2. Development and Findings
Characterization of synthesized ZnO NPs is commonly performed using UV–Visible spectroscopy, XRD, FTIR spectroscopy, and TEM microscopy. These techniques provide the information on the formation, size, structure, and elemental composition of nanoparticles. Optical properties of nanosized particles is being commonly assessed by UV–visible absorption spectroscopy
[22]. The absorption peak (380 nm) found in the present study obviously demonstrates the presence of ZnO NPs in the reaction mixture and it is in agreement with the earlier results of
[23][24]. The FTIR results demonstrated a band around 3400 cm
−1, potentially resulting from OH stretching vibrations; meanwhile, the peak at 2078 cm
−1 for C=H suggests a strong stretch assigned to the alkyl methylene group. The peak at 1634 cm
−1, corresponding to amide I, appears to be caused by carbonyl stretching in proteins
[25]. The peak at 704 cm
−1 was for the C=H bond assigned to strong a mono-substituted aromatic benzene group
[26]. Bearing in mind the FWHM of the plane in (101), the crystalline size of the engineered ZnO NPs was recorded using Scherrer’s formula; and the average particle size of the sample was found to be 351 nm. XRD pattern analysis proved the characteristic hexagonal wurtzite crystalline structure of ZnO NPs, which is in line with the earlier result reported by
[27]. TEM was used to characterize the shape and size of ZnO NPs that were synthesized using
O. arabicus leaf extract. The TEM images clearly showed that the synthesized ZnO NPs were almost hexagonal in shape, with the average diameter of nanoparticles ranging from 10–50 nm approximately, in accordance with the earlier result reported by
[22]. In general, XRD is mostly used to estimate the particle size of nanoparticles, however TEM is the preferable technique for the measurement of nanoparticle size. The Scherrer formula for calculating particle size gives an average value of the entire particle responsible for diffraction,.while when using TEM, besides directly determining particle size, the morphology of the particles can also be noted
[28].
To determine the growth enhancing effects of ZnO NPs applied to the culture medium of
Maerua oblongifolia plant, morphological characteristics such as fresh weigh, dry weight, shoot length, shoot number, and leaf number were studied. Exposure of ZnO NPs to in vitro shoots of the
M.
oblongifolia significantly boosted the vegetative growth in the plant, including improving plant height, number of plants per pot, and plant biomass. This enhancement can most likely be attributed to the role of Zn in the production of tryptophan—the precursor of indole-3-acetic acid phytohormone
[29]. In addition, ZnO NPs can alter the phytohormone biosynthesis of cytokinins and gibberellins, which can drive to an expansion in the number of internodes per plant
[10]. Concentration at 5 mg L
−1 ZnO NPs achieved the greatest growth of all morphological attributes, while the growth was reduced at 10 and 20 mg L
−1. This decrease in growth it might be due to a higher concentration of nanoparticles reaching toxic levels in stem and leaves, which reduced the plant growth; in an earlier study high concentrations of ZnO NPs drastically affected the growth of tomato plants
[7]. The positive effect of ZnO NPs exposed to plants was also reported in wheat
[30], cotton
[26], cluster bean
[31], and ryegrass (
Lolium perenne) seedlings at 2 mg L
−1 [32]. Silver nanoparticles and other nanomaterials’ exposure have also been reported to enhance the growth of
M. oblongifolia [19]. Iron-based NPs improved the growth of maize
[33]. Contrary to our findings, ZnO NPs were reported as being toxic in several plants species such as
Allium cepa roots
[34], rice
[35], and wheat seedlings
[36]. These contradictions might be closely related to the chemical composition, chemical structure, particle size, and surface area of the NPs
[37][38]. The response of the photosynthetic pigments’ content (chl. a, chl. b and carotenoids) to ZnO NP treatment in the present study correlates with earlier reported findings
[39] in which chlorophyll and other photosynthetic pigments in cilantro (
Coriandrum sativum) were remarkably increased after application of ZnO NPs. Similar results were found in several plant species, according to
[16] and
[40], and the exposure of the ZnO NPs improved the photosynthetic pigments and protein in
Phaseolus vulgaris and
Lupinus termis. The reason behind the enhanced chlorophyll and other photosynthetic content in our study is most likely due to the presence of zinc as a vital nutrient for the plants, essential for protochlorophyllide formation. Zn plays an essential role in plant metabolism by influencing the activities of key important enzymes, such as carbonic anhydrase
[39]. In addition, metal nanoparticles are powerful amplifiers of photosynthetic effectiveness that in parallel cause light absorption by chlorophyll, as they cause the transfer of energy from chlorophyll to nanoparticles
[41][42]. Carotenoids act as antioxidant compounds soluble in plant cells. These compounds through a non-enzymatic pathway function to reduce oxidative damage to the plant. Carotenoids represent a vital class of antioxidant molecules, which are known to scavenge harmful free radicals, as well as protecting light-harvesting complex proteins and thylakoid membrane stability. In this study, it seems a certain amount of zinc induced oxidative stress increased carotenoid content synthesis
[43]. Conversely to our findings, there are some studies which reported that ZnO NPs decrease the chlorophyll content in some plants species, such as kidney bean (
Phaseolus vulgaris) and soybean (
Glycine max)
[44][45]. This ambiguity may be because some plant species may have different responses to Zn exposure than others.
Application of ZnO NPs at the concentrations in our study caused an increase in total protein content compared to control: 5 mg L
−1 concentration recorded the highest increase in protein, which may suggest the initiation of de novo synthesis of the enzymes
[46]. On the other hand, protein level started decreasing at doses of 10 and 20 mg L
−1 respectively. The increase in protein at certain concentrations indicates the optimum dose limit for the growth of
M. oblongifolia plants. However, the reduction in protein beyond this concentration indicates the toxic effect of ZnO NPs. Similar results have been reported by a study on lettuce
[47].
Lipids are a key components of cell membranes. They are sensitive to oxidation processes, and generating lipid peroxides is an indicator of an increase in production of toxic oxygen species
[48]. MDA content is considered an indicator of oxidative damage. Our findings showed that MDA was diminished in ZnO NP treated plants. It is known that Zn has the ability to stabilize and protect the biomembranes against peroxidative and oxidative stress, integrity of plasma membrane loss, and change in the permeability of plasma membrane
[49]. Our results at in line with the findings of previous reports in
Vicia faba [50], soybean
[17], and wheat
[30].
The process of oxidative damage is due to the imbalance process of reactive oxygen species (ROS) metabolism in plants. Antioxidant enzymes, such as SOD, CAT, and GR, are known to be the major protective factors protecting against ROS in plants, through which plants can scavenge H
2O
2 (hydrogen peroxide) and O
2− (superoxide radical) and other ROSs
[51][52][53]. The activity of the antioxidant enzymes is significantly enhanced in plants upon exposure to ZnO NPs in a concentration dependent manner. In this study, SOD, CAT, and GR activities of
M. oblongifolia plants exposed to ZnO NPS significantly increased with increasing of the doses. Boosted activity of antioxidant enzymes by supplementation of ZnO NPs may scavenge H
2O
2 and mitigate mineral uptake, reducing plant oxidative stress
[54]. In addition, increasing CAT, SOD, and GR activity, notably with high concentrations of ZnO NPs, probably signals that the antioxidant enzyme system is adapting to counteract excessive production of ROS
[55]. Our findings are consistent with the earlier studies documenting that the SOD, CAT, and GR activities were increased with increasing concentrations of ZnO NPs in rice seed
[56]. Similarly, a previous trial documented that CAT, SOD, and GR activities were enhanced in
Arabidopsis thaliana seedlings treated with gold nanoparticles
[1]. Hence, enhanced antioxidant potential of
M. oblongifolia plants under ZNO NPs treatment signifies better performance. Lu et al.
[57] also observed that SOD, CAT, and GR activities of germinating seeds of soybean exposed to a mixture of nano-SiO
2 and nano-TiO
2 remarkably promoted seed germination and seedling growth. However, the antioxidant enzyme activities of SOD, CAT, and GR in nanoparticle exposed plants vary significantly according to the plant species, nanomaterial type, duration of treatment, and dose.