Indian Mustard Brassica juncea efficiency for the accumulation, tolerance and translocation of zinc from metal contaminated soil

Journal Pre-proof Indian Mustard Brassica juncea efficiency for the accumulation, tolerance and translocation of zinc from metal contaminated soil Hina Chaudhry, Numrah Nisar, Salma Mehmood, Munawar Iqbal, Arif Nazir, Muhammad Yasir PII:

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DOI:

https://doi.org/10.1016/j.bcab.2019.101489

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BCAB 101489

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Biocatalysis and Agricultural Biotechnology

Received Date: 20 November 2019 Revised Date:

27 December 2019

Accepted Date: 30 December 2019

Please cite this article as: Chaudhry, H., Nisar, N., Mehmood, S., Iqbal, M., Nazir, A., Yasir, M., Indian Mustard Brassica juncea efficiency for the accumulation, tolerance and translocation of zinc from metal contaminated soil, Biocatalysis and Agricultural Biotechnology (2020), doi: https://doi.org/10.1016/ j.bcab.2019.101489. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Authors Statement Hina Chaudhry: Investigation: Writing – original draft Numrah Nisar: Conceptualization; Supervision, Project administration Salma Mehmood: Software, Methodology Muhammad Yasir: Resources, Data curation Munawar Iqbal: Visualization, Validation Arif Nazir: Writing – review & editing

Indian Mustard Brassica juncea efficiency for the accumulation, tolerance and translocation of zinc from metal contaminated soil Hina Chaudhry1, Numrah Nisar1, Salma Mehmood1, Munawar Iqbal2, Arif Nazir2*, Muhammad Yasir2 1

Department of Environmental Science, Lahore College for Women University, Lahore, Pakistan 2

Department of Chemistry, The University of Lahore, Lahore, Pakistan *Corresponding Author Email address: [email protected]

Abstract Phytoremediation using hyper accumulators is an emerging and cost-effective option for heavy metal pollution cleanup in soils. There is a dearth of knowledge regarding mechanism of sequestration of heavy metals and their final destination allocated at cellular levels by hyper accumulators. The present study explores the accumulation, tolerance, toxicity, translocation and cellular level accumulation of essential heavy metal Zn in Indian Mustard (Brassica juncea). Accumulation of Zn in roots leaves and stem of Brassica juncea was also quantified by Atomic Absorption spectroscopy (AAS). Plants were grown in sand and soil and were given Zn concentrations (20, 40, 80, and 160 mg/L) for four weeks. The results of this study showed that tolerance index increased with increased concentration of metal. Translocation factor for each concentration of Zn was greater than 1 which showed aerial parts of plants accumulated more amount of metal than their roots. Highest EC50 value was observed in stem and lowest EC50 value was observed in root of Brassica juncea. Zn deposits were found in epidermis, bundle sheet of veins and mesophyll cells of Brassica juncea leaves. Brassica juncea tolerated and grew well even at higher concentrations of Zn. Zinc accumulation in leaf tissues increased as given concentration of Zn increased.

Key words: Brassica juncea; Zinc; cellular accumulation; phytoremediation; hyper accumulator 1. Introduction Plants play pivotal role in nutrient cycling and ecosystem productivity. The phytochemicals produced by the plants exhibit pharmacological effects applicable to the treatment of fungal, and bacterial infections and various other chronic diseases. Medicinal plants have been considered as a huge source of biologically active compounds (Babitha et al., 2019; Bhavani et al., 2018; Chitra et al., 2016; Chitra et al., 2015; Manikandan et al., 2016a, b). Zinc is an essential heavy metal necessary for the growth of plants but it is found to be toxic at higher concentrations, therefore higher levels of Zn in soils warrant remedial measures for ensuring soil health. Hyper accumulators like Brassica juncea (Indian Mustard) show promising outputs in sequestration of heavy metals (Ahmed M. Abu-Dief and Zikry, 2018; Cahyana and Kam, 2016; Chaudhry, 2019; Hamid et al., 2016; Hassen and Asmare, 2019; Ibisi and Asoluka, 2018; M. Alasadi et al., 2019; Nazir et al., 2019). Various studies have focused on the uptake, distribution, accumulation and detoxification of heavy metals. Their concerning mechanisms involving hyper accumulation and tolerance of heavy metal are not well understood. So, the mechanisms of transportation of metal into the cells of plants have gained a lot of importance in research (Abou-Aly et al., 2019; Chowardhara et al., 2019; Kayalvizhi and Kathiresan, 2019; Kumar et al., 2019; Nokman et al., 2019; Rasheed et al., 2019; Singh et al., 2019). For accumulation and tolerance of metal, it is essential to understand the classification and distribution of metal in organs and compartments of cells (Iqbal, M et al., 2019; Iwuoha and Akinseye, 2019; Siddique et al., 2018; Xin et al., 2013). Previous studies have shown that metal was more accumulated in the mesophyll cells in Brassica napus and A. halleri (Carrier et al., 2003). Peng-Jie et al. (2012) have reported the metal localization by using Energy Dispersive X-ray spectrometry in Picris divaricate which

is a hyper accumulator of Zn. His results have shown that metal was distributed in the lower and upper epidermis, bundle sheath cells and trichomes while in mesophyll cells, there was very less accumulation of metal because they are highly tolerant to metal (Ashraf et al., 2018; Ghulam Abbas and Gul Afshan, 2014; Iqbal, Munawar et al., 2019; Kamran et al., 2018; Legrouri et al., 2017; Manikandan et al., 2014; Mouhamad et al., 2017a; Mouhamad et al., 2017b; Mouhamd et al., 2017; Qamar et al., 2017; Tahir et al., 2017; Younas et al., 2017). Present study focusses on the utilization of Brassica juncea for exploring its remediation potential for Zn contaminated soils while understanding the accumulation in stem roots and leaves, tolerance levels, toxicity threshold, translocation factor and transportation of Zn at cellular level of plants. 2. Materials and Methods All the chemicals used in this research work were of analytical grade and purchased from Sigma Aldrich. Seeds of Brassica juncea were collected from Institute of Agricultural Sciences, University of the Punjab, Lahore. Seeds of Brassica juncea were sown in sand and soil separately and were harvested after treating with Zn. 2.1

Zinc Treatment

For the preparation of standard solution Zinc Sulphate (ZnSO4) was used as a source of Zn. Standard solution and their dilutions were prepared in mg/L. For the preparation of standard solution of 200 mg of Zn, 0.247 g of ZnSO4 was weighed out and dissolved in 500 mL of distilled water. After the preparation of standard solution, different concentrations of Zn (20, 40, 80, and 160 mg/L) were prepared. Dilutions of required concentrations of Zn (20, 40, 80, and 160 mg/L) were prepared by taking 5, 10, 20 and 40 mL of stock solution in 50 mL volumetric flask and diluted up to the mark with distilled water. Different concentrations of Zn were given to plants with the age of two

weeks. One plant without Zn addition was considered as control plant. The Zn treatment to plants was continued for two weeks (Fones et al., 2010). 2.2

Preparation of samples for Atomic Absorption Spectrometer

The dried plant material including root, stem and leaves of plants treated with different Zn concentrations were grinded using mortar and pestle. samples of 0.05 g of dried plant material, was taken in 50 mL beaker for acid digestion. 3 mL of nitric acid was added in each sample and left overnight in fume hood for the complete digestion. Acid digested samples were then filtered and diluted up to 10 mL with distilled water (Fones et al., 2010). 2.3

Analysis of Zn content in Plant material by AAS

Zinc content in stems, roots and leaves as well as in soil and sand samples was analyzed by AAS (Thermo Electron Corporation, M series) with three replicates for each sample. Zn concentrations were calculated using the following formula: Metal concentration ( mg⁄kg) =

Concentration ( mg⁄L) ×Solution volume (mL) Sample weight (g)

The tolerance index was calculated to determine the ability of the Brassica juncea to grow in the presence of Zn. The EC50 is the effective concentration of the metal that leads to a 50% decrease in biomass of the plant, as compared to control. EC50 for Zn was determined for the roots and aerial parts using non-linear regressions to fit curves. Bioaccumulation factor shows the capability of the plant to extraxt metal from the contaminated medium. Bioaccumulation factor in plant leaf, stem and root was calculated. The translocation factor (TF) was calculated to determine the efficiency of the plant to translocate the accumulated metal from its roots to the aerial parts (stem and leaf). 2.4

Zinc Accumulation at cellular level by Light Microscopy

2.4.1

Preparation of leaf tissue sections

The leaf samples were alcohol dehydrated by immersing in ethanol, embedded in paraffin wax and then 30 µm sections were cut using microtome. Modified Sulfide-silver method was

used for the histochemical localization of metal by light microscopy (Ayaz et al., 2020; Hu et al., 2009; Ullah et al., 2019). 2.4.2

Preparation of Stain

Stain solution was freshly prepared and it is composed of four different solutions: i.

Gum Arabic (120 mL): 500g of gum arabic was dissolved in 1000 mL of distilled water. It took 3 to 4 days to dissolve and then stored in refrigerator.

ii.

Citrate buffer (pH 3.5) (20 mL): 5.1 g of citric acid and 4.7 g of sodium citrate were dissolved in 20 mL of distilled water and the pH of the solution was adjusted to 3.5 using the pH meter.

iii.

Hydroquinone (60 mL): 3.4g of Hydroquinone was dissolved in 60 mL of distilled water with constant stirring.

iv.

Silver nitrate (1mL): 0.17 g of silver nitrate was dissolved in 1mL of distilled water.

The sections of leaf were carefully placed on glass slides and stained in the dark at 25 oC for 90 min. The slides were then washed gently with distilled water, dried and observed in Light microscope (Camera fitted Light microscope (Nikon ECLIPSE E200). 3. Results and discussion Dry weight of root, stem and leaf of Brassica juncea (Table 1) that were grown in soil gradually decreased with increase in the concentration of Zn. Ghaderian et al. (2007) results show that root and stem biomass of M. flavida (Brassicaceae family) decreased with increase in Zn concentration. Brassica juncea that were grown in sand, their dry weight of root, stem and leaf area gradually increased with increase in the concentration of Zn. In the previous studies, Brassica juncea that were grown in hydroponic system yield high biomass when exposed to increased concentration of Zn (Anjum et al., 2012). Brassica juncea had great capacity to accumulate more amount of Zn from contaminated sites (Table 2). As Zn concentration increased from normal range (100 mg/L), it affects the plant growth (length and

dry weight). If amount of Zn is present in excess amount than it caused reduction in phosphorous and iron availability in Brassica juncea, which may also lead to reduction in growth (length and dry weight) of Brassica juncea. It also reduced the efficiency of this plant for phytoextraction (Hamlin et al., 2003). Normal growth of a plant gets affected if the metal concentration, the plant is taking up is increased from appropriate range. Brassica juncea is known to have the ability to accumulate large amount of heavy metals (Zn, Cu, Au, and Cd) from contaminated sites. Higher concentrations of heavy metals can be toxic to plants as they can hinder the growth of the plant and can cause physiological and structural damage to the plant at higher concentrations (Zhao et al., 2000). Tolerance index of the plant is the ability of plant to grow well and tolerate large amount of metal concentration, when exposed for a long time (Ghosh and Singh, 2005). Tolerance index was calculated for length and biomass of root, stem and leaf of Brassica juncea grown on soil and sand. Tolerance index of root length did not relate to the tolerance index of root biomass (Wu et al., 2007). As concentration of Zn increased from 20 to 160 mg/L, the tolerance index of Brassica juncea grown in soil and sand also increased as compared to control plant which showed it was more tolerant to Zn even on high levels (Table 3). It has been revealed that when M. flavida exposed with large concentration of Zn, its tolerance index increased as concentration of Zn increased from 1 -10 mg/L (Jamali et al., 2014). EC50 value of the plant described the plant’s ability to grow healthy and evaluate the tolerance level of plants that were grown on different concentration of metals (Köhl and Lösch, 1999). EC50 value was calculated for length and biomass of root, stem and leaf of Brassica juncea grown on soil and sand. Highest EC50 value was observed in stem and lowest EC50 value was observed in root of Brassica juncea grown on soil and sand. EC50 value of leaf is greater than

roots and lesser than stem which showed roots of Brassica juncea were more sensitive to Zn treatment than stem of Brassica juncea (Table 4). Accumulation of Zn in root, stem and leaf of Brassica juncea that were grown in soil at various concentrations (0, 20, 40, 80, and 160 mg/L) decreased with increase in concentration level as compared to control plant (Table 2). As results showed that root length and biomass of Brassica juncea decreased with increasing Zn concentration from normal range. For the accumulation of large amount of metal plants should also have large surface area of roots and root biomass in large quantity (Zhu et al., 1999). Brassica juncea that were grown in sand at various concentrations; their Zn accumulation, length and biomass production of root, stem and leaf area increased with increase in concentration level as compared to control. However, Brassica juncea that were grown in soil and sand accumulated more concentration of Zn in above ground tissues as compared to root (Table 2). Lorestani et al. (2011) has reported the similar results. Bioaccumulation factor is defined as the accumulated amount of metal in lower and above ground parts of plants. Various concentration of Zn accumulated in above and ground parts of Brassica juncea that were grown in soil and sand were determined. The maximum and minimum bioaccumulation ratio of Zn was calculated in 20 mg/L and 80 mg/L of Zn in plants that were grown in soil (4.449, 0.663) and 0 and 40 mg/L of Zn in plants that were grown in sand (5.146, 1.419) respectively, as compared to control plant (Table 5). These results indicated that bioaccumulation factor for each concentration was greater than 10 µg/g which showed lower and above ground parts of plants (Brassica juncea) accumulated more amount of metal and plants that accumulate high levels of metals, have great potential to be used for phytoextraction and considered as hyper-accumulator plants (Bu-Olayan and Thomas, 2009). Translocation factor is expressed as the accumulated amount of metals in Brassica juncea that was transferred from soil to Brassica juncea or from roots to its stem or from roots to leaves

(Table 6). Various concentrations of Zn that were transferred from roots to stem and leaves of Brassica juncea that were grown in soil and sand was determined. The maximum and minimum translocation factor of Zn in stem and leaves was recorded in 80 mg/L of Zn in plants that were grown in soil (2524.481, 3532.273) and 160 and 40 mg/L of Zn in plants that were grown in sand (61.50541, 102.3895) respectively, as compared to control plant. These results indicated that translocation factor for each concentration was greater than 1 which showed aerial parts of plants accumulated more amount of metal than their roots, such plants that accumulated more concentration of metals, exhibited great phytoextraction potential (Mojiri et al., 2013). Accumulation of Zn was observed in Brassica juncea at cellular level by light microscope at all given concentrations grown in sand. In leaf tissues, Zn accumulation was shown as black deposits under light microscope. The results showed that Zn deposit in epidermis, bundle sheet of veins and mesophyll cells. Zn accumulation in leaf tissue increased as concentration increased from 0 to 160 mg/L of Zn. Fig. 1 is showing the Zn deposits in control leaf, 20, 40, 80 and 160 mg/L. In the present study, the accumulation of Zn in sections of leaf tissues stained with sulfide silver stain were shown as black deposits along the epidermal walls and mesophyll cells under light microscope which increased with the increase in given concentration of Zn as compared to the control. Similar observations of metal accumulation using the same staining method were reported by Sridhar et al. (2005). At cellular level accumulation of Zn in Brassica juncea was observed by light microscope in all given concentrations (0, 20, 40,80 and 160 mg/L) that were grown in sand. In leaf tissues, Zn accumulation was shown as black deposits under light microscope. The results showed that Zn deposit in epidermis, bundle sheet of veins and mesophyll cells. Zn accumulation in leaf tissue increased as concentration increased from 0 to 160 mg/L of Zn. According to the literature, mostly Zn accumulated in leaf of Brassica

juncea in epidermis and bundle sheet of veins. This same pattern was observed in Thlaspi caerulescens, S. vulgaris and T. praecox (Vogel‐Mikuš et al., 2008). Research shows that in Echinochloa polystachya and Brassica juncea the heavy metals on the cellular and subcellular distribution of metal were mainly accumulated in xylem and xylem was the route via which metals were translocated from roots to leaves. Cellular distribution of heavy metals in plants is said to be linked with metal tolerance in plants. In this study, dark deposits that were presumed to contain metal accumulated less in the xylem vessels and leaves (Zhao et al., 2015). Hu et al. (2009) have used light microscopy along with different techniques in order to determine the distribution of metal in the tissues and cells of Potentilla graffiti H. His results have shown accumulation of metal in rhizodermal and cortex cells while light microscopy has shown metal distributed at the epidermal, cortex, xylem parenchyma and endodermal walls. Accumulation of metal was more in bundle sheath cells and epidermis in the leaves. Accumulation of metal in mesophyll cells was found at the higher concentration of metal i.e. 40 mg/L. 4. Conclusions Finally, it could be concluded that, this study showed Brassica juncea plant was able to grow in heavy metals contaminated soils and also able to accumulate extraordinarily high concentrations of Zn. The metal accumulating ability of this plant, coupled with the potential to rapidly produce large quantities of shoot biomass, makes this plant ideal for phytoextraction. The study demonstrated cellular distribution of Zn in leaves of Brassica juncea. Zn deposited along the epidermal walls and mesophyll cells as observed under light microscope which increased with the increase in given concentration of Zn as compared to the control. References

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Table 1: Dry weight of roots, stem and leaves of B. juncea treated with Zinc Zinc mg/L

Dry weight of Root (g) (Soil)

Dry weight of Root (g) (Sand)

Dry weight of stem (g) (Soil)

Dry weight of stem (g) (Sand)

Dry weight of Leaf (g) (Soil)

Dry weight of Leaf (g) (Sand)

Mean

Mean

Mean

Mean

Mean

Mean

S.D

S.D

S.D

S.D

S.D

S.D

0

0.0226

0.001

0.116

0.004

0.0186

0.003

0.038

0.005

0.050

0.004

0.078

0.002

20

0.0206

0.003

0.066

0.007

0.006

0.002

0.056

0.003

0.046

0.002

0.108

0.004

40

0.035

0.004

0.068

0.008

0.019

0.004

0.058

0.007

0.034

0.002

0.088

0.004

80

0.022

0.002

0.135

0.009

0.040

0.004

0.052

0.003

0.103

0.003

0.062

0.009

160

0.0163

0.001

0.0746

0.006

0.0413

0.003

0.073

0.004

0.073

0.013

0.114

0.01

Table 2: Zinc accumulation in different parts of B. juncea grown in soil and sand Zinc Zinc Accumulation Treatments in Soil (mg/Kg) mg/L Mean S.D

Zinc Accumulation in Root (mg/Kg) (Soil)

Zinc Accumulation in Stem (mg/Kg) (Soil)

Zinc Accumulation in Leaf (mg/Kg) (Soil)

Mean

Mean

Mean

S.D

S.D

S.D

0

1086.333

6.50

267.683

16.45

320.3

12.26

500.666

15.75

20

178.546

6.54

178.233

15.06

238.41

9.71

377.8

11.91

40

326.153

6.64

292.666

4.27

238.796

10.28

299.66

9.54

80

952.9

19.10

10.266

14.32

259.18

16.07

362.646

5.92

160

231.46

25.89

235.063

7.84

144.576

11.75

246.576

10.63

Zinc Zinc Accumulation Treatments in Sand (mg/Kg) mg/L Mean S.D

Zinc Accumulation in Root (mg/Kg) (Sand)

Zinc Accumulation in Stem (mg/Kg) (Sand)

Zinc Accumulation in Leaf (mg/Kg) (Sand)

Mean

Mean

Mean

S.D

S.D

S.D

0

84.333

10.52

151.86

12.69

147.52

13.85

134.663

7.74

20

120.77

5.46

134.873

5.35

108.003

11.63

145.67

9.63

40

290.933

4.91

149.723

7.46

122.39

12.84

140.956

6.80

80

108.156

8.70

182.22

4.51

119.563

8.40

140.953

4.68

160

98.596

9.51

167.683

5.56

155.49

6.27

171.866

3.93

Table 3: Tolerance indices (%) of B. juncea grown in soil and sand Soil

Zinc Treatment (mg/L)

Root Length

Stem Length

Leaf Area

Root Dry Weight

Stem Dry Weight

Leaf Dry Weight

20

70.204

98.4

98.666

91.176

32.142

92.052

40

35.306

96

93.333

294.117

101.785

68.211

80

30

91.2

89.333

98.529

216.071

205.960

160

26.530

90.4

88

72.0588

339.285

145.695

Sand 20

91.095

189.665

104.854

56.733

145.689

310.476

40

92.636

166.666

8.834

58.452

150.862

253.333

80

122.260

137.931

102.912

116.045

134.482

130.476

160

123.458

195.402

113.592

64.183

190.517

391.428

Table 4: EC50 toxicity thresholds for roots, stem and leaves of B. juncea treated with Zinc EC50 (mg/kg) Medium of Growth Roots

Stem

Leaf

Soil

43.19

60

58.58

Sand

79.12

92.28

81.18

Table 5: Bioaccumulation Factor of Zinc in Brassica juncea grown in soil and sand Zn Treatment (mg/L)

Zn in Soil

Zn in Plant

(mg/kg)

(mg/kg)

Bioaccumulation Factor (BAF) (soil)

Zn in Sand

Zn in Plant

(mg/kg) (mg/kg)

Bioaccumulation Factor (BAF) (sand)

0

1086.333 1088.65

1.002

84.333

434.043

5.146

20

178.546

794.443

4.449

120.77

388.546

3.217

40

326.153

831.123

2.548

290.933

413.07

1.419

80

952.9

632.093

0.663

108.156

442.736

4.093

160

231.46

626.216

2.705

98.596

495.04

5.020

Table 6: Translocation Factor of Zinc in B. juncea grown in soil and sand Translocation Factor (TF)

Translocation Factor (TF)

(Soil)

(Sand)

Zn Treatment (mg/L)

Root to stem

Root to leaf

Root to stem

Root to leaf

0

119.656

187.036

97.142

88.675

20

133.762

211.969

80.077

108.005

40

81.593

102.389

81.744

94.144

80

2524.481

3532.273

65.614

77.353

160

61.505

104.898

92.728

102.494

Fig. 1 Light micrographs of Brassica juncea leaves treated with various concentration of Zn (a) Zn deposits in control leaf, (b) Zn deposits in leaf at 20 mg/L, (c) Zn deposits in leaf at 40 mg/L, (d) Zn deposits in bundle sheet of vein of leaf at 40 mg/L, (e) Zn deposits in leaf at 80 mg/L and (f) Zn deposits in leaf at 160 mg/L Darker Parts Show the Zinc Deposits.