Materials and methods

‘Celebrity’ tomato (Solanum lycopersicum L.; Clifton Seed Company, Faison, NC, USA) seedlings were grown in 200-cell trays (TR200A model, Speedling, Ruskin, FL, USA) filled with a growing medium (LM-CB, Lambert Peat Moss Inc., Riviére-Ouelle, QC, Canada) in a greenhouse with supplemental fluorescent lighting (50 μmol·m⁻²·s⁻¹ photosynthetic photon flux density). The seedlings were fertigated once a week with 50 mg N·L⁻¹ using Peters Professional water-soluble fertilizer (20N–8.7P–16.6K; ICL Specialty Fertilizers, Tel Aviv, Israel) and transplanted into square plastic pots (810 cm³; BWI Companies, Inc., Nash, TX, USA) containing 100 g of LM-CB growing media 7 weeks after sowing.

Thereafter, the seedlings were placed in growth chambers (GEN1000, Conviron Ltd., Winnipeg, MB, Canada) set at 26/19 °C (16/8 h, day/night) for optimal and 38/30 °C for heat stress temperatures. Relative humidity was maintained at 60% and light intensity at 300 μmol·m⁻²·s⁻¹ PPFD. Plants were fertigated with 100 mg N·L⁻¹ of 20N–8.7P–16.6K fertilizer for the first 2 weeks, and 200 mg N·L⁻¹ for the final 2 weeks.

A completely randomized design with five plant replicates per treatment was used. Treatments included foliar sprays of nanoparticle water dispersions: 10 ppm CuO (25 to 55 nm), 50 ppm SiO₂ (30 nm) and 50 ppm ZnO (30 to 40 nm), all purchased from US Research Nanomaterials, Inc. (Houston, TX, USA). Distilled water was used as a control, and 0.05% Tween 20 was added as a surfactant to all treatments. Nanoparticle treatments were applied weekly, beginning 3 d after transplanting (DAT), with heat stress imposed at 4 DAT.

Plant growth and physiological measurements were taken at 30 DAT. Shoot fresh weight (FW) and root dry weight (DW) were recorded and leaf transpiration rate was measured using an LI-600 porometer (LI-COR Biosciences, Lincoln, NE, USA). Leaf chlorophyll and carotenoid contents were estimated following the spectrophotometric method of Lichtenthaler and Buschmann (2001). Chlorophyll fluorescence (Fv/Fm), proline and reactive oxygen species (ROS) concentration, nonenzymatic antioxidant capacity (FRAP) and antioxidant enzyme activities were measured as described in Lee et al. (2023), using the second fully expanded leaf.

All experimental data were subjected to a two-way analysis of variance (ANOVA) to examine the effects of temperature, nanoparticles and their interactions using R software (version 4.2.0). Differences between treatment means were determined using Duncan’s multiple range test at a significant level of P = 0.05 with the “agricolae” package. The assumptions of normality and homogeneity of variances were checked using the Shapiro–Wilk test and Levene test (from the “car” package), respectively. The effect size of treatment differences were calculated using Hedge’s g with the “effsize” package.

Results and discussion

The foliar application of CuO, SiO₂, and ZnO nanoparticles had minimal effects on the growth and physiology of tomato seedlings. There were no significant differences between the nanoparticle treatment groups and control plants in shoot FW, leaf pigment and proline content, nonenzymatic antioxidant capacity (FRAP), and superoxide dismutase (SOD) and catalase (CAT) activities under either optimal temperature or heat stress conditions (Table 1). Root DW in SiO₂ nanoparticle-treated seedlings was lower than that of the control at optimal temperature, whereas no significant differences were observed under heat stress. Although many studies have reported enhanced thermotolerance with nanoparticle applications under heat stress, including increased plant biomass, leaf pigment content and antioxidant enzyme activities in tomato, wheat, and sorghum (Azmat et al. 2022; Djanaguiraman et al. 2018; Haghighi et al. 2014), such beneficial effects were not observed in this study, suggesting that low concentrations of CuO, SiO₂, and ZnO nanoparticles may not be effective as foliar treatments for improving plant growth or antioxidant capacity in tomato seedlings.

Table 1.Plant growth, physiology, and antioxidant capacity of tomato seedlings treated with foliar application of CuO, SiO₂, and ZnO nanoparticles under heat stress.

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The only significant benefits observed from nanoparticle application in this study were on leaf chlorophyll fluorescence (Fv/Fm) and ROS concentration. Under heat stress, Fv/Fm values were significantly higher in all nanoparticle-treated groups compared with the control (Hedge’s g: CuO = 1.45, SiO₂ = 1.13, ZnO = 2.14), with a significant decrease in the control under heat stress compared with optimal conditions (P = 0.024 for temperature × nanoparticle interaction), whereas ROS concentration was significantly lower in SiO₂ nanoparticle-treated seedlings than in the control (Hedge’s g: 1.75) (Fig. 1A and C). These results indicate that CuO, SiO₂, and ZnO nanoparticles may help protect photosystem II (PSII) from heat stress damage, with SiO₂ nanoparticle specifically showing potential in promoting ROS scavenging. Similar results have been reported that Si (47 ppm) and ZnO (60 and 90 ppm) nanoparticles improved Fv/Fm in wheat and alfalfa seedings, respectively, under heat stress (Kareem et al. 2022; Younis et al. 2020). Also, SiO₂ nanoparticles (50 ppm) reduced hydrogen peroxide concentration in potato and pea plants under drought stress (Al-Selwey et al. 2023; Sutulienė et al. 2021) and in rice plants under salinity stress (Abdel-Haliem et al. 2017). On the basis of our findings, it is speculated that the decreased ascorbate peroxidase (APX) and guaiacol peroxidase (POD) activities in SiO₂ nanoparticle-treated seedlings under heat stress (Hedge’s g: APX = 2.54, POD = 2.39) (Fig. 1D and E) could be attributed to reduced ROS levels, which inactivated the upregulation of these enzyme activities. However, this study could not identify the specific antioxidants responsible for ROS scavenging, indicating the need for further research to clarify these mechanisms.

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In contrast to the beneficial effects, nanoparticle treatments adversely affected the leaf transpiration rate under heat stress; seedlings treated with CuO and SiO₂ nanoparticles had significantly lower transpiration rates than the control (Hedge’s g: CuO = 2.15, SiO₂ = 1.77) (Fig. 1B). Under heat stress, transpiration contributes to leaf temperature regulation via evaporative cooling (Sharma et al. 2015), suggesting that decreased transpiration in nanoparticle-treated seedlings may have intensified heat stress. Because stomata are primary pathways for foliar nanoparticle uptake (Sidhu et al. 2024), repeated nanoparticle applications may have physically blocked stomatal pores (Hong et al. 2021), leading to reduced transpiration rates. Alternatively, nanoparticles may have induced stomatal closure by regulating related genes and metabolites under heat stress (Guo et al. 2024).

Overall, the heat stress responses observed in tomato seedlings treated with CuO, SiO₂, and ZnO nanoparticles were inconclusive, making it difficult to determine whether the nanoparticles mitigated or exacerbated heat stress. The positive and negative effects of nanoparticle applications observed in Fig. 1 also appeared insufficient to have a significant impact on plant growth (Table 1). However, the effects of nanoparticles can vary depending on factors such as plant species, developmental stages, nanoparticle characteristics (e.g., ion type, size, synthesis method), and application methods. Also, the relatively low nanoparticle concentrations and the limited number of plant replicates in this study could have restricted a thorough understanding of their effects. Therefore, further studies are needed to identify the specific conditions under which nanoparticles enhance plant thermotolerance, as well as to evaluate a wider range of nanoparticle concentrations and plant developmental stages.

Conclusion

Tomato plants are highly susceptible to heat stress, yet the potential of nanoparticles for enhancing their thermotolerance remains unclear. This study demonstrated that foliar application of low concentrations of CuO, SiO₂, and ZnO nanoparticles failed to enhance overall thermotolerance in tomato seedlings, as evidenced by similar levels of plant biomass, leaf pigment, proline, and antioxidant capacity, along with reduced leaf transpiration compared with the control. Although all nanoparticle treatments improved chlorophyll fluorescence (Fv/Fm) and SiO₂ nanoparticles reduced ROS accumulation, these physiological benefits were insufficient to mitigate heat stress damage. To assess fully the effect of nanoparticles on thermotolerance, additional studies are required, particularly by selecting different application methods, higher nanoparticle concentrations, and their long-term effects during the reproductive stage.