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Article

Influence of the Application of Different Zinc Oxide Nanoparticles on a Lettuce Crop Grown in an Acidic Mediterranean Soil

by
Marina de Francisco
1,2,
Pedro Fernandes-Silva
3,
Luisa Durães
3,
Andreia Romeiro
3,4,
Silvia Álvarez-Torrellas
5 and
Patricia Almendros
1,2,*
1
Department of Chemical and Food Technology, Agronomic, Food and Biosystems Engineering School, Universidad Politécnica de Madrid (UPM), 28040 Madrid, Spain
2
Research Centre for the Management of Agricultural and Environmental Risks (CEIGRAM), Universidad Politécnica de Madrid (UPM), 28040 Madrid, Spain
3
University of Coimbra, Chemical Engineering and Renewable Resources for Sustainability (CERES), Department of Chemical Engineering, 3030-790 Coimbra, Portugal
4
University of Coimbra, Centre for Mechanical Engineering Materials and Processes, ARISE, Department of Chemical Engineering, Rua Silvio Lima-Polo II, 3030-790 Coimbra, Portugal
5
Department of Chemical Engineering and Materials, Catalysis and Separation Processes Group, Complutense University of Madrid (UCM), 28040 Madrid, Spain
*
Author to whom correspondence should be addressed.
Horticulturae 2024, 10(7), 681; https://doi.org/10.3390/horticulturae10070681
Submission received: 3 June 2024 / Revised: 19 June 2024 / Accepted: 21 June 2024 / Published: 27 June 2024
(This article belongs to the Section Plant Nutrition)
Browse Figures
Versions Notes

Abstract

:
Zinc (Zn) is a crucial micronutrient essential for the growth and development of crops. Recently, there has been growing interest in harnessing its benefits through the application of zinc oxide (ZnO) nanoparticles (NPs) as an alternative to conventional fertilizers. Different types of ZnO NPs were synthesized in the laboratory by a co-precipitation method using different precursor metal (Zn(NO3)2 for ZnO-1 and ZnSO4 for ZnO-2) and sources of hydroxyl groups (NaOH for ZnO-1 and NH4OH for ZnO-2) or by a sol–gel method, using ZnC4H6O4 (ZnO-3) or ZnSO4 (ZnO-4) as precursor metal. This study focused on the effect of these Zn sources on the impact on lettuce (Lactuca sativa L.) cultivation under acidic and Zn-deficient soil conditions. The efficacy of these Zn sources was evaluated by measuring the lettuce fresh weight, the stem diameter, the Zn concentrations in young leaves and mature leaves, the photosynthetic pigment content (chlorophyll and carotenoid), and the overall Zn status in the soil and soil pore water. The ZnO NPs with particle sizes of 76–104 nm positively affected the stem thickness (with an increase of up to 1.4 times that of the control) and crop biofortification with Zn (up to 3.2 and 12.6 times the Zn in young leaf and mature leaf, compared to the control). The smaller ZnO NPs (ZnO-1 and ZnO-3) showed the highest concentrations of bioavailable Zn and Zn in pore water at the end of the cultivation period (with an average increase of 41% compared to larger sources), resulting in high biofortification levels in both mature and young leaves. Peak concentrations of dissolved Zn in soil pore water were observed at 18 days after planting, followed by a decline attributed to the retention of Zn in more insoluble forms in the soil. The difference in Zn concentration between mature leaves and young leaves indicated its limited mobility in the plant, with more Zn accumulating in mature leaves.

1. Introduction

Lettuce (Lactuca sativa L.) is one of the most widely consumed and cultivated leafy vegetables worldwide. It is a commercially important salad crop, available all year round on the market, and is easily stored. Lettuce contains several therapeutic and health-promoting components such as flavonoids, phenolic acids, antioxidants and nutrients [1,2]. The ideal temperature range for seed germination is between 18 and 21 °C. The best soil types for lettuce cultivation are sandy loam or silt loam with an optimal soil pH range for commercial production between 5.8 and 6.6 [3].
Food security is an increasing priority in the face of a growing world population, so improving the nutritional quality and productivity of crops is essential [4]. Traditionally, fertilizer use has been focused on increasing crop yields. This fertilization has focused on providing macronutrients to the soil, which has led to an increase in micronutrient deficiencies [5]. However, according to the Hidden Hunger Index [6], some countries located in Africa, East Asia, and the Pacific have seen an improvement, attributed to an increased focus on producing micronutrient-rich crops rather than just increasing yields. According to Alloway [7], there are large micronutrient deficiencies in soils; for example, it is estimated that 49% of the world’s soils are deficient in zinc (Zn). Consuming these crops that do not meet nutritional or dietary requirements can lead to malnutrition. Hidden hunger has been defined as micronutrient deficiencies (particularly iron, zinc, iodine, and vitamin A) and it is estimated to impact over two billion people globally [8]. Micronutrient inadequacies are to be found in the developed world as well as in the developing world [9]. A viable strategy to address micronutrient deficiencies in crops and combat hidden hunger is crop biofortification [9].
Zinc is often deficient in the human diet, so fortification of crops with this nutrient is essential [10,11]. Daily dietary intakes of this nutrient are considered to be between 8.5 and 12.9 mg for children under 7 years and adult males, respectively [12]. This approach has been reported in numerous publications that have examined the effect of the application of different Zn sources in the biofortification of cereal [13] but also in horticultural crops [14,15,16,17,18,19,20,21]. This Zn deficiency leads to significant economic losses, primarily due to reduced biomass [22,23]. Lettuce is considered a crop with a moderate sensitivity to zinc deficiency [22].
In plants, Zn plays a crucial role in several physiological and metabolic processes [24,25]. The ideal Zn concentration range for plants is narrow, falling between 20 and 100 mg kg−1 dry matter [26], which underlines the importance of precise dosage and controlled application. Zinc deficiency in plants can cause symptoms such as chlorosis, smaller leaves, and the sterility of spikelets, due to the disruption of functions such as photosynthesis and protein synthesis [24,25,27]. On the other hand, an excess of Zn can cause toxicity in plants, affecting yield or resulting in poor leaf growth or the inhibition of root development [28]. Additionally, symptoms of Zn deficiency in lettuce have been widely reported. These deficiency symptoms include general chlorosis, the development of spots on the margins of mature leaves, or the appearance of dark lesions between the veins [29]. Zinc is a poorly mobile nutrient within the plant and the transfer of Zn from old to young tissue is further reduced in deficient plants [18]. Deficiency symptoms appear first on mature leaves and rarely appear on very young leaves, even under very severe Zn deficiency [29]. The concentration of Zn in lettuce leaves is therefore variable, depending on the position of the leaf, its development, and the availability of Zn in the soil in which it has been grown.
Zinc availability in soil varies widely [22], influenced by factors such as pH or soil organic matter content. Heavily leached sandy and acidic soils often have a low level of plant-available Zn [30]. In addition, mineral soils with a low organic matter content are also deficient in Zn. Other characteristics, such as the concentration of carbonates, clays, and Fe oxides, or the P content, are essential for an accurate estimation of Zn availability to plants [31]. Low Zn availability in soil can lead to a deficiency in plants, affecting their growth [32]. Therefore, the use of micronutrient sources like Zn has gained increasing importance over the past decade [4]. Nanotechnology has enabled the development of Zn nanoparticles (NPs) as nanofertilizers, with advantages in their uptake by plants due to their small size [33,34]. Although large-scale production of nanoagrochemicals and experimentation on crops are ongoing, early results indicate enhanced uptake, improved use efficiency, targeted delivery, and reduced contamination [34]. Furthermore, different studies have reported that the physical and chemical characteristics of different nanoparticles influence their effect on plant development [35,36,37,38,39].
Therefore, the general objective of the present work was to study the influence of different ZnO NPs applied in a Mediterranean acid agricultural soil on the yield and quality of a lettuce crop (Lactuca sativa L.). For this purpose, the Zn biofortification of lettuce, morphometric parameters, and photosynthetic pigments of the plant have been evaluated.

2. Materials and Methods

2.1. Synthesis and Characterization of ZnO Nanoparticles (NPs)

The Zn sources used in this study were synthesized in the laboratory (Table 1). Two chemical synthesis approaches were accomplished: co-precipitation (ZnO-1 and ZnO-2) and sol–gel (ZnO-3 and ZnO-4) methods. ZnO-1 was synthesized following the procedure described by Akhil et al. [40]. For this purpose, 180 mL of the aqueous solution and source of hydroxyl group (NH4OH, 0.3 M) (Merck, Darmstadt, Germany) were added to 180 mL of the metal precursor solution (Zn(NO3)2 0.15 M) (PANREAC, Barcelona, Spain). The mixture was stirred vigorously for 180 min at room temperature. The appearance of a milky-white color indicates the formation of solid particles of Zn compounds. The solution was rinsed three times with distilled water and dried at 80 °C for 24 h. For ZnO-2, the method outlined by Huy et al. [41] was followed with some modifications, including the replacement of the metallic precursor with ZnSO4 (Spectrum Chemical, Gardena, CA, USA) and the hydrogen source with NH4OH. To obtain ZnO-2, the synthesis process consisted of adding 180 mL of the aqueous NH4OH solution (0.3 M) dropwise over 180 mL of the metal precursor solution (ZnSO4 0.15 M). The mixture was stirred vigorously for 180 min at room temperature. The solution was rinsed three times with distilled water and dried at 80 °C for 24 h. ZnO-3 was synthesized using the method described by Lee et al. [42], in which 20 mL of the aqueous solution and source of hydroxyl group (NaOH, 0.3 M) (Merck, Darmstadt, Germany) were added to 100 mL of the metal precursor solution (ZnC4H6O4 0.1 M) (Alfa Aesar, Kandel, Germany). The mixture was mixed slowly with vigorous magnetic stirring at 70 °C. The reaction mixture was kept at 70 °C for 1 h, and then allowed to stand for 7 days at room temperature to produce a sol–gel. The sol–gel was rinsed three times with distilled water and dried at 60 °C for 24 h. ZnO-4 was obtained following the procedure described by Chandrasekaran et al. [43], with a modification of the metallic precursor. For this purpose, 30 mL of the aqueous solution and source of hydroxyl group (NaOH, 0.3 M) were added to 100 mL of the metal precursor solution (ZnSO4 0.1 M). TEA buffering agent (10 mL 0.2 M) (Merck, Darmstadt, Germany) was immediately added to the cloudy white solution. The mixture was stirred vigorously for 1 h at 70 °C and aged for 24 h. The sol–gel was rinsed three times with distilled water and dried at 60 °C for 24 h. The main characteristics of the nanoparticles used in this study are shown in Table 1.
The hydrodynamic diameter and Zeta potential of NPs were recorded using dynamic light scattering (DLS) and electrophoretic migration techniques, respectively, in a Zetasizer Nano ZS equipment (Malvern Panalytical Ltd., Malvern, UK) and Zetaziser Software 7.13. The hydrodynamic diameter refers to the size of a hypothetical sphere with the same diffusion coefficient as the particle under measurement, considering the hydration layer around the particle. Consequently, the hydrodynamic diameter values are always higher than the real diameter of the particles. Moreover, as it is not an individual particle measurement, the agglomeration effects tend to also bias the results to higher values. The hydrodynamic diameter of ZnO-2 sample was not measured due to its larger particle size distribution, which prevents an accurate characterization by the DLS technique. This method only works optimally for analyzing samples with a monodisperse particle size distribution. The ZnO-4 NPs had the largest particle hydrodynamic diameter of all the tested samples (573.2 ± 10 nm), which may indicate a higher difficulty to diffuse in the plant.
The particle size distribution (Figure 1) was accomplished by transmission electron microscopy (TEM) using a JEOL JSM 6335F microscope (JEOL Ltd., Tokyo, Japan) and Image J 1.53e software.
The particle size of all the synthesized ZnO samples measured by TEM indicated that a significant amount of these particles is in the nanoscale range. ZnO-1 presented a similar size to that obtained by the followed synthesis method [40], which was 65 ± 1 nm. On the other hand, both ZnO-2 and ZnO-3 NPs showed higher values than those obtained in the followed synthesis method [41,43], with particles size ranges of 20–70 nm and 25–30 nm, respectively. As for ZnO-4, its size is within the range obtained using the synthesis method followed [42], from 50 to 200 nm.
Synthesized NPs with zeta potential values exceeding +30 mV or below −30 mV typically show high stability in solution, as suspended particles tend to repel each other in this case, preventing agglomeration. However, all samples showed a zeta potential value within this range, and not very high absolute values, indicating that the NPs lack stability against agglomeration.

2.2. Pot Experiment

For this experiment, an acidic soil from Castilla y Leon, Spain (41°13′ N, 5°17′ W), was used. Samples were collected from the Ap horizon (0–20 cm), air dried, sieved to less than 2 mm, and subsequently analyzed following the Spanish official methodology [44]. This soil was classified as Cambisols [45]. The main characteristics of the soil were the following: pH, 5.9 acidic; sand, 803 g kg−1; silt, 116 g kg−1; clay, 81 g kg−1; texture, sandy loam; water-holding capacity (33 kPa), 107 g kg−1; electrical conductivity, 67.2 μS cm−1; extractable P, 18.3 mg kg−1; oxidizable OM, 4.1 g kg−1; total N, 0.6 g kg−1; C:N ratio, 3.7; exchange Na, 58 mg kg−1; exchange K, 115 mg kg−1; exchange Ca, 805 mg kg−1; and exchange Mg, 263 mg kg−1. According to the concentration of available Zn (DTPA–triethanolamine (TEA)-extractable Zn) (0.38 mg kg−1), the original soil was Zn deficient. This soil is mainly characterized by its acidic pH and low organic matter content.
Pots (1 L capacity) with 1 kg soil dry weight were fertilized with the macronutrients N, P, and K, following the fertilization recommendations for this crop [46]. This fertilization involved applying 100 kg N ha−1 in the form of urea, 50 kg P2O5 ha−1 in the form of K2HPO4 and 160 kg K2O ha−1 in the form of K2HPO4 and K2SO4. Therefore, 0.246 g urea, 0.14 g K2HPO4, and 0.20 g K2SO4 were applied per kg potted soil. The different synthesized ZnO NPs were surface-applied at a dose of 8 mg Zn kg−1 soil, and subsequently, the topsoil was mixed. A control treatment with NPK fertilization but without Zn supply was included. Each treatment was replicated three times, resulting in a total of 15 pots arranged in a randomized complete block design.
Lettuce plants (Lactuca sativa L., Romana Verano, Ramiro Arnedo) at 25 days of growth were individually placed in pots and transferred to a controlled greenhouse environment on the Universidad Politécnica de Madrid Campus (Madrid, Spain) in April 2023. The temperature (from 4 °C at night to 38 °C during the day) and relative humidity (between 20 and 85%) conditions were selected to simulate real growing conditions in a Mediterranean climate. Due to the low water-holding capacity of this soil, the soil moisture was controlled by weighing, three times a week, at 100% of its water-holding capacity using tap water.

2.3. Plant Analysis

Forty-seven days after planting (AP), the lettuces reached the requirements for Class II according to 543/2011/UE [47]. Then, the lettuce plants were cut at the neck and the stem diameter was measured, subsequently weighed, and then rinsed with deionized water. In accordance with Ortiz et al. [18], the leaves were then divided into young leaves (the fifth and sixth youngest leaves) and mature leaves (the second and third oldest leaves) for further analyses. The chlorophyll and carotenoid content in the mature fresh leaves were determined according to the method described in AOAC Official Methods of Analysis [48] and Lichtenthaler [49], using 0.2 g of fresh leaf with 50 mL of acetone (Panreac, Castellar del Vallès, Spain, 99.5%) until the leaves acquired a whitish color. The concentration of soluble Zn (MES) in mature leaves was analyzed by mixing 0.5 g of fresh leaf with 8 mL of 1 mM MES [2-(N-morpholino) ethane sulfonic acid)] at pH 6 [50]. Leaves were subsequently dried (in an oven at 60 °C) until reaching a stable weight. The total Zn concentrations in young and mature leaves were determined by adding 0.5 g of dry matter to an acid mixture (5 mL HNO3 [65%], 2 mL HF [48%] and 3 mL H2O) by wet digestion in Teflon vessels using a sample preparation block system (SPB Probe, Perkin-Elmer, Waltham, MA, USA).

2.4. Soil Pore Water Analysis

Rhizon samplers (Rhizosphere Research Products, Wageningen, The Netherlands) were used to extract pore water using a special membrane and vacuum force. After fertilization and over the course of the pot experiment, six extractions were carried out to evaluate the total dissolved Zn concentration in the soil pore water. Zn concentration in the extracts was quantified using flame atomic absorption spectrometry (FAAS) (AAnalyst 900, Perkin Elmer, Waltham, MA, USA).

2.5. Soil Analysis

Following the harvest, the soil from each pot was naturally dried and homogenized. Bioavailable Zn concentration for the plant was assessed using a mixture of low molecular weight organic acids (LMWOAs) (10 mM combination of organic acids solution containing acetic, lactic, citric, malic, and formic acids in a molar ratio of 4:2:1:1:1, respectively) [51]. Next, 2 g of soil was mixed with 20 mL of LMWOAs solution, then shaken for 16 h and centrifuged for 10 min at 5200× g rpm. The extracts were filtered, and Zn concentration values were determined by flame atomic absorption spectrophotometry (FAAS) (AAnalyst 900, Perkin Elmer, Waltham, MA, USA).

2.6. Statistical Analysis

Statistical analysis was accomplished by using Statgraphics Centurion software (v.19, Manugistic Inc., Rockville, MD, USA). ANOVA analyses were performed for comparison of mean values (Fisher LSD test at 95.0% confidence level). Multivariate analyses were conducted to analyze correlations.

3. Results

3.1. Plant Analysis

To evaluate lettuce production, the total fresh-matter weight and the stem diameter were measured at 47 days AP (Figure 2A). No significant differences in the fresh matter between treatments were obtained. Regarding the stem diameter (Figure 2B), all ZnO NPs treatments showed a significant increase in diameter with respect to the control. ZnO-3 treatment showed the largest diameter, with an increase of 39%, compared to the control treatment.
Figure 3 shows the Zn biofortification of lettuce in terms of total Zn concentration in mature leaves and young leaves and the soluble Zn concentration in the leaves, depending on the sources added. No significant differences were observed between the Zn treatments in terms of the total Zn concentration in the young leaves (Figure 3A). However, there were significant differences (p value: 0.0003) between the Zn treatments (mean concentration of 92.4 mg kg−1) and the control (31.4 mg kg−1). In the case of the mature leaves (Figure 3B), there were significant differences (p value: 0.0000) between the experimental factors. The highest total Zn concentration in mature leaves was obtained with the ZnO-1 source, reaching to 654 mg kg−1. Also, significant differences were found between ZnO-1 and ZnO-4 samples, which reached Zn concentrations of 12.5 and 9.0 times the Zn concentration of the control treatment, respectively.
All treatments showed Zn concentrations in mature leaves that exceeded those accumulated in young leaves, with 6.5, 6.3, 5.7, and 5.5-fold increments for ZnO-1, ZnO-2, ZnO-3, and ZnO-4, respectively.
Leaves’ soluble Zn concentration (Figure 3C) showed significant differences (p value: 0.0001) between the Zn treatments and the control. The treatments that showed the highest leaf-soluble Zn concentrations were ZnO-1, ZnO-2, and ZnO-3, with an average increase of 32.7 times compared to the control and no significant differences between them.
Photosynthetic pigments (chlorophylls and carotenoids) contents are shown in Figure 4A,B. No significant differences in total chlorophyll content or carotenoid concentration were observed in any of the treatments compared to the control.

3.2. Soil Analysis

Figure 5 shows the evolution of the dissolved Zn concentration in soil pore water as a function of time since Zn fertilization. The highest Zn concentration values were observed in the first two extractions, at 5- and 18-days AP, in all treatments, showing significant differences compared to the Zn concentration in soil pore water on days 26, 33, 40, and 47 AP (except for the ZnO-3 treatment on day 5, which showed very low Zn concentration). In the day 5 extraction, significant differences were observed between the ZnO-1, ZnO-2, and ZnO-4 treatments and the control, but not between the three treatments. In the day 18 AP extraction, there were significant differences between the ZnO-1 and ZnO-2 treatments and both the control and the ZnO-3, but not between the two treatments. In the remaining extractions, there was an increase in Zn concentration in all treatments compared to the control, with no significant differences among the Zn treatments.
Plant-bioavailable (LMOWAs-method) Zn concentration values measured in soil after the harvest of the lettuce are shown in Figure 6. All the treatments showed higher Zn concentrations compared to the control treatment. Significant differences were observed among the treatments, specifically, between the ZnO-4 treatment (17.18 mg kg⁻¹) and the ZnO-1 and ZnO-3 treatments (28.93 and 30.63 mg kg⁻¹, respectively).

4. Discussion

Lettuce (Lactuca sativa L.) is a crop that shows a positive response to the addition of Zn fertilizers [52,53,54]. However, our results showed that the application of this Zn rate in the form of synthesized ZnO NPs for this acidic- and Zn-deficient soil, had no effect on lettuce yield. Our results are not in line with those reported by Song [54], who observed a maximum increase in the fresh weight of lettuce leaves grown in an acidic soil (pH 5.5) of 65.9 g above the control with a 20 mg kg−1 commercial NPs ZnO treatment.
Our results showed a positive effect of Zn NPs application on morphometric parameters or on Zn biofortification. All Zn treatments exhibited significantly higher stem diameters and total and soluble Zn concentrations in leaves than the control treatment. Our results are in line with those reported by other authors who indicated increases in stem diameter with the application of an adequate dose of the nutrients N, P, K [55], or Zn [56,57]. An increased stem diameter indicates adequate plant growth. Hong et al. [55] reported that increases in N, P, or K doses above the optimum dose resulted in a decrease in stem diameter. On the other hand, Lima et al. [56] reported an increase in plant diameter with an application between 0.3 and 2.4 mg Zn L−1 in the form of ZnSO4 in hydroponic cultivation. Also, Graciano et al. [57] reported increases in the head and stem diameters of lettuces foliar fertilized with Zn in the form of ZnSO4, up to a dose of 800 g Zn ha−1.
Marschner [58] reported that the critical Zn concentrations in leaves are below 15–20 mg kg−1 Zn (dw). Different crops exhibit varying sensitivities to Zn deficiency, with lettuce being moderately sensitive [59]. In our study, all treatments exceeded this Zn range, including the control. In addition, all Zn treatments showed an increase in leaf (mature and young) Zn concentration, which indicates that the application of ZnO NPs leads to a biofortification of the crop in this nutrient. In this aspect, the effect of ZnO-1, followed by ZnO-3 and ZnO-2, NPs on Zn concentration in mature leaves was remarkable. However, in young leaves, all Zn treatments showed the same effect. Values of Zn concentration in lettuce leaves without toxic effects, published in other studies, show a high variability, influenced by the varieties and growing conditions tested [15,16,18,21,52,54,57,60,61].
On the other hand, in our study, an increase in Zn concentration was observed in mature leaves compared to the concentration in young leaves. This ratio between the Zn concentrations of mature leaves and young leaves depended on the treatment used, and ranged from 1.5 (control) to 5.7 (ZnO-3 and ZnO-4), 6.3 (ZnO-2), and 6.5 (ZnO-1). These results are in agreement with those reported by Longnecker and Robson [61] and Ortiz et al. [18], which indicated that when a high concentration of available Zn is applied, it tends to accumulate in the more mature leaves of the plants, increasing the concentration in these older leaves compared to the younger leaves. Ortiz et al. [18] reported a 1.52-fold increase in Zn concentration in the mature leaf relative to the Zn concentration in the young leaf with a Zn-glycine treatment in an alkaline Mediterranean soil. Our high ratio values can be attributed to the soil type, as the response of lettuce crops to Zn application varies according to the type of soil in which they are grown [15]. In the study carried out by Ortiz [18], it was found that in alkaline Mediterranean soils (pH 8.3), Zn treatments did not reach such high levels in lettuce mature leaves. Fontes et al. [21] reported that in a study conducted in pots with acidic soil (pH 5.3) and different doses of ZnSO4 (2.0, 6.0, 18.0 and 36.0 mg dm−3), a 3-fold increase in Zn concentration was observed in mature lettuce leaves compared to young leaves.
The soluble Zn concentration in a plant is a reliable indicator of its nutritional status [62]. However, the assimilability of this nutrient in humans depends on the presence of other substances, which can reduce it by combining with Zn in the intestinal lumen to form a non-absorbable complex [63]. In our study, the highest soluble Zn concentrations were obtained with ZnO-1, ZnO-2, and ZnO-3, with no significant differences among them. This concentration of soluble Zn in mature leaves ranged from 6.3% (control) to 18.4% (average for ZnO-1, ZnO-2, and ZnO-3) of the total Zn extracted in mature leaves.
Additionally, the photosynthetic pigments content of plants is a representative indicator of their physiological state [64]. A reduction in photosynthetic pigments (chlorophyll and carotenoids) affects photosynthesis and the electron transport chain. Zinc deficiency significantly reduces photosynthetic pigments; therefore, it is essential to maintain adequate levels [65]. In our study, the chlorophyll content in lettuce did not show significant differences among the treatments. These results are not in line with those reported by different authors. Balashouri et al. [66] obtained higher amounts of chlorophyll with Zn fertilization, since Zn acts as a cofactor for the normal development of pigment biosynthesis. Landa [67] also reported positive effects of metal NPs on photosynthetic pigments. In this line, Song [54] stated that the use of ZnO NPs in lettuce increases the chlorophyll content with respect to the control but independently of the dose.
Regarding dissolved Zn concentrations in the soil pore water, it was observed that all treatments showed a maximum Zn availability over time, at 18 days AP, followed by a decrease in dissolved Zn. This pattern suggests that the application of ZnO NPs initially increases the concentration of dissolved Zn concentrations in the soil pore water, peaking in the following days, and then decreasing significantly to stabilize. This reduction may be due to the retention of Zn in more insoluble forms in the soil. Furthermore, correlating bioavailable Zn concentrations in soil with soluble Zn concentration in mature leaves at two key points in the experiment, at 18 days AP, when the highest concentrations was observed, and at 47 days AP, at the end of the pot experiment, several findings can be affirmed (Table 2). On the one hand, the soluble Zn concentration in mature leaves correlated positively and significantly with both the soil pore water Zn concentration obtained at 18 days AP (maximum pore water Zn concentration) and the available Zn concentration estimated using the weak extractant (LMWOAs). This suggests that the plant absorbs higher amounts of Zn when there is a peak concentration in the soil (18 days AP), which is reflected in its leaf content. Also, these results indicate that both methods correctly predict the concentration of soluble Zn in the crop. According to Almendros et al. [36], weak extractants demonstrate higher predictive capabilities for Zn concentration in various plant parts compared to stronger extractants.
On the other hand, there was a higher correlation between bioavailable Zn concentration (LMWOAs) and soil pore water concentration at 47 days AP than at 18 days AP. This is explained by the fact that the soil was analyzed at the end of the experiment, which makes the soil Zn status estimated with both methods more comparable at the same time of the trial.

5. Conclusions

The ZnO NPs used in this experiment, with mean particle sizes (TEM) of 76–104 nm, have a positive effect on morphometric parameters such as stem thickness and the Zn biofortification of the crop. The different physico-chemical characteristics of the synthesized nanoparticles influence the efficacy of the treatments. Nanoparticles characterized by their lower particle size (TEM) and hydrodynamic diameter (DLS) values (ZnO-1 and ZnO-3) provide the highest concentrations of bioavailable Zn and Zn in the pore water at the end of the cultivation time. This has a positive effect on the levels of biofortification (both in mature and young leaves) and the leaf-soluble Zn concentration. Particles with a larger particle size (ZnO-2) or with a higher hydrodynamic size (ZnO-4) produce a rapid dissolution of Zn and a low concentration of available Zn in the soil at the end of the crop. The larger hydrodynamic size of the NPs has a negative effect on the concentration of soluble Zn in leaves, showing a clear difficulty of Zn from these larger particles to be absorbed by the plant. The difference in Zn concentration between mature leaves and young leaves indicates a limited mobility of the element in the plant, with more Zn tending to accumulate in the more mature leaves of lettuce.

Author Contributions

Conceptualization, M.d.F., P.A. and L.D.; methodology, M.d.F., P.A. and L.D.; validation, M.d.F., P.A. and L.D.; formal analysis, M.d.F. and P.A.; investigation, M.d.F., L.D., P.F.-S., A.R., S.Á.-T. and P.A.; resources, P.A. and L.D.; data curation, M.d.F., L.D., A.R., S.Á.-T. and P.A.; writing—original draft preparation, M.d.F.; writing—review and editing, M.d.F., P.F.-S., L.D., A.R., S.Á.-T. and P.A.; supervision, P.A. and L.D.; funding acquisition, P.A. and L.D. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Comunidad de Madrid through the call Research Grants for Young Investigators from Universidad Politécnica de Madrid (project: ECOnanoZn, reference APOYO-JOVENES-21-FUF0C0-61-VOXTPR). The work performed at CERES (Chemical Engineering and Renewable Resources for Sustainability) was supported by the European Regional Development Fund (ERDF), through COMPETE 2020—Operational Programme for Competitiveness and Internationalization, combined with Portuguese National Funds, through Fundação para a Ciência e Tecnologia, I.P. [POCI-01-0145-FEDER-006910; UID/EQU/00102/2020].

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Particle size distribution measured by transmission electron microscopy (TEM).
Figure 1. Particle size distribution measured by transmission electron microscopy (TEM).
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Figure 2. Lettuce production. (A) Total fresh matter. (B) Stem diameter. Statistical differences at p ≤ 5% (LSD test) are indicated by different letters. * indicates significance at the 5% level. The vertical line at each of the data represents the standard deviation from the mean.
Figure 2. Lettuce production. (A) Total fresh matter. (B) Stem diameter. Statistical differences at p ≤ 5% (LSD test) are indicated by different letters. * indicates significance at the 5% level. The vertical line at each of the data represents the standard deviation from the mean.
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Figure 3. Zn biofortification of lettuce. (A) Total Zn concentration in young leaves. (B) Total Zn concentration in mature leaves. (C) Mature leaves’ soluble Zn concentration. Statistical differences at p ≤ 5% (LSD test) are indicated by different letters. *** and ** indicates significance at the 0.01 and 0.1% levels. The vertical line at each of the data represents the standard deviation from the mean.
Figure 3. Zn biofortification of lettuce. (A) Total Zn concentration in young leaves. (B) Total Zn concentration in mature leaves. (C) Mature leaves’ soluble Zn concentration. Statistical differences at p ≤ 5% (LSD test) are indicated by different letters. *** and ** indicates significance at the 0.01 and 0.1% levels. The vertical line at each of the data represents the standard deviation from the mean.
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Figure 4. Total photosynthetic pigments contents in fresh weight. (A) Chlorophylls. (B) Carotenoids. The vertical line at each of the data represents the standard deviation from the mean.
Figure 4. Total photosynthetic pigments contents in fresh weight. (A) Chlorophylls. (B) Carotenoids. The vertical line at each of the data represents the standard deviation from the mean.
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Figure 5. Evolution of dissolved Zn concentration in the soil pore water. DAF: days after fertilization. Statistical differences at p ≤ 5% (LSD test) are indicated by different letters. Capital letters indicate differences between treatments for the same time, and lower-case letters indicate differences in time for the same source. ** and * indicate significance at the 0.1 and 5% levels.
Figure 5. Evolution of dissolved Zn concentration in the soil pore water. DAF: days after fertilization. Statistical differences at p ≤ 5% (LSD test) are indicated by different letters. Capital letters indicate differences between treatments for the same time, and lower-case letters indicate differences in time for the same source. ** and * indicate significance at the 0.1 and 5% levels.
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Figure 6. Soil bioavailable Zn concentration (LMWOAs). Statistical differences at p ≤ 5% (LSD test) are indicated by different letters. *** indicates significance at the 0.01% level. The vertical line at each of the data represents the standard deviation from the mean.
Figure 6. Soil bioavailable Zn concentration (LMWOAs). Statistical differences at p ≤ 5% (LSD test) are indicated by different letters. *** indicates significance at the 0.01% level. The vertical line at each of the data represents the standard deviation from the mean.
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Table 1. Main characteristics and physico-chemical properties of ZnO nanoparticles.
Table 1. Main characteristics and physico-chemical properties of ZnO nanoparticles.
ZnO-1ZnO-2ZnO-3ZnO-4
Synthesis method co-precipitation [40]co-precipitation [41]sol–gel [42]sol–gel [43]
Metal precursorZn(NO3)2ZnSO4ZnC4H6O4ZnSO4
Hydroxyl group sourceNaOHNH4OHNaOHNaOH
Hydrodynamic diameter by DLS (nm)312.4 ± 7.4-183.3 ± 1.1573.2 ± 10.0
TEM particle size (nm)76 ± 23104 ± 2483 ± 1795 ± 18
Zeta potential (mV)6.3 ± 0.5−7.4 ± 5.711.4 ± 0.916.5 ± 3.5
Table 2. Linear correlation coefficient (r) for relationships between bioavailable Zn concentration, soluble Zn concentration in mature leaves and Zn concentration in soil pore water (18 and 47 days AP).
Table 2. Linear correlation coefficient (r) for relationships between bioavailable Zn concentration, soluble Zn concentration in mature leaves and Zn concentration in soil pore water (18 and 47 days AP).
Bioavailable Zn Concentration (LMWOAs)Soluble Zn Concentration in Mature Leaves
Zn concentrations in the soil pore water (18 days AP)0.533 *0.598 *
Zn concentrations in the soil pore water (47 days AP)0.722 *NS
Bioavailable Zn concentration (LMWOAs)-0.808 **
** and *: significant at 0.1 and 5% levels; NS: no significant.
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Francisco, M.d.; Fernandes-Silva, P.; Durães, L.; Romeiro, A.; Álvarez-Torrellas, S.; Almendros, P. Influence of the Application of Different Zinc Oxide Nanoparticles on a Lettuce Crop Grown in an Acidic Mediterranean Soil. Horticulturae 2024, 10, 681. https://doi.org/10.3390/horticulturae10070681

AMA Style

Francisco Md, Fernandes-Silva P, Durães L, Romeiro A, Álvarez-Torrellas S, Almendros P. Influence of the Application of Different Zinc Oxide Nanoparticles on a Lettuce Crop Grown in an Acidic Mediterranean Soil. Horticulturae. 2024; 10(7):681. https://doi.org/10.3390/horticulturae10070681

Chicago/Turabian Style

Francisco, Marina de, Pedro Fernandes-Silva, Luisa Durães, Andreia Romeiro, Silvia Álvarez-Torrellas, and Patricia Almendros. 2024. "Influence of the Application of Different Zinc Oxide Nanoparticles on a Lettuce Crop Grown in an Acidic Mediterranean Soil" Horticulturae 10, no. 7: 681. https://doi.org/10.3390/horticulturae10070681

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