Green synthesis of sulfur nanoparticles using Ocimum basilicum leaves and its prospective effect on manganese-stressed Helianthus annuus (L.) seedlings

Ecotoxicology and Environmental Safety 191 (2020) 110242

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Green synthesis of sulfur nanoparticles using Ocimum basilicum leaves and its prospective effect on manganese-stressed Helianthus annuus (L.) seedlings

T

Gehad A. Ragab, Khalil M. Saad-Allah∗ Botany Department, Faculty of Science, Tanta University, Tanta, 31527, Egypt

A R T I C LE I N FO

A B S T R A C T

Keywords: Sulfur nanoparticles Green synthesis Sunflower Manganese stress Osmoprotectants

A novel green approach was utilized to fabricate sulfur nanoparticles (SNPs) with the aid of Ocimum basilicum leaves extract. The effective formation of the synthesized SNPs was examined and approved using UV–visible spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier transform infrared (FT-IR) spectroscopy. The average particle size was 23 nm with spherical shape and crystalline in nature. In the pot experiment, the synthesized SNPs were applied with different concentrations (12.5, 25, 50, 100 and 200 μM) as pre-soaking to Helianthus annuus seeds and irrigated with 100 mM MnSO4. As a result of manganese (Mn) exposure, the harvested 14-day sunflower seedlings showed a significant decline in the growth parameters (shoot length, leaf area and the relative water content of both shoot and root), photosynthetic pigments, mineral content (N, P, K, Ca, and Mg), and protein content compared to the control. The root length, electrolyte leakage, Na and Mn levels, metabolites content (amino acids, protein, glycine betaine, proline, and cysteine) were greatly raised as affected by Mn stress. Mn toxicity reduction using SNPs was demonstrated, as the medium doses enhanced seedlings growth, photosynthetic pigments, and mineral nutrients. Application of SNPs decreased Mn uptake and enhanced S metabolism through increasing cysteine level. Likewise, SNPs elevated seedlings water content and eliminated physiological drought via increasing osmolytes such as amino acids and proline. It can be concluded that green-synthesized SNPs had the potential to limit the deleterious effects of Mn stress.

1. Introduction Being an essential microelement, manganese (Mn) was reported to have structural and catalytic roles involved in normal plant growth and development as well as stress tolerance (Rajpoot et al., 2018). It is one of the most abundant metals in the earth's crust with available concentrations in soil ranges from 0.45 to 4 g/kg, while these levels could be triggered by decreasing soil pH (Liu et al., 2019). In acidic soils that comprise over 50% of the world's potentially arable lands, Mn has become a limiting factor for plant productivity due to its higher availability (Li et al., 2010). In recent years, the Mn level in soil has escalated due to rapid industrialization, mining practices and widespread use of Mn-containing fertilizers and sewage sludge (Sheng et al., 2016). However, Mn with adequate levels is critical for normal plant growth, the excessive concentrations could be toxic for biological systems and threaten both human health and crop production (Nagajyoti et al., 2010). Mn toxicity can be morphologically observed as chlorosis and necrosis, crinkled leaves and brown spots and eventually growth inhibition (Liu et al., 2019). At the physiological level, Mn higher doses might cause various metabolic disorders including disruption of

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nutrient assimilation and translocation, impaired photosynthetic electron transport chain and respiratory machinery (Sheng et al., 2015); besides disrupting protein structures, inactivating enzymes by binding to thiols and displacing other essential metals (Sheng et al., 2016). In addition, Mn is known to generate oxidative stress by stimulating the accumulation of reactive oxygen species exposing plant cells to lipid peroxidation, macromolecule destruction, membrane leakage and dismantling, and DNA cleavage (Liu et al., 2019). Sulfur (S) is considered a macronutrient participating with an indispensable role in plant growth and regulating plant responses to many biotic and abiotic stresses (Sheng et al., 2016). The sufficient application of mineral nutrients such as S could boost plant tolerance and contribute to the alleviation of plant stresses (Dixit et al., 2015). The effective role of sulfur may be attributed to its incorporation in many amino acids like cysteine and methionine besides many defense compounds including glutathione, phytochelatins, glucosinolates, and vitamins (Gill and Tuteja, 2011). Therefore, S is widely supplied in agricultural fields as a mineral nutrient additive and antifungal toxic agent. Though, the reduction of the applied amount is mainly desirable in order to minimize the cost and restrict the resistance of target

Corresponding author. E-mail address: [email protected] (K.M. Saad-Allah).

https://doi.org/10.1016/j.ecoenv.2020.110242 Received 26 November 2019; Received in revised form 19 January 2020; Accepted 21 January 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.

Ecotoxicology and Environmental Safety 191 (2020) 110242

G.A. Ragab and K.M. Saad-Allah

(0.2 M) solution was prepared by dissolving 5 g in 100 ml basil leaves extract with mild stirring for 20 min at room temperature. Sulfur particles were precipitated by the dropwise addition of 10% HCl under stirring, then collected by centrifugation at 10000 rpm for 10 min. The supernatant was discarded, and the precipitate was repeatedly washed with deionized water five-times, and finally with absolute ethanol. The product was collected and dried in a vacuum oven at 60 °C for 5 h yielding a yellowish-white powder that subsequently used for characterization.

pathogens (Salem et al., 2016). In this context, S in nanoscale can be considerably used in an appropriate amount as nanoparticles (SNPs) to enhance plant growth and mitigation of stressful conditions. In a similar way, previous investigations revealed the prominent effect of NPs in improving plant tolerance to various stresses (Kasim et al., 2017). The nanoparticle synthesis using chemical methods such as precipitation, vapor transport, and hydrothermal processes are environmentally unfriendly and expensive. However, the green biogenic methods are rapidly developing as it is simple, convenient, ecofriendly, and safe to handle (Fouad et al., 2017; Ibrahim et al., 2019; Ogunyemi et al., 2019). As previously mentioned, SNPs were successfully synthesized using Melia azedarach leaves aqueous extract (Salem et al., 2015). In the present study, a new green and simple method have been successfully achieved using leaf extract of basil (Ocimum basilicum L). Various studies demonstrated the presence of many bio-molecules in basil extracts including polyphenols such as flavonoids and anthocyanins which act as capping agents of the biosynthesized nanoparticles (Pirtarighat et al., 2019). Correspondingly, the extract of in vitro-grown basil plant was effectively utilized for the biosynthesis of Ag NPs (Pirtarighat et al., 2019). Sunflower (Helianthus annuus L.) is known as a globally important oilseed, food, and ornamental crop. Its seed-derived oil contains soluble vitamins (A, D, E, and K) (Rawashdeh, 2017) and contributes to approximately 10% of the world's edible oil production (Seiler et al., 2017). Sunflower has been identified as a phytoremediator due to its ability to accumulate many heavy metals (Cd, Pb, Zn, Ni, Cu, Hg), hydrocarbons, and other radionuclides (Chauhan and Mathur, 2018). Despite its phytoremediation potential, sunflower cannot tolerate medium levels of Mn exhibiting oxidative stress and inhibited biomass production (Saidi et al., 2014). Thus, the present work aimed to investigate the potential of the innovatively phyto-fabricated SNPs in alleviating Mn stress in sunflower by assessing their effect on the growth rate, pigmentation, nitrogen metabolism, and mineral content.

2.4. Characterization of SNPs The biosynthesis of SNPs was confirmed by the light absorption using a UV–visible spectrophotometer (Shimadzu 240 UV–Visible spectrophotometer). The X-ray diffraction patterns of the dried SNPs were determined by an X-ray diffractometer (GNR-APD 2000 pro, Central Lab at Tanta University). The size and morphology of SNPs were determined using a transmission electron microscopy (TEM) (JEOL model JEM 100 SX electron microscope, Tanta University unit) by placing a drop of the SNPs water suspension on a carbon-coated copper TEM grid. The chemical composition of the synthesized nano powder was studied using the FT-IR spectrophotometer (Bruker Tenor 27, Central Lab, Tanta University). The resultant FT-IR spectrum used to characterize the chemical bonds and molecular structures of the produced SNPs powder and basil leaves extract. 2.5. Growth conditions and treatments 2.5.1. Plant material and growth conditions Seeds of sunflower (Helianthus annuus L.) were obtained from the Agricultural Research Centre, Giza, Egypt, and selected for apparent uniformity of size and shape. The seeds were sterilized in 0.1% HgCl2 for 2 min with stirring then washed in distilled water. The disinfected seeds were separated into 2 groups; the first was soaked in distilled water for 18 h as a control and the second was primed by soaking either in 12.5, 25, 50, 100, or 200 μM of green synthesized SNPs solutions for 18 h. Thereafter, 10 of the primed seeds of every treatment were sown in plastic pots (20 cm diameter and 15 cm depth) filled with 5 kg of clay-sandy soil (2:1 w/w) and three pots were used as a replica for each treatment. Seeds were irrigated with tap water and left to grow in the greenhouse for 7 days, before the treatment with Mn stress, under normal environmental conditions (10 h photoperiod at 28/16 °C ± 2 day/night and 62% relative humidity). The Mn stress was applied as manganese sulfate (100 mM MnSO4 solution), while the 7-days old seedlings were irrigated once at 60% field capacity with tap water (the sub-lethal concentration of MnSO4 (100 mM) was previously determined in a preliminary experiment). The freshly harvested 14-days old seedlings were separated into roots and shoots, washed thoroughly with tap water several times followed by deionized water, and stored at −80 °C. For dry matter analysis, the plant samples were oven-dried at 60 °C till constant weight. Three replicates were utilized for each treatment of all the experiments.

2. Materials and methods 2.1. Preparation of Ocimum basilicum aqueous extract The basil (Ocimum basilicum L.) fresh leaves were collected from the botanical garden of the Faculty of Science, Tanta University, Tanta, Egypt. The leaves were rinsed several times under running tap water to get rid of any debris and dust followed by distilled water 3–4 times. To prepare the aqueous extract, 10 g of fresh basil leaves were ground with 100 ml deionized water using an electric mixer, then filtered through Whatman No.1 filter papers followed by centrifugation at 4000 rpm for 5 min to remove any heavy biomaterials. The freshly prepared aqueous extract was immediately used for the SNPs synthesis. 2.2. Chemicals All chemicals used were of analytical grade and were used as received without any further purification. Sodium thiosulfate, potassium iodide, crystalline iodine, sodium hydroxide, phenol phethalin and sodium hypochlorite were purchased from Elnasr company (Cairo, Egypt). However, ammonium molybdate, stannous chloride, H2SO4, K2HPO4, NH4Cl, HCl, ethanol, HgCl2, MnSO4, sulfosalicylic acid, proline, ninhydrin, glycerol, acetone, citric acid, sodium citrate, glycine, cysteine, perchloric acid, glycine betaine, sodium nitroprusside and 1,2 dichloroethane were purchased from Sigma_Aldrich (St. Louis, MO, USA). 2.3. Green synthesis of sulfur nanoparticles (SNPs)

2.5.2. Growth parameters and photosynthetic pigments The collected 14-days old seedlings were photographed to assess Mn toxicity before measuring growth criteria (lengths, fresh and dry weights, as well as the water content of both roots and shoots). The second expanded leaf in each seedling was selected to estimate chlorophyll a and b by Arnon (1949) method and carotenoids by Horvath et al. (1972) method. Briefly, 0.1 g of fresh leaves were extracted in 80% cold acetone, whereas the absorbance was measured using a UV/ visible spectrophotometer and expressed as mg/g FM.

Based on the disproportionation reaction of sodium thiosulfate in an acid medium to sulfur and sulfonic acid, SNPs were synthesized using sodium thiosulfate pentahydrate (Na2S2O3.5H2O). Sodium thiosulfate

2.5.3. Minerals content and protein quantification The dry plant leaf samples were digested with a mixture of 70% HNO3 and 30% H2O2 (5:2 v/v). The concentrations of K, Mn, Ca, and 2

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of sulfur at 111, 113, 222, 026, 311, 040, 313, 044, 137, and 317, respectively. The prominent positions and intensities of the diffraction peaks are well-matched with standard sulfur (International Centre for Diffraction Data (ICDD), No 00-008-0247). The progress in the formation and morphological structure of SNPs was also monitored using a transmission electron microscope (TEM). TEM micrographs revealed that SNPs were spherical in shape with sizes ranging from 18.6 to 36.3 nm and an average size of 25.9 nm (Fig. 1c). FT-IR analysis was carried out to identify the possible biomolecules present in the Ocimum basilicum leaves extract that responsible for the capping and stabilization of the synthesized sulfur nanoparticles. The obtained FT-IR chart of the basil leaves extract (Fig. 1d) clearly showed a variety of absorption peaks at 3426, 2925, 1640, 1456, 1240, 1160, 1119, 1022, 875, 661, and 468 cm−1. The major peaks in the Ocimum basilicum leaves IR-spectrum were similarly represented in the SNPs IRspectrum (Fig. 1e) with a slight shift (3451, 1637, and 665). Moreover, SNPs IR-spectrum exhibited a new absorption band at 2078.

Mg in the digested solutions were determined using an inductively coupled plasma-optical spectrophotometer (Polyscan 61 E, Thermo Jarrell-Ash Corp., Franklin, MA, USA) at the Central Lab of Tanta University. Nitrogen and phosphorus were detected in the digested samples due to the colorimetric assay of Allen et al. (1974) using Rochelle reagent for N and molybdenum blue method for P against their standard calibration curves. Crude protein content was determined by multiplying N content in a conversion factor of 6.25 (AOAC, 1990), and expressed as mg/g DM. 2.5.4. Electrolytes leakage The electrolyte leakage (EL) was determined by measuring the solute leakage or electrical conductivity (EC) (μmohs/cm) (Sairam et al., 2005). The fresh leaves were rinsed 3 times with deionized water, cut into uniformed leaf discs and incubated in 20 ml distilled water on a rotary shaker. The EC was determined after 1 h as EC1 and after 24 h as EC2. EL was expressed as a percentage (EC1/EC2 × 100). 2.5.5. Stress markers (proline, amino acids, cysteine and glycine betaine) Proline was estimated in the powdered dry leaves while homogenized in 3% sulfosalicylic acid according to Bates et al. (1973) using ninhydrin reagent. The resultant chromophore was extracted with toluene and measured at 520 nm. The proline content was calculated as mg/g DM using a prepared calibration curve by proline. For free amino acids determination, 0.1 g of dry leaf powders was extracted with 95% ethanol. According to Lee and Takahashi (1966), 0.1 ml of the plant extract was boiled for 12 min with a ninhydringlycerol citrate buffer mixture. The amino acids content was calculated as mg/g DM against a prepared calibration curve by glycine. The concentration of cysteine (Cys) was determined according to Gaitonde (1967). The dry leaf samples were extracted in 5% perchloric acid and the supernatant was incubated with acid/ninhydrin reagent for 10 min at 100 °C, then 1 ml of ethanol is added to the mixture and measured at 560 nm. The Cys concentration was detected as mg/g MD using a standard curve by cysteine. Glycine betaine (GB) was extracted within the water extract of the dry leaf powders using the method reported by Grieve and Grattan (1983). The extracted samples were diluted (1:1) with 2 N HCl and then mixed with cold KI-I2 reagent under continuous stirring. The formed periodate crystals were dissolved in 1,2 dichloroethane and then photometrically measured at 365 nm against a standard curve created by GB and the content was expressed as μg/g DM.

3.2. Results of the pot experiment 3.2.1. Growth parameters In the present study, sunflower seedlings treated with 100 mM MnSO4 exhibited evident toxicity symptoms reflected as a reduction in growth parameters as shown in Fig. 2. As compared with the control, Mn stress resulted in a significant reduction in shoot length, leaf area and the relative water content of both shoot and root; however, the root length was significantly increased. The presoaking of sunflower seeds in different SNPs concentrations was considerably effective and alleviated the growth reduction mediated by Mn; whereas the degree of alleviation was dose-dependent. Compared to Mn treatment, priming in S3 and S4 was the most valuable and significantly increased shoot length and leaf area of sunflower seedlings. Concerning the water content of both shoot and root, all the SNPs pre-soaking treatments enhanced the Mn-induced reduction relative to Mn-treatment, where their values became approximately comparable to those of the control. 3.2.2. Photosynthetic pigments The irrigation with MnSO4 was intensely destructive concerning photosynthetic pigments as observed in Fig. 3. The leaves content of Chl a, Chl b and carotenoids were significantly declined with percentages of 45.96, 39.02, and 39.42%, respectively compared to the control. The priming with SNPs triggered an enhanced response and ameliorated Mn-stress by raising the level of photosynthetic pigments to a significant level as compared to Mn treatment. Compared to Mn stress, S3 had a more pronounced impact by increasing chlorophylls (a and b) content; however, S2 was responsible for the highest retrieval impact concerning carotenoids level.

2.6. Statistical analysis The experimental procedures were conducted in triplicates, and the results were expressed as mean ± standard deviation (SD). The data were subjected to ANOVA analysis using Costat under Windows software (CoHort, V. 6.311) and differences (P˂0.05) between means were determined using the LSD test. Each of the experimental values was compared to its corresponding control.

3.2.3. Minerals content Treatment of sunflower seedlings with Mn sulfate resulted in a significant and huge accumulation of Mn ions in the seedlings tissues (2180%) compared to the control. As compared with Mn treatment, seeds priming with all SNPs concentrations markedly decreased Mn content; whereas S1 and S4 treatments possessed the lowest Mn content. Relative to the control, the content of N, P, K, Ca, Mg, in addition to the K/Na ratio, were highly declined as affected by Mn application. Nonetheless, Na level showed a significant increase following exposure to Mn stress (Table 1). Priming with SNPs successfully diminished Mntoxic effects and showed a significant increase in N, P, K, Ca, Mg, and K/Na ratio than their corresponding samples in the stress treatment and reached values close to that of the control. Moreover, the Mn-mediated increase in Na content was reversed by all SNPs presoaking treatments and matched the control level.

3. Results 3.1. Characterization of the green synthesized SNPs The greenly synthesized SNPs using basil leaf extract appeared as yellowish-white powder, and its successive formation was monitored and confirmed by UV–visible spectrophotometer as shown in Fig. 1a. The resultant UV–visible spectrum of SNPs in the range of 250–900 nm clearly showed one peak at 272 nm indicating the successful formation of SNPs. The XRD diffraction pattern (Fig. 1b) exhibited many sharp peaks demonstrating the polycrystalline nature of the biosynthesized SNPs. The characteristic peaks at 2 θ values of 11.7, 15.5, 23.19, 25.97, 26.75, 27.86, 28.84, 31.53, 34.32 and 37.2° are attributed to the crystal planes

3.2.4. Electrolyte leakage The data in Fig. (4) represent the hazard impact of Mn treatment on 3

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Fig. 1. Characterization of greenly synthesized SNPs: a) UV–Vis spectrum, b) XRD pattern, c) TEM micrograph, d) FT-IR profile of Ocimum basilicum leaves extract, e) FT-IR profile of green synthesized SNPs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

content by 45.60 and 20.03% respectively, relative to Mn treatment. The higher SNPs (S4 and S5) treatments slightly declined cysteine content of sunflower seedlings in comparison with Mn-stressed seedlings. The quaternary ammonium osmoprotectant glycine betaine (GB) showed a slight increment (6.66%) relative to the un-stressed control as a result of sunflower seedlings exposure to 100 MnSO4. Controversially, all the used SNPs treatments decreased GB content as compared to Mn stress. The most pronounced decrement was obtained when sunflower seeds were pre-treated with the medium dose (S3) of SNPs, as it caused a 30.11% decline in GB content relative to the stressed treatment.

the electrolyte leakage (EL) of sunflower leaves, resulting in a significant increase by 65.26% compared to the unstressed control. On the contrary, a recovery effect was documented with all SNPs treatments that diminished EL compared to the stress treatment, where EL at S2 and S3 treatments reached close to that of the non-treated seedlings. 3.2.5. Metabolites (protein, amino acids, proline, cysteine, and GB) Relative to the control, the application of Mn had resulted in a significant reduction in the crude protein level with a percentage of 17.0% (Fig. 5). Nevertheless, SNPs pre-soaking had resulted in a pronounced increase in the crude protein values in Mn-stressed sunflower seedlings. The most effective doses were S1, S4, and S5, as they resulted in a significant enhancement (20.06, 14.09 and 23.57%, respectively) in the protein content over Mn treatment. The total amino acids content was significantly boosted (49.12%) as a result of Mn exposure compared to the unstressed control (Fig. 5). All the used SNPs pre-treatments significantly increased the total free amino acids content, compared to the stressed and unstressed treatments. S2 and S4 pre-treatments were the most effective in increasing amino acids content, where they markedly increased the amino acids by 81.41 and 82.05%, respectively with respect to the control treatment. As shown in Fig. (5), treatment with Mn caused a significant increase in proline and cysteine contents (64.31 and 18.51%, respectively) compared with their control counterparts. Irrespective of its level, SNPs treatments downregulated proline level in sunflower seedlings compared to Mn stress, with S2 as the most lowering treatment, but still higher than the control value. Concerning cysteine, the low (12.5 and 25 μM) and the medium (50 μM) doses raised cysteine level over that of the stressed treatment. S2 treatment provoked the highest cysteine level followed by S3, as they stimulated an upregulation in its

4. Discussion In this investigation, a new approach for SNPs biosynthesis was developed using basil (Ocimum basilicum) leaves extract. The obtained UV–visible spectrum approved the successive formation of SNPs due to the resultant maximum peak at 272 nm. It has been demonstrated that SNPs showed an optical absorption spectra in the range of 245–300 nm, with a maximum absorbance at 280 nm wavelength (Chaudhuri and Paria, 2011). In a similar way, Suryavanshi et al. (2017) and Shankar et al. (2018) demonstrated that the synthesized SNPs exhibited a maximum absorption peak at 290 and 280, respectively. Moreover, Tripathi et al. (2018) revealed the presence of a maximum absorption peak at 274 nm due to the greenly synthesized-SNPs. The results of the XRD pattern revealed characteristic diffraction peaks that coincided with the standard sulfur and proved the polycrystalline structure of the green biosynthesized SNPs. The average crystallite size of the synthesized sulfur nanoparticles was found to be 23 nm and calculated using Debye-Scherrer's equation (Klug and 4

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Fig. 2. Growth parameters of Mn-stressed and pre-soaked in SNPs (0.0, 12.5, 25, 50, 100 and 200 μM) sunflower seedlings. Different letters indicate significant differences at 5% level.

more characteristic of eugenol, linalool, flavonoids, tannins, proteins, and terpenes, which are abundant in it. This result is in accordance with the findings of Rasaee et al. (2016). On the other side, the resultant IRchart of SNPs contained the major peaks in the basil leaves with a slight shift, indicating the effective presence of biomolecules in the obtained sample. At the IR spectrum of the leaves extract, the intense peak at 3426 cm−1 can be attributed to the O–H groups of alcohols and N–H of amines; meanwhile, the absorption peaks around 2925 cm−1 might be assigned to C–H stretching of the alkanes (Akintelu and Folorunso, 2019). In the synthesized SNPs, the first peak shifted to 3451 cm−1 and expanded to a broad peak. So, it can be concluded that flavonoids and polyphenols present in Ocimum basilicum leaves were responsible for the capping of SNPs. The band at 1640 cm−1 in the leaves spectrum arose from the vibration of carbonyl groups in the amide proteins (Anuradha

Alexander, 1974): D = K λ/β cos θ, where, D is the mean diameter of nanoparticles, β is the full width at half-maximum value of XRD diffraction lines, λ is the wavelength of X-ray radiation source (0.15405 nm), θ is the half diffraction angle, and K is the Scherrer constant with a value of 0.9. The unassigned peaks in the XRD pattern are thought to be related to crystalline and amorphous organic biomolecules present in the extract of Ocimum basilicum. Similar reports on XRD analysis of bio-synthesized SNPs were reported earlier (Kouzegaran and Farhadi, 2017). Moreover, the TEM micrographs revealed that the synthesized SNPs possessed a spherical shape with an average size of 26 nm and totally matched with the XRD analysis results. FT-IR results of the basil leaves extract showed many absorption peaks representing its complex nature and many biomolecules. The aqueous extract of Ocimum basilicum was reported to exhibit IR peaks 5

Ecotoxicology and Environmental Safety 191 (2020) 110242

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Fig. 3. Photosynthetic pigments of Mn-stressed and pre-soaked in SNPs (0.0, 12.5, 25, 50, 100 and 200 μM) sunflower seedlings. Different letters indicate significant differences at 5% level.

(Malapermal et al., 2017) and for SNPs by (Tripathi et al., 2018). In the pot experiment, Mn application resulted in a remarkable reduction in sunflower growth as manifested in the reduction of shoot length, leaf area and the relative water content of both shoot and root. The evident inhibition in the growth due to Mn stress could be a direct result of Mn accumulation in plant tissues and the disturbance in the photosynthesis, nutrient elements uptake and other vital metabolic processes (Sheng et al., 2015). Application of SNPs as priming treatments was distinctly effective in limiting Mn hazard effects on growth parameters, and retaining normal homeostasis as manifested by raising shoot and root water content than Mn treatment to match the control. A number of studies revealed that exogenous exposure to S as a mineral additive could overcome plant stresses including salinity (de Andrade et al., 2018) and heavy metals

et al., 2014). This peak is shifted to 1637 in the SNPs spectrum confirming the involvement of proteins in NPs synthesis (Malapermal et al., 2017). The absorption bands at 661 and 665 cm−1 for the leaves extract and SNPs respectively could be attributed to the aromatic C–H bending (Pirtarighat et al., 2019). The emergence of a new absorption band at 2078 cm−1 in the SNPs spectrum proved the adsorption of carbonyl groups (C]O) on the sulfur particles. Therefore, FT-IR analysis of synthesized SNPs confirmed characteristic peaks corresponding to vibrational bands of hydroxyl (-OH), amine (-N-H) and carbonyl groups (C]O) as functional groups. Depending on the above observation, it can be assumed that the reduction of S ions into SNPs and their stabilization are achieved by waters soluble aromatic compounds such as phenolic and flavonoids besides proteins. Similar reports were accounted for silver NPs by

Table 1 Mineral analysis of Mn-stressed and pre-soaked in SNPs (0.0, 12.5, 25, 50, 100 and 200 μM) sunflower seedlings. Different letters indicate significant differences at 5% level. Treatment

Mineral ions (mg/g DM) N

Control Mn S1+ Mn S2+ Mn S3+ Mn S4+ Mn S5+ Mn

2.26 1.87 2.25 1.98 1.76 2.14 2.31

P ± ± ± ± ± ± ±

ab

0.15 0.08cd 0.05ab 0.03c 0.00d 0.09b 0.07a

0.28 0.15 0.25 0.29 0.36 0.31 0.24

K ± ± ± ± ± ± ±

bc

0.03 0.02d 0.04c 0.00bc 0.02a 0.03b 0.03c

4.09 2.54 3.55 2.93 4.04 3.59 4.40

Ca ± ± ± ± ± ± ±

b

0.03 0.00f 0.03d 0.01e 0.04b 0.04c 0.02a

1.30 0.97 1.07 1.00 1.12 1.05 1.20

Mg ± ± ± ± ± ± ±

a

0.007 0.025e 0.032d 0.002e 0.001c 0.011d 0.002b

6

0.39 0.25 0.34 0.28 0.36 0.33 0.42

Na ± ± ± ± ± ± ±

b

0.006 0.001g 0.006d 0.006f 0.002c 0.000e 0.004a

0.25 0.61 0.22 0.20 0.19 0.20 0.26

Mn ± ± ± ± ± ± ±

b

0.006 0.006a 0.006c 0.006d 0.002d 0.004d 0.014b

0.02 0.46 0.20 0.30 0.29 0.15 0.30

K/Na ratio ± ± ± ± ± ± ±

f

0.000 0.004a 0.001d 0.002b 0.001c 0.002e 0.001b

16.13 ± 0.31cd 4.18 ± 0.05f 15.67 ± 0.41d 14.52 ± 0.47e 21.23 ± 0.19a 18.17 ± 0.23b 16.67 ± 0.93c

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decreased EL compared to Mn stress and enhanced the membrane stabilization, which could be due to the recorded reduction in Mn contents and its triggered oxidative injury. The enhanced membrane permeability by SNPs would account for increased minerals uptake. Therefore, the current results demonstrated the SNPs capability in boosting plant tolerance via enhancing minerals uptake and water status, which directly translated into the recovered plant growth. Similar results were reported in buckwheat exposed to Cd stress (Lu et al., 2019). In this concern, Rais and Masood (2013) reported that the increased availability of S regulated the activity of nitrate reductase and increased the accumulation of N. In the present results, SNPs reduced Na than stress treatment and increased K/Na ratio and thus enhanced efficient uptake of other essential elements. In this context, a higher K/ Na ratio was advocated as an important physiological attribute for mitigating stress deleterious impacts (Alqarawi et al., 2014). As a result of Mn exposure, total amino acids, proline, Cys, and GB were greatly increased than the control, which agreed with previous results of Lei et al. (2007). It can be suggested that the accumulation of amino acids including proline, and cysteine might participate in plant induced metal tolerance (Khan et al., 2000). Thus, it can be supposed that sunflower seedlings respond to Mn-induced water deficit by producing enormous quantities of osmolytes, but not to the limit to cope with Mn toxicity as marked by the reduced growth. On the other side, SNPs could alleviate Mn hazard impacts by increasing total amino acids over the Mn stress. The increase in total amino acids and proline by SNPs could be involved in osmoregulation, metal chelation and antioxidant defense (Wu et al., 2004). Therefore, osmotic adjustment maintained by SNPs pre-treatment could regulate stomatal opening and restrict metal uptake (Lei et al., 2007) as revealed by the depleted Mn content. The present data validated that SNPs application (S2 and S3) improved S metabolism and caused over-production of cysteine compared to stress treatment as similarly reported before by Zhang et al. (2018) in Cr-stressed polish wheat. High cysteine content could enhance the biosynthesis of compounds with high-affinity thiol groups like glutathione and phytochelatins, thus induce metal chelation and antioxidative machinery (Lu et al., 2019).

Fig. 4. Electrolytes leakage (EL) of Mn-stressed and pre-soaked in SNPs (0.0, 12.5, 25, 50, 100 and 200 μM) sunflower seedlings. Different letters indicate significant differences at 5% level.

(Zhang et al., 2018). Nevertheless, this is the first work investigating the capability of SNPs in alleviating Mn stress. Owing to their tiny size and larger specific surface area, NPs can be rapidly absorbed and transferred ensuring an appropriate mineral quantity for effective utilization in plant growth and development (Saxena et al., 2016). A significant decline in photosynthetic pigments; Chl a, Chl b and carotenoids, was recorded with Mn treatment. This finding is in agreement with Rajpoot et al. (2018) and Sheng et al. (2015) in Mnstressed rice and wheat, respectively. The reduction in photosynthetic pigments might be ascribed to the recorded depletion in Mg ions induced by Mn stress. It is well-known that Mg involved in chlorophyll synthesis, thylakoid synthesis, and chloroplast development in leaves (Sheng et al., 2015). Moreover, Mn stress accounted for impaired photosynthesis via the reduction of leaf area compared to the untreated plants due to their dominant role in capturing light and achieving photosynthesis (Shu et al., 2012). However, SNPs application greatly enhanced photosynthesis and the content of photosynthetic pigments than Mn stress treatment, which reflected on the overall growth as previously reported by Sheng et al. (2016) and Dixit et al. (2015). These positive impacts could be directly related to the maintenance of Mg level and reducing the disruption in chlorophyll biosynthesis. The abovementioned results revealed that SNPs enhanced water content and recovered the Mn-mediated water stress which might improve stomatal conductance with a positive impact on photosynthetic capacity (Lu et al., 2019). The membranes' integrity was substantially damaged due to heavy metals toxicity, including Mn stress as recorded here, with the increased EL compared to the control. That membrane destabilization might commonly be attributed to higher Mn accumulation and its substitution of essential metal ions within biomolecules (Ghori et al., 2019), or disrupting redox homeostasis and generating oxidative stress (Sheng et al., 2016). The loss of membrane integrity and ion leakage contributed directly with the obtained Mn-mediated mineral nutrients (N, P, K, Ca and Mg) deficiency, and disturbance of water balance resulting in growth inhibition, which coincides with results of Shi and Zhu (2008). Like other heavy metals, Mn could have a negative impact on N level via inhibiting the involved enzymes in the translocation and absorption of nitrate Nagajyoti et al. (2010). Therefore, the decreased protein content with Mn application might be demonstrated by the reduction in protein synthesis as a result of N depletion induced by heavy metals (Kasim et al., 2017). In this context, Mn stress was demonstrated to increase protein degradation due to oxidative modification (Rajpoot et al., 2018). On the contrary, Mn exposure caused an increased uptake of Na ions that disturbed the uptake of important ions like K and Ca mobility within the plant and hence reduced K/Na ratio and disrupting osmotic potential (Hashem et al., 2016). Interestingly, SNPs (particularly the medium doses) significantly

5. Conclusion In conclusion, SNPs suggested to be green-fabricated using water extract of Ocimum basilicum leaves with an average size of 23 nm. Moreover, the present results exhibited that seed presoaking with medium doses of SNPs improved sunflower tolerance against Mn toxicity via reducing Mn uptake, maintaining of membrane stability, balanced mineral uptake and photosynthetic pigments levels. Also, the positive impact of SNPs could be attributed to the overproduction of osmolytes and enhanced S metabolism via upregulated cysteine. Overall, these results presented priming with SNPs as an efficacious application for the alleviation of Mn stress.

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement Gehad A. Ragab: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. Khalil M. Saad-Allah: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing - review & editing. 7

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Fig. 5. Osmolytes content (proteins, amino acids, proline, cysteine, and glycine betaine) of Mn-stressed and pre-soaked in SNPs (0.0, 12.5, 25, 50, 100 and 200 μM) sunflower seedlings. Different letters indicate significant differences at 5% level.

Declaration of competing interest

Alqarawi, A.A., Abd Allah, E.F., Hashem, A., 2014. Alleviation of salt-induced adverse impact via mycorrhizal fungi in Ephedra aphylla Forssk. J. Plant Interact. 9, 802–810. Anuradha, G., Sundar, B.S., Kumar, J.S., Ramana, M.V., 2014. Synthesis and characterization of silver nanoparticles from Ocimum basilicum L . var . thyrsiflorum. Eur. J. Acad. Essays 1, 5–9. AOAC, 1990. Official Methods of Analysis, fifteenth ed. Association of Official Analytical Chemists, Inc., Arlington, TX, USA AOAC, Washington, DC. Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. Chaudhuri, R.G., Paria, S., 2011. Growth kinetics of sulfur nanoparticles in aqueous surfactant solutions. J. Colloid Interface Sci. 354, 563–569. Chauhan, P., Mathur, J., 2018. Potential of Helianthus annuus for phytoremediation of

Both authors declare they don't have any personal or institutional conflict of interest with any third party. References Akintelu, S.A., Folorunso, A.S., 2019. Characterization and antimicrobial investigation of synthesized silver nanoparticles from Annona muricata leaf extracts. J. Nanotechnology, Nanomedicine Nanobiotechnology 6, 1–5. Allen, S.E., Grimshaw, H.M., Parkinson, J.A., Quarmby, C., 1974. Chemical Analysis of Ecological Materials. Blackwell Scientific Publications.

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toxicity for plants: a review. Environ. Chem. Lett. 8, 199–216. Ogunyemi, S.O., Abdallah, Y., Zhang, M., Fouad, H., Hong, X., Ibrahim, E., Masum, M.M.I., Hossain, A., Mo, J., Li, B., 2019. Green synthesis of zinc oxide nanoparticles using different plant extracts and their antibacterial activity against Xanthomonas oryzae pv. oryzae. Artif. Cells. Nanomedicine Biotechnol 47, 341–352. Pirtarighat, S., Ghannadnia, M., Baghshahi, S., 2019. Biosynthesis of silver nanoparticles using Ocimum basilicum cultured under controlled conditions for bactericidal application. Mater. Sci. Eng. C 98, 250–255. Rais, L., Masood, A., 2013. Sulfur and nitrogen co-ordinately improve photosynthetic efficiency, growth and proline accumulation in two cultivars of mustard under salt stress. J. Plant Biochem. Physiol. 1, 1–6. Rajpoot, R., Rani, A., Srivastava, R.K., Pandey, P., Dubey, R.S., 2018. Protective role of Mentha arvensis aqueous extract against manganese induced toxicity by reducing Mn translocation and promoting antioxidative defense in growing indica rice seedlings. J. Crop Sci. Biotechnol. 21, 353–366. Rasaee, I., Ghannadnia, M., Honari, Hosein, 2016. Antibacterial properties of biologically formed chitosan nanoparticles using aqueous leaf extract of Ocimum basilicum. Nanomedicine J 3, 240–247. Rawashdeh, H.M., 2017. Sunflower seed yield under trickle irrigation using treated wastewater. Afr. J. Agric. Res. 12, 1811–1816. Saidi, I., Nawel, N., Djebali, W., 2014. Role of selenium in preventing manganese toxicity in sunflower (Helianthus annuus) seedling. South Afr. J. Bot. 94, 88–94. Sairam, R.K., Srivastava, G.C., Agarwal, S., Meena, R.C., 2005. Differences in antioxidant activity in response to salinity stress in tolerant and susceptible wheat genotypes. Biol. Plant. (Prague) 49, 85–91. Salem, N.M., Albanna, L.S., Awwad, A.M., 2016. Green synthesis of sulfur nanoparticles using Punica granatum peels and the effects on the growth of tomato by foliar spray applications. Environ. Nanotechnology, Monit. Manag. 6, 83–87. Salem, N.M., Albanna, L.S., Awwad, A.M., Ibrahim, Q.M., Abdeen, A.O., 2015. Green synthesis of nano-sized sulfur and its effect on plant growth. J. Agric. Sci. 8, 188–194. Saxena, R., Tomar, R.S., Kumar, M., 2016. Exploring nanobiotechnology to mitigate abiotic stress in crop plants. J. Pharmaceut. Sci. Res. 8, 974–980. Seiler, G.J., Qi, L.L., Marek, L.F., 2017. Utilization of sunflower crop wild relatives for cultivated sunflower improvement. Crop Sci. 57, 1083–1101. Shankar, S., Pangeni, R., Park, J.W., Rhim, J.W., 2018. Preparation of sulfur nanoparticles and their antibacterial activity and cytotoxic effect. Mater. Sci. Eng. C 92, 508–517. Sheng, H., Zeng, J., Liu, Y., Wang, X., Wang, Y., Kang, H., Fan, X., Sha, L., Zhang, H., Zhou, Y., 2016. Sulfur mediated alleviation of Mn toxicity in polish wheat. Front. Plant Sci. 7, 1–13. Sheng, H., Zeng, J., Yan, F., Wang, X., Wang, Y., Kang, H., Fan, X., Sha, L., Zhang, H., Zhou, Y., 2015. Effect of exogenous salicylic acid on manganese toxicity, mineral nutrients translocation and antioxidative system in polish wheat (Triticum polonicum L.). Acta Physiol. Plant. 37, 32–43. Shi, Q., Zhu, Z., 2008. Effects of exogenous salicylic acid on manganese toxicity, element contents and antioxidative system in cucumber. Environ. Exp. Bot. 63, 317–326. Shu, X., Yin, L., Zhang, Q., Wang, W., 2012. Effect of Pb toxicity on leaf growth, antioxidant enzyme activities, and photosynthesis in cuttings and seedlings of Jatropha curcas L. Environ. Sci. Pollut. Res. 19, 893–902. Suryavanshi, P., Pandit, R., Gade, A., Derita, M., Zachino, S., Rai, M., 2017. Colletotrichum sp.-mediated synthesis of sulphur and aluminium oxide nanoparticles and its in vitro activity against selected food-borne pathogens. LWT - Food Sci. Technol. (Lebensmittel-Wissenschaft -Technol.) 81, 188–194. Tripathi, R.M., Rao, R.P., Tsuzuki, T., 2018. Green synthesis of sulfur nanoparticles and evaluation of their catalytic detoxification of hexavalent chromium in water. RSC Adv. 8, 36345–36352. Wu, F.B., Chen, F., Wei, K., Zhang, G.P., 2004. Effect of cadmium on free amino acid, glutathione and ascorbic acid concentrations in two barley genotypes (Hordeum vulgare L.) differing in cadmium tolerance. Chemosphere 57, 447–454. Zhang, X., Kang, J., Pang, H., Niu, L., Lv, J., 2018. Sulfur mediated improved thiol metabolism, antioxidant enzymes system and reduced chromium accumulation in oilseed rape (Brassica napus L.) shoots. Environ. Sci. Pollut. Res. 25, 35492–35500.

multiple pollutants in the environment: a Review. J. Biol. Sci. Med. 4, 5–16. de Andrade, J.J., de Oliveira, F.J.M., Pessoa, L.G.M., dos Nascimento, S.A., de Souza, E.S., Barros Júnior, G.B., Miranda, M.F.A., Oliveira, A.C., dos Santos Freire, M.B.G., 2018. Effects of elemental sulfur associated with gypsum on soil salinity attenuation and sweet sorghum growth under saline water irrigation. Aust. J. Crop. Sci. 12, 221–226. Dixit, G., Singh, A.P., Kumar, A., Dwivedi, S., Deeba, F., Kumar, S., Suman, S., Adhikari, B., Shukla, Y., Trivedi, P.K., Pandey, V., Tripathi, R.D., 2015. Sulfur alleviates arsenic toxicity by reducing its accumulation and modulating proteome, amino acids and thiol metabolism in rice leaves. Sci. Rep. 5, 1–16. Fouad, H., Hongjie, L., Yanmei, D., Baoting, Y., El-Shakh, A., Abbas, G., Jianchu, M., 2017. Synthesis and characterization of silver nanoparticles using Bacillus amyloliquefaciens and Bacillus subtilis to control filarial vector Culex pipiens pallens and its antimicrobial activity. Artif. Cells, Nanomedicine Biotechnol. 45, 1369–1378. Gaitonde, M.K., 1967. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem. J. 104, 627. Ghori, N., Ghori, T., Hayat, M., Imadi, S., Gul, A., Altay, V., Ozturk, M., 2019. Heavy metal stress and responses in plants. Int. J. Environ. Sci. Technol. 16, 1807–1828. Gill, S.S., Tuteja, N., 2011. Cadmium stress tolerance in crop plants: probing the role of sulfur. Plant Signal. Behav. 6, 215–222. Grieve, C.M., Grattan, S.R., 1983. Rapid assay for determination of water soluble quaternary ammonium compounds. Plant Soil 70, 303–307. Hashem, A., Abd-Allah, E.F., Alqarawi, A.A., Egamberdieva, D., 2016. Bioremediation of adverse impact of cadmium toxicity on Cassia italica Mill by arbuscular mycorrhizal fungi. Saudi J. Biol. Sci. 23, 39–47. Horvath, G., Kissimon, J., Faludi-Dániel, Á., 1972. Effect of light intensity on the formation of carotenoids in normal and mutant maize leaves. Phytochemistry 11, 183–187. Ibrahim, E., Fouad, H., Zhang, M., Zhang, Y., Qiu, W., Yan, C., Li, B., Mo, J., Chen, J., 2019. Biosynthesis of silver nanoparticles using endophytic bacteria and their role in inhibition of rice pathogenic bacteria and plant growth promotion. RSC Adv. 9, 29293–29299. Kasim, W., AboKassem, E., Ragab, G., 2017. Ameliorative effect of yeast extract, IAA and green-synthesized nano zinc oxide on the growth of Cu-stressed Vicia faba seedlings. Egypt. J. Bot. 57, 1–16. Khan, A.A., McNeilly, T., Collins, J.C., 2000. Accumulation of amino acids, proline, and carbohydrates in response to aluminum and manganese stress in maize. J. Plant Nutr. 23, 1303–1314. Klug, H.P., Alexander, L.E., 1974. X-ray diffraction procedures: for polycrystalline and amorphous materials. X-Ray Diffr. Proced. Polycryst. Amorph. Mater. In: Harold P. Klug, Leroy E. Alexander, second ed. Wiley-VCH, 0-471-49369-4, pp. 992. Kouzegaran, V., Farhadi, K., 2017. Green synthesis of sulphur nanoparticles assisted by a herbal surfactant in aqueous solutions. Micro Nano Lett 12, 329–334. Lee, Y.P., Takahashi, T., 1966. An improved colorimetric determination of amino acids with the use of ninhydrin. Anal. Biochem. 14, 71–77. Lei, Y., Korpelainen, H., Li, C., 2007. Physiological and biochemical responses to high Mn concentrations in two contrasting Populus cathayana populations. Chemosphere 68, 686–694. Li, Q., Chen, L.S., Jiang, H.X., Tang, N., Yang, L.T., Lin, Z.H., Li, Y., Yang, G.H., 2010. Effects of manganese-excess on CO2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase, carbohydrates and photosynthetic electron transport of leaves, and antioxidant systems of leaves and roots in Citrus grandis seedlings. BMC Plant Biol. 10, 1–16. Liu, P., Huang, R., Hu, X., Jia, Y., Li, J., Luo, J., Liu, Q., Luo, L., Liu, G., Chen, Z., 2019. Physiological responses and proteomic changes reveal insights into Stylosanthes response to manganese toxicity. BMC Plant Biol. 1–21. Lu, Y., Wang, Q., Li, J., Xiong, J., Zhou, L., He, S., Zhang, J., Chen, Z., He, S., Liu, H., 2019. Effects of exogenous sulfur on alleviating cadmium stress in tartary buckwheat. Sci. Rep. 9, 1–12. Malapermal, V., Botha, I., Krishna, S.B.N., Mbatha, J.N., 2017. Enhancing antidiabetic and antimicrobial performance of Ocimum basilicum, and Ocimum sanctum (L.) using silver nanoparticles. Saudi J. Biol. Sci. 24, 1294–1305. Nagajyoti, P.C., Lee, K.D., Sreekanth, T.V.M., 2010. Heavy metals, occurrence and

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