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. 2018 Aug 6;8(8):362. doi: 10.1007/s13205-018-1386-9

Assessment of the effects of metal oxide nanoparticles on the growth, physiology and metabolic responses in in vitro grown eggplant (Solanum melongena)

Venkidasamy Baskar 1,#, Safia Nayeem 1,#, Sree Preethy Kuppuraj 1, Thiruvengadam Muthu 2, Sathishkumar Ramalingam 1,✉
PMCID: PMC6081843  PMID: 30105187

Abstract

Nanoparticles (NPs) are widely used in various domestic products and their usage is constantly increasing which in turn can raise several environmental health issues. Like other abiotic stresses, nanomaterials also affect the growth of crop plants. Solanum melongena is a common vegetable crop grown in the tropics and subtropics regions with medicinal properties. In this study, S. melongena was analyzed for its response to three commercially important metallic nanoparticles, namely NiO, CuO, and ZnO, at four different concentrations (100, 250, 500 and 1000 mg/L). The growth of the eggplant seedlings was suppressed by all the NPs in a concentration-dependent manner and among them, NiO was shown to be more toxic as it suppressed the root and shoot growth effectively. Total chlorophyll contents were decreased in the NP-treated plants compared to control plants. Significant changes were found in the secondary metabolites such as anthocyanins, total phenolic and total flavonoid contents in the NP-treated plants. A dose-dependent increase in the reactive oxygen species (ROS) generation was noticed in the NP-treated plants which are evidenced by the 4-nitro blue tetrazolium chloride (NBT) and 3,3′-diamiobenzidine (DAB) histochemical staining. The DNA damage imposed by the NP in the seedlings of eggplants may be due to the elevated ROS and MDA (malondialdehyde) production. NiO NP was found to be more toxic comparable to CuO and ZnO NPs in the present study. Apart from the toxic effects, nanoparticles also showed profound effects on the production of important secondary metabolites such as phenolics and flavonoid compounds.

Electronic supplementary material

The online version of this article (10.1007/s13205-018-1386-9) contains supplementary material, which is available to authorized users.

Keywords: Nanoparticles, Eggplant, Reactive oxygen species, Malondialdehyde, Anthocyanin, Phenolics, Flavonoids

Introduction

Nanometer-sized metal particles have attracted intensive attention due to their wide application in biomedical, agriculture, electronics, telecommunications, and renewable energy (Ma et al. 2015). Their unique features and properties allow them to be used in various fields such as reproductive science, disease prevention, conversion of agricultural and food wastes to energy through nanobioprocessing, chemical sensors and plant treatment using various nanocides (Nair et al. 2010). On the other hand, this expanding field does raise concerns about the toxicity and environmental impact of nanomaterials. Nanofertilizers can be used effectively on crops for controlled release of chemicals, thereby enhancing target activity and plant growth. Atmospheric deposition, agricultural application, rain erosion, and surface runoff are the main pathways through which nanoparticles (NPs) enter the soil. Their weak migration ability gradually will result in the accumulation of NPs in soil with the advance in time. This could result in changes in the soil-based food crop quality and yield (Priester et al. 2012).

The effect of NPs on plants depends on the plant species, seed size, growth stage, growth medium and the nanoparticle-coating material (Yang et al. 2017). Nanoparticles have been found to exert profound changes on the physiological indices in plants, namely the germination percentage, root elongation, biomass and leaf number (Lee et al. 2010). Nanoparticles are found to change the content of amino acids, fatty acids, non reducing sugars and phenolics in plants (Rico et al. 2014). Production of plant hormones can be influenced by NPs (Yang et al. 2017). Rice seedlings, when exposed to carbon nanotubes, demonstrated decreased phytohormone concentrations (Hao et al. 2016). Studies on TiO2 NPs in cucumber leaves showed the increased content of total chlorophyll and catalase (CAT) and decreased ascorbate peroxidase (APX) content in leaves (Servin et al. 2013). Rare earth oxide NPs (Gd2O3, La2O3, CeO2 and Yb2O3) when added to roots at high concentrations was found to exert detrimental effects on plant growth in radish, tomato, cabbage, cucumber, lettuce, rape, wheat, and corn (Ma et al. 2010; López-Moreno et al. 2010). Nickel oxide (NiO) NP has been reported to induce cellular oxidative stress by a strong increase in reactive oxygen species (ROS) formation (Oukarroum et al. 2009). NiO NP treatment enhanced the activities of antioxidant enzymes, namely CAT, superoxide dismutase (SOD), and glutathione (GSH) in tomato (Faisal et al. 2013). Magaye et al. (2012) reported that NiO NPs can easily be transported into biological systems and hence can induce both cytotoxic and genotoxic effects. Zinc (Zn) is an essential micronutrient crucial for plant development. It is an essential component of a majority of plant enzymes and proteins and forms a part of structural ions in transcription factors (Broadley et al. 2007). Zinc in higher doses is toxic to plants (Broadley et al. 2007). Zinc nanoparticles exhibit biological activities such as antibacterial, antifungal, antioxidant (Santhoshkumar et al. 2017), anti-diabetic, anti-inflammatory, wound healing (Agarwal et al. 2017), anti-corrosive and UV filtering properties. They have potential application in drug delivery and diagnosis of diseases (Santhoshkumar et al. 2017). The photocatalytic activity of zinc oxide (ZnO) NPs promotes the level of ROS production under irradiation with energy at/or above its band gap energy and induces phytotoxicity (Ma et al. 2013). Oryza sativa when exposed to ZnO NPs showed a reduction in the leaf surface area (Salah et al. 2015). Phytotoxicity studies with ZnO NPs on Lolium perenne (rye-grass) show that concentrated ZnO NPs absorbed on the root surface and in the rhizosphere solution caused potential effects on the plant’s growth (Lin and Xing 2008). The potent antibiotic activity of nano-sized copper oxides (CuO) has found wide application in nanobiocide products available in the markets (De Rosa et al. 2010; Nair et al. 2010). CuO NP can cross plant cell walls and cell membranes, thereby distributing themselves around nucleus and organelles, forming agglomerates within the root cells (Yuan et al. 2016). Detrimental effects of nano-sized CuO on plants include symptoms such as growth inhibition (Perreault et al. 2014a), modified antioxidant enzyme activity (Kim et al. 2012; Dimkpa et al. 2012), metabolic disturbances (Hong et al. 2015), macromolecular damages (Shaw and Hossain 2013), photosynthesis interferences (Perreault et al. 2014b), decreased chlorophyll content (Shi et al. 2011, 2014) and induced DNA lesions (Atha et al. 2012). The toxicogenomic effects of CuO have been attributed to the Cu2+ ions released from CuO NPs (Tang et al. 2016). Studies in zucchini and maize showed that CuO NPs did not affect the germination but suppressed root elongation (Stampoulis et al. 2009; Wang et al. 2012). Yasmeen et al. (2015) have reported that wheat (Triticum aestivum) seeds when soaked in copper NPs and inoculated in distilled water resulted in increased shoot growth but with severe reduction in seedling vigor. CuO NPs can induce DNA damage by the accumulation of oxidatively modified, mutagenic DNA lesions (7,8-dihydro-8-oxoguanine; 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 4,6-diamino-5-formamidopyrimidine) (Areeba et al. 2016). CuO NPs, when exposed to wheat plants, inhibited their growth and changed the structure of the roots (Dimkpa et al. 2012; Tang et al. 2016). CuO NPs were found to significantly reduce the germination rate and biomass of rice seeds, fresh weights and root length of Arabidopsis seedlings (Shaw and Hossain 2013).

In this study, the most economically viable vegetable crop Eggplant (Solanum melongena) was exposed to different concentrations of various metallic nanoparticles such as NiO, CuO and ZnO NP to investigate their growth stimulative and the toxic effects in eggplant. Growth parameters, chlorophyll and anthocyanin levels, were determined in response to NP treatments. Furthermore, antioxidant potential, ROS generation, and genotoxicity were investigated in eggplants with response to nanoparticle treatment. The influence of NPs on the contents of the total phenolics and flavonoids was also examined.

Materials and methods

Seed treatment and data observation

Surface sterilization of S. melongena seeds was done using 70% ethanol and 0.1% mercuric chloride as described previously by Ray et al. (2011). The seeds were rinsed well with distilled water after each treatment to remove traces of ethanol and mercuric chloride. Metal oxide nanoparticles such as ZnO (18 nm, 99.95%), CuO (25–55 nm, 99%) and NiO NPs (10–20 nm, 99%) were obtained from US research Nanomaterials. Four different concentrations (100, 250, 500 and 1000 mg/L) of nanoparticles were added aseptically to culture vessels lined with sterile filter paper. The seedling germination was inhibited by all the respective bulk materials such as CuCl2, ZnSO4, NiSO4 at > 150 mg/L concentration. However, except NiO NP, no significant growth suppression was found in the CuO and ZnO NP treatment up to 250 mg/L. Therefore, we proceeded further to study the impact of metal oxide nanoparticles at various higher concentrations. Around 20–30 seeds were inoculated in each concentration of nanoparticles and kept for 15 days, at 25 °C under a 16/8-h photoperiod. Similar experiments with distilled water were conducted as a control. After 15 days, seedlings were harvested. Seedling growth in terms of shoots and root length was recorded using a ruler.

Total chlorophyll estimation

The total chlorophyll content of NP (NiO, CuO, and ZnO)-treated and control seedlings of eggplants was estimated as described previously by Ma et al. (2013). Using a scalpel, the harvested fresh seedlings (50 mg) were cut into small bits into eppendorfs. To each Eppendorf, 95% (v/v) ethanol was added until the samples were soaked completely. This was left undisturbed for 3 days under dark conditions at 4 °C. The resulting supernatant was recorded at 664.2 and 648.6 nm. The following formula was used for total chlorophyll estimation, total chlorophyll = Chl a + Chl b, Chl a = 13.36 A664.2 − 5.19 A648.6, Chl b = 27.43 A648.6 − 8.12 A664.2. Total chlorophyll content was expressed as milligram per gram per fresh weight (FW).

Determination of malondialdehyde (MDA)

The procedure of Heath and Packer (1968) was followed for the measurement of malondialdehyde (MDA) content in seedlings using the thiobarbituric acid (TBA) test. Approximately 100 mg of NP-treated and control seedling tissues was ground to a fine powder using liquid nitrogen. The samples were homogenized and extracted with 1 mL of 1% (w/v) trichloroacetic acid (TCA). Centrifugation was done at 13,000×g for 15 min and 0.5 mL of the resultant extract was added to 1 mL of 20% (w/v) TCA and 1 mL of 0.5% (w/v) TBA solution. The extract was heated at 95 °C for 30 min followed by centrifugation at 10,000×g for 10 min. The resultant supernatant absorbance was recorded at 532 and 600 nm. The amount of MDA was calculated using the formula C¼ = D/EL. The content of MDA was expressed as micromoles units per g/FW.

Determination of anthocyanin content

Anthocyanin concentration was determined as described previously by Baskar et al. (2015). Fifty micrograms of NP-treated and untreated S. melongena seedlings was powdered with liquid nitrogen. The powdered samples were mixed with 1 mL of 1% (v/v) HCl in methanol and incubated in the dark at 4 °C overnight. In this extract, 1 ml of 500 µL chloroform and 500 µL distilled H2O were added and centrifuged at 13,000×g for 2 min at 4 °C, and the absorbances of the supernatant were recorded at 530 and 657 nm by UV–Vis spectrophotometer. Anthocyanin quantity was determined using A530 − 1/4 A657 formula and the results were presented as milligram per grams FW.

Antioxidant capacity

The antioxidant capacity of nanoparticle-treated and control samples was determined by diphenyl-2-picriylhydrazil (DPPH) radical scavenging assay using the method described by Brand-Williams et al. (1995). Total phenolic contents (TPC) were determined by Folin–Ciocalteu spectroscopic method using gallic acid as standard and the results were represented as mg gallic acid equivalent (GAE) per gram of sample (mg GAE/g). Total flavonoid content (TFC) estimation was done following the calorimetric method of Bao et al. (2005) with catechin as standard. The results of TFC were expressed as mg catechin equivalent (CE) per gram (mg CE/g).

In vivo ROS detection

The roots of eggplant seedlings from NP-treated (NiO, CuO and ZnO) and control samples were cut with a scalpel followed by nitro blue tetrazolium chloride (NBT) and 3,3′-diaminobenzidine (DAB) histochemical staining (Kumar et al. 2014) for the qualitative detection of ROS generation. Staining was performed for 2 h duration with 0.5 mg/mL NBT solution and 8 h duration with 2 mg/mL DAB solution. Decolourisation was performed using boiling ethanol for 2 h after DAB staining. The stained root samples of NP treated and control were placed on a white sheet and the images were taken using a digital camera (Canon, Japan).

DNA extraction and electrophoresis

DNA fragmentation assay was performed to detect the apoptotic DNA cleavage in the eggplant seedlings exposed to different concentrations of NiO, CuO and ZnO nanoparticles. Approximately 100 mg of NP-treated and control seedling tissues was ground to a fine powder with a mortar and pestle. Cetyl trimethyl ammonium bromide (CTAB) method was employed to isolate DNA from the NP-treated and control samples (Rogers and Bendich 1989). The ground samples were treated with CTAB buffer and incubated at 65 °C for 20 min. This was followed by centrifugation and the addition of phenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation, ice-cold isopropanol was added to precipitate the DNA. Finally, DNA was dissolved in TE buffer after ethanol wash. The extracted genomic DNA (5 µg) was run on an agarose gel stained with ethidium bromide and visualized under UV transilluminator.

Statistical analysis

All samples were analyzed in triplicates. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (P < 0.05) with the help of SPSS ver. 17 statistical package program. The values are expressed as means of triplicate analysis of the samples (n = 3) ± SD.

Results and discussion

Nanoparticles play a profound role in influencing plant growth and development. They have been found to impact several factors such as seed germination, biomass production, shoot growth, root growth, physiological and biochemical activities (Siddiqi and Husen 2017). Hence the present work focused on the phytotoxicity of three important metal oxide nanoparticles, such as NiO, CuO and ZnO NPs, recording their enhancive and inhibitive effects on S. melongena seedlings. This edible fruit is a good source of dietary fiber, vitamin B1, and copper and is widely used in the cuisine of many countries.

Effects of metal oxide NP on the growth characteristics of eggplant seedlings

The eggplant seedlings were treated with NiO, CuO and ZnO NPs at different sub-lethal concentrations (100, 250, 500 and 1000 mg/L) to study various physiological and biochemical parameters. The plant root and shoot growth of treated and control eggplant seedlings were evaluated after 2 weeks to investigate the possible growth retardation effects of nanoparticles. There was a significant decline in seed germination and plant growth with elevated concentrations of nanoparticles. Interestingly, at 100 mg/L lower concentration of CuO NP root and shoot length of the eggplant seedlings was higher compared to control plants (Figs. 1, 2). The increased length of the seedlings at this concentration indicates the growth promoting ability of CuO NP at lower concentrations (Fig. 2b). However, at higher concentrations (500 and 1000 mg/L) CuO NP exhibited the repression of shoot and root length (Figs. 1, 2). In accordance with our results, a significant reduction in shoot growth and biomass was observed at higher concentrations (200 and 500 mg/L) of CuO NP in Vigna radiata (Nair et al. 2014). In Brassica juncea, ZnO NP showed dose-dependent growth inhibition and a significant inhibition of root and shoot length was found in higher concentration (1000 and 1500 mg/L) (Rao and Shekhawat 2014). In our study, ZnO NP revealed dose-dependent growth retardation. The root and shoot length of eggplant seedlings were sequentially decreased from lower to higher concentrations of ZnO NP (Fig. 2c). In the case of NiO NP, all the four concentrations showed suppression of root and shoot length with maximum inhibition of seedlings in terms of root and shoot growth at the highest concentration (1000 mg/L) compared to control (Fig. 2a). In agreement with our results, Faisal et al. (2013) found that the root growth profile of tomato was inhibited in a dose-dependent manner. Although all the NP showed growth inhibition, NiO NP displayed significant toxicity to the eggplant seedlings.

Fig. 1.

Fig. 1

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The figure showing the morphological features of Solanum melongena seedlings treated with different concentrations (100, 250, 500 and 1000 mg/L) of a nickel oxide (NiO) nanoparticles, b copper oxide (CuO) nanoparticles, c zinc oxide (ZnO) nanoparticles

Fig. 2.

Fig. 2

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Effects of metal oxide nanoparticles (NiO, CuO and ZnO) on the root and shoot growth of the Solanum melongena seedlings. a Total length of root and shoot (cm) of S. melongena at different concentrations (100, 250, 500 and 1000 mg/L) of nickel oxide (NiO), b copper oxide (CuO), c zinc oxide (ZnO) nanoparticles. Each value in the graph is represented as mean ± SD (n = 3). Significantly different values (P < 0.05) showed in different alphabet letters

Effects of metal oxide NP on total chlorophyll content

Several previous studies reported that nanoparticle exposure significantly altered the total chlorophyll content and photosynthetic performance in various plants. In our study, the exposure of metal oxide NP (NiO, CuO and ZnO) to eggplant seedlings showed a decline in the total chlorophyll content. The reduction in chlorophyll content varied between the NP treatment and their concentrations (Fig. 3). In the case of NiO and CuO NP treatment, chlorophyll contents exhibited a gradual decrease in a dose-dependent manner compared to control plants (Fig. 3a, b). A strong decline in chlorophyll content was found at the highest concentration (1000 mg/L) in CuO NP treatment. Compared to NiO and CuO NPs, ZnO NP treatment recorded reduction in total chlorophyll contents for all the tested concentrations with a significant decline in the chlorophyll reduction at even the lowest concentration of 100 mg/L (Fig. 3c). In agreement with our results, NiO, CuO and ZnO NPs were shown to decrease the chlorophyll contents and photosynthetic performance in various crops, including aquatic plants in a concentration-dependent manner (Gong et al. 2011; Nair and Chung 2015; Rao and Shekhawat 2014). It has been reported that in ZnO NP-treated Arabidopsis plants, the expression of chlorophyll biosynthesis genes and photosystem-related genes were reduced, exhibiting reduced chlorophyll a and b in treated plants (Wang et al. 2016). Loss of chlorophyll is associated with irreversible damage of the chloroplast structure due to ROS formation impacted by combining of electrons with molecular oxygen (Foyer et al. 1994). Thus, nanoparticle could affect the crop yield by influencing the net rate of photosynthesis caused by reduced chlorophyll contents.

Fig. 3.

Fig. 3

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Dose-dependent effects of three different nanoparticles (NiO, CuO and ZnO) on total chlorophyll content (mg/g FM) in Solanum melongena. a Total chlorophyll content of S. melongena at different concentrations of nickel oxide (NiO) nanoparticles (100, 250, 500 and 1000 mg/L). b Copper oxide (CuO) nanoparticles (100, 250, 500 and 1000 mg/L). c Zinc oxide (ZnO) nanoparticles (100, 250, 500 and 1000 mg/L). Each value in the graph is represented as mean ± SD (n = 3). Significantly different values (P < 0.05) showed in different alphabet letters

Determination of metal oxide NP-induced malondialdehyde (MDA) production

In the present study, we observed the formation of MDA in eggplant seedlings with response to NP treatments. The increased accumulation of lipid peroxide in treated samples compared to control is an indication of enhanced ROS production. A concentration-dependent enhancement in MDA formation was found in all the NP (NiO, CuO and ZnO)-treated eggplant seedlings (Fig. 4). Significant MDA level was found in the ZnO NP-treated seedlings, followed by CuO and NiO NP treatment (Fig. 4). Rao and Shekhawat (2014) reported a dose-dependent enhancement of MDA formation in the root, shoot and leaf tissues of B. juncea plants upon exposure to ZnO NP. Nair and Chung (2015) also reported an increased lipid peroxidation at high concentrations of CuO NP-treated B. juncea seedlings. In our study, a sequential increase in MDA generation was found in the CuO NP treatment from lower to higher concentrations. Thus, all the three metal oxide nanoparticles could induce dose-dependent MDA accumulation in the eggplant seedlings.

Fig. 4.

Fig. 4

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Dose-dependent effects of three different nanoparticles (NiO, CuO and ZnO) on concentration of MDA (Micromole/g FM) in Solanum melongena. a Concentration of MDA in S. melongena at different concentrations of nickel oxide (NiO) nanoparticles (100, 250, 500 and 1000 mg/L), b copper oxide (CuO) nanoparticles (100, 250, 500 and 1000 mg/L), c zinc oxide (ZnO) nanoparticles (100, 250, 500 and 1000 mg/L). Each value in the graph is represented as mean ± SD (n = 3). Significantly different values (P < 0.05) showed in different alphabet letters

Effects of metal oxide NP on anthocyanin production

Anthocyanins are stress-responsive molecules, protecting plants from damage induced by ROS. Anthocyanin production was measured in both the NP-treated (NiO, CuO and ZnO) and untreated seedlings of eggplants. In all the three NPs used, changes in anthocyanin content were directly proportional to the concentration of NPs used up to 500 mg/L (Fig. 5). However, anthocyanin contents showed a decreased in all the NP-treated eggplant seedlings at 1000 mg/L concentration. Interestingly, CuO NP treatment revealed the accumulation of significant levels of total anthocyanin followed by ZnO and NiO NP treatment (Fig. 5). Similarly, an enhanced anthocyanin production was noticed in the Arabidopsis plants treated with CuO NP (Nair and Chung 2014). However, compared to control, a slightly decreased anthocyanin content was found at lower concentration (100 mg/L) followed by an increase at higher concentration (250 and 500 mg/L) of ZnO NP-treated eggplants. In accordance with our results, the exposure of ZnO NP resulted in a slight decrease of anthocyanin production at 100 ppm compared to control and then increased at 300 and 500 ppm in the potato plants (Raigond et al. 2017). Therefore, the enhanced anthocyanin production on NP exposure (up to 500 mg/L) might serve for the protection against the oxidative stress induced by the nanoparticles (Thiruvengadam et al. 2015).

Fig. 5.

Fig. 5

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Dose-dependent effects of three different nanoparticles (NiO, CuO and ZnO) on total anthocyanin content (mg/g FM) in Solanum melongena. a Total Anthocyanin content of S. melongena at different concentrations (100, 250, 500 and 1000 mg/L). a Nickel oxide (NiO) nanoparticles, b copper oxide (CuO) nanoparticles, c zinc oxide (ZnO) nanoparticles. Superscript letters with same alphabets are non-significant and those with different alphabets indicates that they are significantly different (P < 0.05) analyzed by Duncan’s multiple range test

Effects of metal oxide NP on antioxidant capacity in eggplants

Nanoparticles have been reported to cause oxidative stress in plants (Rico et al. 2013) owing to their interaction with the cellular components resulting in the formation of ROS (Krishnaraj et al. 2012). Phenolic compounds, flavonoids, and antioxidant enzymes act as defense molecules in mitigating oxidative stress caused by excess ROS (Corral-Diaz et al. 2014). The widely used radical scavenging assay 2,2-diphenyl-1-picryl hydrazyl (DPPH) was carried out in NP-treated (NiO, CuO and ZnO) and non-treated eggplant seedlings to determine the antioxidant potential. The observed radical scavenging values (%) were lesser in NP-treated plants than that of control plants (Fig. S1). Antioxidant capacity followed a decreasing order with the increasing concentration of NP treatment. Among the different NP treatment, ZnO NP showed higher DPPH activity compared to NiO and CuO NPs (Fig. S1). DPPH activity was more pronounced at lower concentrations (100, 250 mg/L) of ZnO NP-treated eggplant seedlings (Fig. S1c). However, at higher (1000 mg/L) concentrations all NPs reported lower DPPH activity. NiO at 1000 mg/L showed the least antioxidant capacity compared to CuO and ZnO NP treatment (Fig. S1). These results suggest that lower radical scavenging activity observed in NiO and CuO NPs indicate that they are more toxic compared to ZnO NP. The previous study by Zafar et al. (2016) in Brassica nigra also reported that at lower concentrations ZnO NP showed an increased DPPH activity followed by a decrease at higher concentrations. Flavonoids and phenolic acids are highly correlated with the total antioxidant activity as they are potent antioxidants (Zhao et al. 2014). Total phenolic contents (TPC) at all the four concentrations of NP treatment were found to be higher than the control eggplants. A gradually increasing order of TPC values with increasing NP concentration was observed in NiO, CuO and ZnO NPs till 500 mg/L and then declined at 1000 mg/L (Fig. 6). A significant reduction in the TPC contents was observed at 1000 mg/L of all the NP-treated eggplant seedlings. TPC contents were higher in ZnO (180 µg/g) treatment followed by NiO (130 µg/g) and CuO NP (120 µg/g) (Fig. 6). Similar to TPC, total flavonoid content (TFC) was also higher in all NP-treated samples compared to control (Fig. 7). TFC level followed a concentration-dependent increase up to 500 mg/L in CuO and ZnO NPs (Fig. 7). A drastic reduction in the TFC was found at 1000 mg/L concentration of all the NP investigated in this study. TFC contents were higher in ZnO (302 µg/g) followed by NiO (200 µg/g) and CuO NP (185 µg/g)-treated eggplant seedlings at 500 mg/L. Previous studies have also shown that TPC and TFC contents were significantly altered in several plants treated with the nanoparticles in a dose-dependent manner. In line with our results, Zafar et al. (2016) also reported the TPC and TFC contents were enhanced in the ZnO NP-treated B. nigra plants in a concentration-dependent manner. Similarly, the bulk and nano-ZnO, and CuO treatment was shown to increase the contents of TPC and TFC in Glycyrrhiza glabra seedlings (Oloumi et al. 2015). They concluded that nano- and bulk-sized ZnO and CuO could affect the secondary metabolite production. Furthermore, the contents of flavonoids and phenolics were increased in Amaranthus caudatus L. when treated with the biologically synthesized silver nanoparticles and they suggested that antioxidant activity and phytochemical contents can be enhanced by nanoparticles in a dose-dependent manner (Azeez et al. 2017). The above results clearly indicate that NPs could have an impact on the secondary metabolite production.

Fig. 6.

Fig. 6

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Dose-dependent effects of three different nanoparticles (NiO, CuO and ZnO) on total phenolic content (µg/g) in Solanum melongena. Total phenolic content in S. melongena at different concentrations (100, 250, 500 and 1000 mg/L) of a nickel oxide (NiO) nanoparticles, b copper oxide (CuO) nanoparticles, c zinc oxide (ZnO) nanoparticles. Each value in the graph is represented as mean ± SD (n = 3). Significantly different values (P < 0.05) showed in different alphabet letters

Fig. 7.

Fig. 7

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Dose-dependent changes in the total flavonoid content (µg/g) in Solanum melongena treated with the three different metal oxide nanoparticles (NiO, CuO and ZnO). a Total flavonoid content in S. melongena at different concentrations of nickel oxide (NiO) nanoparticles (100, 250, 500 and 1000 mg/L), b copper oxide (CuO) nanoparticles (100, 250, 500 and 1000 mg/L), c zinc oxide (ZnO) nanoparticles (100, 250, 500 and 1000 mg/L). Each value in the graph is represented as mean ± SD (n = 3). Significantly different values (P < 0.05) showed in different alphabet letters

Detection of metal oxide NP-induced ROS generation

Plants respond to various stresses, including abiotic and biotic stresses through the production of reactive oxygen species (H2O2, OH−, and O2− molecules). The ROS will be effectively scavenged by the innate-evolved plants’ enzymatic and nonenzymatic antioxidant mechanisms. Several previous studies reported that diverse factors, namely size and shape, solubility and particle dissolution, metal ions released from metal and metal oxide NPs, biotransformation of NPs, light, etc., may cause the ROS production and phytotoxicity (Yang et al. 2017). In this study, NBT and DAB histochemical stainings were performed in the roots of eggplant seedlings exposed to the metal oxide NP (NiO, CuO, and ZnO NP) for visual detection of the presence of O2− and H2O2 radicals. ROS generation was increasing with increasing concentrations of nanoparticles. A concentration-dependent increased dark blue coloration was noticed in the roots of NP-treated plants after staining with NBT (Fig. S2). Similarly, NP-treated and control plant roots stained with the DAB solution resulted in a gradual increase in the dark brown color formation in NP-treated roots exhibiting a dose-dependent phenomenon (Fig. S3). Among the different NP used, NiO NP treatment enhanced the ROS production even at low concentrations (100 mg/L) compared to CuO and ZnO NP which is evident from the NBT and DAB staining. Followed by the NiO, CuO recorded higher ROS production compared to ZnO NP-treated plant roots. In accordance with our results, NiO was shown to induce ROS production in tomato in a concentration-dependent manner, which was detected through 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining. Similarly, ZnO NP-treated Allium cepa roots exhibited a concentration-dependent increase in ROS generation which was detected in vivo by staining the roots with DCFH-DA (Ahmed et al. 2017). Nair et al. (2014) also reported a concentration-dependent ROS generation in the Vigna radiata L. seedlings treated with the CuO NP.

Determination of metal oxide NP-induced genotoxicity

Nanoparticles have been reported to cause DNA strand break (Rim et al. 2013) and chromosomal aberrations (Ghosh et al. 2012). Nanoparticle-mediated genotoxicity is primarily triggered by ROS-mediated oxidative stress. Mitochondrial depolarization, down-regulation of DNA repair genes, chromosomal instability, damage of cellular components, oxidative stress-induced apoptosis, and mitosis inhibition are some of the factors involved in NP-induced toxicity (Rim et al. 2013). It has been reported that abiotic stresses induce DNA damage in various plants (Kumari et al. 2009). DNA laddering assay was performed for the qualitative determination of the extent of DNA damage in the seedlings of eggplants treated with different metal oxide (NiO, CuO and ZnO) nanoparticles. Compared to untreated plants, NP-treated eggplants revealed the presence of DNA damage (Fig. S4). The genomic DNA of control plant samples was undamaged as evidenced by a thick band on the agarose gel while DNA damage was found in all the NP-treated samples at all the concentrations used which indicated these engineered nanoparticles could induce DNA damage. In agreement with our results, DNA damage imposed by the various nanoparticles in several plants has been detected through several techniques which include DNA fragmentation assay, tunnel assays and RAPD (Baskar et al. 2015; Thiruvengadam et al. 2015; Lee et al. 2013).

Conclusion

In the present investigation, we examined the eggplants (15-day-old seedlings) responses to different metal oxide NPs such as CuO, ZnO, and NiO at various concentrations. A strong suppression of plant root and shoot growth was observed in all the tested NP in a dose-dependent manner. NiO NP was found to repress the shoot and root growth even at the lowest concentration (100 mg/L). A similar concentration-dependent decrease in the total chlorophyll content was observed in the NP-treated seedlings with ZnO showing the significant reduction in chlorophyll contents. The decreased anthocyanin accumulation was found at highest concentrations (1000 mg/L) of NP-treated samples. The increase in anthocyanin levels at lower concentrations of NP exhibits significant tolerance against oxidative stress. Furthermore, the nonenzymatic antioxidants such as flavonoids and phenolics were altered in NP-treated plants and these changes occurred depending on the concentrations of NP used. The increased intracellular ROS formation and MDA production were prominent at higher concentrations (500 and 1000 mg/L) of metal oxide NP treatments. ROS generation in the roots of NP-treated S. melongena seedlings was evident with DAB and NBT histochemical staining. Altogether, our results suggest that the higher concentration (500 and 1000 mg/L) of all the subjected metal oxide nanoparticles induces phytotoxicity via the inhibition of growth features in eggplants which is greatly correlated with the detected different physiological and biochemical changes. It is also suggested that further studies would be necessary to study the effects of NP-induced phytotoxicity at different growth stages of plants.

Electronic supplementary material

Below is the link to the electronic supplementary material.

13205_2018_1386_MOESM1_ESM.jpg (117.1KB, jpg)

Supplementary material 1 Figure S1: Concentration-dependent changes in the total radical scavenging activity (%) of the seedlings of Solanum melongena treated with three different nanoparticles (NiO, CuO and ZnO). a) Total radical scavenging activity (%) of S.melongena at different concentrations (100 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L) of (a) Nickel oxide (NiO) nanoparticles, (b) Copper Oxide (CuO) nanoparticles, (c) Zinc oxide (ZnO) nanoparticles. Each value in the graph is represented as mean ± SD (n = 3). Significantly different values (P < 0.05) showed in different alphabet letters (JPG 118 kb)

13205_2018_1386_MOESM2_ESM.jpg (2.4MB, jpg)

Supplementary material 2 Figure S2: Figure showing the in vivo generation of gradual reactive oxygen species (ROS) formation in the roots of Solanum melongena seedlings treated with different concentrations (100 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L) of (a) Nickel oxide (NiO) (b) Copper oxide (CuO) (c) Zinc oxide (ZnO) nanoparticles were examined using Nitroblueterazolium (NBT) staining (JPG 2476 kb)

13205_2018_1386_MOESM3_ESM.jpg (3.2MB, jpg)

Supplementary material 3 Figure S3: Dose-dependent effects of metal oxide nanoparticales (NiO, CuO and ZnO) on reactive oxygen species (ROS) generation in the two week old Solanum melongena roots at different concentrations (100 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L) (a) Nickel oxide (NiO) nanoparticles (b) Copper oxide (CuO) nanoparticles (c) Zinc oxide (ZnO) nanoparticles was determined using 3,3’-diaminobenzidine (DAB) staining (JPG 3274 kb)

13205_2018_1386_MOESM4_ESM.jpg (906.3KB, jpg)

Supplementary material 4 Figure S4: Dose dependant effects of metal oxide nanoparticles (NiO, CuO and ZnO) on DNA fragmentation analysis of Solanum melongena treated with the three different concentrations such as 100 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L. DNA fragmentation analysis of (a) Nickle oxide (NiO) nanoparticles, Lane 1- control, Lane 2- NiO NP (100mg/L), Lane 3- NiO NP (250 mg/L), Lane 4- NiO NP (500 mg/L), Lane 5 – NiO NP (1000 mg/L), (b) Copper oxide (CuO) nanoparticles, Lane 1- CuO NP (100mg/L), Lane 2- CuO NP (250 mg/L), Lane 3- CuO NP (500 mg/L), Lane 4- CuO NP (1000 mg/L), Lane 5 – control, (c) Zinc oxide (ZnO) nanoparticles, Lane 1- ZnO NP (100mg/L), Lane 2- ZnO NP (250 mg/L), Lane 3- ZnO NP (500 mg/L), Lane 4- ZnO NP (1000 mg/L), Lane 5 – control (JPG 907 kb)

Acknowledgements

This study was supported by a Grant (Sanction no. PDF/2016/000750) from the Department of Science and Technology—Science and Engineering Research Board, Government of India.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

13205_2018_1386_MOESM1_ESM.jpg (117.1KB, jpg)

Supplementary material 1 Figure S1: Concentration-dependent changes in the total radical scavenging activity (%) of the seedlings of Solanum melongena treated with three different nanoparticles (NiO, CuO and ZnO). a) Total radical scavenging activity (%) of S.melongena at different concentrations (100 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L) of (a) Nickel oxide (NiO) nanoparticles, (b) Copper Oxide (CuO) nanoparticles, (c) Zinc oxide (ZnO) nanoparticles. Each value in the graph is represented as mean ± SD (n = 3). Significantly different values (P < 0.05) showed in different alphabet letters (JPG 118 kb)

13205_2018_1386_MOESM2_ESM.jpg (2.4MB, jpg)

Supplementary material 2 Figure S2: Figure showing the in vivo generation of gradual reactive oxygen species (ROS) formation in the roots of Solanum melongena seedlings treated with different concentrations (100 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L) of (a) Nickel oxide (NiO) (b) Copper oxide (CuO) (c) Zinc oxide (ZnO) nanoparticles were examined using Nitroblueterazolium (NBT) staining (JPG 2476 kb)

13205_2018_1386_MOESM3_ESM.jpg (3.2MB, jpg)

Supplementary material 3 Figure S3: Dose-dependent effects of metal oxide nanoparticales (NiO, CuO and ZnO) on reactive oxygen species (ROS) generation in the two week old Solanum melongena roots at different concentrations (100 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L) (a) Nickel oxide (NiO) nanoparticles (b) Copper oxide (CuO) nanoparticles (c) Zinc oxide (ZnO) nanoparticles was determined using 3,3’-diaminobenzidine (DAB) staining (JPG 3274 kb)

13205_2018_1386_MOESM4_ESM.jpg (906.3KB, jpg)

Supplementary material 4 Figure S4: Dose dependant effects of metal oxide nanoparticles (NiO, CuO and ZnO) on DNA fragmentation analysis of Solanum melongena treated with the three different concentrations such as 100 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L. DNA fragmentation analysis of (a) Nickle oxide (NiO) nanoparticles, Lane 1- control, Lane 2- NiO NP (100mg/L), Lane 3- NiO NP (250 mg/L), Lane 4- NiO NP (500 mg/L), Lane 5 – NiO NP (1000 mg/L), (b) Copper oxide (CuO) nanoparticles, Lane 1- CuO NP (100mg/L), Lane 2- CuO NP (250 mg/L), Lane 3- CuO NP (500 mg/L), Lane 4- CuO NP (1000 mg/L), Lane 5 – control, (c) Zinc oxide (ZnO) nanoparticles, Lane 1- ZnO NP (100mg/L), Lane 2- ZnO NP (250 mg/L), Lane 3- ZnO NP (500 mg/L), Lane 4- ZnO NP (1000 mg/L), Lane 5 – control (JPG 907 kb)

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