Biochar alleviates fluoride toxicity and oxidative stress in safflower (Carthamus tinctorius L.) seedlings

Biochar alleviates fluoride toxicity and oxidative stress in safflower (Carthamus tinctorius L.) seedlings

Chemosphere 223 (2019) 406e415 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Biochar ...

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Chemosphere 223 (2019) 406e415

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Biochar alleviates fluoride toxicity and oxidative stress in safflower (Carthamus tinctorius L.) seedlings Kazem Ghassemi-Golezani*, Salar Farhangi-Abriz Department of Plant Eco-physiology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Fluoride toxicity reduced safflower seedling growth via enhancing oxidative stress.  Fluoride solubility and uptake were reduced by biochar application.  Fluoride toxicity and oxidative stress were alleviated by biochar treatment.  Biochar application improved safflower seedling growth under fluoride toxicity.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 December 2018 Received in revised form 20 January 2019 Accepted 13 February 2019 Available online 14 February 2019

An original research was laid out as factorial to evaluate the possible effects of biochar (0, 25 and 50 g kg1 soil) on mitigating fluoride toxicity (non-contamination, 100, 200, 400 and 800 mg NaF kg1 soil) in safflower seedlings. Increasing fluoride toxicity up to 200 mg NaF kg1 soil did not decrease the safflower growth. However, the growth of plants under 400 and 800 mg NaF kg1 was reduced by enhancing soluble fluoride concentration in the soil. This growth reduction was the consequence of an increase in fluoride content of plant tissues, generation of super oxide radicals and hydrogen peroxide, lipid peroxidation, misbalancing potassium and calcium ions, and a decrease in synthesis of photosynthetic pigments including chlorophylls, carotenoids, anthocyanin, flavonoids and xanthophyll's and photochemical efficiency of photosystem II. Application of biochar decreased the fluoride solubility, fluoride content of plant tissues, oxidative stress and antioxidant enzymes activities, leading to an increase in cation exchange capacity of soil and the pH, calcium and potassium uptakes, maximum efficiency of photosystem II, synthesis of photosynthetic pigments, and plant growth. In general, addition of 50 g biochar to 1 kg soil was the best treatment for alleviation of the fluoride toxicity in safflower plants. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: T Cutright Keywords: Antioxidant enzymes Biochar Fluoride toxicity Oxidative stress Photosynthetic pigments

1. Introduction

* Corresponding author. E-mail addresses: [email protected] (K. Ghassemi-Golezani), [email protected] hotmail.com (S. Farhangi-Abriz). https://doi.org/10.1016/j.chemosphere.2019.02.087 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

Fluoride is the 13th most plentiful element in the soil and is one of the most extensive elements in the pedosphere. This element can be increased through industrial activities related to aluminum extraction, irrigation and high rate of phosphorus fertilizers in the

K. Ghassemi-Golezani, S. Farhangi-Abriz / Chemosphere 223 (2019) 406e415

 soil (Jha et al., 2009; Alvarez-Ayuso et al., 2011). Fluoride is found in many soils, ranging from 20 to 1000 mg g1 (Davison, 1983). The availability of fluoride in soil is generally controlled through the soil pH and fluoride adsorption by mineral parts of the soil (Samal et al., 2015). In usual form, the fluoride is adsorbed to the soil and  therefore plant uptake of fluoride is usually minimal (AlvarezAyuso et al., 2011; Ropelewska et al., 2016), however, in soils polluted with fluoride and/or under low pH, fluoride availability and solubility were heightened and plants may take up it in  excessive quantities (Alvarez-Ayuso et al., 2011). The adverse effect of fluoride on plants is revealed, for example, by necrosis and chlorosis of leaves and a decrease in photosynthetic pigments, leading to a reduction in plant growth rate and biomass (Gupta et al., 2009; Yadu et al., 2018). High level of fluoride inside the plant cells has been shown to influence the physiological cycles, disturbance of nutrients, water usage and revealing toxicity symptoms, such as chlorophyll degradation, low seedling establishment, growth rate and photosynthetic activities, high reactive oxygen species (ROS) generation and consequently membrane damage (Panda, 2015; Yadu et al., 2016, 2017). Most of the pollutants activate the generation of ROS inside the plant cells, make oxidative stress and cause redox imbalances of cells (Yadu et al., 2016; Farhangi-Abriz and Ghassemi-Golezani, 2018; Liu et al., 2018). ROS are naturally created through the cellular metabolic reactions such as photochemical activities of photosystems I and II. The level of ROS can be easily influenced by the environmental stress. The chief cellular mechanisms susceptible to injury by ROS are phospholipids, nucleic acids, carbohydrates and proteins (You and Chan, 2015). Plant cells are secure against the adverse effects of ROS by several antioxidant mechanisms, including low molecular mass compounds and enzymes regenerations such as superoxide dismutase (SOD), peroxidases (POX) and catalase (CAT) (Farhangi-Abriz and Ghassemi-Golezani, 2018; Ghassemi-Golezani and Nikpour-Rashidabad, 2017). Several strategies such as rising soil carbon content by biochar have been established to decrease the toxic effects of pollutants in plants (Ali et al., 2017; Farhangi-Abriz and Torabian, 2017; Ramzani et al., 2017; Abbas et al., 2017). Biochar is the carbon-rich material that is obtained from organic resources such as wood, manure or leaves, when they are heated under low or anoxia conditions. In other words, biochar is produced by so-called thermal decomposition of organic material under limited supply of oxygen (O2) at temperatures up to 700  C (Lehmann and Joseph, 2015). Biochar is added to the soil for improving organic carbon storage, soil structure and crop productivity. The physical characteristics of biochar directly or indirectly are related with the way affecting soil systems (Xu et al., 2016). Since biochars are manufactured from biomass, they contain a range of plant macro and micro-nutrients (Glaser et al., 2015; Lehmann and Joseph, 2015; Ali et al., 2017). Biochar not only improves plant growth and productivity under non-stress situations, but also enhances crop yield under stressful conditions such as drought (Ali et al., 2017), salinity (Farhangi-Abriz and Torabian, 2017) and heavy metal stress (Abbas et al., 2017). Biochar application reduces the oxidative stress in plants, modifies the antioxidant enzyme activities, and reduces the bioavailable cadmium (Abbas et al., 2017). Up to date, there was no any report concerning the biochar effect on bioavailability of fluoride and oxidative stress of fluoride toxicity in plants. Therefore, the aim of this research was to evaluate the effects of biochar on bioavailability of fluoride in the soil and oxidative stresses of fluoride toxicity on safflower (Carthamus tinctorius L.) seedlings.

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2. Materials and methods 2.1. Experimental conditions A pot experiment with three replications was conducted in a glass greenhouse with the day and night temperatures of 25 and 22  C, respectively, 140 W m2 light intensity, and about 13 h photoperiod. This research was laid out as factorial based on randomized complete block design. The soil samples were collected from the upper layer (0e20 cm) of the Agricultural Research Farm of the University of Tabriz and analyzed according to Carter and Gregorich (2008) (Texture: silty loam; pH: 5.9; EC: 1.68 dSm1; Organic carbon: 13.1 g kg1; Total N: 0.08%; Available P: 37 mg kg1; Available K: 157 mg kg1 and Cation exchange capacity: 17.1 cmol kg1). The biochar was purchased from a local company (Total N: 0.75%; Total C: 32.96%; Total H: 1.7%; Total O: 28.43%; Total Na: 8.3 mg kg1; Total K: 3210 mg kg1; Total Ca: 3470 mg kg1; Total Mg: 960 mg kg1; cation exchange capacity: 20.8 cmol kg1; pH: 7.8). The carbon, hydrogen, nitrogen and oxygen contents of biochar were measured by an elemental analyzer (Elementar, Germany), and other nutrients were analyzed by a flame photometer (Corning flame photometer, 410). Five levels of fluoride [0, 100, 200, 400 and 800 mg NaF kg1 soil (About 4.5, 11.5, 22, 45.5 and 73.5 mg soluble fluoride kg1 soil)] and three levels of biochar (0, 25 and 50 g kg1 soil) were applied. The soil was contaminated with sodium fluoride (NaF) and then thoroughly mixed with biochar according to the treatments. Each polyethylene pot (20  22 cm) was filled with 2.7 kg untreated (control) or treated soil, using 45 pots in general. Subsequently, three seeds of Carthamus tinctorius L were sown in each pot and irrigated with tap water to achieve 100% field capacity (FC). Seven days after sowing (early seedling emergence), 10 g of a fertilizer (Master 20-20-20-Valagro-Italy) was dissolved in a liter of water (EC ¼ 0.8 dS m1, pH ¼ 7.1) and added to the pots (300 mL per pot). Plants within all pots were harvested at 6 leaves stage, and biochemical and physiological measurements were performed. The cation exchange capacity of soil in each pot was determined by the ammonium acetate method (Chapman, 1965) and the total soluble fluoride of soil was assayed by the method of Larsen and Widdowson (1971). The soil pH was also measured by a pH meter (Model: HI 99121, Hanna Instrument, USA). 2.2. Cation analysis Each powdered sample of dried leaf (200 mg) was separately burned at 560  C for 7 h in an electric furnace and the ashes digested in 10 mL 1 M HCl at 25  C for 24 h. The Kþ (Potassium) and Ca2þ (Calcium) contents in the digested samples were measured by a flame photometer (Corning flame photometer, 410). 2.3. Fluoride content of plant tissues Plant tissues were oven-dried at 80  C for 48 h and then powdered. About 500 mg of the powdered root or shoot tissues were separately digested in 0.1 N HCl at 25  C for 24 h. Subsequently, the samples were transferred to a hot plate (120  C) and kept there for 1 h. The samples were then centrifuged at 25  C in 5000 rpm for 15 min. The supernatant was collected and mixed with a total ionic strength adjustment buffer (TISAB buffer) and distilled water, and assayed by the ion selectivity method (Orion 9609, Thermo Scientific, USA). The fluoride content in plant tissues was estimated by NaF 99.99% standard (Sigma-Aldrich, United States) as mg kg1 dry weight (DW) (Cui et al., 2012). Fluoride uptake by root was calculated as a ratio of total plant fluoride content per root dry weight. Fluoride translocation from root to

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shoot was determined as the ratio of shoot fluoride content/root fluoride content. 2.4. Plant growth Root and shoot lengths and dry weights of safflower were determined after oven drying at 80  C for 48 h. Then, the means of these growth parameters for each pot were calculated. 2.5. Soluble protein and antioxidant enzymes The soluble protein contents of root and leaf samples were assayed according to the method of Bradford (1976). Initially 1 g of root and leaf tissues homogenized in 4 mL Na-phosphate buffer, then the homogenized samples centrifuged at 12.000 g for 10 min. The supernatants were collected and the absorbance was recorded at 595 nm, using a UVevisible spectrophotometer (Dynamica, Halo DB-20 eUVeVisible Spectrophotometer, United Kingdom). The enzyme was extracted from 1 g of each plant sample (root and leaf) by potassium phosphate buffer (pH 7.0). The homogenate sample was centrifuged at 15,000 g for 15 min at 4  C and the supernatant was used as the source of enzyme. The activity of POX was measured according to Gueta-Dahan et al. (1997). The activity of SOD was assayed by the nitro blue tetrazolium method as described by Giannopolitis and Ries (1977). The activity of CAT was assayed by changing absorbance at 240 nm and described as Ug1 FW (Singh et al., 2010). 2.6. Malondialdehyde, H2O2, and O2 

Lipid peroxidation was assayed by the level of oxidation of membrane polyunsaturated fatty acids producing malondialdehyde (MDA), as described by Cakmak and Horst (1991). The plant tissues (0.5 g) were homogenized in 5 mL of 5% trichloroacetic acid and centrifuged for 10 min at 1800 g. The supernatant was collected and added to 2-thiobarbituric acid. Then the tubes were heated in a Bain Marie at 98  C for 10 min. Then, the reaction was stopped in ice bath and absorbance of the supernatant was recorded at 532 and 600 nm. For measuring H2O2 content in roots and leaves of safflower, about 1000 mg of plant tissues were homogenized in 5 mL of 0.1% trichloro-acetic acid at 4  C and centrifuged at 12,000 g for 10 min. 0.5 mL of the supernatant was collected and added to the 0.5 mL of potassium phosphate buffer (pH 7) plus 1 mL of potassium iodide (1 M) and the absorbance was read at 390 nm (Hsu and  Kao, 2007). The O2 generation was measured by the method of Wang and Jiao (2000). 2.7. Leaf area and chlorophyll fluorescence Leaf area (LA) was measured by a portable area meter (model ADC-AM 300 UK) as cm2 per plant. Maximum efficiency of photosystem II [Variable fluorescence (Fv)/maximum fluorescence (Fm)] was estimated by a portable chlorophyll fluorometer (OS-30, OPTISCIENCES, USA). Dark-adapted leaves (30 min) were initially exposed to a weak modulate measuring beam, followed by exposure to saturated white light to estimate the initial (F0) and maximum (Fm) fluorescence values, respectively. Variable fluorescence (Fv) was calculated by subtracting F0 from Fm. 2.8. Photosynthetic pigments About 1 g of each leaf sample was homogenized in 4 mL acetone (80%), and then centrifuged at 12,000 g for 20 min at 4  C. The supernatant was collected and the absorbance was recorded at 645 and 663 nm (for chlorophylls) and 480 and 510 nm (for

carotenoids), using a spectrophotometer (Arnon, 1949; Maclachlan and Zalik, 1963). For anthocyanin, 1 g of fresh leaf sample was homogenized in 3 mL extraction mixture (0.6 mL water, 2.37 mL methanol and 0.03 mL HCl) followed by centrifuging at 12,000 g for 20 min at 4  C. The absorbance of supernatant was read at 530 and 657 nm (Mancinelli, 1984). Total flavonoid content was measured according to the methods of Zhishen et al. (1999). The xanthophyll content was measured by a spectrophotometer according to Lawrence (1990), using 50 mg dried leaf sample and recording the absorbance at 474 nm. 2.9. Statistical analysis Data were analyzed by MSTATC software according to the experimental design, and means were compared with Duncan multiple range test at p  05. All figures were drawn, using the Microsoft Excel 2016. 3. Results 3.1. Soil pH and EC The interaction of fluoride toxicity  biochar was significant for the soil pH and CEC (p  0.01). In general, increasing fluoride toxicity considerably decreased these soil characters. The soil pH and CEC did not change significantly up to 200 mg NaF kg1 and 100 mg NaF kg1 soil, respectively (Table 1). The biochar treatment enhanced soil pH by 7e13% and CEC by 4e9%, but this improvement was more evident under high level of fluoride toxicity, although there was no significant difference between 25 and 50 g kg1 biochar (Table 1). 3.2. Soluble fluoride concentration The soluble fluoride concentration of soil increased with increasing fluoride toxicity, yet decreased with the addition of biochar to the soil. The difference of non-biochar and biochar treatments was more evident under 400 and 800 mg NaF kg1 soil. The reducing effects of 25 and 50 g biochar per 1 kg soil on soluble fluoride concentration under 800 mg NaF kg1 soil were 18% and 23%, respectively (Fig. 1). 3.3. Kþ and Ca2þ contents Significant interaction of fluoride toxicity and biochar was observed for Kþ and Ca2þ contents of safflower leaves (p  0.01). Increasing fluoride toxicity up to 200 mg NaF kg1 soil did not alter the Kþ and Ca2þ contents of safflower leaves, but these cations contents reduced under 400 and 800 mg NaF kg1 soil. Biochar applications under all levels of fluoride contamination and noncontamination conditions significantly improved the Kþ and Ca2þ contents of safflower leave (Table 1). 3.4. Plant growth Fluoride toxicity and biochar had significant effects on dry weights of the roots, shoots, as well as their lengths in safflower. Increasing fluoride toxicity up to 200 mg NaF kg1 soil did not alter the root and shoot dry weights and their lengths, but these parameters were decreased with further increase of fluoride. Mixing biochar with the soil increased the dry weights of the roots and shoots and their lengths under fluoride-free condition and all levels of fluoride toxicity. In general, treatment of soil with 50 g biochar kg1 soil had better effect than 25 g biochar kg1 soil on increasing

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Table 1 Means of soil pH, cation exchange capacity of soil (CEC), Kþ and Ca2þ contents of leaves, root and shoot dry weights and length of safflower seedlings under different levels of fluoride toxicity and biochar treatments. Fluoride (mg NaF kg1 soil)

Biochar (g kg1 soil)

Soil pH

Soil CEC (cmol kg1 Soil)

Kþ content (mg g1 DW)

Ca2þ Content (mg g1DW)

Root DW (g) Shoot DW (g)

Root length (cm)

Shoot Length (cm)

0

0 25 50 0 25 50 0 25 50 0 25 50 0 25 50

5.63 ± 0.06e 6.16 ± 0.05abc 6.09 ± 0.07abc 5.60 ± 0.03e 6.13 ± 0.08abc 6.33 ± 0.08a 5.56 ± 0.06e 6.24 ± 0.04 ab 6.23 ± 0.05 ab 5.43 ± 0.03e 5.98 ± 0.05cd 5.85 ± 0.06d 5.20 ± 0.03f 5.80 ± 0.03d 5.70 ± 0.08d

19.20 ± 0.10cde 20.10 ± 0.16b 20.46 ± 0.12 ab 18.93 ± 0.06def 20.70 ± 0.26a 20.86 ± 0.36a 18.46 ± 0.18g 19.53 ± 0.26c 19.33 ± 0.15cd 18.06 ± 0.22h 18.80 ± 0.17cfg 18.86 ± 0.11efg 17.33 ± 0.13i 18.60 ± 0.20 fg 18.73 ± 0.14 fg

19.34 ± 0.52b 22.21 ± 0.55a 21.95 ± 0.74a 19.29 ± 0.66b 21.45 ± 0.81a 22.31 ± 0.72a 19.65 ± 0.47b 22.03 ± 0.65a 22.36 ± 0.64a 15.32 ± 0.41e 17.02 ± 0.34d 18.10 ± 0.59c 12.21 ± 0.57g 14.44 ± 0.33f 14.36 ± 0.45f

10.59 ± 0.22cd 11.43 ± 0.29b 12.13 ± 0.33a 10.70 ± 0.19cd 11.63 ± 0.19 ab 11.76 ± 0.21 ab 11.01 ± 0.61c 12.12 ± 0.27a 11.86 ± 0.18 ab 8.43 ± 0.12f 9.14 ± 0.31e 10.33 ± 0.31d 6.12 ± 0.25i 6.78 ± 0.11h 7.36 ± 0.14g

0.77 ± 0.02c 0.82 ± 0.03 ab 0.83 ± 0.02 ab 0.74 ± 0.02d 0.82 ± 0.08 ab 0.82 ± 0.02 ab 0.75 ± 0.03cd 0.81 ± 0.02b 0.84 ± 0.01a 0.60 ± 0.01h 0.73 ± 0.03e 0.75 ± 0.01d 0.43 ± 0.01i 0.64 ± 0.04g 0.67 ± 0.02f

6.33 ± 0.15b 7.23 ± 0.22a 7.50 ± 0.17a 6.06 ± 0.17b 7.20 ± 0.11a 7.36 ± 0.14a 6.13 ± 0.07 b 7.40 ± 0.17a 7.56 ± 0.14a 4.13 ± 0.12e 5.16 ± 0.08c 5.00 ± 0.05cd 3.93 ± 0.09e 4.70 ± 0.21d 5.06 ± 0.17cd

22.10 ± 0.32 d 24.36 ± 0.19bc 25.16 ± 0.42 ab 21.83 ± 0.21d 24.10 ± 0.20c 25.36 ± 0.24a 21.86 ± 0.29d 24.46 ± 0.18abc 25.06 ± 0.65 ab 16.67 ± 0.13g 18.00 ± 0.39f 19.34 ± 0.35e 14.65 ± 0.26h 16.71 ± 0.24g 18.67 ± 0.41ef

100

200

400

800

2.04 ± 0.09c 2.30 ± 0.04b 2.44 ± 0.08a 2.15 ± 0.06c 2.36 ± 0.03b 2.47 ± 0.09a 2.11 ± 0.05c 2.37 ± 0.06b 2.50 ± 0.08a 1.63 ± 0.04f 1.77 ± 0.03e 1.87 ± 0.04d 1.17 ± 0.08h 1.48 ± 0.09g 1.60 ± 0.06f

Data represents the average of three replicates (n ¼ 3) ± standard error. Different letters between the Fluoride and biochar treatments indicate significant difference at p  0.05. DW: dry weight.

Soluble fluoride (mg kg-1 Soil)

80 70 60 50 40 30 20 10 0 0

100

200

400

800

Fluoride added (mg NaF kg-1 Soil) 0

25

50

Fig. 1. Changes in soluble fluoride concentration of soil in response to the biochar (0, 25 and 50 g biochar kg1 soil) under fluoride toxicity of soil. Data represents the average of three replicates.

plant growth under the high level of fluoride toxicity (Table 1). 3.5. Fluoride content of plant tissues Fluoride toxicity and biochar significantly influenced the fluoride content of roots and leaves (p  0.01). The fluoride content in roots and leaves increased with rising fluoride concentration of soil, but decreased under the fluoride contaminated soils with the addition of biochar to the soil (Fig. 2). Treatment of soil with biochar under non fluoride contaminated soil did not show any effect on fluoride content of plant tissues of safflower. There was no significant difference between 25 and 50 g biochar kg1 soil, in fluoride content of safflower roots and leaves under the non-contaminated, 100 and 200 mg NaF kg1 soil, but under 400 and 800 mg NaF kg1

soil, biochar treatment with 50 g biochar kg1 soil showed a better effect than 25 g biochar kg1 soil. Similar to fluoride content of plant tissues, fluoride uptake by the plants increased with rising fluoride levels of soil. All of the biochar treatments in fluoride contaminated soils, significantly decreased the uptake of fluoride from the soil to the plants. However, under non-contaminated soil, addition of biochar did not show a tangible effect on fluoride uptake. There was no noticeable difference between the 25 and 50 g biochar kg1 soil in fluoride uptake of safflower plants under non-contaminated, 100, 200 and 400 mg NaF kg1 soil, but under the high level of fluoride toxicity, biochar treatment with 50 g biochar kg1 soil showed the superior effect than 25 g biochar kg1 soil. The safflower plants up to 100 mg NaF kg1 soil showed a better effect in diminishing fluoride translocation to the shoots, but with further rising in fluoride levels of the soil, translocation of fluoride from the roots to the shoots was increased. Biochar application under 200, 400 and 800 mg NaF kg1 soil, significantly reduced the translocation of fluoride to the shoots of safflower plants, but the addition of biochar to the soil under non-contaminated soils and 100 mg NaF kg1 did not show any significant effect on fluoride translocation to safflower plants. Under all levels of fluoride toxicity, both biochar treatments showed a similar effect on decreasing translocation of fluoride (Fig. 2).

3.6. Antioxidant activities Fluoride toxicity and biochar treatments had significant effects on antioxidant activities of the root and leaf of safflower (p  0.01). Increasing the fluoride level up to 100 mg NaF kg1 soil did not alter the activities of antioxidant enzymes in roots and leaves, but further rising in fluoride concentration of soil caused an increase in CAT, SOD and POX activities in safflower plants, compared with the non-contaminated and 100 mg NaF kg1 soil. Maximum rise in activity of antioxidant enzymes was observed under 800 mg NaF kg1 soil. Biochar addition to the soil significantly decreased CAT, SOD and POX activities in safflower roots and leaves, compared with the non-biochar treatment. In most cases, the lowest activity of CAT, POX and SOD under the highest level of fluoride toxicity was observed in 50 g biochar kg1 soil. Treatment of soil with biochar under non-contaminated soil did not show any considerable effect on CAT, SOD and POX activities in safflower plants (Fig. 3).

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Fluoride transloca on (Shoot fluride/ Root fluride)

a 800

ab bcd bcd

1 a

1000

Fluoride uptake by root (mg per root DW)

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bc de cd

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de de e

100

de de de

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0.6 0.4 0.2 0

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Fluoride added (mg NaF kg-1 Soil) 0

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Fig. 2. The content of fluoride in roots and leaves of safflower in response to the biochar applications (0, 25 and 50 g biochar kg1 soil) under fluoride toxicity of soil. Data represents the average of three replicates (n ¼ 3) ± standard error. Different letters between the fluoride and biochar treatments indicate significant difference at p  0.05.

3.7. ROS generation and lipid peroxidation Fluoride toxicity and biochar treatments showed a significant interaction for ROS generation and lipid peroxidation in safflower roots and leaves (p  0.01). Fluoride level of 100 mg NaF kg1 soil did not affect ROS generation and lipid peroxidation, but with further increase of fluoride in the soil, the concentrations of H2O2,  O2 and MDA were also increased in roots and leaves. Maximum increase in lipid peroxidation and ROS generation was observed under 800 mg NaF kg1 soil. When compared to no-biochar treatment, any biochar addition to the soil reduced the ROS generation and lipid peroxidation in safflower plants. In most cases, both levels of biochar under all levels of fluoride toxicity showed statistically similar effects on reducing ROS generation and lipid peroxidation in roots and leaves. However, treatment of soil with 50 g biochar kg1 soil revealed a superior effect than 25 g biochar kg1 soil on  reducing O2 generation in safflower roots. The application of biochar to the soil under non-contaminated and 100 mg NaF kg1 soil  did not show any significant effect on H2O2, O2 and MDA contents in safflower plants (Fig. 4).

3.8. LA and Fv/Fm Significant interaction of fluoride toxicity and biochar treatments was observed for LA and Fv/Fm of safflower leaves (p  0.01). Increases in fluoride level to 100 mg NaF kg1 soil did not affect the LA and Fv/Fm of safflower leaves, but these parameters were

decreased due to a further increase in fluoride levels. In general, biochar treatments under all levels of fluoride contaminations and non-contamination condition significantly improved the LA and Fv/ Fm of safflower. However, application of biochar to the soil under 100 mg NaF kg1 soil did not show a tangible effect. Treatment of soil with 50 g biochar kg1 soil exposed better effect than 25 g biochar kg1 soil on increasing LA and Fv/Fm of safflower (Table 2).

3.9. Photosynthetic pigments The interaction of fluoride toxicity and biochar was significant for all of the photosynthetic pigments of safflower (p  0.01). Fluoride level of 100 mg NaF kg1 soil did not affect the contents of Chl a, b, and Chl a/b ratio in safflower leaves, but with further increase in fluoride level of soil, the contents of Chl a and b were decreased in leaves. Adding biochar to the soil significantly increased chlorophyll content of safflower plants under all fluoride levels. However, Chl b was not affected by biochar under 200 mg NaF kg1 soil. In high level of fluoride toxicity (800 mg NaF kg1 soil), biochar treatment with 50 g biochar kg1 soil showed a better effect than 25 g biochar kg1 soil. With the exception of nonfluoride treatment, the addition of biochar to the soil increased Chl a/b ratio in safflower leaves (Table 2). The anthocyanin and carotenoid contents were not significantly affected by 100 mg NaF kg1 soil, but with increasing fluoride level in soil, significantly reduced these pigments. Biochar addition to the soil in non-fluoride, 100 and 200 mg NaF kg1 soil treatments,

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POX Leaf (U g-1 FW)

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50

Fig. 3. Changes in antioxidant enzymes activities in safflower roots and leaves under different rates of biochar applications (0, 25 and 50 g biochar kg1 soil) and fluoride toxicity. Data represents the average of three replicates (n ¼ 3) ± standard error. Different letters between the fluoride and biochar treatments indicate significant difference at p  0.05. CAT: catalase; POX: peroxidase; SOD: Superoxide dismutase.

did not significantly affect anthocyanin content of leaves, but increased the anthocyanin content in other levels of fluoride toxicity. Biochar treatments significantly improved the carotenoid content of leaves under all levels of fluoride contamination and non-contamination conditions (Table 2). Increasing fluoride toxicity up to 200 mg NaF kg1 soil did not significantly change leaf flavonoid content, but it was reduced under 400 and 800 mg NaF kg1 soil. The flavonoid content was also significantly improved by biochar treatment under 400 and

800 mg NaF kg1 soil, but no significant improvement was observed under low levels of fluoride toxicities. The xanthophyll content was not significantly varied under 100 mg NaF kg1 soil, but increasing fluoride level in the soil, significantly reduced this pigment. Biochar treatments under 200, 400 and 800 mg NaF kg1 soil significantly enhanced the xanthophyll content of safflower leaves (Table 2).

K. Ghassemi-Golezani, S. Farhangi-Abriz / Chemosphere 223 (2019) 406e415

0.8

b b e e

d 0

100

f f

0.2

0

0 0 100 200 400 800 Fluoride added (mg NaF kg-1 Soil) 50

0

0.4

0.4

0.1 0

25

50

0.2

bc c

b

0.3

de ef

a b bc

b d de

de ef

c g g g

g fg g

0.2

800

a

0.5

Leaf H2O2 (μmol g-1 FW)

0.5

0.3

400

d efg fgh

25

200

Fluoride added (mg NaF kg-1 Soil)

h gh h fgh gh h

0

Root H2O2 (μmol g-1 FW)

c

0.4 g g g

f f

g g g

0.2

e

e

e

d

0.4

0.6

g g g

c

b

0.6

a

0.8

Leaf O2 (μmol g-1FW h-1)

1

a

1

g g g

Root O2 (μmol g-1 FW h-1)

412

0.1 0

0

100

200

400

800

0

Fluoride added (mg NaF kg-1 Soil) 0

25

100

200

400

800

Fluoride added (mg NaF kg-1 Soil)

50

0 6

5

5

50

1 0

b b c

4

f f

2

g g g

e

d d

3 g g g

e f fg

fg fg fg

2

d de

3

Leaf MDA (mmol g-1 FW)

a b b c

4

g fg fg

Root MDA (mmol g-1 FW)

a

6

25

1 0

0

100

200

400

800

Fluoride added (mg NaF kg-1 Soil) 0

25

50

0 100 200 400 800 Fluoride added (mg NaF kg-1 Soil) 0

25

50

(O2 

Fig. 4. The generation of reactive oxygen species and H2O2) and lipid peroxidation (MDA) in safflower roots and leaves under different rates of biochar usage (0, 25 and 50 g biochar kg1 soil) and fluoride toxicity. Data represents the average of three replicates (n ¼ 3) ± standard error. Different letters between the fluoride and biochar treatments indicate significant difference at p  0.05.

3.10. Principal component analysis (PCA) PCA was used to assess the relation of fluoride toxicity with growth, physiological and biochemical properties of the safflower plants. The PCAs were contributed up to 92% of variance in root and leaf characters. According to the PCA of leaf and root, Fluoride content, ROS generation and antioxidant enzymes were in the opposite direction, but Ca, K, photosynthetic pigments, LA, soil pH and soil CEC were in the positive direction with root and shoot

growth of safflower (Fig. 5). Shoot dry weight of safflower had a strong positive correlation with Ca, K and flavonoids. The contents of fluoride and Chl a in leaf tissues were also highly correlated with shoot dry weight. Chl a and Chl b were more effective than anthocyanin and carotenoids in improving Fv/Fm. Xanthophyll showed a positive correlation with chlorophyll biosynthesis in safflower leaves. Plant growth was decreased with increasing fluoride uptake by roots, while fluoride translocation had no strong effect in decreasing plant growth.

K. Ghassemi-Golezani, S. Farhangi-Abriz / Chemosphere 223 (2019) 406e415

413

Table 2 Means of photosynthetic pigments, efficiency of photosystem II and leaf area of safflower seedlings under different levels of fluoride toxicity and biochar treatments. Fluoride (mg NaF kg1 soil)

Biochar (g kg1 soil)

LA (cm2)

Fv/Fm

Chl a (mg g1DW)

Chl b (mg g1DW)

Chl a/b

Ant (mg g1DW) Car (mg g1DW)

Fla (mg g1DW)

Xan (mg g1DW)

0

0 25 50 0 25 50 0 25 50 0 25 50 0 25 50

82.66 ± 2.81c 95.00 ± 1.82b 101.33 ± 1.83a 84.00 ± 1.89c 94.03 ± 1.72b 103.66 ± 2.11a 77.02 ± 1.82de 85.12 ± 3.10c 92.01 ± 2.11b 67.14 ± 0.91h 75.00 ± 1.04ef 79.23 ± 1.52d 62.66 ± 1.12i 71.00 ± 1.22g 73.33 ± 0.76 fg

0.79 ± 0.01e 0.82 ± 0.01c 0.87 ± 0.01a 0.81 ± 0.02cd 0.85 ± 0.01b 0.85 ± 0.01b 0.73 ± 0.01 fg 0.79 ± 0.01de 0.81 ± 0.01c 0.72 ± 0.01gh 0.78 ± 0.02e 0.81 ± 0.01cd 0.63 ± 0.02i 0.71 ± 0.01h 0.74 ± 0.01f

1.91 ± 0.03bc 1.94 ± 0.02b 2.02 ± 0.02a 1.88 ± 0.02c 1.94 ± 0.02b 2.00 ± 0.01a 1.82 ± 0.01d 1.88 ± 0.03c 1.88 ± 0.02c 1.52 ± 0.03f 1.69 ± 0.03e 1.73 ± 0.02e 1.22 ± 0.01h 1.45 ± 0.02g 1.51 ± 0.03f

0.95 ± 0.02bc 0.97 ± 0.01bc 1.01 ± 0.02a 0.95 ± 0.01c 0.97 ± 0.01b 0.96 ± 0.01bc 0.86 ± 0.02d 0.85 ± 0.02d 0.85 ± 0.01d 0.69 ± 0.01f 0.73 ± 0.01e 0.73 ± 0.02e 0.52 ± 0.02i 0.61 ± 0.01h 0.63 ± 0.02g

1.98 ± 0.03gh 2.00 ± 0.01g 2.00 ± 0.01g 1.98 ± 0.01h 2.00 ± 0.01g 2.08 ± 0.01f 2.10 ± 0.02f 2.20 ± 0.01e 2.23 ± 0.01e 2.20 ± 0.02e 2.33 ± 0.01b 2.40 ± 0.02a 2.30 ± 0.01c 2.40 ± 0.02a 2.40 ± 0.02a

0.053 ± 0.002 ab 0.057 ± 0.004 ab 0.050 ± 0.003 ab 0.050 ± 0.003 ab 0.060 ± 0.008a 0.050 ± 0.006 ab 0.030 ± 0.009 cd 0.047 ± 0.009abc 0.047 ± 0.010abc 0.020 ± 0.005d 0.040 ± 0.002bc 0.43 ± 0.007abc 0.017 ± 0.005d 0.040 ± 0.008bc 0.040 ± 0.009bc

0.52 ± 0.02de 0.54 ± 0.02cd 0.58 ± 0.01a 0.54 ± 0.01cd 0.55 ± 0.01bc 0.54 ± 0.01cd 0.55 ± 0.01bc 0.51 ± 0.02e 0.56 ± 0.02 ab 0.41 ± 0.02g 0.47 ± 0.01f 0.47 ± 0.01f 0.32 ± 0.02i 0.40 ± 0.01gh 0.38 ± 0.01h

0.69 ± 0.02 ab 0.71 ± 0.02a 0.70 ± 0.02a 0.69 ± 0.01a 0.66 ± 0.02b 0.69 ± 0.01a 0.61 ± 0.02c 0.70 ± 0.01a 0.70 ± 0.02a 0.43 ± 0.02 fg 0.56 ± 0.02e 0.59 ± 0.01d 0.33 ± 0.02h 0.41 ± 0.01g 0.44 ± 0.01f

100

200

400

800

0.44 ± 0.01e 0.52 ± 0.02c 0.56 ± 0.01b 0.46 ± 0.01d 0.53 ± 0.02c 0.59 ± 0.01a 0.35 ± 0.01g 0.39 ± 0.02f 0.38 ± 0.01f 0.32 ± 0.02hi 0.39 ± 0.02f 0.39 ± 0.01f 0.24 ± 0.02j 0.31 ± 0.01i 0.33 ± 0.01h

Data represents the average of three replicates (n ¼ 3) ± standard error. Different letters between the Fluoride and biochar treatments indicate significant difference at p  0.05. LA: Leaf area; Fv/Fm: Efficiency of photosystem II; Chl a: Chlorophyll a; Chl b: Chlorophyll b; Ant: Anthocyanin; Car: Carotenoid; Fla: Flavonoid; Xan: Xanthophyll.

Increasing CEC of soil, improved the root growth. A strong relation between ROS generation and fluoride content of root and leaf was observed.

4. Discussion The results clearly indicated that safflower plants can tolerate up to 200 mg NaF kg1 soil, but further increase in fluoride content of soil can considerably reduce safflower growth (Table 1). Reduction in safflower growth under fluoride toxicity could be attributed to enriched fluoride content of plants (Fig. 2), increased oxidative stress of roots and leaves (Fig. 4) and diminished Kþ and Ca2þ contents in plant tissues (Table 1). Improving growth of safflower by biochar applications (Table 1) was the result of a decrease in the soluble fluoride concentration of soil and fluoride content of roots and leaves (Figs. 1 and 2), an increase in photosynthetic pigments, Kþ and Ca2þ contents in plant tissues, leaf area and Fv/Fm (Tables 1 and 2), and also a decrease in oxidative stresses caused by fluoride toxicity (Fig. 4). Increasing the fluoride content of root and leaf in safflower plant with increasing fluoride toxicity (Fig. 2) may be caused by rising soluble fluoride concentration of soil. Increasing fluoride toxicity of the soil reduced the pH, and in low pH, the solubility of fluoride was increased. However, in another study by Elrashidi and Lindsay (1987) addition of fluoride increased the soil pH. Fluoride in alkaline soils at a pH of 6.5 and above is almost completely fixed in the soils as calcium fluoride, if sufficient calcium carbonate is available. But in a soil with low pH, increasing fluoride content enhances the separation of Aluminum Fluoride and the separated aluminum ions can adhere to soil sulfate and create aluminum sulfate. Aluminum sulfate can decrease the soil pH. It seems this response is related to soil properties such as organic carbon content, soil acidity and/or other parameters. Decreasing cation exchange capacity of soil under fluoride toxicity could be related to decreasing pH of soil (Table 1). In most cases, cation exchange capacity of soil and soil pH showed a positive correlation with each other. Decreasing soluble fluoride concentration of soil with biochar treatments was associated with high pH of soil. The previous reports suggested that the addition of carbon rich materials to the soil can considerably improve the pH and cation exchange capacity of the soil (Puga et al., 2015; Lehmann and Joseph, 2015). The fluoride content of plant tissues was reduced due to the reduction in soluble fluoride concentration of the soil (Fig. 1). A decline in fluoride

uptake and translocation in safflower plants due to biochar treatments were associated with high soil pH and low solubility of fluoride in the soil. The generation of reactive oxygen species in root and leaf tissues was increased with increasing fluoride toxicity (Fig. 4). This could be related to the decline in efficiency of photosystem II and energy transfer through the photosynthetic components due to fluoride elevation. Although, Chl a and b are the main pigments in the photosynthetic process, other pigments such as carotenoid, flavonoid, anthocyanin and xanthophyll have also important protective roles in adjusting energy and electron transports through the photosystems. These pigments are involved in photosystem assembly and contribute to light harvesting by absorbing light energy in a region of the visible spectrum where chlorophyll absorption is lower and by transferring the energy to chlorophyll. They also protect plants from excess light energy via energy dissipation, free radical detoxification and membrane stability (Silva et al., 2016; Young and Pallett, 2017). Reductions in photosynthetic pigments, and photosystem II efficiency (Table 2) were led to an increase in ROS generation and antioxidant enzymes activities under fluoride toxicity of soil. Addition of biochar to the soil decreased ROS generation, lipid peroxidation (Fig. 4) and antioxidants activation (Fig. 5) through limiting fluoride absorption and increasing the synthesis of photosynthetic pigments and efficiency of photosystem II (Table 2) under fluoride toxicity of soil. Chlorophyll as well as flavonoid and xanthophyll showed the highest correlation with decreasing lipid peroxidation in leaf tissues. Enhancing maximum quantum yield of photosystem II by biochar application is also related to improving the synthesis of chlorophylls and other photosynthetic pigments (Fig. 5). Fluoride toxicity of soil decreased the contents of Kþ and Ca2þ ions in leaves of safflower (Table 1) by reducing the cation exchange capacity (Table 1), but the addition of biochar to the soil increased these cations in plant tissues through enhancing cation exchange capacity of soil. The soil with a high level of cation exchange capacity has a great potential to supply the nutrients to plants in adequate amounts (Puga et al., 2015). The superior effects of biochar in enriching plant tissues with essential nutrients and diminishing the generation of ROS under the fluoride toxicity of soil are led to the synthesis of more photosynthetic pigments, which can potentially improve safflower plant performance under this toxic condition.

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Variables of leaf (axes F1 and F2: 92.66 %)

Biplot of leaf (axes F1 and F2: 92.66 %)

1

30

0.75

F transloca on

25

F transloca on

20 0.5 15 Fla

F2 (4.61 %)

Shoot dry weight C Xan Chl a Shoot length Fv/Fm Chl b

10

K 0

H2O Leaf F POX SOD O2 F MDA Soluble F CAT

Leaf area

-0.25

F2 (4.61 %)

0.25

5

Ca

Shoot dry weight

K Xan Shoot length

0

H 2O

Chl a

Fv/Fm

Car

Leaf F POX

SOD O2 MDACAT F Uptake

Chl b

-5

Ant

-0.5

Fla

Soluble F

-10

Leaf area

-0.75

Car

-15 Ant

-1 -1

-0.75

-0.5

-0.25

0

0.25

0.5

0.75

1

-20 -10

-5

0

F1 (88.05 %)

5

10

15

F1 (88.05 %) Biplot of root (axes F1 and F2: 91.29 %)

Variables in root (axes F1 and F2: 91.29 %) 20

1

Soil PH 0.75

Soil PH

15

0.5

F2 (8.23 %)

0.25

Soluble F H2O2 SOD MDA CAT POX O2 F Root F

Root length Root dry weight

0

F2 (8.23 %)

Soil CEC

10

Soil CEC

5

Root length

Soluble F

H2O2 CAT

Root dry weight

O2

0

SOD MDA POX F Uptake Root F

-0.25 -5 F transloca on

-0.5

F transloca on

-10

-0.75

-1

-15 -1

-0.75

-0.5

-0.25

0

0.25

0.5

0.75

1

F1 (83.06 %)

-10

-5

0

5

10

F1 (83.06 %)

Fig. 5. Principal component analysis (PCA) of the leaf and root parameters of safflower plants. Fv/Fm: Efficiency of photosystem II; Chl a: Chlorophyll a; Chl b: Chlorophyll b; Ant: Anthocyanin; Car: Carotenoid; Fla: Flavonoid; Xan: Xanthophyll; F translocation: Fluoride translocation; Soluble F: Soluble fluoride; SOD: Superoxide dismutase; CAT: Catalase;  POX: Peroxidase; F uptake: Fluoride uptake; Root F: Root fluoride; MDA: Malondialdehyde; O2: Oxygen radicals; H2O2: Hydrogen peroxide; Leaf F: Leaf fluoride; K: Potassium; Ca: Calcium.

5. Conclusions The results indicated that the growth of safflower was not significantly affected by fluoride toxicity up to 200 mg NaF kg1 soil. However, further increase in fluoride toxicity was led to an increase in oxidative stress and misbalancing of cations, thereby retarding seedling growth. These deleterious effects of fluoride toxicity were considerably diminished by biochar application,

through adjusting pH and cation exchange capacity and also decreasing the solubility of fluoride in the soil. The biochar treatment also enhanced nutrients uptakes and synthesis of photosynthetic pigments, and reduced the ROS generation in safflower plants subjected to fluoride toxicity. Addition of 50 g kg1 biochar to the soil produced the best results in alleviating the harmful effects of fluoride toxicity on safflower seedlings.

K. Ghassemi-Golezani, S. Farhangi-Abriz / Chemosphere 223 (2019) 406e415

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