Impact of Biologically and Chemically Synthesized Zinc Oxide Nanoparticles on Seed Germination and Seedlings’ Growth

Abstract

Zinc oxide nanoparticles have gained attention in the last decades due to their versatile applications; in agriculture, they have been used for their multiple benefits. In this study, the effects of zinc oxide nanoparticles, obtained via chemical and biological synthesis and of 70–80 nm in size, on the germination and seedling growth of Capsicum annuum and Solanum lycopersicum seed were determined. The physiological parameters, photosynthetic pigments, the content of total phenols, total flavonoids, as well as the antioxidant capacity of 2,2-diphenyl-1-picrylhydrazyl (DPPH), and the enzymatic activity of catalase, peroxidase, and polyphenol oxidase were evaluated. The results indicated that treatment with zinc oxide nanoparticles obtained via green synthesis improved seed germination rate, while chemically synthesized nanoparticles at higher concentrations decreased germination percentage. In general, the parameters of enzymatic and nonenzymatic antioxidants in treated plants showed significant differences with respect to the control. However, the treatments with the bionanoparticles resulted in more beneficial parameters. Zinc oxide nanoparticles obtained via green synthesis are more effective in generating bioactive compounds and activating the enzyme defense system due to being more biocompatible.

1. Introduction

The use of nanoparticles in agriculture has increased due to their wide range of applications as nanofertilizers, nanocarriers, and nanosensors [1,2]. Nanoparticles (NPs) are particles with an average size of less than 100 nm. In agriculture, zinc oxide nanoparticles (ZnONPs) are used due to their catalytic, physical, nutritional, and antimicrobial properties. They have a positive effect on plants [3], such as chili [4], wheat [5], and other crops [6,7,8]. NPs, including ZnONPs, are used as a priming method to enhance seed germination and suppress dormancy [9,10,11,12]. Several studies suggest that ZnONPs strengthen the growth of different plant species [13] by providing Zn⁺² as a micronutrient. At the same time, ZnONPs can act as elicitors, which force the plant to raise its defense system. Thus, the presence of ZnONPs affects antioxidant enzymes such as superoxide dismutase, peroxidase, and catalase. Consequently, the plant produces bioactive compounds such as phenolic compounds, increasing plants’ antioxidant capacity [14]. However, the above benefits depend on the size, size distribution, and morphology of the zinc nanoparticles, which are related to the synthesis method through which NPs are prepared [15].

Zinc ion reduction reactions result in nanocrystalline zinc oxide nanoparticles (ZnONP). The syntheses use metal precursors, reducing agents, and often stabilizing molecules [16]. If the reaction is chemically based, there are drawbacks, such as the production of toxic byproducts [17] that are harmful to the environment. In addition, these reactions typically require high energy consumption and often have high costs associated with purchasing the chemicals [18]. Biological syntheses have emerged as a substitute and a solution for the problems related to chemical processes. Green syntheses use bio-extracts, such as plant or microbial extracts, during the reaction. The extracts contain secondary metabolites and antioxidant substances capable of reducing ions to nanoparticles, replacing toxic reagents [19,20,21]. With the use of bio-extracts, not only is there a reduction in the environmental impact, but various compounds can form a layer on the surface of the formed nanoparticle and, through that, improve the NPs’ characteristics and activity [22]. Thus, the NPs obtained via green synthesis are preferred over those that are traditionally synthesized [23].

Recent studies show that the use of ZnONPs obtained via green synthesis at concentrations of 10–100 mg L⁻¹ improved seed germination, the size of roots, shoots, and leaves, the number of roots, and the total fresh biomass in various plants: Triticum aestivum [24], Phaseolus vulgaris, Vigna angularis [25], and Hordeum vulgare [26]. In addition, ZnONPs obtained via green synthesis do not generate toxicity in concentrations of up to 100 mg L⁻¹ in tomato seeds [27]. Sharma et al. established that the concentration of ZnONPs at 20 mg L⁻¹ improved the germination rate, the vigor of the seedlings, the production of reactive oxygen species (ROS), and the metabolic process of starch [28].

On the other hand, Tymoczuk and Wojnarowicz tested the effects of chemically prepared submicron ZnO particles and ZnONPs on the in vitro germination and seedling growth of onion (Allium cepa L. ‘Sochaczewska’) seeds, showing that the germination was enhanced but unaffected by the size of the particles. Also, there was no significant effect on the size of young plants [29]. Biosynthesized nanoparticles can more easily penetrate the cover of the seeds due to the bio-coating on the surface; this could activate the enzymatic and nonenzymatic defense system, protecting the plant from oxidative damage, which causes increased growth in young plants [30]. However, there is no clear comparison between the NPs obtained traditionally (via chemical synthesis) and using biological methods.

Based on the above, the objective of this study was to compare the effects of zinc oxide nanoparticles of the same size, obtained using two different methods (biological synthesis and chemical synthesis), on physiological indices, photosynthetic pigments, bioactive compounds, and enzymatic activity in seeds and seedlings of Capsicum annuum and Solanum lycopersicum. We hypothesized that the biosynthesized nanoparticles are more effective in increasing physiological indices, photosynthetic pigments, bioactive compounds, and enzymatic activity in Capsicum annuum and Solanum lycopersicum seeds and seedlings compared to those obtained via chemical synthesis.

2. Materials and Methods

Zinc oxide nanoparticles obtained via green biological synthesis (BZnONP) had an average size of 70 nm, as reported in our previous work [21]. The BZnONPs were prepared using Larrea tridentata extract at 20 mg mL⁻¹, pH of 13, 70 °C, and four hours of reaction. Zinc oxide nanoparticles obtained via Carbomex^® physical/mechanical process (CZnONP) were purchased from Carbomex S.A. de CV (Independencia 632 Col. 16 de septiembre Sur. Puebla, México). CZnONPs have an average size of 80 nm and no coating on the surface; thus, throughout the manuscript, we refer to them as chemically synthesized. Both nanoparticle types had spherical morphology and homogeneous particle size distribution as determined by dynamic light scattering and scanning electron microscopy (provided by the producer of CZnONPs and determined in the previous investigation for BZnONPs) [21].

2.1. Seed Germination Test

The tests were carried out using seeds of the chili (Capsicum annuum) variety Jalapeño and tomato (Solanum lycopersicum) variety Saladette, from Rancho Los Molinos SA de CV (Cuernavaca-Tepoztlán, Morelos, México). For disinfection, all seeds were washed with 70% ethanol for 2 min, then washed thrice with distilled water. The germination tests were performed by placing 10 seeds in a Petri dish (diameter 8 cm) filled with filter paper, and the seeds were incubated with 5 mL of each prepared solution of ZnONPs.

Since our preliminary evaluation [21] showed no significant difference for the lowest ZnONPs concentration, the treatments applied here were 100, 200, 300, and 400 mg L⁻¹ of BZnONP or CZnONP. The control was seeds treated only with distilled water. Each trial was performed in triplicate (27 experimental units per seed type with 10 seeds/Petri dish) for a total of 30 seeds/seedlings per treatment. The Petri dishes were sealed with Parafilm and placed in a Novatech artificial growth chamber CA-550 (NOVATECH, San Pedro, Tlaquepaque, México) at a constant temperature of 26 °C and a 12 h day/night cycle for 15 days [31].

Physiological Parameters of Seedlings

After 7 days, 3 plants were taken randomly from each treatment to determine the physiological parameters. At 7 days, the following measurements were taken: seed vigor index, germination percentage (%), root length (mm), plumule length (mm), and weight of fresh plumule (epicotyl) (mg) and fresh root (mg). The remaining plants were allowed to grow for 15 days for the other analyses (enzymatic and biocompounds activity). All seeds were considered for the percentage of germination and measured at 7 days (100% was 30 seeds in each treatment). The fresh weights of the plumule and root were weighed on an analytical scale and reported in milligrams per plumule. The vigor of seedlings was calculated as a germination percentage by the length of seedlings in cm (root bud) [32]. The germination percentage (G%) was determined as the ratio of germinated seeds to the total number of seeds incubated. The length of the plumule was measured from the intersection of the radicle with the hypocotyl to the base of the cotyledon. The length of the radicle was determined from the base of the hypocotyl to the apex of the radicle [33].

2.2. Determination of Photosynthetic Pigments

Total chlorophyll and carotenoids were determined after the appearance of 3 to 4 true leaves, 15 days after seedling development, using the method of Lichtenthaler [34], suspending 1 g of fresh sample (the whole plant) in 5 mL of pure acetone. The homogenized plant matter was filtered and diluted to 10 mL with pure acetone; absorbance readings were performed at 665, 645, and 470 nm in a spectrophotometer UV-Visible Jenway 7305 (TEquipment, Long Branch, NJ, USA). The content was reported according to the equations of Lichtenthaler:

Total Chlorophyll = 7.05 × (A₆₆₅) + 18.09 × (A₆₄₅)

Chlorophyll a (Ca) = 11.24 × (A₆₆₅) − 2.04 × (A₆₄₅)

Chlorophyll b (Cb) = 20.13 × (A₆₄₅) − 4.19 × (A₆₆₅)

Carotenoids = (1000 × A₄₇₀ − 1.90Ca − 63.14Cb)/214

2.3. Content of Bioactive Compounds

Preparation of the extract: 1 g of fresh sample (true leaves) was mixed in 10 mL of 80% methanol and left with constant stirring for 24 h at 70 rpm at room temperature. The extract was centrifuged at 5000 rpm for 5 min, and the supernatant was used for the following content of bioactive compounds and enzymatic activity analyses.

Total phenolic compounds were quantified using the method of Folin and Ciocalteu [35]. An amount of 300 μL of the extract was added to 1080 μL of deionized water, followed by an addition of 120 μL of Folin–Ciocalteu reagent and stirred in a vortex for 10 s. After 10 min of rest in darkness, 0.9 mL of 7.5% (w/v) Na₂CO₃ was added and vortexed for 10 s. Samples were placed at room temperature for 30 min. Finally, absorbance was measured at 765 nm in a spectrophotometer Jenway 7305 UV-Vis. The standard was prepared with gallic acid (GA), and the results are reported in mg of gallic acid equivalents per 100 g fresh weight (mg AG 100 g⁻¹ FW).

Total flavonoids were determined using the aluminum chloride colorimetric assay [36]. An amount of 250 μL of methanolic extract was mixed with 1.25 mL of deionized water and 75 μL of 5% NaNO₂. After 5 min, 150 μL of 10% AlCl₃ was added. After 6 min, 500 μL of NaOH 1 M and 275 μL of deionized water were added and vortexed for a minute. The absorbances at 510 nm were measured using a spectrophotometer Jenway 7305 UV-Vis. The standard curve was prepared with catechin dissolved in absolute ethanol, and the results are expressed in mg CE 100 g⁻¹ fresh weight.

The antioxidant capacity of the extracts was determined using the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay according to Brand-Williams [37]. A solution of DPPH in ethanol at a concentration of 0.025 mg m L⁻¹ was prepared; 50 μL of methanolic extract was mixed with 1950 μL of DPPH solution; and, after 30 min, the absorbance of the samples was read using a Jenway 7305 UV-Vis spectrophotometer at 517 nm. The standard curve was prepared with Trolox, and the results are expressed in μM equivalent of Trolox in 100 g⁻¹ fresh weight (μM Trolox 100 g⁻¹).

2.4. Enzymatic Activity

The catalase enzymatic activity (CAT 1.11.1.6) was measured according to the method of Aebi [38]. CAT activity was measured spectrophotometrically (Jenway 7305 UV-Vis) at room temperature by measuring the decrease in absorbance at 240 nm resulting from the decomposition of H₂O₂. The extinction coefficient (ε240 = 43.6 M⁻¹ cm⁻¹) and protein content were used [39] to calculate enzyme activity. The activity was expressed in protein U mg⁻¹, where catalase activity unit (U) is defined as the amount of enzyme that caused an absorbance change of 0.001 per minute under assay conditions.

The enzymatic activity of peroxidase (POD 1.11.1.7) was measured using guaiac as a hydrogen donor. POD activity was measured spectrophotometrically (Jenway 7305 UV-Vis) by monitoring the increase in absorbance at 470 nm resulting from the oxidation of the guaiac by H₂O₂. The extinction coefficient (ε470 = 5.57 mM⁻¹ cm⁻¹) and protein content were used [39] to calculate enzyme activity. The activity was expressed in protein U mg⁻¹, where one unit (U) of enzyme activity was defined as 0.001 changes in absorbance per minute under assay conditions [40].

The enzymatic activity of polyphenol oxidase (PPO 1.14.18.1) was measured according to the method of Oktay [41]. PPO activity was measured spectrophotometrically (Jenway 7305 UV-Vis) at room temperature by recording the increased absorbance at 420 nm resulting from catechol decomposition. The extinction coefficient (ε420 = 3450 M⁻¹ cm⁻¹) and protein content were used [39] to calculate enzyme activity. The activity was expressed in protein U mg⁻¹, where one unit (U) of enzyme activity was defined as a 0.001 change in absorbance per minute under assay conditions [41].

2.5. Statistical Analysis

All results are the average values of 3 repetitions. Before the analysis of variance, the variables reported in percentage (vigor and germination) were normalized by applying the arcsine and square root transformation. The variance analysis and means comparison were carried out with the non-parametric LSD Fisher test (p ≤ 0.05) with the InfoStat statistical package version 2020.

3. Results and Discussion

3.1. Physiological Parameters

Compared to the control, the exposure to 100 and 200 mg L⁻¹ ZnONPs boosted the percent germination of seeds regardless of seed type (Figure 1). In chili Capsicum annuum seeds, the germination increased to 90% in 200 mg L⁻¹ of BZnONPs treatment compared to control at 50%. Here, also, 300 mg L⁻¹ of BZnONPs improved the percent germination (70%). The exposure of tomato seeds (Solanum lycopersicum) to nanoparticle solutions improved germination percent for 100 and 200 mg L⁻¹ of BZnONPs (72% and 81%, respectively) compared to the control (48%). The tomato seeds are more sensitive to high concentrations of ZnONPs and experience germination suppression at 300 and 400 mg L⁻¹. In addition, biosynthesized BZnONPs showed higher germination percentages (increasing by 20% for 100 and 200 mg L⁻¹).

However, there is a significant difference between the percent germination in tomato seeds treated with 300 and 400 mg L⁻¹ solutions, where CZnONPs show a substantial decrease in germination (decreasing by 38% and 46%, respectively, in comparison to the control). The nanoparticles influence seed germination by penetrating the seed coat and increasing its permeability [42]. Thus, the size and dissolution rate of the nanoparticles are essential parameters for high NPs absorption. The CZnONPs are less effective than BZnONPs in promoting seed germination via penetrating the seed coat at lower concentrations, suggesting that the coating on BZnONPs helps the penetration. On the other hand, at higher concentrations (300 and 400 mg L⁻¹), CZnONPs cause phytotoxicity (significant in tomato seeds and slight in chili seeds). Because the nanoparticles obtained via chemical synthesis do not have a biological coating, at high NPs concentrations, they possibly form aggregates [43], increasing effective nanoparticle size and local Zn ion concentrations, causing a more substantial toxicity effect. In the case of BZnONPs, the biocoating prevents aggregation; thus, the concentration effect is equally distributed. The aforementioned agrees with several authors. Itroutwar and collaborators found that immersing maize seeds in ZnONP, biosynthesized with seaweed, in concentrations of 5 to 100 mg L⁻¹ improves the germination percentage while, at higher concentrations, the germination percentage decreases [44].

On the other hand, wheat seeds treated with nanoparticles obtained chemically showed strong phytotoxicity, even at low NPs concentrations [45]. Thus, the germination percent is affected by mechanical (seed penetration) and chemical (zinc ion concentration) effects upon exposure to ZnONPs. The mechanical effects are predominant at lower concentrations, and at higher concentrations, chemical effects lead to toxicity.

The vigor index is a measure of seed viability [46]. It is calculated by multiplying the percentage of germination by the length of the seedling in centimeters. It determines seeds’ potential for rapid, uniform emergence and development under various conditions [47]. The vigor index showed a growth increase of 230% and 225% compared to the control (Figure 2) for chili and tomato seeds treated with 200 mg of L⁻¹ BZnONPs solution, respectively. Most treatments applied to chili seeds maintained or increased seed vigor. However, there was a difference in how nanoparticles prepared chemically vs. biologically behaved. CZnONPs boost the seed vigor index in chili seeds at 100, 200, and 300 mg L⁻¹ treatments but not in tomato seeds.

Nonetheless, in tomato seeds, the increase in vigor index is seen only for the 100 mg L⁻¹ concentration for these CZnONPs. Improving physiological parameters is typically attributed to free radical quenching by zinc oxide nanoparticles that penetrate the seeds through breaks in the seed coating [48]. So, the biocoating in BnONPs plays an important role in the delivery of NPs into the seeds and the equivalent distribution of Zn ion concentration inside the seeds. One of the possibilities is that the coating protects the stability of the nanoparticle and reduces the zinc ions’ dissolution [43]. The 400 mg L⁻¹ treatment showed a decrease in the vigor index by 70% with respect to the control in tomato seeds for both types of NPs. Again, the 400 mg L⁻¹ concentration represents the ZnONP level to display phytotoxicity [49]. We can conclude that seed exposure to ZnONPs counts as a priming method to improve seed vigor and germination [50].

The seeds’ exposure to 100, 200, and 300 mg L⁻¹ of ZnONP increased the fresh weight of the plumula and roots compared to the control. The fresh weight indicated the growth potential of the young plants. The seeds exposed to 200 mg L⁻¹ of ZnONP had longer plumules and roots for both seeds (

Table 1. Effects of varying concentrations of zinc oxide nanoparticles obtained via biological (BZnONPs) and chemical synthesis (CZnONPs) on the fresh weight and length of the plumule and root for Capsicum annuum seeds.

Concentration of ZnONP	Fresh Weight of Plumule		Fresh Weight of Root		Lenght of the Plumule		Length of the Roots
(mg L−1)	(mg)		(mg)		(mm)		(mm)
100B	11.24 ± 0.29	b	1.831 ± 0.046	bc	22.60 ± 0.15	b	11.61 ± 0.46	d
200B	13.41 ± 0.51	a	1.651 ± 0.048	c	25.71 ± 0.47	a	22.71 ± 0.30	a
300B	11.77 ± 0.15	b	2.171 ± 0.082	a	20.60 ± 1.10	c	19.22 ± 0.20	b
400B	9.54 ± 0.33	c	1.912 ± 0.024	b	19.72 ± 1.10	c	10.38 ± 0.20	de
control	9.85 ± 0.41	c	1.840 ± 0.067	bc	19.37 ± 0.17	c	9.52 ± 0.43	e
100C	11.20 ± 0.40	b	1.982 ± 0.064	b	20.38 ± 0.90	d	16.79 ± 1.05	c
200C	11.33 ± 0.42	b	1.294± 0.045	d	21.20 ± 1.54	bc	15.89 ± 0.88	c
300C	9.20 ± 0.13	c	1.512 ± 0.055	c	24.22 ± 1.97	ab	12.74 ± 0.63	d
400C	9.44 ± 0.30	c	1.296 ± 0.058	d	14.87 ± 0.38	e	9.043 ± 0.25	e

Values are average of three repetitions ± standard deviation. Bolded values are the significantly highest in the set. Different letters indicate significant differences, according to Fisher’s exact test (p ≤ 0.05).

and

Table 2. Effects of varying concentrations of zinc oxide nanoparticles obtained via biological (BZnONPs) and chemical synthesis (CZnONPs) on the fresh weight and length of the plumule and root for Solanum lycopersicum seeds.

Concentration of ZnONP	Fresh Weight of Plumule		Fresh Weight of Roots		Length of Plumule		Length of Roots
(mg L−1)	(mg)		(mg)		(mm)		(mm)
100B	23.26 ± 0.29	b	8.49 ± 0.05	b	26.96 ± 0.15	b	8.60 ± 0.46	d
200B	24.43 ± 0.85	a	10.64 ± 0.62	a	30.07 ± 0.47	a	27.01 ± 1.61	a
300B	23.79 ± 0.15	b	8.83 ± 0.08	b	24.96 ± 1.10	c	24.81 ± 1.07	bc
400B	11.79 ± 0.76	e	7.90 ± 0.02	c	8.45 ± 0.98	e	9.61 ± 0.50	d
control	20.10 ± 1.01	c	7.83 ± 0.07	c	25.06 ± 0.55	c	8.20 ± 0.41	d
100C	23.22 ± 0.40	b	8.64 ± 0.06	b	24.74 ± 0.90	c	26.83 ± 1.40	b
200C	23.35 ± 0.42	b	7.95 ± 0.04	c	25.56 ± 0.46	bc	12.96 ± 0.54	c
300C	15.89 ± 0.55	d	8.17 ± 0.05	b	11.91 ± 0.34	d	9.07 ± 1.21	d
400C	19.86 ± 0.15	c	4.89 ± 0.31	d	19.23 ± 0.38	cd	6.033 ± 0.25	e

Values are an average of three repetitions ± standard deviation. Bolded values are the significantly highest in the set. Different letters indicate significant differences, according to Fisher’s exact test (p ≤ 0.05).

). This increase may be related to the fact that zinc is involved in the biosynthesis of endogenous hormones, such as auxins and gibberellins [4], which are responsible for cell division and generating root elongation.

The Capsicum annuum seeds treated with 200 mg L⁻¹ BZnONP resulted in the plumule’s highest fresh weight and length, an increase of 36% and 32%, respectively, compared to the control (Table 1). The treatment with CZnONP at the same concentration (200 mg L⁻¹) only increased the plumule’s fresh weight by 15% and length by 9.4% with respect to the control. Thus, the growth boost indicates that BZnONPs are more efficient growth promoters than CZnONP, possibly because bio-nanoparticles are coated with phytochemicals [22]. These phytocompounds functionalize the nanoparticles, giving them superior stability and biocompatibility [19] so that their application is more homogeneous and with better attraction to plant cell walls. At 400 mg L⁻¹ levels, the seedling growth is not improved (BZnONP) or suppressed (CZnONP); thus, the Capsicum annuum plants experience toxic zinc levels.

Similar trends are present in treatments of Solanum lycopersicum seeds. The exposure to 100 and 200 mg L⁻¹ treatments increased the fresh weight of the plumule and roots compared to the control, regardless of the nanoparticle type. However, at higher concentrations, a decrease in the growth parameters can be observed, especially in the fresh plumule weight, which is lowered by 49% compared to the control for seeds when treated with BZnONPs (Table 2). Again, the tomato seeds exposed to 200 mg L⁻¹ of ZnONP had longer plumules and roots. This increases zinc participation in the biosynthesis of endogenous hormones such as auxins and gibberellins [4], which are responsible for the cell division that facilitates the elongation of the roots. Similar, strong growth suppressions due to phytotoxicity were previously described in maize and rice treated with high concentrations of chemical ZnONP (2000 mg L⁻¹) [51] and in Capsicum annuum seeds treated with ZnONP concentrations of 200 to 500 mg L⁻¹ [33].

3.2. Determination of Photosynthetic Pigments

3.2.1. Total Chlorophyll

The total chlorophyll content was influenced by the presence of ZnONPs in the seeds regardless of the type (Figure 3). The biosynthesized nanoparticles increased the levels of chlorophyll, whereas the chemical ones showed toxicity at 300 and 400 mg L⁻¹ concentrations. Surprisingly, these concentrations of BZnONPs were the best treatment for Capsicum annuum, and for the seeds of Solanum lycopersicum, the best treatment proved to be the 300 mg L⁻¹ level of biosynthesized nanoparticles. The increased chlorophyll levels can be attributed to the fact that zinc is involved in the synthesis of chlorophyll to form chloroplasts, which indicates that the higher the zinc concentration in the plant, the higher the chlorophyll concentration that could be obtained. Plants’ high total chlorophyll levels are essential because it is the primary photosynthetic photoreceptor [52], so a high concentration of chlorophylls will increase the content of photoassimilates and, consequently, lead to higher biomass [53]. However, for CZnONPs treatments, a drastic decrease in the total chlorophyll concentration in both seedlings can be observed. The zinc accumulation causes damage to the subcellular organization [54], specifically in the chloroplast, generating its rupture [55], which could be responsible for the chlorophyll decrease. Similar effects were reported by Alsuwayyid et al. in wheat (Triticum aestivum) seeds and seedlings, where a reduction in chlorophyll concentration as a function of the increase in the ZnONPs concentration was observed [56]. Since the size and shape are comparable and the nanoparticle concentrations are the same, the difference between biological and chemical nanoparticles could be explained by the biocoating’s presence [19,21]. As mentioned above, the biocoating slows the zinc ions’ dissolution into the surrounding and protects the seedling from toxic concentrations [43]. Also, BZnONPs, due to the well-homogenized and well-distributed synthesis, are well-packed nanoparticles with limited porosity [57], which would influence their dissolving process.

3.2.2. Carotenoids

The total carotenoid levels showed a somewhat different trend than the total chlorophyll concentrations. In this evaluation, the best treatments were 100 and 200 mg L⁻¹ BZnONP for both treated seeds. In the Capsicum annuum, the increase was 110% (2.7 mg L⁻¹), and in Solanum lycopersicum seeds, 36% (1.9 mg L⁻¹) in comparison to the control (1.3 and 1.4 mg L⁻¹, respectively). The rest of the treatments with BZnONPs presented carotenoid concentrations slightly increasing or even decreasing by 67% (Solanum lycopersicum at 400 mg L⁻¹) with respect to the control. The best treatments with CZnONPs to increase carotenoid levels were 300 and 400 mg L⁻¹ for both seedling types.

At high zinc concentrations, the synthesis of chlorophylls is altered due to the breakdown of chloroplasts [55], which, in turn, causes oxygen to accept electrons and become reactive oxygen species, ROS [58]. The presence of ROS influences the generation of carotenoids, increasing their concentration [59]. Carotenoids are antioxidant compounds produced through a nonenzymatic route that reduces plant oxidative damage. They are present in the plastum of plant tissues and exert a photoprotective action on photosynthetic tissues [60]. The results obtained in this work suggest that CZnONPs generate a more significant rupture of chloroplasts at high concentrations, lowering chlorophyll levels, which leads to a higher content of protective carotenoids (Figure 4). These effects agree with the results seen in Arabidopsis plants [60,61] and tomatoes [62] treated with high concentrations of nanoparticles obtained via traditional synthesis, generating more significant amounts of carotenoids and lower chlorophyll concentrations.

On the other hand, the results obtained in plants treated with BZnONP suggest better biocompatibility by not affecting the total chlorophyll content at high concentrations and increasing the carotenoid levels. Similar effects were observed in cotton [63] and Vicia faba [64] plants treated with nanoparticles obtained via green synthesis. In these, at lower NPs concentrations, the photosynthetic pigments increased to high chlorophyll concentrations, indicating no rupture of chloroplasts.

3.3. Bioactive Compounds

Applying ZnONPs increases the concentrations of total phenols, flavonoids, and antioxidant capacity, as reported in the literature [14]. Thus, these nanomaterials have great potential to be new abiotic promoters effective in inducing the biosynthesis of secondary metabolites. After penetrating plant cells, zinc oxide nanoparticles interact with the cell’s components, molecules, organelles, and intracellular structures. The nature of the interaction between nanoparticles and target cell organelles, such as chloroplasts and mitochondria, may provoke chemical and physical changes [65].

3.3.1. Total Phenols Content, TPC

TPC levels show similar trends for 100 to 300 mg L⁻¹ concentrations for both types of nanoparticles (CZnONP and BZnONP in Figure 5). In chili seedlings, the best treatment was 300 mg L⁻¹ of BZnONPs (352.79 mg of GA/100g FW, an increase of 73% with respect to the control). Simultaneously, the level of TPC in seedlings treated with CZnONPs at the same concentration experienced a rise of only 44% with respect to the control. In the case of tomato Solanum lycopersicum seedlings, the highest concentration of total phenols was determined in seedlings treated with bionanoparticles at 400 mg L⁻¹, resulting in a TPC concentration of 466.58 mg of GA/100g FW (an increase of 21%). However, as observed in other parameters, at that concentration of CZnONPs, the seedlings presented a decrease of 40% in TPC with respect to the control. The treatment of Capsicum annuum seeds with CZnONPs at 400 mg L⁻¹ also showed a decrease in TCP by 40%.

In general, the treatments with BZnONP induce a synthesis of TPC, which could be related to a better ability of BZnONP to penetrate seeds [42] and generate a greater expression of the genes responsible for the production of the nonenzymatic defense system metabolites [66]. This system is activated in situations of stress in plants where an excess of ROS is produced [59,67], which subsequently affects cellular structures, organules, DNA, proteins, lipids, and carbohydrates [60,68]. The primary function of TPC is to protect plants from oxidative damage [58]. At the same time, TPC can improve metabolic pathways, leading to larger plants, better nutritional content [69], and increased resistance to biotic and abiotic stresses [70].

The response of the seedlings, with respect to TPC, was similar for both nanoparticles; nonetheless, BZnONP increased the content of secondary metabolites, significantly affecting other characteristics of seedlings, eventually leading to products with added value. These findings are consistent with what has been reported in the literature, where biosynthesized ZnONP enhanced plant growth, sprouts, chlorophyll content, total protein content, total phenol content, flavonoids, and enzymatic activity in Juniperus procera [71] and Brassica oleracea var italic [72]. While nanoparticles obtained via chemical syntheses can generate high concentrations of TPC, they negatively influence physiological characteristics. Capsicum annuum L. treated with various chemically synthesized ZnONP concentrations showed a higher TPC content in the roots of the seedlings exposed to the highest ZnONP levels, but their root length decreased by 50.0% [33]. A similar trend was observed by Iziy et al., where TPC concentrations increased as a function of ZnONP concentrations, but these treatments presented low fresh and dry weight. The authors attributed the physiological declination to the interruption of the biosynthesis and transfer of growth regulators, such as gibberellic acid and auxin [73].

3.3.2. Flavonoids

Flavonoids are secondary metabolites with antioxidant activity, whose potency depends on the number and position of free OH groups [72]. The treatment with BZnONPs and CZnONPs increased the content of flavonoids in both types of seedlings, recording the most significant increase for 300 and 400 mg L⁻¹ of BZnONPs treatments of Solanum lycopersicum and 300 mg L⁻¹ BZnONPs treatment of Capsicum annuum. As the concentration of BZnONPs increases, the flavonoid level also rises in Capsicum annuum seedlings; nevertheless, an opposite trend is seen in applying chemical nanoparticles. An increase in the concentration of CZnONP slowly decreases the flavonoid level (the greatest concentration is recorded for 100 mg L⁻¹ treatment). Solanum lycopersicum seedlings generally have higher levels of flavonoids with a different response to both types of ZnONP. BZnONP treatments resulted in higher levels of flavonoids than CZnONP treatments (maximum increase of 39% at 100 mg L⁻¹ of CZnONP). Applying 300 and 400 mg L⁻¹ of BZnONP increased flavonoid levels by about 70% (Figure 6).

The results prove the hypothesis that plants’ response to nanomaterials could differ depending on the physicochemical characteristics of nanoparticles [74,75]. These factors include size, shape, chemical composition, physicochemical stability, crystalline structure, surface area, surface energy, surface roughness, and surface coating [43]. All these are manipulated during nanoparticle formation, which, in turn, change the absorption, biomolecular interactions, signaling cascades, and biological systems of nanoparticles. Thus, there is a strong suggestion that BZnONPs have a different external surface, regardless of having the same size range as CZnONPs, allowing them to interact with the plant cells in a specific favorable manner.

It has been shown that the flavonoids contained in the vacuole can be used in the flavonol–peroxidase cleaning system to purge active oxygen species, especially H₂O₂ [76]_. The activity of such systems in plants seems necessary because it provides optimal conditions for growth. Our results are consistent with the findings of Iziy et al., who studied the effect of ZnONP on the content of phenols and flavonoids in Portulaca oleracea L. [73]. The authors explained the physiological response to the defense system activation. The CZnONP trends presented in Figure 6A are similar to the findings of Javed et al., who studied the effect of CZnONP on the content of phenols and flavonoids of Stevia rebaudiana [77]. They showed a flavonoid content decrease in plants treated with 100 and 1000 mg L⁻¹ of CZnONP. This decrease in flavonoid content under high nanoparticle concentrations is seen in the imbalance between antioxidant activity and oxidative stress, reducing antioxidant activity. However, the current study showed no such toxicity symptoms in seedlings treated with BZnONP, which could explain the higher biocompatibility of BZnONPs.

3.3.3. Antioxidant Activity

Figure 7 shows the results of the antioxidant capacity determined using the DPPH method. The most significant increase in the antioxidant capacity of DPPH occurs in both seeds treated with BZnONP, with a concentration of 400 mg L⁻¹ for Capsicum annuum and 300 mg L⁻¹ for Solanum lycopersicum. Overall, CZnONPs show a negative trend with increasing concentration of NPs, except for treatment with 400 mg L⁻¹ in Capsicum annuum, which was determined to have a high antioxidant activity. This behavior is similar to that observed with TPC and flavonoids for seedlings treated with BZnONP. The increase in antioxidant activity of plants exposed to NP is related to the increase in phenolic compounds [78], which are ROS sequestrants [58,79].

Our results are consistent with the findings of Ushahra et al. on the effect of BZnONP on the antioxidant capacity of Eruca sativa [80]. The trend obtained in the seedlings of Solanum lycopersicum treated with CZnONP is similar to that obtained by Çekiç et al., who determined that antioxidant capacity decreases under very high concentrations of nanoparticles because Zn, as a heavy metal, is toxic to many plants in too high concentrations [81,82]. The decrease in antioxidant capacity, as seen under CZnONPs treatment of Solanum lycopersicum, might be related to the excessive production of free radicals, which, in turn, suppresses plant growth [83].

3.4. Enzymatic Activity

The ZnONPs influenced the plant’s ability to activate the enzyme defense system, presenting statistically significant differences (p ≤ 0.05) in the different variables measured: catalase (CAT 1.11.1.6), peroxidase (POD 1.11.1.7), and polyphenol oxidase (PPO 1.14.18.1).

In general, all plants treated with ZnONPs presented higher CAT enzyme activity regardless of seed and nanoparticle type compared to the control. The highest concentration of CAT was obtained with the doses of 100 mg L⁻¹ of BZnONPs (6.67 U CAT /mg of protein, an increase of 90% with respect to the control). Overall, with the rise in BZnONPs concentration in Capsicum annuum seedlings, the enzymatic activity of CAT decreases. The opposite is seen in treatment with CZnONPs, in which the highest CAT level was measured under 300 mg L⁻¹ CZnONPs treatment (70% higher than control but 20% lower than the best treatment of BZnONPs). Similar behavior was recorded in Solanum lycopersicum seedlings, where the best treatment was 200 mg L⁻¹ for BZnONPs (a 71% increase with respect to control,

Table 3. Effects of different concentrations of ZnONPs obtained via biological (BZnONPs) and chemical synthesis (CZnONPs) on the enzymatic activity of CAT, POD, and PPO in Capsicum annuum and Solanum lycopersicum seeds.

Concentration of ZnONP	Catalase				Peroxidase				Polyphenol Oxidase
	Units of CAT mg−1 of Protein				Units of POD mg−1 of Protein				Units of PPO mg−1 of Protein
(mg L−1)	Capsicum annuum		Solanum lycopersicum		Capsicum annuum		Solanum lycopersicum		Capsicum annuum		Solanum lycopersicum
100B	6.67 ± 0.66	a	7.02 ± 0.93	b	10.19 ± 0.41	cd	13.06 ± 0.58	a	0.26 ± 0.01	a	0.24 ± 0.07	ab
200B	5.11 ± 0.16	c	7.79 ± 0.17	a	10.32 ± 0.57	bc	11.21 ± 0.68	b	0.26 ± 0.01	a	0.22 ± 0.03	b
300B	6.14 ± 0.31	b	5.13 ± 0.37	d	10.01 ± 0.80	cd	10.79 ± 0.92	bc	0.21 ± 0.06	cd	0.22 ± 0.02	b
400B	4.31 ± 0.29	d	5.22 ± 0.32	d	11.33 ± 1.12	a	12.27 ± 1.00	ab	0.22 ± 0.01	e	0.25 ± 0.02	a
control	3.32 ± 0.10	e	4.57 ± 0.09	e	9.47 ± 0.29	cd	10.35 ± 0.30	c	0.16 ± 0.01	e	0.15 ± 0.01	d
100C	4.73 ± 0.28	d	5.93 ± 0.32	c	8.26 ± 0.33	d	8.96 ± 0.46	d	0.23 ± 0.02	bc	0.23 ± 0.02	b
200C	4.94 ± 0.61	cd	5.48 ± 0.33	cd	8.97 ± 1.15	d	9.68 ± 1.13	d	0.19 ± 0.07	d	0.21 ± 0.04	c
300C	5.83 ± 0.82	b	6.93 ± 0.96	b	10.73 ± 0.45	b	11.63 ± 0.51	b	0.25 ± 0.06	a	0.22 ± 0.02	b
400C	4.90 ± 0.43	c	5.59 ± 0.15	c	10.39 ± 0.69	bc	11.38 ± 0.77	b	0.21 ± 0.03	c	0.21 ± 0.02	c

Values are an average of three repetitions ± standard deviation. Bolded values are the significantly highest in the set. Different letters indicate significant differences, according to Fisher’s exact test (p ≤ 0.05).

).

For the enzymatic activity of POD, the best treatment turned out to be 400 mg L⁻¹ of BZnONPs, with an increase of 19% with respect to the control. However, for CZnONPs, a decrease in peroxidase activity is observed in 100 and 200 mg L⁻¹ treatments in the seedlings of Capsicum annuum (8.26 and 8.97 U of POD mg L⁻¹, respectively, compared with the control 9.47 U of POD mg⁻¹). Similar effects are shown in the results for Solanum lycopersicum seedlings; all treatments with BZnONPs showed an increase with respect to the control, obtaining the best results for seedlings treated with 100 mg L⁻¹ (a 20% increase, 13.06 U of POD mg ⁻¹, compared to the control, 10.35 U of POD mg⁻¹). The seeds treated with 100 mg L⁻¹ CZnONPs show a decrease in the enzyme activity of the peroxidase with 8.96 U of POD mg⁻¹, which represents 13% of the control (Table 3).

On the other hand, the highest enzymatic activity of polyphenol oxidase (PPO) was determined in Capsicum annuum for 100 and 200 mg L⁻¹ BZnONPs treatments, which are statistically equal to 300 mg L⁻¹ CZnONPs treatment. In the case of tomato seedlings, the best treatment was 400 mg L⁻¹ BZnONPs. The application of CZnONP to Solanum lycopersicum seeds also showed an increase in PPO activity, although not as high as BZnONP treatments (Table 3).

An increase in the activity of antioxidant enzymes is due to the adaptive defense mechanism of plants against the harmful effects of ZnONP [79]. The results obtained here indicate that the biosynthesized nanoparticles improve the enzymatic activity in a more significant percentage in both seeds for all the enzymes measured. Hence, they are more efficient in activating the enzymatic defense system at lower concentrations compared to CZnONPs. The results indicate that applying BZnONPs induced a greater peroxidase enzymatic activity in both plants (Table 3), especially in Capsicum annuum seeds. A higher concentration of BZnONPs increases the enzymatic activity of POD; this may be related to the fact that ZnONPs induce a greater synthesis of phenolic compounds (Figure 5) and because POD is an enzyme that, in addition to sequestering H₂O₂, catalyzes the oxidative interaction reactions of a wide range of phenolic compounds [84]. Thus, POD activity could increase by increasing the concentration of phenolic compounds in the plant.

Our results are consistent with those reported by Pejam et al., where the ZnONP application improved the effectiveness of the ROS elimination system acting as positive regulators of the genes of the defense system and involved in the production of secondary metabolism [85], which presented itself as increased activity in the enzymes. PPO is an enzyme that catalyzes the hydroxylation of monophenols to o-diphenols, which are then oxidized to quinones [86]. POD catalyzes oxidation reactions and restores H₂O, using H₂O₂ as an electron receptor to catalyze different oxidative reactions [31]. Catalase efficiently breaks down high concentrations of H₂O₂ and reduces the damage by OH; therefore, the level of hydrogen peroxide is controlled by CAT in plant cells [51]. El-Zohri et al. showed that tomato plants respond to the foliar application of ZnONP at low concentrations. They suggested that nonenzymatic antioxidants and phenolic compounds worked in coordination with antioxidant enzymes to reduce the negative effects of oxidative stress [86]. Abdel et al. mentioned that the decrease in CAT with respect to peroxidase activity could be due to the increasing rate of ROS clearance by the other antioxidant enzymes [87]. Similar effects were reported in safflower plants where increased polyphenol oxidase activity was a function of higher concentrations of zinc oxide nanoparticles [88]. Oxidative stress occurs when plants experience stress from an abiotic cause that produces ROS [89], which are metabolized by enzymes such as CAT, POD, and PPO. The presence of BZnONPs, more effectively than CZnONPS, causes stress and stimulates enzymatic activity, as demonstrated in this study.

A comparison of the two types of zinc oxide nanoparticles showed benefits in the use of bio-coated nanoparticles. The germination and vigor index of the seeds increased upon exposure to 200 mg L⁻¹ BZnONPs. This priming concentration of BZnONPs promoted the seedlings’ largest fresh mass and length. At the same time, the tomato seeds showed more sensitivity to the treatments, especially to CZnONPs. This was evident in the strong suppression of the vigor index treated with 300 and 400 mg L⁻¹ CZnONPs solutions. Overall, BZnONPs were a better primer for both seeds. We deduce that the ZnONPs have dual action on the seeds. First, the NPs penetrate the seeds, prompting their cracking. This mechanical influence is then suppressed by a chemical effect related to the release of Zn ions. The chemical toxicity at higher concentrations is significant for CZnONPs, which, without biocoating, might aggregate and dissolve faster, increasing local concentration. Bionanoparticles are more stable and possibly dissolve slower due to the surface molecules’ protective action. The 200 mg L⁻¹ BZnONPs concentration promotes plant growth due to increased cell division and tissue growth.

The difference between bio and chemical ZnONPs is also visible in the chlorophyll and carotenoid levels. Firstly, chlorophyll increases under treatment with bionanoparticles but significantly decreases under CZnONPs exposures. The chlorophyll damage is the chemical effect of zinc accumulation that destroys the chloroplast’s subcellular organization. Simultaneously, the rupture of chloroplasts at high CZnONPs concentrations lowers chlorophyll levels, which leads to a higher content of protective carotenoids. Since the ZnONPs’ size and shape are comparable, and the nanoparticle concentrations are the same, the difference between biological and chemical nanoparticles could be explained by the biocoating’s presence, suggesting the better biocompatibility of BZnONPs. Biocoating slows down the zinc dissolution, controlling local Zn²⁺ concentration.

The high concentrations of ZnONPs, 300 and 400 mg L⁻¹, show phytotoxicity, suppressing growth and destroying chlorophyll, increasing plant stress, and boosting amounts of bioactive compounds, polyphenols, and flavonoids. The bionanoparticles increased the TPC in chili and tomato seedlings, while the treatment with 400 mg L⁻¹ of CZnONPs decreased TPC. As the concentration of BZnONPs increases, the flavonoid level also rises in Capsicum annuum seedlings; nevertheless, an opposite trend is seen upon applying chemical nanoparticles. The same trends are seen in the antioxidant capacity of DPPH. The highest activity occurs in both seeds treated with BZnONP at 400 mg L⁻¹ for Capsicum annuum and 300 mg L⁻¹ for Solanum lycopersicum. Overall, CZnONPs show a negative trend in antioxidant activity. The decrease in antioxidant capacity, as seen under CZnONPs treatment of Solanum lycopersicum, might be related to the excessive production of free radicals, which, in turn, suppresses plant growth. Since the polyphenols and flavonoids protect the plants from ROS and lead to larger plants, the uncoated nanoparticles do not perform this function sufficiently and only suppress plant growth. Again, the growth burst in treatments with BZnONPs suggests biocompatibility based on coating interaction with seeds.

In a similar fashion, enzymatic activity is better triggered in the presence of BZnONPs. Thus, we conclude that the biocoating plays an essential role in the ZnONPs’ interaction with seeds and young plants.

4. Conclusions

This study compared the influence of zinc oxide nanoparticles of the same size, 70–80 nm, obtained via chemical and biological synthesis in chili and tomato seeds and seedlings. The results showed that the biosynthesized nanoparticles showed a more beneficial impact than the chemically produced ZnONPs, proving that the set hypothesis is correct. Without significant phototoxicity, BZnONPs increased the germination percentage, vigor index, length, and biomass of roots and seedlings. These NPs also proved to be more effective in activating the defense system in plants, increasing the amounts of bioactive compounds and enzymatic activity even at low concentrations compared to CZnONP. All these improved parameters indicate that the biocoating can enhance the ZnONP biocompatibility and penetration into the seeds. BZnONPs have been shown to be a successful primer for seeds and a potential nanofertilizer. Thus, applying biosynthesized nanoparticles could be an excellent ecological alternative to improve seedlings’ quality, proving sustainable nanoparticle production’s validity.