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Article

Can Zinc Oxide Nanoparticles Alleviate the Adverse Effects of Salinity Stress in Coffea arabica?

by
Jegnes Benjamín Meléndez-Mori
1,2,
Yoiner K. Lapiz-Culqui
1,
Eyner Huaman-Huaman
1,
Marileydi Zuta-Puscan
1 and
Manuel Oliva-Cruz
1,*
1
Research Group on Stress Biochemistry in Plants, Instituto de Investigación para el Desarrollo Sustentable de Ceja de Selva (INDES-CES), Universidad Nacional Toribio Rodríguez de Mendoza (UNTRM), Chachapoyas 01001, Peru
2
Kasvaa Group SAC, Research & Development Department, Chachapoyas 01001, Peru
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(5), 1239; https://doi.org/10.3390/agronomy15051239
Submission received: 20 April 2025 / Revised: 15 May 2025 / Accepted: 18 May 2025 / Published: 20 May 2025
(This article belongs to the Special Issue Regulation of Nanomaterials in Crop Growth and Physiology Under Abiotic Stress)
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Abstract

:
Salinity is one of the main limiting factors for agricultural production worldwide. Nanotechnology has emerged as a possible tool to improve plant tolerance to salt stress. However, the application of zinc oxide (ZnO) nanoparticles in agriculture raises questions about their safety and long-term impact. The objective of this study was to investigate the effects of foliar application of ZnO nanoparticles on the physiology and defense systems of coffee plants in the presence/absence of NaCl (150 mM). A foliar spray of ZnO-NPs (0, 50, and 100 mg L−1) was applied to coffee plants individually and in combination with simulated stress conditions. The results showed that the application of ZnO-NPs to plants under salt stress had both positive and negative effects. An increase in proline content ranging from 33% to 77% was detected in stressed plants treated with ZnO-NPs, in contrast to stressed plants that did not receive the application. CAT activity increased by 69.4% to 152.8% with the application of ZnO-NPs compared to plants under salt stress that did not receive the treatment. Additionally, the application of ZnO-NPs decreased H2O2 levels by up to 18.7% with respect to the control group. On the other hand, 45% higher Na+ accumulation was observed in NaCl-stressed seedlings treated with ZnO-NPs (50 mg L−1). MDA levels in stressed plants treated with ZnO-NPs increased by 3% to 50%. Furthermore, the combined effect of ZnO-NP (100 mg L−1) and salt resulted in a significant reduction in carotenoids, limiting their photoprotective function. The results obtained indicate the complex interaction between the application of ZnO-NPs and various physiological processes in coffee plants, including photosynthesis, antioxidant enzyme activity, and the generation of reactive oxygen species. This phenomenon requires detailed analysis to fully understand the response of coffee plants to ZnO-NPs’ application.

1. Introduction

Coffee (Coffea arabica L.) is a key and transcendental crop for more than 50 countries in what is known as the “coffee belt”, due to its influence in economic indicators and its participation in the social, cultural, institutional, and political structure [1,2].
In 2022/2023, global production was estimated to reach 168.2 million bags (60 kg bags), representing a stagnant growth rate of 0.1% compared to the 2021/2022 coffee year and 1.5% less compared to 2020/2021 [3]. These figures show that coffee cultivation depends on a series of climatic factors, in addition to location, soil types, shade, and management practices. As shown, coffee production has experienced losses in recent years due to diseases, pests, droughts, heat stress, and salinity [4,5]. Abiotic stresses significantly influence plant physiology at different levels (molecular, biochemical, and morphological), which can result in damage and losses in agricultural production [6]. Although the response to these stresses is a relevant topic for coffee cultivation, its research has been less exhaustive compared to other topics. However, since 2006, work has been done to understand these mechanisms in coffee and to develop strategies to reduce their impact [7].
Salinity is one of the most significant abiotic stress factors limiting plant growth and crop production [8,9]. Ionic toxicity, nutrient uptake imbalance and osmotic stress are some of the ways in which salt stress can negatively affect plant growth and development, due to excessive mineral (Na and Cl) accumulation in shoots [10]. Thus, the expansion of coffee cultivation to new areas with the presence of salt pans could significantly affect the characteristics of the plant [11]. In coffee plants, salinity causes structural damage to mesophyll cells and alters the organization of pectins, hemicelluloses, and lignin composition in the cell wall [12]. In addition, high salt concentrations inhibit growth, manifesting in reduced leaf area, plant height, and stomata number [13]. Despite the paucity of studies of this effect on coffee, salinization could accelerate with climate change as water availability changes [14]. Therefore, further research is crucial to understand the mechanisms of coffee’s response to salinity and to develop early solutions.
To combat the effects of salt stress, it is essential to develop tolerant crops, especially in places where agricultural land is exposed to salt stress [15]. In this framework, the key agronomic characteristics to be prioritized for sustainable coffee crop are resistance to pathogens and pests, high yield, superior cup quality (including low caffeine content), and tolerance to drought and salinity [8].
Declining crop production due to abiotic and biotic stresses, as well as climate change, has been a significant concern over the years. Consequently, production processes exert a significant impact on both land and socioeconomic systems. Recently, nanotechnology has proven to be an accepted and practical tool to increase crop yields and ensure plant sustainability [16]. In the agricultural field, the use of nanoparticles in plants has been reported to control the detrimental impacts of environmental stress and plant adaptation processes, in addition to regulating seed germination [17,18], growth, and other positive benefits [19]. In this context, ZnO nanoparticle treatment has been widely used in agriculture to improve agricultural production, as it can offer effective solutions to a variety of agricultural problems due to its active role in the interaction between atomic or molecular structures [20,21]. ZnO nanoparticles have been shown to significantly improve the growth attributes of Vigna radiata and Cicer arietinum [22]. ZnO nanoparticles have been shown to enhance chlorophyll, carotenoids, and proteins levels, as well as the efficiency of antioxidant enzyme [23], and to regulate photosynthetic processes and the formation of active oxygen such as superoxide and hydroxide anion [24].
However, it is important to note that ZnO nanoparticles can also have adverse effects on plant growth and metabolism. These responses are conditioned by the characteristics and anatomy of the host plant, factors that modulate the uptake, accumulation, and translocation of ZnO nanoparticles [25]. In other words, there is a knowledge gap regarding the effects of excessive use of zinc nano-agrochemicals, as they can potentially be toxic and decrease crop productivity.
Understanding how coffee plants respond to biotic and abiotic stress factors is essential to maintain the sustainability of coffee farming. This study aimed to determine the effects of foliar application of ZnO nanoparticles on the physiological and antioxidant system of coffee seedlings in the presence/absence of NaCl. It was hypothesized that the application of ZnO nanoparticles could improve salinity tolerance by increasing chlorophyll content, improving mineral nutrition and strengthening plant antioxidant mechanisms.

2. Materials and Methods

2.1. Experimental Site and Materials

This study took place from May to October 2024 at the facilities of the Laboratory of Research in Plant Physiology and Biotechnology of the Universidad Nacional Toribio Rodríguez de Mendoza (Latitude: −6.2331027 and Longitude: −77.8514271; Amazonas–Peru). The coffee seeds of the Geisha variety (viability 94%) were provided by ROMANS PERU SAC, a company based in the district of Chirinos (Cajamarca, Peru) and made up of coffee-growing families from that district. Zinc oxide nanoparticles (ZnO-NPs) were purchased from Sigma-Aldrich (product number 721077) and had the following technical specifications: concentration 20 wt. % in H2O, particle size <100 nm (TEM), avg. part. size ≤40 nm (APS), pH 7.5 ± 1.5, and density 1.7 g/mL ± 0.1 g/mL at 25 °C).

2.2. Experiment Set-Up and Experimental Design

Seeds were subjected to surface disinfection with Benomyl 50% WP (2 g L−1) for 5 min and then rinsed with distilled water. The seeds were then hydrated for 24 h and allowed to germinate under natural conditions in washed river sand. After 60 days, the seedlings were transplanted into pots filled with substrate based on agricultural soil, peat, and sand in a 4:2:2 ratio (Supplementary Table S1). The plants were watered twice a week with Hoagland solution [26]. After 60 days of transplanting, two levels of salt (NaCl 0, 150 mM) were applied. The pot was watered with saline water regularly twice a week until the end of the experiment. Plants were grown under natural photoperiod conditions in a greenhouse with 50% shade (mean photon flux density: 1052.75 µmol m2 s−1). During the experiment, the average temperature was 20 °C and the average humidity was 80%. The concentration of NaCl 150 mM was added in 75 mM parts to avoid salt shock. Two foliar sprays of ZnO-NPs at different concentrations (0, 50, 100 mg L−1) were performed: the first one 10 days after the beginning of irrigation with saline solution, and the second 15 days after the first application of ZnO-NPs.
The salt stress experiment comprised the following treatments: (i) control (double distilled water), (ii) NaCl (150 mM), (iii) ZnO-NPs (50 mg L−1), (iv) ZnO-NPs (100 mg L−1), (v) ZnO-NPs (50 mg L−1) + NaCl (150 mM), and (vi) ZnO-NPs (100 mg L−1) + NaCl (150 mM). The experiment was conducted under a completely randomized experimental design (CRD), the number of replicates for each treatment was 10 plants, and sampling was performed 30 days after the treatment. The assignment of treatments to pots was performed completely randomly using the sample() function of the R software (version 4.3.0). To mitigate any effects derived from position, the pots were also randomly redistributed in the experimental space.

2.3. Relative Chlorophyll Content and Stomatal Conductance

Stomatic conductance (mmol/m2S) was measured with a leaf porometer SC-1 (Decagon Devices, Pullman, WA, USA). Chlorophyll content index (SPAD) was determined using a chlorophyll meter SPAD-502 (Konica Minolta, Tokyo, Japan). Data collection was performed on all plants in the experiment. Two apical leaves per plant were selected, and the average value of the measurements obtained 30 days after the onset of stress was recorded.

2.4. Determination of Photosynthetic Pigments

Photosynthetic pigments were determined in 0.2 g of fresh leaves immediately isolated from the plant. The samples were crushed with a mortar placed on an ice bed using 0.2 g of magnesium carbonate and 6 mL of ice-cold 80% acetone. The process was carried out under indirect light in a Protector Basic 47 fume hood (Labconco Corp., Kansas City, MO, USA). The homogenate was transferred into amber Eppendorf tubes and centrifuged for 10 min at 2800 rpm (12 °C). Then, 2 mL of the supernatant was taken, and the absorbance values were read at 663.2, 646.8, and 470 nm in quartz cuvettes using a Genesys 180 UV/Vis spectrophotometer (Thermo Scientific™, Madison, WI, USA). Photosynthetic pigment concentrations were calculated according to the equations proposed by Lichtenthaler and Buschmann [27], and were expressed as µg g−1 FW:
  • Chl-a (µg/mL): 12.25 A663.2 − 2.79 A646.8
  • Chl-b (µg/mL): 21.50 A646.8 − 5.10 A663.2
  • Carotenoids (µg/mL): (1000 A470 − 1.82 Chl-a − 85.02 Chl-b)/198

2.5. Na+ and Nutrients Content

The dried samples (0.2 g) were digested in a nitric-chloride acid solution [28]. Subsequently, the solution was filtered and volumetrically made up to 25 mL with ultrapure water. Quantification of macronutrients (P, K, Ca, Mg), micronutrients (Fe, Zn, Cu, Mn), and Na was performed using an Agilent 4100 Microwave Plasma-Atomic Emission Spectrometer (MP-AES) (Agilent Technologies, Santa Clara, CA, USA). The results are expressed in µg g−1 DW and were obtained by interpolation on calibration curves constructed with standard solutions of various concentrations. Total C and N contents were determined in 0.2 g of sample through a LECO CN928 combustion analyzer (LECO Corp., St Joseph, MI, USA).

2.6. Determination of Proline Content

To determine the proline content in the tissues the ninhydrin reaction method was followed [29]. For this purpose, 200 mg of tissue was homogenized with 2 mL of 3% aqueous sulfosalicylic acid and centrifuged at 10,000 rpm for 30 min at 4 °C. Then, 1 mL of the supernatant was removed and mixed with 1 mL of 0.1 M acid ninhydrin and 1 mL of 99.9% glacial acetic acid (warm) in amber falcon tubes. The reaction mixture was incubated at 90 °C for 60 min and cooled rapidly in an ice bath to stabilize the reaction. For the final chromophore extraction, 3 mL of toluene was added. The absorbance of the obtained upper phase was measured at 520 nm, using toluene as a blank, in a Genesys 180 UV/Vis spectrophotometer (Thermo Scientific™, Madison, WI, USA). Proline content was calculated using a standard L-proline curve and expressed as μg g−1 FW.

2.7. Lipid Peroxidation

Lipid peroxidation was quantified through the measurement of malondialdehyde (MDA) using the 2-thiobarbituric acid (TBA) assay, following the methodology of Velikova et al. [30], with minor modifications. Fresh leaves or roots (200 mg) were homogenized with 2 mL of 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 14,000 rpm for 10 min. Then, 1 mL of the supernatant was mixed with 2 mL of 0.5% thiobarbituric acid solution (TBA) in 20% TCA. Subsequently, the mixture was incubated at 90 °C for 30 min and cooled sharply in an ice bath. Once cooled, the sample was centrifuged at 14,000 rpm for 5 min. Absorbance (optical density, OD) readings of the supernatant were taken at 450, 532, and 600 nm on a Genesys 180 UV/Vis spectrophotometer (Thermo Scientific™, Madison, WI, USA). The MDA content was calculated by the following formula [31]: MDA (µmol g−1 FW) = [6.45 × (OD532 − OD600) – 0.56 × OD450] × V/W, where V: volume of extraction, W: fresh weight of sample.

2.8. Hydrogen Peroxide (H2O2)

Quantification of H2O2 content was performed spectrophotometrically by reaction with potassium iodide (KI) according to the method of Loreto and Velikova [32]. Tissue (200 mg) was homogenized on an ice bath with 2 mL of 0.1% (w/v) trichloroacetic acid (TCA). After centrifugation at 12,000 rpm for 15 min, 1 mL of the supernatant was taken and mixed with 1 mL of 10 mM potassium phosphate buffer (pH 7.0) and 2 mL of 1 M potassium iodide (KI). The reaction mixture was incubated for 60 min at room temperature, and the absorbance was measured at 390 nm on a Genesys 180 UV/Vis spectrophotometer (Thermo Scientific™, Madison, WI, USA). The H2O2 content was determined using a standard calibration curve prepared with known concentrations of H2O2.

2.9. Determination of Antioxidant Enzyme Activity

Fresh leaves and roots (0.5 g) were ground using liquid nitrogen and homogenized with 5 mL of potassium phosphate buffer (10 mM, pH 7.0) with 4% (w/v) polyvinylpyrrolidone. The resulting mixture was centrifuged (12,000 rpm) at 4 °C for 20 min and the supernatant was obtained for the determination of catalase (CAT).
CAT enzyme activity was quantified using the method of Kato and Shimizu [33]. The 3 mL CAT assay mixture contained potassium phosphate buffer (10 mM, pH 7.0), 0.1 mL of enzyme extract, and 0.035 mL of 3% H2O2 (freshly prepared and stored at 4 °C). A decrease in absorbance was recorded at 240 nm. Enzyme activity was calculated using the extinction coefficient of H2O2 (40 mM−1 cm−1 at 240 nm) and is reported as μmol of H2O2 oxidized per minute per gram of FW.

2.10. Data Analysis

All data collected were tabulated in Excel spreadsheets, and processed and analyzed by analysis of variance (ANOVA) and using a comparison of means test (Tukey, p ≤ 0.05). Additionally, a Pearson’s correlation test was performed on the variables. R software v. 4.4.2 was used to perform this analysis.

3. Results

3.1. Relative Chlorophyll Content and Stomatal Conductance

The SPAD responses and stomatal conductance of coffee seedlings treated with foliar application of ZnO-NPs in the presence/absence of NaCl are shown in Figure 1. It is observed that the foliar application of ZnO-NPs at a concentration of 50 mg L−1 increased (p ≤ 0.05) the SPAD index only compared to the 100 mg L−1 ZnO-NP treatment (Figure 1a), while the SPAD results did not experience significant changes in the other treatments. On the other hand, the results show that ZnO-NP spraying (in the presence/absence of NaCl) significantly (p ≤ 0.05) decreased stomatal conductance compared to control plants (Figure 1b).

3.2. Determination of Photosynthetic Pigments

Figure 2 shows the effect of ZnO nanoparticles on the photosynthetic pigment content of coffee seedlings in the presence/absence of NaCl. The Chl-a content presented a significant increase (p ≤ 0.05) when coffee seedlings were sprayed with the highest concentration of ZnO-NPs (100 mg L−1), showing a value of 940.57 ± 20.60 µg g−1 FW. Under salt stress, the contents were significantly reduced, showing values between 856 and 905 µg g−1 FW (Figure 2a). It is observed that seedlings treated with 150 mM NaCl presented a marked reduction in Chl-b concentration (438.40 ± 22.92 µg g−1 FW); however, its level was largely enhanced (964.99 ± 34.95 µg g−1 FW) when seedlings received an exogenous administration of ZnO-NPs at a concentration of 100 mg L−1 (Figure 2b). Additionally, it was determined that total chlorophyll content was significantly higher (1870.88 ± 59.84 μg g−1 FW) in salinity-stressed plants treated with ZnO-NPs at 100 mg L−1 (Figure 2c), mainly explained by the high level of Chl-b recorded in this experimental group. The highest carotenoid concentration was observed in seedlings that were treated with ZnO-NPs (50 mg L−1) + NaCl, and then those treated with NaCl alone, exhibiting values of 318.14 ± 11.09 and 309.33 ± 11.17 μg g−1 FW, respectively (Figure 2d).
Regarding the ratio between Chl-a and Chl-b, we observed that Chl a/b values were significantly higher in seedlings treated with 150 mM NaCl (1.95 ± 0.10) (Figure 2e). On the other hand, it was observed that the ratio of total chlorophyll to carotenoids was significantly higher in seedlings treated with ZnO-NPs (100 mg L−1) + NaCl, with a value of 15.58 ± 1.36 (Figure 2f). The lowest Chl a+b/carotenoid ratio was recorded in seedlings treated with 150 mM NaCl (4.19 ± 0.29).

3.3. Na+ and Nutrients Content

Figure 3a shows significant differences (p ≤ 0.05) in Na+ levels in leaves and roots according to the applied treatments. In leaves, it was observed that seedlings under NaCl stress and treated with ZnO-NPs (50 mg L−1) accumulated 45% more Na+ than those under NaCl stress but without nanoparticle treatment (6902.23 ± 55.74 µg g−1 DW). Overall, Na+ accumulation in all treatment increased by 27% (NaCl 150 mM + ZnO-NPs 100 mg L−1) to 100% (NaCl 150 mM + ZnO-NPs 50 mg L−1) compared to the control group (5035.82 ± 29.78 µg g−1 DW). In roots, the application of ZnO-NPs increased the Na+ content in a range from 24% (ZnO-NPs 50 mg L−1) to 180% (NaCl 150 mM + ZnO-NPs 100 mg L−1) compared to the control (2422.4 ± 38.52 µg g−1 DW).
Figure 3b shows that K+ concentration in leaves was significantly higher (18%) in seedlings treated only with 50 mg L−1 of ZnO-NPs (9509.99 ± 73.67 µg g−1 DW) compared to the control group (8046.62 ± 10.08 µg g−1 DW). The K+ determined in the root of ZnO-NP-treated seedlings (in the presence/absence of NaCl) exceeded by at least 8% the K+ content determined in control seedlings (6584.21 ± 54.38 µg g−1 DW) and those grown under salt stress without ZnO-NP application (6327.35 ± 52.27 µg g−1 DW).
The Na+/K+ ratio in leaves showed a considerable increase both in plants grown in the presence of salinity and treated with ZnO-NPs, and in those treated with the highest concentration of ZnO-NPs (100 mg L−1) without salt stress (Figure 3c), because of the reduction in K+ content (Figure 3b). At the root level, an increase in the Na+/K+ ratio was observed both in the NaCl (150 mM) + ZnO-NPs (100 mg L−1) treatment and in plants under salt stress without ZnO-NP application.
Figure 3d,e show that Mg2+ and Ca2+ concentrations in leaves of seedlings stressed with NaCl and treated with ZnO-NPs (50 mg L−1) increased significantly (75% for Mg2+ and 35% for Ca2+) compared to seedlings stressed only with NaCl, and even more (103% for Mg2+ and 61% for Ca2+) when compared to control seedlings. At the root level, seedlings treated with ZnO-NPs (100 mg L−1) under salt stress (NaCl) accumulated the highest concentrations of Mg2+ and Ca2+.
Figure 3f shows a significant increase in leaf Zn2+ concentration after ZnO-NP application, with increases ranging from 2466% to 3108% compared to treatments without nanoparticles. This high accumulation in leaf tissue could trigger toxicity symptoms such as chlorosis (yellowing) and necrosis (tissue death), which represents a major challenge. In roots, application of the highest dose of nanoparticles (100 mg L−1) resulted in an increase in Zn2+ accumulation of more than 259% compared to the control.
Table 1 shows a significant reduction (p < 0.0001) in leaf P content in the ZnO-NP treatments, with a decrease of between 62% and 82% compared to the control group (152.35 ± 5.90 µg g−1 DW). In contrast, root P concentration remained between 122 to 139 µg g−1 DW without showing significant differences. Regarding C and N contents, the results show that there were no significant differences among treatments, with values ranging between 39.89 and 42.01% for C and between 2.71 and 3.33% for N.
Table 2 shows significant differences in Fe2+, Cu2+, and Mn2+ contents in leaves and roots among treatments. In leaves, Fe2+ content ranged from 104 to 140 µg g−1 DW, with the highest peak (p < 0.005) in seedlings stressed by NaCl and treated with 50 mg L−1 of ZnO-NPs. In roots, control seedlings presented the highest Fe2+ content, exceeding the other experimental groups by more than 60%. On the other hand, it was observed that leaf Cu2+ content decreased by 38.79% in plants under salt stress treated with ZnO-NPs at 100 mg L−1 compared to the control. At the root level, both the control group and the treatments with the lowest concentration of ZnO-NPs (50 mg L−1) presented Cu2+ levels more than 60% lower with respect to the other experimental groups. The average Mn2+ concentration in leaves ranged from 23 to 43 µg g−1 DW, with the lowest value observed in the control group. In roots, Mn2+ content was found to range from 9 to 14 µg g−1 DW.

3.4. Determination of Proline Content

The application of ZnO-NPs significantly altered proline concentrations in both leaves (p < 0.0001) and roots (p < 0.0001) of coffee seedlings grown in the presence/absence of NaCl (Figure 4). In leaf tissue, seedlings subjected to salt stress (NaCl) and treated with ZnO-NPs (100 mg L−1) showed a 234% increase in proline content (302.16 ± 10.115 µg g−1 FW) compared to control seedlings (90.33 ± 4.95 µg g−1 FW) (Figure 4a). In root tissue, ZnO-NP treatments increased proline levels compared to control seedlings (32.18 ± 2.39 µg g−1 FW) and those only treated with 150 mM NaCl (41.88 ± 4.55 µg g−1 FW) (Figure 4b). The proline level in the root tissue of seedlings treated with ZnO-NPs ranged from 60 to 105 μg g−1 FW.

3.5. Lipid Peroxidation

The results presented in Figure 5 show the MDA content in foliar and root tissue of coffee seedlings treated with ZnO-NPs, both in the presence and absence of NaCl. NaCl-stressed seedlings treated with ZnO-NPs (100 mg L−1) increased leaf MDA content (2.93 ± 0.25 µmol g−1 FW) by 70% compared to control plants (1.72 ± 0.07 µmol g−1 FW). However, it was observed that single spraying with ZnO-NPs (100 mg L−1) reduced MDA by 25% relative to NaCl-stressed plants without nanoparticle treatment (1.47 ± 0.14 µmol g−1 FW) (Figure 5a). In root tissue, the MDA content recorded in seedlings sprayed with ZnO-NPs (without salt stress) showed statistically similar values to that of control seedlings, with levels ranging from 0.53 to 0.66 µmol g−1 FW (Figure 5b). In addition, significantly lower MDA values were observed in seedlings subjected only to salt stress (0.07 ± 0.006 µmol g−1 FW) and in those that combined the application of ZnO-NPs at 50 mg L−1 (0.18 ± 0.04 µmol g−1 FW).

3.6. Hydrogen Peroxide (H2O2)

Figure 6 shows the H2O2 content of both leaves and roots of coffee seedlings treated with ZnO-NPs grown under in the presence/absence of NaCl. It was observed that the use of ZnO-NPs significantly reduced H2O2 levels in leaf and root tissue. At the foliar level, application of ZnO-NPs resulted in a 10–20% reduction in H2O2 levels (equivalent to a decrease of 0.7 to 1.3 nmol g−1 FW) compared to control (6.36 ± 0.44 nmol g−1 FW) and NaCl-only stressed plants (6.51 ± 0.71 nmol g−1 FW) (Figure 6a). In root tissue, the highest concentration of H2O2 (0.51 ± 0.02 nmol g−1 FW) was observed in seedlings subjected to salt stress without ZnO-NP treatment. In contrast, plants with ZnO-NP application presented H2O2 levels of between 0.13 and 0.24 nmol g−1 FW, reaching a level like that of the control group (Figure 6b).

3.7. Determination of Antioxidant Enzyme Activity

Differences in CAT activity in leaves were significant, in contrast to CAT activity in roots, which showed no statistical difference (Figure 7). The mean CAT activity in leaves of NaCl-stressed seedlings treated with ZnO-NPs (100 mg L−1) increased by 97% and 152% with respect to control and NaCl-only stressed seedlings, respectively (Figure 7a). Foliar application of ZnO-NPs at a concentration of 100 mg L−1 increased CAT activity by 86% compared to the control. The mean CAT activity in roots showed values between 1.17 and 1.42 µmol/min/g (Figure 7b).

3.8. Correlation Analysis

Figure 8 shows the linear relationships between the parameters evaluated in the experiment according to Pearson’s correlation analysis. The results revealed a moderate positive correlation between proline and MDA levels in leaves with Na concentration in roots (r = 0.695 and r = 0.601, respectively). On the other hand, a moderate negative correlation was found between H2O2 levels in leaves and Na concentration in leaves (r = −0.595). Additionally, a strong positive correlation was observed between proline and MDA levels in leaves (r = 0.799), while CAT activity in leaves presented a moderate negative correlation with H2O2 levels in leaves (r = −0.528).

4. Discussion

Several studies have examined the application of ZnO-NP as a tool to reduce salinity stress in several crops, including Mangifera indica [34], Brassica napus [35], Solanum lycopersicum [36], and Pisum sativum [37], which could be related to the enhancement of antioxidant enzyme activities, enhancement of chlorophyll pigments, maintenance of nutrient balance in the cell, and maintenance of cell membrane integrity. However, for controlled delivery of nanomaterials, a thorough understanding of their interaction with plants, both positive and negative effects, is required [38,39].
Abiotic environmental stressors, such as salinity, cause significant changes in photosynthetic elements such as enzymes, chlorophylls, and carotenoids, reducing photosynthetic efficiency and inhibiting plant growth and development [40,41]. In that sense, the results observed in this study show that high doses of ZnO-NPs (100 mg L−1) reduced the relative chlorophyll content (SPAD readings), suggesting a possible inhibitory effect. This differs from the results reported by Rossi et al. [42], who reported that foliar application of ZnO-NPs did not alter the SPAD index in coffee plants. Rossi et al. [42] supported their finding by the absence of changes in the light energy use efficiency (Fv/Fm) of plant photosystem II.
To obtain a deeper understanding of the effects of ZnO-NP application on plant physiology, quantification of photosynthetic pigments was performed. The results showed that coffee plants exposed to salt stress (150 mM NaCl) presented a significant increase in Chl a+b content when treated with 100 mg L−1 ZnO-NPs (Figure 2c), mainly due to the increase in Chl-b. This increase in Chl-b under the treatment resulted in a reduction in the Chl a/b ratio. A study in lettuce crops reported a decrease in the Chl a/b ratio in response to foliar application of ZnO-NPs [43]. Yoshihara et al. [44] reported that the decrease in the Chl a/b ratio can be explained because NPs stimulate the activity of the enzyme chlorophyll a oxygenase, which catalyzes the synthesis of Chl-b from Chl-a. Moreover, the reduction in the Chl a/b ratio and the increase in Chl-b suggest a higher ratio of PSII to PSI [45], which could imply more efficient capture and utilization of solar radiation in leaves.
Chl and Car are essential pigments that play a crucial role in both light harvesting for photosynthesis and photoprotection of photosynthetic structures against oxidative damage, especially under stress conditions. Their depletion under salt stress can severely compromise plant growth and productivity [46]. In this sense, the photosynthetic apparatus is one of the main sites of action of Zn in plants, which could explain why Zn application improved Chl content in plants [47,48]. However, the increase in the Chl a+b/Car ratio (Figure 2f) is an indicator that stressful conditions accelerated Car degradation and disrupted its photoprotective function [49], which is further supported by the high MDA content in leaves (Figure 4a). In that sense, knowing that lipid peroxidation is one of the indicators of oxidative stress, the increase in MDA in certain treatments that used ZnO-NPs could indicate a decrease in the efficiency of the antioxidant system, which is unable to cope with the increase in ROS production [50,51,52]. In our case, a positive correlation was observed between MDA content in leaves and proline content. While proline accumulation can alleviate membrane damage and reduce MDA levels [53], this mechanism, which manifests with a rapid increase in proline levels to suppress lipid peroxidation and accumulate osmoprotective agents [54], should be interpreted with caution. The contribution of proline to the prevention of lipid peroxidation varies significantly among species and phenological stages [55].
On the other hand, the results showed that coffee plants exposed only to salt stress (150 mM NaCl) presented a significant increase in hydrogen peroxide (H2O2) levels; however, this adverse effect was significantly attenuated in plants treated with ZnO-NP. These findings align with those of Mustafa et al. [37], who reported a decrease in H2O2 content in Pisum sativum plants subjected to salt stress (50 mM NaCl) and exposed to 50 ppm ZnO-NP, highlighting the impact of ZnO-NPs on this specific characteristic, particularly when combined with salt. In this context, a negative correlation between CAT activity and H2O2 concentration was expected, considering the fundamental role of CAT in H2O2 detoxification.
Salinity stress is characterized by osmotic stress, which hinders water uptake, and ionic stress, associated with the accumulation of toxic ions [56]. In this scenario, our findings indicate that upon application of ZnO-NPs along with salt stress, the foliar proline level is significantly elevated compared to plants that were exposed to salt stress alone. Under such conditions, the results suggest, at least in part, that the application of ZnO promotes the synthesis of organic osmolytes, such as proline. According to Zhang et al. [57], an increase in proline concentration is a response/adaptation reaction of plants to salinity stress, as it favors osmotic adjustment in plant cells. Furthermore, it should be emphasized that proline also plays a role in reducing stress-mediated photoinhibition and thus improves photosynthetic efficiency during stress [58].
Along with the previously cited responses, coffee plants treated with 50 mg L−1 ZnO-NPs, both in the presence and absence of NaCl, experienced greater foliar Na+ accumulation compared to plants that were only subjected to salt stress, exacerbating salt toxicity symptoms as evidenced by increased marginal leaf necrosis. Under salt stress conditions, Na+ competes with K+ for uptake; therefore, excessive Na+ accumulation can alter the Na+/K+ ratio and ionic homeostasis [59,60]. The increase in the Na+/K+ ratio generated by the accumulation of Na+ causes a lower K+ absorption and also a cytosolic K+ efflux, which can cause alterations in the biochemical metabolism and affect the normal development of the plant [61]. Therefore, the stability of the Na+/K+ ratio in the plant is an important indicator of its tolerance to salinity [62]. In this context, the results of the present study indicate that, although ZnO-NPs can mitigate some effects of salt stress, their interaction with Na+ could generate adverse effects under certain conditions. In the current study, these effects are manifested as a decrease in stomatal conductance.
In general, it has been reported in the scientific literature that nanoparticles can induce both positive and negative responses in plants, the nature of which depends primarily on the size and concentration of the nanoparticles, as well as on the plant species treated [63,64]. Along these lines, multiple studies have evidenced the significant ability of ZnO-NPs to alleviate certain biotic and abiotic stresses, in addition to the beneficial effects of ZnO-NPs on plant growth and productivity, which could be linked to the accumulation of ZnO-NPs in various plant tissues [20]. However, an inordinate use of ZnO-NPs (>150 mg L−1) can cause moderate toxicity in plants, which could result from a modification in Zn homeostasis and its impact on the uptake of other elements and mutual elemental interactions [65]. In coffee, the remarkable increase in Zn after ZnO-NP application is attributed to the adherence of the nanoparticles to the leaf surface, leading to a continuous long-term release of Zn ions [42]. Naseer et al. [66] indicate that foliar applications allow better dispersion and absorption on the leaf surface, facilitating translocation from the point of application to other parts of the plant. This sustained release can lead to Zn accumulation in phytotoxic concentrations, which are capable of compromising plant health and yield. According to Siddiqi et al. [67] and Yang et al. [68], a possible mechanism of toxicity of ZnO-NPs lies in their photocatalytic activity, which could elevate the concentration of ROS in plant cells, ultimately leading to the disruption of the cell membrane, biomolecule interactions, and cell death. On the other hand, it is important to consider that the dispersion of ZnO-NPs in the environment during their large-scale application could have effects on non-target organisms, such as beneficial insects, soil microorganisms, and other plants. This raises the need for comprehensive ecotoxicity studies to assess these risks.

5. Conclusions

The results of this study have shown that the application of ZnO-NPs can have both favorable and negative effects on coffee plants. When combined with salt stress, ZnO-NPs showed the potential to mitigate the adverse effects of salinity, which could be due to increased proline content, improved CAT activity, and reduced H2O2 levels in leaves and roots. However, adverse responses were also observed by increasing Na accumulation and MDA levels, possibly indicating toxicity of the nanoparticles. These findings highlight the importance of optimizing the dosage and application conditions of nanoparticles to maximize their benefits and minimize their adverse effects. The complex interaction between zinc oxide nanoparticles (ZnO-NPs) and salt stress in coffee plants indicates the need for more detailed studies that encompass the molecular underpinnings of plant protection mechanisms and phytohormone communication.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15051239/s1, Table S1: Chemical and physical characteristics of the substrate used for the experiment.

Author Contributions

Conceptualization, J.B.M.-M.; methodology, J.B.M.-M.; formal analysis, M.Z.-P.; investigation, Y.K.L.-C. and E.H.-H.; resources, E.H.-H.; data curation, M.Z.-P.; writing—original draft preparation, J.B.M.-M.; writing—review and editing, J.B.M.-M.; project administration, M.O.-C.; funding acquisition, M.O.-C. All authors have read and agreed to the published version of the manuscript.

Funding

The research was funded by the project SNIP No. 352439/CUI No. 2314883, “Creación de los Servicios del Centro de Investigación, Innovación y Transferencia Tecnológica de Café—CEINCAFÉ”, executed by the Research Institute for Sustainable Development of the Jungle Edge at the National University Toribio Rodríguez de Mendoza of Amazonas, Peru. The APC was funded by Vicerrectorado de Investigación of the National University Toribio Rodríguez de Mendoza of Amazonas.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

The authors wish to acknowledge the support of the Laboratorio de Investigación de Suelos y Aguas (LABISAG) in the completion of the elemental analysis of the samples.

Conflicts of Interest

Author Jegnes Benjamín Meléndez-Mori was employed by the company Kasvaa Group SAC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflicts of interest.

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Agronomy 15 01239 g001
Figure 1. Effects of ZnO nanoparticles on (a) SPAD value and (b) stomatal conductance of coffee seedlings in the presence/absence of NaCl (150 mM). The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
Figure 1. Effects of ZnO nanoparticles on (a) SPAD value and (b) stomatal conductance of coffee seedlings in the presence/absence of NaCl (150 mM). The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
Agronomy 15 01239 g001
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Figure 2. Effects of ZnO nanoparticles on photosynthetic pigments of coffee seedlings in the presence/absence of NaCl (150 mM). (a) Chlorophyll a, (b) Chlorophyll b, (c) Chlorophyll a+b, (d) Carotenoid, (e) Chl a/b ratio, and (f) Chl a+b/Carotenoid ratio. The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
Figure 2. Effects of ZnO nanoparticles on photosynthetic pigments of coffee seedlings in the presence/absence of NaCl (150 mM). (a) Chlorophyll a, (b) Chlorophyll b, (c) Chlorophyll a+b, (d) Carotenoid, (e) Chl a/b ratio, and (f) Chl a+b/Carotenoid ratio. The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
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Figure 3. Effects of ZnO nanoparticles on the content of (a) Na+, (b) K+, (c) Na+/K+ ratio, (d) Mg2+, (e) Ca2+, and (f) Zn2+ in leaves and roots of coffee seedlings under conditions of presence/absence of NaCl (150 mM). The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
Figure 3. Effects of ZnO nanoparticles on the content of (a) Na+, (b) K+, (c) Na+/K+ ratio, (d) Mg2+, (e) Ca2+, and (f) Zn2+ in leaves and roots of coffee seedlings under conditions of presence/absence of NaCl (150 mM). The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
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Figure 4. Effects of ZnO nanoparticles on proline of coffee seedlings in the presence/absence of NaCl (150 mM). (a) Leaves and (b) roots. The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
Figure 4. Effects of ZnO nanoparticles on proline of coffee seedlings in the presence/absence of NaCl (150 mM). (a) Leaves and (b) roots. The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
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Figure 5. Effects of ZnO nanoparticles on MDA content of coffee seedlings in the presence/absence of NaCl (150 mM). (a) Leaves and (b) roots. The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
Figure 5. Effects of ZnO nanoparticles on MDA content of coffee seedlings in the presence/absence of NaCl (150 mM). (a) Leaves and (b) roots. The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
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Figure 6. Effects of ZnO nanoparticles on H2O2 content of coffee seedlings in the presence/absence of NaCl (150 mM). (a) Leaves and (b) roots. The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
Figure 6. Effects of ZnO nanoparticles on H2O2 content of coffee seedlings in the presence/absence of NaCl (150 mM). (a) Leaves and (b) roots. The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
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Figure 7. Effects of ZnO nanoparticles on CAT activity of coffee seedlings in the presence/absence of NaCl (150 mM). (a) Leaves and (b) roots. The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
Figure 7. Effects of ZnO nanoparticles on CAT activity of coffee seedlings in the presence/absence of NaCl (150 mM). (a) Leaves and (b) roots. The standard deviation (±SD) is shown by the error bars. Significant differences (p ≤ 0.05) between treatments are indicated by different letters above these bars.
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Figure 8. Pearson’s correlation between the physiological and nutritional parameters evaluated in the experiment. The evaluated variables include the following: SPAD: chlorophyll index, Chl a: chlorophyll a, Chl b: chlorophyll b, Chl a+b: total chlorophyll, Chl a/b: chlorophyll a to chlorophyll b ratio, MDA: malondialdehyde, H2O2: hydrogen peroxide, CAT: catalase activity, Na: sodium, C: carbon, N: nitrogen, P: phosphorus, K: potassium, Mg: magnesium, Ca: calcium, Zn: zinc, Fe: iron, Cu: copper, Mn: manganese. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 8. Pearson’s correlation between the physiological and nutritional parameters evaluated in the experiment. The evaluated variables include the following: SPAD: chlorophyll index, Chl a: chlorophyll a, Chl b: chlorophyll b, Chl a+b: total chlorophyll, Chl a/b: chlorophyll a to chlorophyll b ratio, MDA: malondialdehyde, H2O2: hydrogen peroxide, CAT: catalase activity, Na: sodium, C: carbon, N: nitrogen, P: phosphorus, K: potassium, Mg: magnesium, Ca: calcium, Zn: zinc, Fe: iron, Cu: copper, Mn: manganese. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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Table 1. Contents of P, C, and N in coffee seedlings treated with ZnO nanoparticles, grown in the presence/absence of NaCl (150 mM).
Table 1. Contents of P, C, and N in coffee seedlings treated with ZnO nanoparticles, grown in the presence/absence of NaCl (150 mM).
TreatmentP (µg g−1 DW)C (%) N (%)
LeavesRoots
Control152.35 ± 5.90 a139.94 ± 5.61 a42.00 ± 1.60 a2.71 ± 0.60 a
NaCl (150 mM)161.5 ± 11.90 a122.34 ± 18.75 a41.15 ± 2.14 a2.83 ± 0.92 a
ZnO-NPs (50 mg L−1)57.41 ± 11.21 b131.06 ± 12.57 a41.85 ± 1.75 a3.27 ± 0.41 a
ZnO-NPs (100 mg L−1)26.00 ± 8.13 c133.52 ± 12.30 a42.01 ± 1.20 a3.33 ± 0.64 a
NaCl (150 mM) + ZnO-NPs (50 mg L−1) 48.56 ± 10.66 bc134.96 ± 20.20 a40.23 ± 3.07 a3.26 ± 0.36 a
NaCl (150 mM) + ZnO-NPs (100 mg L−1)34.84 ± 4.2 bc123.10 ± 11.32 a39.89 ± 4.03 a3.33 ± 0.85 a
CV (%)12.0310.246.0221.14
p-value<0.00010.5710.8210.753
Means within a column followed by the same letter are not significantly different according to Tukey’s test at p ≤ 0.05.
Table 2. Contents of Fe2+, Cu2+, and Mn2+ in coffee seedlings treated with ZnO nanoparticles, grown in the presence/absence of NaCl (150 mM).
Table 2. Contents of Fe2+, Cu2+, and Mn2+ in coffee seedlings treated with ZnO nanoparticles, grown in the presence/absence of NaCl (150 mM).
TreatmentFe2+ (µg g−1 DW)Cu2+ (µg g−1 DW)Mn2+ (µg g−1 DW)
LeavesRootsLeavesRootsLeavesRoots
Control104.72 ± 20.99 b384.83 ± 18.87 a16.01 ± 3.36 ab7.18 ± 0.92 b23.65 ± 3.93 b9.45 ± 1.73 b
NaCl (150 mM)107.64 ± 11.15 b239.97 ± 10.58 b19.64 ± 3.43 ab19.57 ± 2.89 a30.35 ± 4.98 ab14.06 ± 2.61 ab
ZnO-NPs (50 mg L−1)132.21 ± 8.93 ab182.19 ± 12.03 d26.65 ± 5.01 a7.34 ± 1.68 b36.08 ± 6.04 ab9.72 ± 1.75 b
ZnO-NPs (100 mg L−1)127.32 ± 7.14 ab200.10 ± 4.97 cd23.74 ± 2.77 a19.47 ± 3.30 a32.96 ± 5.13 ab14.03 ± 2.07 ab
NaCl (150 mM) + ZnO-NPs (50 mg L−1) 140.52 ± 5.56 a205.88 ± 5.36 cd27.29 ± 6.87 a7.38 ± 0.96 b43.56 ± 8.23 a10.18 ± 1.04 b
NaCl (150 mM) + ZnO-NPs (100 mg L−1)104.35 ± 3.33 b215.42 ± 10.06 bc9.80 ± 1.05 b21.85 ±3.21 a33.36 ± 2.88 ab16.17 ± 3.17 a
CV (%)9.284.7520.2817.2816.3917.7
p-value0.005<0.00010.002<0.00010.0180.010
Means within a column followed by the same letter are not significantly different according to Tukey’s test at p ≤ 0.05.
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Meléndez-Mori, J.B.; Lapiz-Culqui, Y.K.; Huaman-Huaman, E.; Zuta-Puscan, M.; Oliva-Cruz, M. Can Zinc Oxide Nanoparticles Alleviate the Adverse Effects of Salinity Stress in Coffea arabica? Agronomy 2025, 15, 1239. https://doi.org/10.3390/agronomy15051239

AMA Style

Meléndez-Mori JB, Lapiz-Culqui YK, Huaman-Huaman E, Zuta-Puscan M, Oliva-Cruz M. Can Zinc Oxide Nanoparticles Alleviate the Adverse Effects of Salinity Stress in Coffea arabica? Agronomy. 2025; 15(5):1239. https://doi.org/10.3390/agronomy15051239

Chicago/Turabian Style

Meléndez-Mori, Jegnes Benjamín, Yoiner K. Lapiz-Culqui, Eyner Huaman-Huaman, Marileydi Zuta-Puscan, and Manuel Oliva-Cruz. 2025. "Can Zinc Oxide Nanoparticles Alleviate the Adverse Effects of Salinity Stress in Coffea arabica?" Agronomy 15, no. 5: 1239. https://doi.org/10.3390/agronomy15051239

APA Style

Meléndez-Mori, J. B., Lapiz-Culqui, Y. K., Huaman-Huaman, E., Zuta-Puscan, M., & Oliva-Cruz, M. (2025). Can Zinc Oxide Nanoparticles Alleviate the Adverse Effects of Salinity Stress in Coffea arabica? Agronomy, 15(5), 1239. https://doi.org/10.3390/agronomy15051239

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