Impact of Phytomediated Zinc Oxide Nanoparticles on Growth and Oxidative Stress Response of In Vitro Raised Shoots of Ochradenus arabicus
Biogenic nanoparticles have potential roles in the growth and development of plants and animals as they are ecofriendly and free of chemical contaminants. In this study, we assessed the effects of phytomediated zinc oxide nanoparticles (ZnONPs) on shoot growth, biochemical markers, and antioxidant system response in Ochradenus arabicus, which is a medicinal plant. The shoot length and fresh and dry weights were found to be higher in groups with 5 and 10 mg/L ZnONPs than in the control. At high concentrations of ZnONPs (50, 100, and 300 mg/L), biomass was decreased in a concentration-dependent manner. The shoot number was observed to be highest at 50 mg/L among all applied concentrations of ZnONPs. The levels of the stress markers proline and TBARS were found to be higher in shoots treated with 100 and 300 mg/L ZnONPs than in the control as well as NP-treated shoots. The levels of antioxidant enzymes were significantly increased at high concentrations of nanoparticles compared with the control. Thus, synthesized phytomediated ZnONPs from shoots of O. arabicus and their application to the same organ of O. arabicus in vitro were found to be effective as a low concentration of nanoparticles promoted shoot growth, resulting in high biomass accumulation. Thus, using green nanotechnology, such endemic plants could be conserved in vitro and multiple shoots could be produced by reducing the phytohormone concentration for multiple uses, such as the production of potential secondary metabolites.
The application of nanoparticles (NPs) in agriculture has contributed to increased crop quality, the regulation of crop production, and improved stress tolerance in various plant species. At present, various methods are available for synthesizing NPs, such as chemical and physical methods. However, they can be expensive, environmentally damaging, and labor-intensive. Nanoparticles have both benefits and risks for flora and fauna. Substantial work has been carried out on the synthesis of nanoparticles and their use in biological systems [1, 2]. However, work related to nanoparticle applications on plant systems has been limited to the observation of physiological, biochemical, and morphological parameters. The phytomediated synthesis of NPs is nontoxic, inexpensive, facile, ecofriendly, and beneficial not only to human health but also to different types of microorganisms. Zinc nanoparticles (used as micronutrients in agriculture) have potential to boost the yield and growth of medicinal as well as food crops. Zinc oxide nanoparticles (ZnONPs) are among the most commonly synthesized NPs worldwide, next to carbon nanotubes and gold, silver, and titanium dioxide NPs . In the last few years, ZnONPs have received significant attention due to their purported ability to enhance nutrient accumulation by plants in order to augment the quality of crops [4, 5]. Some nanoparticles have been used for various biological purposes, such as gene and drug delivery. Various metallic oxide nanoparticles have been used for growth promotion as well as secondary metabolite production from candy leaf [6, 7].
Zinc is considered an important micronutrient for both animals and plants . It maintains the integrity of the cell membrane, ensures chloroplast synthesis, and enhances enzyme activities. According to Cakmak , Zn exerts effects on membrane function, protein synthesis, cell elongation, and environmental stress tolerance. Zinc is a necessary micronutrient and plays potential roles in the activities of various enzymes, such as tryptophan synthetase, dehydrogenases, transphosphorylases, aldolases, superoxide dismutase, isomerases, and DNA and RNA polymerases [10, 11]. Farmers use zinc fertilizers to enhance crop yield. Zinc can also control some diseases caused by bacteria and fungi. Moreover, Zn plays important roles in the regulation of phytohormones such as auxin, protein synthesis, carbohydrate metabolism, and stress-alleviating responses [12, 13]. Various nanoparticles synthesized by plant extracts have many promoting effects on seed germination, morphological markers, yield traits, and biomass .
Ochradenus arabicus (family: Resedaceae) is a medicinal plant used locally and found mostly in desert regions of Saudi Arabia. Different compounds have been reported from the genus Ochradenus, such as quercetin-3-O-p-coumaryl(1-6)-β-glucosyl(1-6)-β-glucoside-7-O-α-rhamnoside, quercetin-3-O-β-glucosyl(1-2)-α-rhamnoside-7-O-α-rhamnoside, quercetin glycosides, isoquercitrin, quercetin-3-gentiobioside, and others including astragalin, kaempferol, glycosides, and afzelin . Various solvent extracts of O. arabicus were reported to show antibacterial, anticancer, and antifungal activities . The plant has seed dormancy, which may be due to the hard seed coat, presence of some inhibitors, underdeveloped embryos, or low internal hormone levels. The germination rate of O. arabicus is very low at 2.6%, as reported by Nadeem et al. .
A high concentration of ZnONPs produces oxidative stress, which is common in plants; however, in response to NPs, plants produce antioxidant enzymes and secondary metabolites to cope with oxidative stress. There are a few reports on the application of NPs for in vitro plant growth and development. However, there is a need to investigate the effects of such NPs on plant growth and plant regeneration as plants have potential applications to cure various health problems due to the presence of different secondary metabolites. Unfortunately, O. arabicus is endemic to Saudi Arabia and found in only a few places within the country , so our aim is to produce multiple O. arabicus shoots with high biomass by applying phytomediated ZnONPs. Therefore, in the present study, we investigated the effect of phytomediated ZnONPs synthesized from O. arabicus and their application to regenerated shoots of this species for the first time.
2. Materials and Methods
2.1. Seed Germination
Seeds of wild plant Ochradenus arabicus were collected from the Central Region of Saudi Arabia in 2010, under the project (Grant No: 10-BIO1289-02) approved by the National Plan for Science, Technology, and Innovation. The plant was identified by a taxonomist, Dr. Jacob Thomas Pandalayil, at the Department of Botany and Microbiology, College of Science, King Saud University, and identification was also performed by internal transcribed spacer sequence of ribosomal DNA (GenBank accession No: JQ899053). Mature seeds were washed with tap water for 30 min to remove dust and microbes. The washed seeds were kept in 50% bleach for 20 min for surface disinfection, after which they were washed with autoclaved distilled water three or four times. Seeds were kept for germination on 0.8% agar in the dark at °C.
2.2. Shoot Proliferation from Germinated Seeds
Shoots were produced on MS medium  with cytokinin (1 mg/L BA). The cultures were grown in a culture room set at relative humidity 55%–60% and temperature 25°C–28°C under PPFD of 40 μmol m-2 s-1 for a 16 h photoperiod. The shoots were cultured for 45 days on MS medium to obtain more shoots.
2.2.1. Plant Extract Preparation for ZnONP Synthesis
Young shoots of O. arabicus obtained in vitro on MS medium were used for nanoparticle synthesis. Five grams of fresh shoots was chopped with a razor into small pieces to obtain a good amount of extract. To prepare the shoot extract, these small pieces were added to 100 mL of double-distilled water (in a 250 mL conical flask) and kept in a water bath at 100°C for 20 min. The extract was taken out from the water bath and cooled down. This extract was then filtered with Whatman filter paper No. 1 twice and kept at 4°C until use.
2.2.2. Phytomediated Synthesis of ZnONPs
A total of 100 mL of fresh O. arabicus shoot extract was added to 0.05 M Zn(NO3)2 solution at 60°C, followed by continuous stirring for 12 h. A brown whitish color appeared upon the addition of 0.1 N NaOH, as upon the synthesis of nanoparticles. Centrifugation was performed at 8000 rpm, 4°C for 10 min for pellet formation. The pellet was washed with double-distilled water. Further, the pellet was washed with absolute alcohol and dried at 40°C for 12 h. Calcination was performed at 500°C for 3 h.
2.3. Characterization of ZnONPs
The biologically synthesized ZnONPs were observed as precipitation of brown whitish particulates and confirmed by UV-visible spectroscopy. Particle shape, size, and texture were analyzed using Fourier transform infrared (FTIR) and transmission electron microscopy (TEM). Dynamic light scattering (DLS) was also used to measure the size of the synthesized nanoparticles.
2.3.1. Optimization of ZnONP Concentration
The stability of the synthesized ZnONPs was evaluated using UV-Vis spectroscopy before their application on shoots. Various concentrations of ZnONPs, including 5, 10, 25, 50, 100, and 300 mg/L, were applied to the regenerated shoots of O. arabicus cultured on MS medium to determine the optimum growth and shoot proliferation with phytohormone (0.1 mg/L BA). As a control, the culture without nanoparticles but the same concentration of BA (0.1 mg/L BA) was performed. Shoot explants of the same size for the control as well as the treatments were kept on MS medium. Each magenta jar contained 100 mL of MS medium, and four explants were kept to determine the reproducibility of the results. The cultures were grown in a culture room set at relative humidity 55%–60% and temperature 25°C–28°C under PPFD of 40 μmol m-2 s-1 for a 16 h photoperiod. The shoots were cultured for 45 days on MS medium to investigate growth parameters.
2.3.2. Biomass Determination
The fresh and dry weights of treated and untreated shoots were measured after 45 days of culture on MS medium. Each treatment was performed in triplicate to ensure reliability of the results.
2.3.3. Shoot Length and Number
Shoot length and number were measured after 45 days of culture on MS medium and were compared with the control and among the treated shoots.
2.3.4. Estimation of Total Chlorophyll
The method reported by Arnon  for estimating total chlorophyll was used. Fresh leaves (0.1 g) were washed in DW, chopped into small pieces, and extracted in DMSO. Incubation was performed at 65°C for 120 min, after which the samples were taken out and absorbance was measured immediately at 663 nm and 645 nm using a UV-Vis spectrophotometer. The calculated chlorophyll content was expressed as mg/g FW.
2.3.5. Proline Estimation
Fresh leaves (0.5 g) were used for the estimation of proline using the method developed by Hanson et al. . The fresh samples were ground in 10 mL of 3% aqueous sulfosalicylic acid. The samples were centrifuged for 15 min at , and the obtained supernatant (2 mL) was taken out and placed in another tube. Two milliliters of each acid ninhydrin and acetic acid was added to the above samples. The samples were incubated in boiling water for 1 h, and the reaction was terminated by putting the samples in an ice bath. Toluene (4 mL) was added to the above samples and vortexed. The aqueous phase was separated from chromatophore-containing toluene. The proline content was estimated in samples by measuring the absorbance of chromatophore-containing toluene at 520 nm (model UB-1800; Shimadzu, Japan).
2.3.6. Thiobarbituric Acid Reactive Substances (TBARS)
TBARS content of fresh leaves was estimated using the method of Cakmak and Horst . The leaf samples (0.5 g) were homogenized in 5 mL of trichloroacetic acid (TCA, 0.1% ()). The supernatant was collected at for TBARS estimation. Four milliliters of 0.5% () thiobarbituric acid (TBA) in 20% () TCA was added to 1 mL of supernatant taken from the above step and placed for 30 min at 90°C in a water bath. The reaction was stopped by placing the sample in an ice bath, followed by centrifugation at , after which the supernatant was collected. TBARS content was measured from the absorbance measured at wavelengths of 532 and 600 nm with a spectrophotometer:
Here, A532 represents absorbance at 532 nm, A600 represents absorbance at 600 nm, is extraction volume, and is fresh weight of tissue.
2.3.7. Catalase (EC 126.96.36.199)
The method developed by Aebi  was used to record the activity of catalase (CAT). A total of 0.25 g of fresh leaves was ground in phosphate buffer (0.5 M, pH 7.3) containing 0.3 mM EDTA, 1% Triton ×100 (), and 1% PVP (). The mixture was centrifuged, and the supernatant was collected for an enzymatic assay. The enzyme activity assay was performed in 2 mL of reaction buffer containing 0.1 mL of 3 mM EDTA, 0.1 mL of enzyme extract, and 0.1 mL of 3 mM H2O2 for 3 min. CAT was recorded at 240 nm using a UV-Vis spectrophotometer, with enzyme unit expressed in mg−1 protein min−1.
2.3.8. Superoxide Dismutase (EC 188.8.131.52)
The superoxide dismutase (SOD) activity was measured using the method reported by Dhindsa et al. . The fresh leaf samples (0.25 g) were ground in 2 mL of phosphate buffer containing 1% Triton ×100 (), 0.3 mM EDTA, and 1% PVP (). The sample was centrifuged at for 10 min, after which the supernatant was collected for assaying SOD activity. The enzyme assay was performed in 1.5 mL of reaction buffer containing 0.2 mL of methionine and 0.1 mL of each of 1 M NaCO3, 2.25 mM NBT solution, riboflavin, 3 mM EDTA, enzyme extract, and 1 mL of DDW incubated in the light. The blank was kept in the dark while containing all components as in the treated samples. The sample absorbance along with the blank was recorded at 560 nm using a UV-Vis spectrophotometer (Model UB-1800; Shimadzu, Japan). A 50% reduction in color was considered one enzyme unit (EU), and activity of SOD was calculated in mg−1 protein min−1.
2.3.9. Ascorbate Peroxidase (EC 184.108.40.206)
The ascorbate peroxidase (APX) activity was checked using the method of Nakano and Asada . Fresh leaves (0.25 g) were ground in 1 mL of extraction buffer (50 mM phosphate buffer), 1% Triton ×100 (), 1% PVP (), and 0.3 mM EDTA. The supernatant was obtained after centrifugation for assaying enzyme activity at 290 nm using a UV-Vis spectrophotometer. The extinction coefficient () 2.8 mM-1 cm-1 was used for calculating APX activity and expressed as EU mg-1 protein min-1.
2.3.10. Glutathione Reductase (EC 220.127.116.11)
The glutathione reductase (GR) activity was measured in fresh shoots at a wavelength of 340 nm following the protocol of Rao . The fresh leaves (0.25 g) were ground in extraction buffer, and the supernatant was collected after centrifugation for the enzymatic assay. The reaction mixture (1 mL) contained 0.05 mL of each of 0.2 mM NADPH and 0.5 mM GSSG and 0.1 mL of enzyme extract. The GR activity was calculated using the molar absorptivity constant of NADPH (6.2 mM-1 cm-1) and expressed as EU mg-1 protein min-1.
2.4. Statistical Analysis
The experimental design was based on triplicates, repeated twice, and was conducted under controlled conditions. ANOVA (post hoc Duncan) was used to test the experimental data using Origin Pro software (v.8.5). All values are presented as the .
3.1. Characterization of Phytomediated ZnONPs
Fresh shoots with leaves (45 days old) raised on MS medium  were used for the preparation of ZnONPs, as shown in Figure 1. The optical absorption spectrum of the synthesized ZnONPs was recorded in the range of 200–800 nm using a UV-Vis spectrophotometer (Shimadzu). The UV spectrum was taken at different time intervals to evaluate the synthesis of nanoparticles. The white precipitation in the suspension indicated the synthesis of ZnONPs, and a further sharp peak of the UV spectrum at 280 nm showed the pure synthesis of ZnONPs (Figure 2(a)). The sharp peak indicated the synthesis of nanoparticles with a nanoscale size and distribution . The powder of ZnONPs was used for obtaining Fourier transform infrared (FTIR) spectra (as pellets in KBr) in the range of 4000–400 cm−1 with a resolution of 1 cm−1 (Figure 2(b)). The FTIR results revealed the presence of functional groups in the shoot extract, which are responsible for the reduction and stabilization of zinc oxide nanoparticles. FTIR spectra obtained for ZnONPs showed shifts and a number of peaks, which indicated their complex nature.
An absorption band was identified as a broad and intense peak at 3449.21 cm-1, resulting from stretching and vibration mode of O-H bond . The peak at 2346.56 cm-1 corresponded to CO2 that was adsorbed on the surface of the sample . The peak at 1633.25 cm-1 was assigned to a β-sheet, as a secondary structure of the protein . The peak at 1061.53 cm-1 was attributed to stretching of the carbonyl functional groups in carboxylic acids, ketones, and aldehydes . The band at 452.77 cm-1 was assigned to the stretching vibration mode of the Zn-O bond in ZnO nanoparticles. These results confirmed the presence of functional groups in the shoot extract of O. arabicus which are responsible for reduction and stabilization of synthesized zinc oxide nanoparticles.
The average zeta potential value of ZnONPs was observed to be −14.5 mV with conductivity of 0.0148 mS/cm (Figure 2(c)), which indicated their stability. From the TEM micrograph of ZnONPs, they were observed to be in the size range of 16.56–19.65 nm (Figure 2(d)).
3.2. Measurement of Morphometric Traits and Biomass
Morphometric traits including shoot length and number varied at different concentrations of ZnONPs, and some of them were found to be better than control as well as other shoot treatments (Figure 3). The highest biomass at 5 and 10 mg/L ZnONPs and shoot length at 10 and 25 mg/L ZnONPs were observed upon comparison with the control as well as other NP-treated shoots (Figures 4 and 5). As the concentrations of phytomediated ZnONPs increased in MS medium (50, 100, and 300 mg/L), the shoot length was affected more than the shoots treated with low concentrations of ZnONPs (5 and 10 mg/L) and the control shoots. The shoot number was higher significantly than the control with ZnONP concentrations 10, 25, 50, 100, and 300 mg/L as compared to control. Among all treatments, the shoot number was found to be highest at 50 mg/L ZnONPs (Figure 6).
3.3. Biochemical Parameters
Chlorophyll content decreased at ZnONP concentrations, 25, 50, 100, and 300 mg/L, in a concentration-dependent manner (Figure 7). The level of chlorophyll content was increased with 5 and 10 mg/L of ZnONPs as compared to control and other shoot treatments. The levels of biochemical markers including proline and TBARS were found to be highest at 100 and 300 mg/L ZnONPs among the treatments and control (Figures 8 and 9). Both marker levels were increased as the concentration of ZnONPs increased in MS medium as compared to control. The antioxidant enzyme activities including CAT, SOD, APX, and GR were found to be higher with 300 mg/L ZnONPs than for the control (Figure 10) and other treatments. APX activity was found to be very low at low concentrations of ZnONPs (5, 10, and 25 mg/L), showing no significant difference compared with the control.
The application of an optimal nanoparticle concentration is important to improve the growth and development of plants, as negative and/or positive effects might be observed at various concentrations. The optimal concentration of NPs induces growth and increases biomass in treated plants, as reported by many researchers. The impact of NPs also depends on the plant species, seed size, growth medium, growth stage, and material used for coating of nanoparticles . Nanoparticles have been found to induce pronounced variations in the physiological indices in plants, such as percentage germination, root biomass, leaf number, and elongation . Nanoparticles affect many factors, such as seed germination, biomass production, root growth, shoot growth, and biochemical and physiological activities .
In the current study, different concentrations of phytomediated ZnONPs were applied on regenerated shoots of O. arabicus cultured on MS medium. All culture conditions and the size of the explants were kept the same for the ZnONP-treated and control shoots. Cytokinin (BA) was used at a concentration of 0.1 mg/L on MS medium with ZnONPs, instead of BA (1 mg/L) used for shoot multiplication without NPs. Fresh weight and dry weight at 5 and 10 mg/L ZnONPs and shoot length at 10 and 25 mg/L ZnONPs were found to be higher significantly than that in the control (Figures 4 and 5). Our results are supported by a previous study  in which a low concentration of ZnONPs (1.5 ppm) produced the maximal promoting effect on shoot dry weight in Cicer eritenum. Our results are also supported by Zafar et al. , who found that 10 mg/L ZnONPs proved very effective for shoot emergence on MS medium in Brassica nigra. The application of 10 mg/L ZnONPs was also shown to induce callus and shoot regeneration in rapeseed  and plantlet growth in Linum usitatissimum . The shoot proliferation rate in date palm was also found to be higher from buds cultured with ZnONPs on MS medium . Moreover, the application of ZnONPs at 10 ppm on roots was shown to help to enhance the growth and yield of tomato plants . ZnONPs promoted the shoot and root lengths and biomass in Vigna radiata .
A low concentration of nanoparticles improves growth and development in plants in vivo as well as in vitro, as reported above. The number of shoots was significantly increased in O. arabicus compared with control with ZnONP concentrations 10, 25, 50, 100, and 300 mg/L. The highest number of shoots was recorded with 50 mg/L ZnONPs, which was significantly different compared with all treatments (Figure 6). However, shoot length decreased in a concentration-dependent manner at 50–300 mg/L ZnONPs. Similarly, a high concentration of metal oxide nanoparticles caused phytotoxicity in plants, as reported by other researchers [42–44]. As the concentration of ZnONPs increased in MS medium, the biomass accumulation decreased, which might have been due to the production of reactive oxygen species. Our results are supported by the work of Zafar et al. , who showed that high concentrations of ZnONPs (500 to 1500 mg/L) adversely affected Brassica nigra seedling growth and seed germination and also led to increases in nonenzymatic antioxidants and antioxidative activities. The suspensions of zinc oxide nanoparticles at concentrations of 200–1000 mg/L affected seed germination in Triticum aestivum and decreased the level of photosynthetic pigments .
The photosynthetic rate depends on the chlorophyll content in the chloroplast, which plays an important role in plant growth and development. The chlorophyll content increased in shoots of O. arabicus treated with 5 and 10 mg/L ZnONPs compared with the level in the control; thereafter, it significantly decreased in a concentration-dependent manner (Figure 7). Thus, enhanced chlorophyll content in O. arabicus leaves at a low concentration of ZnONPs (5 and 10 mg/L) might play an important role in various growth parameters, such as shoot length and biomass production. Zinc deficiency was also reported to decrease chlorophyll synthesis in Zea mays L. . Zinc plays important roles in many structural components of proteins and enzymes as a cofactor for pigment biosynthesis . For example, in Arabidopsis thaliana (L.) Heynh, seedling growth, chlorophyll content, and rate of photosynthesis were reduced upon exposure to a high concentration of ZnONPs . Moreover, ZnONPs were shown to inhibit the expression of genes involved in chlorophyll synthesis and photosystem structure . An exposure of Solanum melongena to different concentrations of nanoparticles, namely, NiO, CuO, and ZnO, decreased its chlorophyll content .
Proline is an osmolyte that protects plant cells from biotic and abiotic stresses. It stabilizes subcellular structures in the cell and scavenges free radicals under various stress conditions . The level of proline in O. arabicus changed with increasing ZnONP concentration on MS medium, compared with that in the control. The highest proline content was found to be 746.74 μg/g FW in shoots treated with 100 mg/L ZnONPs, which was significantly higher than that in the other treatments as well as control (Figure 8). Helaly et al.  performed an experiment on banana regeneration in which proline content increased in the callus and shoots upon exposure to nanozinc and ZnONPs. Similarly, proline was increased in tomato plants upon exposure to ZnONPs at 8 mg/L, compared with the level in control plants . Moreover, foliar treatment with various concentrations of nano-ZnO or ZnO to flax plants gradually increased proline to a level significantly higher than that in the control [53, 54]. Saradhi and Mohanty  reported that proline protects cells against free radical- and singlet oxygen-induced damage resulting from excess reactive oxygen species (ROS).
Thiobarbituric acid reactive substances (TBARS) are produced as byproducts of lipid peroxidation, resulting from oxidative stress, and can be measured by the TBARS assay using thiobarbituric acid as a reagent . The TBARS content increased in all shoots treated with ZnONPs compared with the level in the control, in a concentration-dependent manner. The highest TBARS (12.063 nM/g FW) accumulation was found to occur with 300 mg/L ZnONPs, which was higher than in the other treatments and the control (Figure 9). However, no significant difference was found in TBARS accumulation in shoots of O. arabicus upon the application of 5 mg/L ZnONPs compared with the control. Our results are in line with the findings of Kumari et al. , who performed an experiment on Allium cepa with ZnONPs (nonbiogenic) and noted the accumulation of TBARS in a concentration-dependent manner.
Antioxidant defense systems play potential roles in plant protection under various stresses. The scavenging of ROS is necessary to avoid potential oxidative damage, for which various mechanisms play important roles in the cell. Enzymatic and/or nonenzymatic mechanisms have been shown to be involved in the antioxidant defense systems of plants [56, 57]. The activity of antioxidant enzymes was shown to be increased in in vitro shoot culture of Arabidopsis thaliana  upon the application of NPs. Moreover, the enzyme activities of CAT and SOD significantly increased in shoots of O. arabicus compared with those in the control at all concentrations of ZnONPs. The changes in activity of CAT and SOD were observed to be nonsignificant between the treatments at 10 and 25 mg/L ZnONPs; however, they remained significantly higher than that in the control. The activity of APX was observed to be very low in the shoots treated with 5, 10, and 25 mg/L ZnONPs, showing no significant differences compared with the control. In the same way, changes in GR activity were found to be very low upon treatment with 5 and 10 mg/L ZnONPs, showing no significant differences compared with the control. This might have been due to the involvement of ZnONPs in various metabolic activities of the cell, and application of low concentration of it played an important role in cell protection from ROS and induced growth and biomass accumulation. Several studies have reported that ZnONPs induced positive effects on the growth and development of crop plant species. ZnONPs have been widely used because zinc is an essential micronutrient and participates in various metabolic reactions . Similarly, a low concentration of ZnONPs reduced the adverse effects generated by Cd in Lycopersicon esculentum and increased the activities of nitrate reductase and carbonic anhydrase, as well as protein content, compared with the levels in the control in both unstressed and stressed plants . It has also been reported that the toxicity of ZnONPs on crop plants is much lower than that of bulk ZnO or Zn2+ particles .
The CAT, SOD, APX, and GR activities increased in a concentration-dependent manner with 50, 100, and 300 mg/L ZnONPs. The highest enzyme activities were observed in the shoots treated with 300 mg/L ZnONPs, with CAT, SOD, APX, and GR showing activities of 12.857, 2.132, 1.332, and 0.436 U/mg/min protein, respectively (Figure 10). The increased levels of enzymes at a high concentration of ZnONPs may counteract oxidative stress . SOD plays an important role in this as it catalyzes the dismutation of superoxide anion to H2O2 , and its increased activity may protect plants against oxidative damage [62, 63]. Our results are supported by the work of Zaeem et al. , who showed that superoxide dismutase activities were higher in shoots of Linum usitatissimum treated with 500 mg/L ZnONPs. Our results are also supported by Faizan et al. , who found that the activities of POX, CAT, and SOD increased upon the application of ZnONPs (at 8 mg/L) at 45 days after sowing as compared with the levels in control plants. Application of ZnO-NPs to As-stressed soybean plants resulted in the increase of SOD, catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) and improved the growth of the plants . Similarly, foliar spray of ZnO-NPs mitigates the negative impact of salt stress as the level of dismutase (SOD) and catalase (CAT) was increased and improved the growth of the Lycopersicon esculentum Mill. . ZnONPs protect the photosynthetic apparatus in plants by enhancing the activities of antioxidative enzymes as well as increasing biochemical markers. ZnONPs added to MS medium induced SOD, CAT, and POX activity in banana and enhanced its tolerance to biotic stress .
Zinc oxide nanoparticles have become one of the most important metal oxide NPs in biological applications due to their numerous benefits. In the present study, synthesized phytomediated nanoparticles from O. arabicus and their application on the shoot of O. arabicus at a low concentration proved to be very effective for promoting the proliferation of multiple shoots and the production of high biomass. Moreover, low concentrations of ZnONPs with reduced cytokinin concentration promoted shoot proliferation in O. arabicus. However, high concentrations of ZnONPs caused toxicity, as revealed by analyses of biochemical and morphological markers which might be associated with greater production of ROS. Low concentrations of ZnONPs on in vitro raised shoots of O. arabicus could be used to boost the production of various secondary metabolites as plants have substantial medicinal value to cure various diseases. Thus, phytomediated synthesized nanoparticles are environmentally safe and could be used in vivo as well as in vitro for crops as well as medicinal plants for the improvement of their yield and quality traits.
|TBARS:||Thiobarbituric acid reactive substances|
|MS:||Murashige and Skoog|
|FTIR:||Fourier transform infrared|
|TEM:||Transmission electron micrograph|
|NBT:||Nitro blue tetrazolium chloride|
|NADPH:||Nicotinamide adenine dinucleotide hydrogen phosphate|
Data is available on request.
Conflicts of Interest
The authors declare no conflicts of interest.
The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education Kingdom of Saudi Arabia, for funding this research work through the project number IFKSURG-014.
F. Rahmani, A. Peymani, E. Daneshvand, and P. Biparva, “Impact of zinc oxide and copper oxide nano-particles on physiological and molecular processes in Brassica napus L,” Indian Journal of Plant Physiology, vol. 21, no. 2, pp. 122–128, 2016.View at: Publisher Site | Google Scholar
M. B. Homaee and A. A. Ehsanpour, “Physiological and biochemical responses of potato (Solanum tuberosum) to silver nanoparticles and silver nitrate treatments under in vitro conditions,” Indian Journal of Plant Physiology, vol. 20, no. 4, pp. 353–359, 2015.View at: Publisher Site | Google Scholar
L. R. Khot, S. Sankaran, J. M. Maja, R. Ehsani, and E. W. Schuster, “Applications of nanomaterials in agricultural production and crop protection: a review,” Crop Protection, vol. 35, pp. 64–70, 2012.View at: Publisher Site | Google Scholar
A. Hussain, S. Ali, M. Rizwan et al., “Zinc oxide nanoparticles alter the wheat physiological response and reduce the cadmium uptake by plants,” Environmental Pollution, vol. 242, no. Part B, pp. 1518–1526, 2018.View at: Publisher Site | Google Scholar
M. Rizwan, S. Ali, M. Zia ur Rehman et al., “Alleviation of cadmium accumulation in maize (_Zea mays_ L.) by foliar spray of zinc oxide nanoparticles and biochar to contaminated soil,” Environmental Pollution, vol. 248, pp. 358–367, 2019.View at: Publisher Site | Google Scholar
R. Javed, M. Usman, B. Yücesan, M. Zia, and E. Gürel, “Effect of zinc oxide (ZnO) nanoparticles on physiology and steviol glycosides production in micropropagated shoots of _Stevia rebaudiana_ Bertoni,” Plant Physiology and Biochemistry, vol. 110, pp. 94–99, 2017.View at: Publisher Site | Google Scholar
R. Javed, B. Yucesan, M. Zia, and E. Gurel, “Elicitation of secondary metabolites in callus cultures of Stevia rebaudiana Bertoni grown under ZnO and CuO nanoparticles stress,” Sugar Tech, vol. 20, no. 2, pp. 194–201, 2018.View at: Publisher Site | Google Scholar
A. Singh, Mineral nutrient requirement, their disorders and remedies in groundnut, Groundnut Research in India, 2004.
I. Cakmak and T. R. No, “Tansley Review No. 111,” New Phytologist, vol. 146, no. 2, pp. 185–205, 2000.View at: Publisher Site | Google Scholar
D. S. Auld, “Zinc coordination sphere in biochemical zinc sites,” in Zinc biochemistry, physiology, and homeostasis, pp. 85–127, Springer, 2001.View at: Publisher Site | Google Scholar
M. R. Broadley, P. J. White, J. P. Hammond, I. Zelko, and A. Lux, “Zinc in plants,” New Phytologist, vol. 173, no. 4, pp. 677–702, 2007.View at: Publisher Site | Google Scholar
A. W. Peck and G. K. McDonald, “Adequate zinc nutrition alleviates the adverse effects of heat stress in bread wheat,” Plant and Soil, vol. 337, no. 1-2, pp. 355–374, 2010.View at: Publisher Site | Google Scholar
V. Tavallali, M. Rahemi, S. Eshghi, B. Kholdebarin, and A. Ramezanian, “Zinc alleviates salt stress and increases antioxidant enzyme activity in the leaves of pistachio (Pistacia vera L.‘Badami’) seedlings,” Turkish Journal of Agriculture and Forestry, vol. 34, no. 4, pp. 349–359, 2010.View at: Google Scholar
T. Prasad, P. Sudhakar, Y. Sreenivasulu et al., “Effect of nanoscale zinc oxide particles on the germination, growth and yield of peanut,” Journal of Plant Nutrition, vol. 35, no. 6, pp. 905–927, 2012.View at: Publisher Site | Google Scholar
H. Barakat, A. El-Mousallamy, A. Souleman, and S. Awadalla, “Flavonoids of _Ochradenus baccatus_,” Phytochemistry, vol. 30, no. 11, pp. 3777–3779, 1991.View at: Publisher Site | Google Scholar
J. Hussain, N. U. Rehman, A. L. Khan et al., “Phytochemical and biological assessment of medicinally important plant Ochradenus arabicus,” Pakistan Journal of Botany, vol. 46, no. 6, pp. 2027–2034, 2014.View at: Google Scholar
M. Nadeem, F. Al-Qurainy, S. Khan, M. Tarroum, and M. Ashraf, “Effect of some chemical treatments on seed germination and dormancy breaking in an important medicinal plant Ochradenus arabicus Chaudhary, Hill C. & AG Mill,” Pakistan Journal of Botany, vol. 44, no. 3, pp. 1037–1040, 2012.View at: Google Scholar
S. Khan, F. Al-Qurainy, M. Nadeem, and M. Tarroum, “Development of genetic markers for Ochradenus arabicus (Resedaceae), an endemic medicinal plant of Saudi Arabia,” Genetics and Molecular Research, vol. 11, no. 2, pp. 1300–1308, 2012.View at: Publisher Site | Google Scholar
T. Murashige and F. Skoog, “A revised medium for rapid growth and bio assays with tobacco tissue cultures,” Physiologia Plantarum, vol. 15, no. 3, pp. 473–497, 1962.View at: Publisher Site | Google Scholar
D. I. Arnon, “Copper enzymes in isolated CHLOROPLASTS. POLYPHENOLOXIDASE INBETA VULGARIS,” Polyphenoloxidase in Beta vulgaris. Plant Physiology, vol. 24, no. 1, pp. 1–15, 1949.View at: Publisher Site | Google Scholar
A. Hanson, C. Nelsen, A. Pedersen, and E. Everson, “Capacity for proline accumulation during water stress in barley and its implications for breeding for drought resistance 1,” Crop Science, vol. 19, no. 4, pp. 489–493, 1979.View at: Publisher Site | Google Scholar
I. Cakmak and W. J. Horst, “Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max),” Physiologia Plantarum, vol. 83, no. 3, pp. 463–468, 1991.View at: Publisher Site | Google Scholar
H. Aebi, “ Catalase _in vitro_,” Methods in Enzymology, vol. 105, pp. 121–126, 1984.View at: Publisher Site | Google Scholar
R. S. Dhindsa, P. Plumb-Dhindsa, and T. A. Thorpe, “Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase,” Journal of Experimental Botany, vol. 32, no. 1, pp. 93–101, 1981.View at: Publisher Site | Google Scholar
Y. Nakano and K. Asada, “Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts,” Plant and Cell Physiology, vol. 22, no. 5, pp. 867–880, 1981.View at: Google Scholar
M. Rao, “Cellular detoxifying mechanisms determine the age dependent injury in tropical trees exposed to SO2,” Journal of Plant Physiology, vol. 140, no. 6, pp. 733–740, 1992.View at: Publisher Site | Google Scholar
A. K. Zak, R. Razali, W. H. Abd Majid, and M. Darroudi, “Synthesis and characterization of a narrow size distribution of zinc oxide nanoparticles,” International Journal of Nanomedicine, vol. 6, p. 1399, 2011.View at: Publisher Site | Google Scholar
M. Zare, K. Namratha, S. Alghamdi et al., “Novel green biomimetic approach for synthesis of ZnO-Ag nanocomposite; antimicrobial activity against food-borne pathogen, biocompatibility and solar photocatalysis,” Scientific Reports, vol. 9, no. 1, pp. 1–15, 2019.View at: Publisher Site | Google Scholar
S. H. Largani and M. A. Pasha, “The effect of concentration ratio and type of functional group on synthesis of CNT–ZnO hybrid nanomaterial by an in situ sol–gel process,” International Nano Letters, vol. 7, no. 1, pp. 25–33, 2017.View at: Publisher Site | Google Scholar
S. Suresh, S. Karthikeyan, and K. Jayamoorthy, “FTIR and multivariate analysis to study the effect of bulk and nano copper oxide on peanut plant leaves,” Journal of Science: Advanced Materials and Devices, vol. 1, no. 3, pp. 343–350, 2016.View at: Publisher Site | Google Scholar
S. O. Ogunyemi, Y. Abdallah, M. Zhang et al., “Green synthesis of zinc oxide nanoparticles using different plant extracts and their antibacterial activity againstXanthomonas oryzaepv. oryzae,” Artificial Cells, Nanomedicine, and Biotechnology, vol. 47, no. 1, pp. 341–352, 2019.View at: Publisher Site | Google Scholar
J. Yang, W. Cao, and Y. Rui, “Interactions between nanoparticles and plants: phytotoxicity and defense mechanisms,” Journal of Plant Interactions, vol. 12, no. 1, pp. 158–169, 2017.View at: Publisher Site | Google Scholar
C. W. Lee, S. Mahendra, K. Zodrow et al., “Developmental phytotoxicity of metal oxide nanoparticles to Arabidopsis thaliana,” Environmental Toxicology and Chemistry: An International Journal, vol. 29, no. 3, pp. 669–675, 2010.View at: Publisher Site | Google Scholar
K. S. Siddiqi and A. Husen, “Plant response to engineered metal oxide nanoparticles,” Nanoscale Research Letters, vol. 12, no. 1, pp. 1–18, 2017.View at: Publisher Site | Google Scholar
U. Burman, M. Saini, and P. Kumar, “Effect of zinc oxide nanoparticles on growth and antioxidant system of chickpea seedlings,” Toxicological and Environmental Chemistry, vol. 95, no. 4, pp. 605–612, 2013.View at: Publisher Site | Google Scholar
H. Zafar, A. Ali, J. S. Ali, I. U. Haq, and M. Zia, “Effect of ZnO nanoparticles on Brassica nigra seedlings and stem explants: growth dynamics and antioxidative response,” Frontiers in Plant Science, vol. 7, p. 535, 2016.View at: Publisher Site | Google Scholar
S. Mousavi Kouhi and M. Lahouti, “Application of ZnO nanoparticles for inducing callus in tissue culture of rapeseed,” International Journal of Nanoscience and Nanotechnology, vol. 14, no. 2, pp. 133–141, 2018.View at: Google Scholar
A. Zaeem, S. Drouet, S. Anjum et al., “Effects of biogenic zinc oxide nanoparticles on growth and oxidative stress response in flax seedlings vs. in vitro cultures: a comparative analysis,” Biomolecules, vol. 10, no. 6, p. 918, 2020.View at: Publisher Site | Google Scholar
K. M. Awad, A. M. Al-Mayahi, M. A. Mahdi, A. S. Al-Asadi, and M. H. Abass, “In vitro assessment of ZnO nanoparticles on Phoenix dactylifera L. micropropagation,” Scientific Journal of King Faisal University, vol. 21, no. 1, 2020.View at: Publisher Site | Google Scholar
M. Faizan, A. Faraz, and S. Hayat, “Effective use of zinc oxide nanoparticles through root dipping on the performance of growth, quality, photosynthesis and antioxidant system in tomato,” Journal of Plant Biochemistry and Biotechnology, vol. 29, no. 3, pp. 553–567, 2020.View at: Publisher Site | Google Scholar
P. Mahajan, S. Dhoke, and A. Khanna, “Effect of nano-ZnO particle suspension on growth of mung (Vigna radiata) and gram (Cicer arietinum) seedlings using plant agar method,” Journal of Nanotechnology, vol. 2011, Article ID 696535, 7 pages, 2011.View at: Publisher Site | Google Scholar
C. Ma, H. Liu, H. Guo et al., “Defense mechanisms and nutrient displacement in Arabidopsis thaliana upon exposure to CeO2and In2O3nanoparticles,” Environmental Science: Nano, vol. 3, no. 6, pp. 1369–1379, 2016.View at: Publisher Site | Google Scholar
N. A. Anjum, V. Adam, R. Kizek et al., “Nanoscale copper in the soil-plant system - toxicity and underlying potential mechanisms,” Environmental Research, vol. 138, pp. 306–325, 2015.View at: Publisher Site | Google Scholar
M. E. Letelier, S. Sánchez-Jofré, L. Peredo-Silva, J. Cortés-Troncoso, and P. Aracena-Parks, “Mechanisms underlying iron and copper ions toxicity in biological systems: pro-oxidant activity and protein-binding effects,” Chemico-Biological Interactions, vol. 188, no. 1, pp. 220–227, 2010.View at: Publisher Site | Google Scholar
A. N. U. Haq, A. Islam, F. Younas et al., “Impact of zinc oxide nanoflowers on growth dynamics and physio-biochemical response ofTriticum aestivum,” Toxicological and Environmental Chemistry, vol. 102, no. 10, pp. 568–584, 2020.View at: Publisher Site | Google Scholar
T. Hisamitsu, O. Ryuichi, and Y. Hidenobu, “Effect of zinc concentration in the solution culture on the growth and content of chlorophyll, zinc and nitrogen in corn plants (Zea mays L),” Journal of Tropical Agriculture, vol. 36, no. 1, pp. 58–66, 2001.View at: Google Scholar
P. Balashouri and Y. Prameeladevi, “Effect of zinc on germination, growth and pigment content and phytomass of Vigna radiata and Sorghum bicolor,” Journal of Ecobiology, vol. 7, pp. 109–114, 1995.View at: Google Scholar
X. Wang, X. Yang, S. Chen et al., “Zinc oxide nanoparticles affect biomass accumulation and photosynthesis in Arabidopsis,” Frontiers in Plant Science, vol. 6, p. 1243, 2016.View at: Publisher Site | Google Scholar
V. Baskar, S. Nayeem, S. P. Kuppuraj, T. Muthu, and S. Ramalingam, “Assessment of the effects of metal oxide nanoparticles on the growth, physiology and metabolic responses in in vitro grown eggplant (Solanum melongena),” Biotech, vol. 8, no. 8, pp. 1–12, 2018.View at: Publisher Site | Google Scholar
S. Hayat, Q. Hayat, M. N. Alyemeni, A. S. Wani, J. Pichtel, and A. Ahmad, “Role of proline under changing environments a review,” Plant Signaling & Behavior, vol. 7, no. 11, pp. 1456–1466, 2012.View at: Publisher Site | Google Scholar
M. N. Helaly, M. A. El-Metwally, H. El-Hoseiny, S. A. Omar, and N. I. El-Sheery, “Effect of nanoparticles on biological contamination of'in vitro'cultures and organogenic regeneration of banana,” Australian Journal of Crop Science, vol. 8, no. 4, pp. 612–624, 2014.View at: Google Scholar
M. Faizan, A. Faraz, M. Yusuf, S. Khan, and S. Hayat, “Zinc oxide nanoparticle-mediated changes in photosynthetic efficiency and antioxidant system of tomato plants,” Photosynthetica, vol. 56, no. 2, pp. 678–686, 2018.View at: Publisher Site | Google Scholar
M. S. Sadak, A. R. Abd el-Hameid, F. S. A. Zaki, M. G. Dawood, and M. E. el-Awadi, “Physiological and biochemical responses of soybean (Glycine max L.) to cysteine application under sea salt stress,” Bulletin of the National Research Centre, vol. 44, no. 1, pp. 1–12, 2020.View at: Publisher Site | Google Scholar
P. P. Saradhi and P. Mohanty, “Involvement of proline in protecting thylakoid membranes against free radical- induced photodamage,” Journal of Photochemistry and Photobiology B: Biology, vol. 38, no. 2-3, pp. 253–257, 1997.View at: Publisher Site | Google Scholar
M. Kumari, S. S. Khan, S. Pakrashi, A. Mukherjee, and N. Chandrasekaran, “Cytogenetic and genotoxic effects of zinc oxide nanoparticles on root cells of _Allium cepa_,” Journal of Hazardous Materials, vol. 190, no. 1-3, pp. 613–621, 2011.View at: Publisher Site | Google Scholar
I. Štolfa, T. Ž. Pfeiffer, D. Špoljarić, T. Teklić, and Z. Lončarić, “Heavy metal-induced oxidative stress in plants: response of the antioxidative system,” in In Reactive Oxygen Species and Oxidative Damage in Plants Under Stress, pp. 127–163, Springer, 2015.View at: Google Scholar
S. Dutta, M. Mitra, P. Agarwal et al., “Oxidative and genotoxic damages in plants in response to heavy metal stress and maintenance of genome stability,” Plant Signaling and Behavior, vol. 13, no. 8, article e1460048, 2018.View at: Publisher Site | Google Scholar
B. Hafeez, Y. Khanif, and M. Saleem, “Role of zinc in plant nutrition-a review,” Journal of Experimental Agriculture International, vol. 3, no. 2, pp. 374–391, 2013.View at: Publisher Site | Google Scholar
S. M. M. Kouhi, M. Lahouti, A. Ganjeali, and M. H. Entezari, “Comparative effects of ZnO nanoparticles, ZnO bulk particles, and Zn 2+ on Brassica napus after long-term exposure: changes in growth, biochemical compounds, antioxidant enzyme activities, and Zn bioaccumulation,” Water, Air, and Soil Pollution, vol. 226, no. 11, pp. 1–11, 2015.View at: Publisher Site | Google Scholar
T. T. Chanu and H. Upadhyaya, “Zinc Oxide Nanoparticle-Induced Responses on Plants: A Physiological Perspective,” in Algae and Microorganisms, pp. 43–64, Elsevier, 2019.View at: Publisher Site | Google Scholar
S. Goswami and S. Das, “Copper phytoremediation potential of _Calandula officinalis_ L. and the role of antioxidant enzymes in metal tolerance,” Ecotoxicology and Environmental Safety, vol. 126, pp. 211–218, 2016.View at: Publisher Site | Google Scholar
Z. Wang, Y. Zhang, Z. Huang, and L. Huang, “Antioxidative response of metal-accumulator and non-accumulator plants under cadmium stress,” Plant and Soil, vol. 310, no. 1-2, pp. 137–149, 2008.View at: Publisher Site | Google Scholar
Y. Gao, C. Miao, L. Mao, P. Zhou, Z. Jin, and W. Shi, “Improvement of phytoextraction and antioxidative defense in _Solanum nigrum_ L. under cadmium stress by application of cadmium-resistant strain and citric acid,” Journal of Hazardous Materials, vol. 181, no. 1-3, pp. 771–777, 2010.View at: Publisher Site | Google Scholar
P. Ahmad, M. N. Alyemeni, A. A. Al-Huqail et al., “Zinc oxide nanoparticles application alleviates arsenic (as) toxicity in soybean plants by restricting the uptake of as and modulating key biochemical attributes, antioxidant Enzymes, Ascorbate-Glutathione Cycle and Glyoxalase System,” Plants (Basel), vol. 9, no. 7, p. 825, 2020.View at: Publisher Site | Google Scholar
M. Faizan, J. A. Bhat, C. Chen et al., “Zinc oxide nanoparticles (ZnO-NPs) induce salt tolerance by improving the antioxidant system and photosynthetic machinery in tomato,” Plant Physiology and Biochemistry, vol. 161, pp. 122–130, 2021.View at: Publisher Site | Google Scholar