlorophyll content analysis, 0.1 g of fresh leaf sample was extracted with 8 mL of 95% acetone for 24 h at 4°C in the dark. The absorbance was measured by a spectrophotometer (UV-2550; Shimadzu, Kyoto, Japan) at 646.6, 663.6 and 450 nm. Chlorophyll content was calculated by the standard method of Arnon [51].

Net photosynthesis (Pn), leaf stomatal conductance (gs), transpiration rate (Ts), and water use efficiency (WUE) were measured from three different plants in each treatment group. On the same day (before sampling) these parameters were measured in between 11:30 and 13:30 with a clear sky. Rates of leaf Pn, gs, Ts, and Ci were measured with an LI–COR gas–exchange system (LI-6400; LICOR Biosciences, Lincoln, NE, USA) with a red-blue LED light source on the leaf chamber. In the LI-COR cuvette, CO2 concentration was set as 380 mmol mol–1 and LED light intensity was set at 1000 mmol m–2 s –1, which is the average saturation intensity for photosynthesis in B. napus [52].

2.7 Determination of oxidative stress indicators

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 by 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 Health and Packer [53].

To estimate H2O2 content of plant tissues (leaf samples), 3 ml of sample extract was mixed with 1 ml of 0.1% titanium sulphate in 20% (v/v) H2SO4 and centrifuged at 6,000 g for 15 min. The yellow colour intensity was evaluated at 410 nm. The H2O2 level was computed by extinction coefficient of 0.28 mmol−1 cm−1. The contents of H2O2 were measured by following the method of Jana and Choudhuri [54].

Stress-induced electrolyte leakage (EL) of uppermost stretched leaves was determined by Dionisio-Sese and Tobita [55] method. The leaves were cut into minor slices (5 mm length) and placed in test tubes having 8 mL distilled water. These tubes were incubated and transferred into water bath at 32°C 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 pH/conductivity meter (model 720, INCO-LAB Company, Kuwait) and calculated as:

EL = (EC1/ EC2) = × 100

2.8 Determination of activities of antioxidant enzymes

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 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 measurements of superoxidase dismutase (SOD) and peroxidase (POD) activities. SOD activity was assayed in 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 by 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.

Peroxidase (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% H2O2 and 0.1 mL of 4% guaiacol solution was prepared. Increases in the absorbance at 470 nm because of guaiacol oxidation was recorded for 2 min.

Catalase (CAT) activity was analyzed according to Aebi [58]. The assay mixture (3.0 mL) was comprised of 100 μL enzyme extract, 100 μL H2O2 (300 mM) and 2.8 mL 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 (APX) activity was measured according to Nakano and Asada [59]. 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).

2.9 Determination of Fe, Zn and Cr concentrations in plant

Digestion of plant samples was carried out through di−acid (HNO3−HClO4) method. Dry samples (0.5 g) of both roots and leaves were added in a flask having 10 mL of HNO3−HClO4 (3:1, v:v), and the mixture was kept overnight. After that, 5 mL HNO3 was added and samples were completely digested by placing on a hot plate [60]. Contents of Fe, Zn and Cr in roots and shoots of the plants was measured by using an atomic absorption spectrophotometer.

2.10 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. Testing showed that all plant related data were approximately normally distributed. Thus, the differences between treatments were determined using analysis of variance, and the least significant difference test (P < 0.05) used for multiple comparisons between treatments. One-way analysis of variance (ANOVA) was used to assess the significance of the variations of Cr among the different plant parts, followed by HSD tests. Graphical presentation was carried out using “Origin pro-2017”. Furthermore, the plots of principal component analysis on B. napus parameters were carried out by using the RStudio 4.3.1 software.

3. Results

3.1 Plant growth and biomass

In the present study, various growth parameters were also studied in B. napus grown in different levels of tannery wastewater (0, 33, 66 and 100%) under the application of Fe and Zn-lys. The results regarding various growth parameters are presented in Fig 1. According to the results, the maximum reduction in the growth (plant height, number of leaves, leaf area, root length) and biomass (fresh/dry weights of roots and leaves) of B. napus were observed at maximum level of tannery wastewater application (100%), compared to the plants grown in the control (without the addition of tannery wastewater). However, combined application of Zn and Fe-lys increased plant height, number of leaves, leaf area, root length, fresh weight of root, fresh weight of leaves, dry root weight and dry leaves weight by 33, 25, 40, 25, 32, 23, 40 and 29%, observed in the plants which were grown in 100% addition of wastewater in the soil, compared to those plants which were grown in 100% addition or irrigation wastewater without the application of Zn and Fe-lys.

Fig 1

Effect different levels of tannery wastewater on plant height (A), number of leaves (B), leaf area (C), root length (D), root fresh weight (E), root dry weight (F), leaf fresh weight (G) and leaf dry weight (H) under the application of Zn and Fe-lys on B. napus. Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed and means differences were tested by HSD (P < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Different levels of tannery wastewater used are as follow: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

3.2 Gas exchange parameters and chlorophyll contents

Gas exchange parameters, carotenoids and total chlorophyll contents of B. napus along with various levels of tannery wastewater treatments and with and without application of Zn-lys (10 mg/L) and Fe-lys (5 mg/L) are given in Fig 2. The addition of tannery wastewater significantly reduced the contents of chlorophyll, carotenoids, and gas exchange parameters of B. napus, in comparison to the plants under control treatment. A 100% application of tannery wastewater reduces the transpiration rate, stomatal conductance, net photosynthesis, water use efficiency, total chlorophyll, and contents of carotenoids by 68, 57, 62, 57, 76 and 60% respectively. Whereas, exogenous application of both Zn-lys (10 mg/L) and Fe-lys (5 mg/L) significantly (P < 0.05) improves gas exchange parameters, contents of carotenoids and total chlorophyll in B. napus even at all levels of tannery wastewater applications. As presented in Fig 2, all abovementioned characteristics of the plants were significantly improved by 46, 30, 30, 42, 34 and 31% respectively under the combined application of both Zn-lys (10 mg/L) and Fe-lys (5 mg/L) in plants irrigated with 100% tannery wastewater.

Fig 2

Effect different levels of tannery wastewater on chlorophyll contents (A), carotenoid contents (B), stomatal conductance (C), transpiration rate (D), net photosynthesis (E) and water use efficiency (F) under the application of Zn and Fe-lys on B. napus. Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed and means differences were tested by HSD (P < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Different levels of tannery wastewater used are as follow: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

3.3 Oxidative stress (electrolyte leakage, MDA and H2O2)

Various oxidative stress indicators i.e., MDA (malondialdehyde) contents, hydrogen peroxide (H2O2) initiation and electrolyte leakage (%) in the roots and leaves of B. napus plants were also measured (Fig 3). Results showed that increasing the concentration of tannery wastewater in the soil enhances the contents of MDA, H2O2 and EL (%) in the roots and leaves of B. napus, while the maximum increased in oxidative stress indicators was observed in the plants which were grown in 100% addition of tannery wastewater in the soil compared to the control treatment. Whereas, exogenous application of both Zn-lys (10 mg/L) and Fe-lys (5 mg/L) significantly (P < 0.05) mitigated the oxidative stress by lowering the contents of MDA, H2O2, EL in both leaves and roots of the plants as compared to the plants grown with zero application of Zn and Fe-lysine. At maximum concentration of tannery wastewater treatment combined with the application of Zn-lys (10 mg/L) and Fe-lys (5 mg/L) reduced the EL by 27 and 32% in roots and leaves of the B. napus, similarly contents of both MDA and H2O2 were declined by 20, 26, 32 and 39% in both roots and leaves of the plant as presented in Fig 3.

Fig 3

Effect different levels of tannery wastewater on MDA contents in the roots (A), MDA contents in the leaves (B), H2O2 contents in the roots (C), H2O2 contents in the leaves (D), EL percentage in the roots (E) and EL percentage in the leaves (F) under the application of Zn and Fe-lys on B. napus. Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed and means differences were tested by HSD (P < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Different levels of tannery wastewater used are as follow: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

3.4 Antioxidant enzymes activities

Activities of various antioxidants (superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were also measured from the roots and leaves of B. napus plants (Fig 4). The results illustrate that a significant decrease was observed in the activities of antioxidant enzymes i.e., SOD, POD, CAT and APX in the roots and leaves of the B. napus grown under different levels of tannery wastewater in the soil, compared to those plants which were not in the control treatment. Results also depicted the maximum decrease in the activities of antioxidant enzymes in the roots and leaves of the B. napus was observed in the plants grown in the 100% addition of tannery wastewater in the soil, compared to those plants which were grown in 0% addition of tannery wastewater in the soil. However, application of Zn and Fe-lys significantly (P < 0.05) increased the activities of SOD, POD, CAT and APX by 74, 57, 56 and 63% in leaves and 50, 47, 60 and 83% in roots of B. napus, compared to those plants which were grown without the application of Zn and Fe-lys (Fig 4).

Fig 4

Effect different levels of tannery wastewater on SOD activity in the roots (A), SOD activity in the leaves (B), POD activity in the roots (C), POD activity in the leaves (D), CAT activity in the roots (E), CAT activity in the leaves (F), APX activity in the roots (G) and APX activity in the leaves (H) under the application of Zn and Fe-lys on B. napus. Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed and means differences were tested by HSD (P < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Different levels of tannery wastewater used are as follow: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

3.5 Uptake and accumulation of Cr, Zn and Fe

The contents of Cr, Zn and Fe were also measured from the roots and shoots of B. napus, when grown in the different levels of tannery wastewater with or without the application of Zn and Fe-lys (Fig 5). Increasing the concentration of tannery wastewater in the soil induces a significant (P < 0.05) increase in the contents of Cr, while a significant decrease in the contents of Zn and Fe in the roots and shoots of B. napus, compared to control plants (Fig 5). Increasing levels of wastewater in the soil increased Cr contents by 53.3 in the roots and 22.5 times in the shoots of the plants grown in 100% addition of tannery wastewater in the soil, compared to the plants grown in the control treatment. Compared to the control treatment, the maximum decrease in the contents of Zn and Fe was 55 and 60% in the roots, while decrease by 50 and 75%, respectively in the shoots of B. napus plants under the irrigation of 100% wastewater in the soil, compared to the control treatment. Although, Cr contents were decreased by the application of Zn and Fe-lys and Cr contents decreased by 30% in the roots and 33% in the shoots in the plants grown in 100% addition of tannery wastewater in the soil, compared to the plants grown in the control treatment. In contrast, Zn and Fe contents were increased by the application of Zn and Fe-lys and were increased by 28 and 32% respectively in the roots and 31 respectively and 33% in the shoots in the plants grown in 100% addition of tannery wastewater in the soil, compared to the plants grown in the control treatment.

Fig 5

Effect different levels of tannery wastewater on the uptake/accumulation of Zn contents in the roots (A), Zn contents in the shoots (B) Fe contents in the roots (C), Fe contents in the shoots (D) Cr contents in the roots (E) and Cr contents in the shoots (F) under the application of Zn and Fe-lys on B. napus. Values are demonstrated as means of three replicates along with standard deviation (SD; n = 3). One-way ANOVA was performed and means differences were tested by HSD (P < 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments. Different levels of tannery wastewater used are as follow: 0 (without irrigation with wastewater), 33 (33% irrigation with wastewater), 66 (66% irrigation with wastewater) and 100 (100% irrigation with wastewater).

3.6 Correlation analysis between growth, physiological traits and Cr uptake in B. napus

A Pearson’s correlation graph was constructed to quantify the relationship between various growth parameters with Cr uptake in different parts of the plant (Fig 6). Cr concentration in the roots was positively correlated with Cr concentration in the shoots, MDA contents, H2O2 initiation and EL, while negatively correlated with root length, plant height, transpiration rate, net photosynthesis, SOD activity in leaves, Zn contents in roots, carotenoid contents, stomatal conductance, APX activity in leaves, water use efficiency, POD activity in leaves, leaves fresh weight, root fresh weight, root dry weight, Fe contents in shoots, total chlorophyll contents, Fe contents in roots, POD activity in roots, number of leaves, leaf area, CAT activity in roots, SOD activity in roots, CAT activity in leaves, APX activity in roots and Zn contents in shoots. Similarly, Cr concentration in the shoots was positively correlated with Cr concentration in the roots, MDA contents, H2O2 initiation and EL while negatively correlated with root length, plant height, transpiration rate, net photosynthesis, SOD activity in leaves, Zn contents in roots, carotenoid contents, stomatal conductance, APX activity in leaves, water use efficiency, POD activity in leaves, leaves fresh weight, root fresh weight, root dry weight, Fe contents in shoots, total chlorophyll contents, Fe contents in roots, POD activity in roots, number of leaves, leaf area, CAT activity in roots, SOD activity in roots, CAT activity in leaves, APX activity in roots and Zn contents in shoots. This correlation is depicted a close connection between Cr uptake and growth in B. napus.

Fig 6

Correlation between morpho-physiological traits, Zn and Fe contents with Cr uptake in different parts of B. napus.

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

A heatmap-histogram analysis was also constructed to explore the relationship between the different growth attributes and Cr uptake (Fig 7). Only significant differences (aquamarine color) were observed in the Cr uptake for 100% addition of tannery wastewater in the soil and without the application of Zn and Fe-lys, while all other traits were showing non-significant differences (blue color) within the used treatments. This histogram depicted a clear difference between the Cr uptake abilities and growth attributes of B. napus.

Fig 7

Heatmap Histogram correlation between morpho-physiological traits, Zn and Fe contents with Cr uptake in different parts of B. napus.

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

3.7 Principal component analysis

Principal component analysis (PCA) was used to evaluate the effect of different levels of tannery wastewater with the exogenous application of Zn and Fe-lys on different attributes of B. napus (Fig 8). Of all the main components, the first two components (Dim1 and Dim2) comprise more than 97% of the whole database and make up the largest portion of all components (Fig 8). Among this, Dim1 contributes 93.4%, and Dim2 contributes 3.6% of the whole dataset. In addition, Fig 8 also shows that Cr contents in roots and shoots, MDA contents, EL and H2O2 initiation in the roots and leaves were positively correlated in the dataset from all the variables. In contrast, Fe and Zn contents in roots and shoots, SOD, POD, CAT and APX activities in leaves and roots, morpho-physiological traits in leaves and roots, photosyntethic parameters and pigment contents were negatively correlated in the dataset from all the variables (Fig 8).

Fig 8

Loading plots of Principal Component Analysis (PCA) on different morpho-physiological traits, Zn and Fe contents with Cr uptake in different parts of B. napus.

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

4. Discussion

Excessive discharge of Cr could be extremely toxic for normal plant growth, morphological and physio-biochemical processes and membrane integrity of plants [61]. Result of present study revealed that morphological traits of the plant including plant growth and biomass were significantly reduced through the addition of tannery wastewater in soil (Fig 1). Cr has the ability to decline the growth attributes as previously reported in different plants including B. napus [62], Zea mays [63], Helianthus annuus [64], and Triticum aestivum [65]. Cr toxicity can cause decrease in plant growth and biomass, as toxic concentration of Cr in the soil makes the uptake essential nutrients unavailable. This has been reported in various studies, i.e., Brassica juncea [66], and Brassica campestris [67]. In the present study, the application of Fe and Zn-lys considerable decrease Cr contents in different parts of the plants, which is also a main factor to decrease plant growth and biomass, when grown under different levels of tannery wastewater in the soil. Both Zn and Fe-lys are widely recognized as the amino-chelated fertilizers and they have the profound capacity to boost up the uptake of necessary nutrients and safeguard the plants against external stresses. Amino acids have shown tremendous potential of making complexes with different toxic metals and eventually reducing their mobility [11, 68]. In the present study, combined application of Fe and Zn-lys increased plant growth and biomass (Fig 1), which was also suggested by previous studies that an application of Zn or Fe-lys can be useful for increasing plant growth and biomass [22, 46, 47]. This is because Zn and Fe-lys enhances photosynthetic processes and provides a substantial growth in a short time under metal stressed environments [69].

Results revealed that gas exchange characteristics and chlorophyll contents of the B. napus were significantly reduced under the application of tannery wastewater. Cr toxicity in the soil caused a significant decrease in photosynthesis and contents of chlorophyll and carotenoids, which were also previously recorded in different plants such as Zea mays, Oryza sativa, Spinacia oleracea and Helianthus annuus [41, 63]. In the present study, a significant (P < 0.05) increased in the gas exchange characteristics and photosynthetic pigments of the B. napus were measured with the combined application of Zn and Fe-lys (Fig 2). Amino acids also take part in the increased leaf photosynthesis and thus increase of photosynthetic pigments of the plants [3, 70]. Results indicate that a combined application of both these micronutrients along with lysine effectively improves the gas exchange parameters and chlorophyll contents of the plants (Fig 2). Similar finding were reported by Rizwan et al. [11], and Bashir et al. [47] in Triticum aestivum and Oryza sativa (respectively) under Cd stress.

PTEs stress is a grave risk for plant growth, toxic concentration of HMs in plants and results in the over generation of reactive oxygen species (ROS) as compared to no stressed plants [15, 22, 71]. Increasing levels of tannery wastewater in the soil causes oxidative stress by over-production of ROS, which is manifested by increasing levels of MDA, H2O2 and EL in the roots and leaves (Fig 3). Previously, Cr toxicity induced oxidative damages in various organs of Brassica juncea, Brassica oleracea botrytis, and Cymbopogon flexuosus [41, 63, 72] have been reported. In our study, the application of Zn and Fe-lys significantly (P < 0.05) decreased the contents of MDA, H2O2 EL (%) in B. napus when grown in various concentrations of tannery wastewater and increased the activities of various antioxidant enzymes like APX, SOD, CAT and POD (Fig 4). It was noted that the application of amino acid chelation with micronutrients have ability to overcome the generation of ROS in the different parts of the plant. In addition, application of Zn or Fe-lys have ability to increase the activities of different enzymatic antioxidants, which was observed in soybean [73]. Similar results were also reported for Oryza sativa and Spinacia oleracea [22, 74]. However, the results are more promising that combined application of Zn and Fe-lys scavenges ROS generation (Fig 3), and offers a better option to enhance antioxidant capacity in various crops under wastewater treatments (Fig 4).

Our results stated that increases in the level of tannery wastewater application in soil resulted in significant accumulation of Cr contents in different parts of the plant by restricting the uptake of essential nutrients like Zn and Fe (Fig 5). High concentrations of Cr contents in different tissues of plants were also detected previously [22, 62, 75, 76]. The main effect of high concentrations of metals on the photosynthetic pigments and gaseous exchange parameters are related to decreases in the net photosynthesis rate, reduced ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) efficiency, and the inhibition of electron transport and PSII activities [37, 77–79]. It was also observed that restriction in the uptake of necessary micronutrients is attributed to the inability of plants roots to successfully absorb these vital nutrients under the uncompromising stress of Cr toxicity [26]. This is because of the higher uptake of Cr in the plant parts significantly affects growth and composition of the plant [25, 30]. Although, our finding recommended that a combined exogenous application of Zn-lys and Fe-lys effectively restricts the accumulation and uptake of Cr and improved the accumulation of both Zn and Fe in various tissues of the plant (Fig 5). It was reported that, amino acids have ability to form complexes with different PTEs and results in immobilizing these metals and thus cause reduction in metal uptake by the plants [11]. In line with the result of this study, application of Zn-lys improves the concentration of Zn in various plants like Triticum aestivum [11], Oryza sativa [46] and Spinacia oleracea [22]. Similarly Fe-lys enhanced the contents of Fe in Oryza sativa [47] and Zea mays [63].

5. Conclusions

Our results confirmed that plant growth, photosynthesis and antioxidant system were adversely affected by Cr stress generated as a result of tannery wastewater application. Although, the oxidative stress and uptake/accumulation of Cr were enhanced with increasing levels of tannery wastewater applications. However, foliar application of Zn and Fe-lys improved plant growth and composition by decreasing the concentration of ROS and Cr concentration in various parts of the plants. Therefore, long-term field studies should be executed to draw parallels amongst plants/crops, metal stress, micronutrient fertigation regimes, nutrients mobility patterns and plant growth in order to gain insights into underlying mechanisms.

Physic-chemical properties of loam soil used in the pot experiment.

(DOCX)

Click here for additional data file.

Characteristics of tannery wastewater used for irrigation of the soil used in the pot experiment.

(DOCX)

Click here for additional data file.

12 Aug 2021

PONE-D-21-21085

Ameliorating the Cr toxicity for rapeseed (Brassica napus L.) growth and biomass through the foliar application of micronutrients chelation with amino acids grown in tannery wastewater

PLOS ONE

Dear Dr. Saleem,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR:

It’s very strange for me, why authors have irrigated the soil with tannery wastewater and why they did not collect wastewater irrigated soil. If wastewater is harmful then soil should be taken where this harmful effluent discharged.

Please re-write the line number 35-36.

Please check the line number 45, is this correct sentence.

Authors need to add some key findings in your abstract section.

Please use PTEs instead of heavy metals throughout the manuscript.

L54-56. How these agricultural practices can contribute metals toxicity in arable lands?.

L65- Revise production of industrial section???

Please remove spaces b/w words in throughout the manuscript.

Authors did experiment about wastewater but I could not found any significant and relevant information in the introduction section.

Article is not properly formatted according to the journal format. E.g., headings font size??

L133- Total organic content of the….. what does it mean??

How temperature was maintained 25 °C and 10 °C average temperature of day and night.???????

L152-156. Please re-write these sentences.

Please check the texts in all figures (especially on y-axis)

Check the superscript and subscript throughout the manuscript.

Its very strange for me that Cr permissible limit is 0.10 and wastewater has just 4

If wastewater has Cr conc… around 4 then how it reaches to 160 in figure 5 E.

==============================

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Saqib Bashir

Academic Editor

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i have read this manuscript very carefully and evaluted from the neutral reviewers. All they have suggested the major revision. Authors needs to revise this current version very carefully .

1- It’s very strange for me, why authors have irrigated the soil with tannery wastewater and why they did not collect wastewater irrigated soil. If wastewater is harmful then soil should be taken where this harmful effluent discharged.

2- Please re-write the line number 35-36.

3- Please check the line number 45, is this correct sentence.

4- Authors need to add some key findings in your abstract section.

5- Please use PTEs instead of heavy metals throughout the manuscript.

6- L54-56. How these agricultural practices can contribute metals toxicity in arable lands?.

7- L65- Revise production of industrial section???

8- Please remove spaces b/w words in throughout the manuscript.

9- Authors did experiment about wastewater but I could not found any significant and relevant information in the introduction section.

10- Article is not properly formatted according to the journal format. E.g., headings font size??

11- L133- Total organic content of the….. what does it mean??

12- How temperature was maintained 25 °C and 10 °C average temperature of day and night.???????

13- L152-156. Please re-write these sentences.

14- Please check the texts in all figures (especially on y-axis)

15- Check the superscript and subscript throughout the manuscript.

16- Its very strange for me that Cr permissible limit is 0.10 and wastewater has just 4

17- If wastewater has Cr conc… around 4 then how it reaches to 160 in figure 5 E.

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Write up is very poor (view comment in attachment). Treatment plan is ambiguous. Figures are not self-explanatory, A, B & C etc. are not explained in description.

Results are not properly presented. Interpretation is not clear. Discussion section must be re-written before re-submission.

Conclusion section must be linked with the quantified results. General statements commonly mislead the readers.

Over all, manuscript is not properly managed. English language is very poor. There are lot of language and grammar mistakes.

Reviewer #2: Respected Editor,

Thanks for choosing me a potential reviewer for the manuscript entitled “Ameliorating the Cr toxicity for rapeseed (Brassica napus L.) growth and biomass through the foliar application of micronutrients chelation with amino acids grown in tannery wastewater” from Zaheer et al. I have read the manuscript carefully and found much novelty in it. In addition, in this research work Authors try to overcome Cr toxicity from wastewater using the application of Zn and Fe-lys and measured various growth, chlorophyll, photosynthesis, oxidative stress and response of antioxidants, nutrients and Cr uptake from different parts of the plants were measured. Although, it’s a novel approach to use Zn or Fe-lys and a cheap method for the developing countries such as Pakistan. Hence, I want to add some suggestions which can increase the readership for the paper. Few suggestions and recommendations are as follow:

On the title: Please correct the scientific name of the plant.

On the abstract: Please add some introductive sentences in the beginning of the abstract and also add some future recommendation in the last of the abstract section. In addition, write your parameters such as oxidative stress or antioxidants, at least once in this part.

On the keywords: These words should not repeat in the title.

On the Introduction: Please cite some latest articles and also try to cite multiple references in each sentence. Also revise some of the minor mistakes such as rapeseed, chromium or ROS. In addition, write your objectives critically.

On the Materials and methods: Please write your protocols in details, also add chemical names and complete description of your analysis.

On the Results and discussions: Please write some details in the legends of the figures and revise some of the results in more descriptive manners. Also revise some sentences about antioxidant capacity in the discussion section.

On the conclusion: Please write at least one future recommendations from your study.

Thanks, and regards

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: PONE-D-21-21085_reviewer.pdf

Click here for additional data file.

30 Sep 2021

Reviewer’s comments

ACADEMIC EDITOR:

1. It’s very strange for me, why authors have irrigated the soil with tannery wastewater and why they did not collect wastewater irrigated soil. If wastewater is harmful then soil should be taken where this harmful effluent discharged.

Response: Respected reviewer, in this study we have used natural soil and collected the wastewater from Kasur District, where the industries directly discharged the wastewater and many formers are using this wastewater to irrigated their land. It’s not strange for anyone, and It’s a normal study and we already published 100 of literatures on this idea. This study will help the formers to learn about the use of wastewater in their irrigated land. And we have noticed from the study that irrigated wastewater is not helpful for the farmer so that we have used the artificial chemical i.e., micro-chelation with amino acids to overcome the Cr toxicity in the plants by minimizing its concentration. This is the main novelty of our study.

2. Please re-write the line number 35-36.

Response: Respected reviewer, done.

3. Please check the line number 45, is this correct sentence.

Response: Respected reviewer, corrected.

4. Authors need to add some key findings in your abstract section.

Response: Respected reviewer, I have re-written the results in the abstract section and re-written most of the key results. Although, I have not written in the form of percentage as we have measured 26 parameters in this study, this will make abstract more complex and difficult for the readers to understand. Firstly, Cr toxicity was decreased growth parameters then application of amino chelation increased these parameters. This is difficult to write in abstract section. Moreover, we have written the details story in the “Results” section. Thanks

5. Please use PTEs instead of heavy metals throughout the manuscript.

Response: Respected reviewer, corrected throughout the MS.

6. L54-56. How these agricultural practices can contribute metals toxicity in arable lands?

Response: Respected reviewer, I have corrected this sentence, although the reason behind this mechanism is that various agricultural practices such as fertilization have many nutrients like Cu, Cd or Fe which required by plants in minute quantity, so that continuous fertilization can cause metal contamination in the soil.

7. L65- Revise production of industrial section???

Response: Respected reviewer, I have rephrased these sentences.

8. Please remove spaces b/w words in throughout the manuscript.

Response: Respected reviewer, I have revised the MS and corrected it accordingly.

9. Authors did experiment about wastewater but I could not found any significant and relevant information in the introduction section.

Response: Respected reviewer, I have added all relevant information about wastewater in this section such as effect on plants, it’s amount and many other general information.

10. Article is not properly formatted according to the journal format. E.g., headings font size??

Response: Respected reviewer, I have revised it according to the journal’s format.

11. L133- Total organic content of the….. what does it mean??

Response: Respected reviewer, all matter is present in Table 1S, and we are discussing about it’s protocol. For better concern please see the Table 1S.

12. How temperature was maintained 25 °C and 10 °C average temperature of day and night.???????

Response: Respected reviewer, the source was not available for this information. I have removed this and replace it with all relevant details of green house like, wires, open air experiment, what we will do during rain. Et.,

13. L152-156. Please re-write these sentences.

Response: Respected reviewer, done.

14. Please check the texts in all figures (especially on y-axis)

Response: Respected reviewer, I have checked it, looks satisfactory for me, and also we added PCA for this study for better understanding the results.

15. Check the superscript and subscript throughout the manuscript.

Response: Respected reviewer, I have cross checked throughout the MS.

16. Its very strange for me that Cr permissible limit is 0.10 and wastewater has just 4

Response: Respected reviewer, this is showing that wastewater contains too high amount of Cr in it compared to other heavy metals. For example: this study is based on Cr stress if it will be Zn or Cd stress then their values will be higher or at least close to the permissible limit. We can see only Cr have too much high quality in it wastewater. Thanks for understanding.

17. If wastewater has Cr conc… around 4 then how it reaches to 160 in figure 5 E.

Response: Respected reviewer, for this please see the M&M section, we have supplied wastewater at different intervals of the experiment, not only one time. For the response of your comments I have used blue colour in the text.

Reviewer #1

Write up is very poor (view comment in attachment). Treatment plan is ambiguous. Figures are not self-explanatory, A, B & C etc. are not explained in description.

Results are not properly presented. Interpretation is not clear. Discussion section must be re-written before re-submission.

Conclusion section must be linked with the quantified results. General statements commonly mislead the readers.

Over all, manuscript is not properly managed. English language is very poor. There are lot of language and grammar mistakes.

Response: Respected reviewer, thanks for your valuable suggestions and time. I have revised my whole manuscript and also reviewed by corresponding author and we have improved it according to your suggestion. Almost all the results have re-written and for better understanding of our results we have added PCA and heat-map. The manuscript is cross-checked and reduced all grammar mistakes especially Brassica napus (scientific name of the crop). All legend of the figures are revised and written in the details even we added the statistical analysis and treatments. In addition, treatments also revised and written in the form of numbered. We also do hard work on discussion section and all introductive sentences are removed and just focuses on main findings with the mechanism. Although, we did not know the dimension of the pots but pots have capacity of 5 kg of soil which is mention in the text. Also, we have revised the conclusion and read the MS many times to make sure that no grammar or spell or scientific error. Thanks for your time and suggestions. For the response of your comments, I have used Red colour in the MS.

Reviewer #2

Respected Editor,

Thanks for choosing me a potential reviewer for the manuscript entitled “Ameliorating the Cr toxicity for rapeseed (Brassica napus L.) growth and biomass through the foliar application of micronutrients chelation with amino acids grown in tannery wastewater” from Zaheer et al. I have read the manuscript carefully and found much novelty in it. In addition, in this research work Authors try to overcome Cr toxicity from wastewater using the application of Zn and Fe-lys and measured various growth, chlorophyll, photosynthesis, oxidative stress and response of antioxidants, nutrients and Cr uptake from different parts of the plants were measured. Although, it’s a novel approach to use Zn or Fe-lys and a cheap method for the developing countries such as Pakistan. Hence, I want to add some suggestions which can increase the readership for the paper. Few suggestions and recommendations are as follow:

On the title: Please correct the scientific name of the plant.

Response: Respected reviewer, corrected.

On the abstract: Please add some introductive sentences in the beginning of the abstract and also add some future recommendation in the last of the abstract section. In addition, write your parameters such as oxidative stress or antioxidants, at least once in this part.

Response: Respected reviewer, we have added an introductive sentence in the beginning and also added a conclusion sentence in the end of the abstract section. Also we added antioxidants in this section.

On the keywords: These words should not repeat in the title.

Response: Respected reviewer, corrected.

On the Introduction: Please cite some latest articles and also try to cite multiple references in each sentence. Also revise some of the minor mistakes such as rapeseed, chromium or ROS. In addition, write your objectives critically.

Response: Respected reviewer, I have made all the changes according to your suggestions.

On the Materials and methods: Please write your protocols in details, also add chemical names and complete description of your analysis.

Response: Respected reviewer, added all relevant information.

On the Results and discussions: Please write some details in the legends of the figures and revise some of the results in more descriptive manners. Also revise some sentences about antioxidant capacity in the discussion section.

Response: Respected reviewer, all relevant information is added.

On the conclusion: Please write at least one future recommendations from your study.

Thanks, and regards

Response: Respected reviewer, already added by the suggestion of another reviewer. For the response of your comments, I have used green colour in the text.

Submitted filename: Reviewers comments.docx

Click here for additional data file.

1 Dec 2021

PONE-D-21-21085R1Combined Application of Zinc and Iron-Lysine on Morpho-Physiological Traits, Antioxidant Capacity and Chromium Uptake in Rapeseed (Brassica napus L.) Under Different Levels of Tannery WastewaterPLOS ONE

Dear Dr. Saleem,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR:please improve grammatical mistakes in the revised version.

==============================

Please submit your revised manuscript by Jan 15 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Saqib Bashir

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Title is not appropriate and very confusing. Kindly revise title accordingly. Write up is still poor. It requires extensive language improvement. I observe a lot of grammar mistakes in the manuscript.

Reviewer #2: Authors have made all significant changes in the MS, therefore paper should be accepted in the current form.

**********

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

8 Dec 2021

ACADEMIC EDITOR:

please improve grammatical mistakes in the revised version.

Response: We have gone through with English Editing company and Certificate is also attached.

Reviewer #1:

Comments: Title is not appropriate and very confusing. Kindly revise title accordingly. Write up is still poor. It requires extensive language improvement. I observe a lot of grammar mistakes in the manuscript.

Response: We have gone through with English Editing company and Certificate is also attached.

Reviewer #2:

Comments: Authors have made all significant changes in the MS, therefore paper should be accepted in the current form.

Response: thanks

Submitted filename: Response letter.docx

Click here for additional data file.

15 Dec 2021

PONE-D-21-21085R2Combined Application of Zinc and Iron-Lysine and its effects on Morpho-Physiological Traits, Antioxidant Capacity and Chromium Uptake in Rapeseed (Brassica napus L.)PLOS ONE

Dear Dr. Saleem,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR:

Please address minor corrections and submit.

==============================

Please submit your revised manuscript by Jan 29 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Saqib Bashir

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

16 Dec 2021

Comment

ACADEMIC EDITOR:

Please address minor corrections and submit.

Response: We had revised the manuscript and corrected it accordingly and go throughout the MS to ensure that there will be no grammar mistake. Thanks for your time and suggestions.

Submitted filename: Reviewersssss.docx

Click here for additional data file.

19 Dec 2021

Combined Application of Zinc and Iron-Lysine and its effects on Morpho-Physiological Traits, Antioxidant Capacity and Chromium Uptake in Rapeseed (Brassica napus L.)

PONE-D-21-21085R3

Dear Dr. Saleem,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Saqib Bashir

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

30 Dec 2021

PONE-D-21-21085R3

Combined Application of Zinc and Iron-Lysine and its effects on Morpho-Physiological Traits, Antioxidant Capacity and Chromium Uptake in Rapeseed (Brassica napus L.)

Dear Dr. Saleem:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Saqib Bashir

Academic Editor

PLOS ONE