Foliar Application of Selenium Enhances Drought Tolerance in Tomatoes by Modulating the Antioxidative System and Restoring Photosynthesis

Abstract

Drought stress can impact the physiological and biochemical properties of crops. However, selenium (Se) can effectively alleviate the abiotic stress experienced by plants. This study aims to investigate how applying selenium to tomato leaves affects their antioxidant system and photosynthetic traits when subjected to drought conditions. The experiment used four different foliar selenium concentrations and three different irrigation levels. The investigation scrutinized the effects of foliar spraying employing different selenium concentrations on the antioxidant system, osmotic adjustment substances, photosynthetic performance, and growth indices of tomatoes under drought stress. The findings indicated that drought stress led to cellular oxidative damage, significantly elevating peroxide, MDA, proline, and soluble sugar content (p < 0.001). Under severe drought stress, malondialdehyde (MDA) and proline levels increased by 21.2% and 110.0% respectively, compared to well-watered conditions. Concurrently, the net photosynthetic rate exhibited a reduction of 26.0% and dry matter accumulation decreased by 35.5%. However, after spraying with a low concentration of selenium, selenium reduced oxidative damage and malondialdehyde content by reducing the content of peroxide in leaves, restoring photosynthesis, and promoting the normal growth of tomato. Compared to the control group, spraying with 2.5 mg·L⁻¹ selenium resulted in a 21.5% reduction in MDA content, a 111.8% increase in net photosynthetic rate, and a 29.0% increase in dry matter accumulation. When subjected to drought stress conditions, foliar spraying of low concentrations of selenium (2.5 mg·L⁻¹) can effectively reduce oxidative damage caused by drought stress and alleviate growth constraints in tomatoes. In addition, treatments with high selenium concentrations exhibited specific toxic effects. These findings offer valuable insights into the mechanisms governing selenium-induced drought tolerance in tomatoes, thus advancing our comprehension of standard tomato production practices.

1. Introduction

As a widely cultivated crop, tomatoes (Solanum lycopersicon L.) can be grown both in greenhouses and in fields. Heatwaves, which are characterized by sustained periods of high temperatures, are a prominent example of extreme weather phenomena that have garnered significant attention from the scientific community. Plant development is significantly impacted by heatwaves within a short period, unlike the slow and continuous effect of global warming [1]. Over the past few decades, heatwaves have become more frequent and more intense across terrestrial regions. Furthermore, according to current climate projections, droughts and heatwaves are expected to occur more frequently and with greater severity [2,3]. In recent years, greenhouse cultivation has become increasingly popular due to its ability to withstand extreme weather conditions, as well as reducing the limitations of the natural environment, and enhancing the growth conditions for crops [4]. A tomato’s canopy and transpiration area are large, but its roots and stems have low hydraulic conductivity [5]. As a result, it is susceptible to drought stress. Although solar greenhouse can withstand extreme climates to some extent, the occurrence of heatwaves during the summer months can also lead to drought stress for plants inside it [6]. Therefore, it is crucial to identify and use effective antioxidants to help tomato plants adapt to drought stress and maintain higher yields. Amidst stressful periods, plants have adopted mechanisms, such as antioxidant production to mitigate the pressure induced by water deficit. Nevertheless, these intrinsic drought-resistant compounds prove insufficient to confer resilience to plants under prolonged drought circumstances [7]. Therefore, the use of external adjuncts is crucial in helping plants to effectively combat drought stress effectively.

Selenium may potentially regulate the production and elimination of reactive oxygen species (ROS) [8,9]. Additionally, for the human body, selenium is an essential micronutrient acquired primarily through the food chain, emphasizing the pivotal role of selenium-rich plants as a key source for human selenium supplementation [10]. Although the complete scope of the importance of selenium to plants is not fully understood, studies have reported that applying suitable doses of selenium can bolster plant resilience against both biotic and abiotic stressors [11,12]. However, a significant proportion of the world’s soils are selenium deficient incapacitating them to supply plants with their required selenium quantities. For example, approximately 51% of the soils in China have inadequate selenium concentrations (<0.125 mg·kg⁻¹) [13]. Moreover, given selenium’s dual role in humans, both essential and potentially toxic, and its narrow range of dietary requirements (40–400 μg·d⁻¹), understanding the mechanisms of selenium biofortification becomes a vital strategy in tackling human selenium deficiency [14,15].

At the present state of knowledge, Se is not classified as a plant essential nutrient, although recent experimental results show that Se can be used as an exogenous substance to alleviate abiotic stresses, including drought stress in plants [16,17]. Under drought stress conditions, Se protects chloroplasts and increases chlorophyll content [18]. As evidenced in selenoproteins, selenium contributes to antioxidant protection, enhances metabolism, regulates redox reactions, particularly under stress conditions [19], and protects plants from oxidative damage [20]. Furthermore, selenium aids in supporting antioxidant enzymatic activity, reducing oxidative stress products such as hydrogen peroxide, superoxide, and malondialdehyde. As a result, it aids in the restoration of plant growth, enhances production, and improves crop quality, particularly under drought stress conditions [21]. However, some studies have argued that selenium does not significantly assist plants [22]. In addition, we found that there is a wide variation in the existing literature regarding the optimal concentration of selenium to be applied to tomato, with recommendations such as the following: 2.5 μM Se was sufficient to positively influence the growth parameters of tomato in the study of Haghighi [23], 50 μM Se was effective in alleviating the drought stress imposed on tomato in the experiment of Ramasamy [24] and in the experiment of Rady [25], and both 20 and 40 mM Se significantly increased the content and activity of osmoprotective and antioxidant defense system components in tomato. In addition, the results of Saleem [26] indicated that 10 mM Se would have a positive effect on tomato, while 40 and 80 mM Se would have a negative effect on tomato.

Therefore, it is crucial to investigate the regulatory mechanisms of foliar spraying with different selenium levels on drought-stressed tomatoes. The objective of this study was (i) to elucidate how selenium improves the drought tolerance of tomatoes, and (ii) to study how the growth of tomatoes is affected by various concentrations of selenium.

2. Materials and Methods

2.1. Experimental Site

This tomato trial, located at the Xinxiang Comprehensive Experimental Base of the Chinese Academy of Agricultural Sciences (35°9′ N, 113°47′ E, altitude 74 m), started from March to June 2023 in a greenhouse facility. The mean annual temperature was 14 °C, with 2398.8 sunny hours and 201 days without frost a year. The greenhouse used for the experiment is 60 m long and 8.5 m wide, with a total area of 510 m², oriented in an east–west direction with a southern exposure. The upper part of the greenhouse is covered with a 0.2 mm non-drip polyethylene film, overlaid with 5 cm of insulating cotton, while insulating materials are embedded in the side and rear walls. The experiment was conducted under potting conditions, using potting soil taken from field topsoil (0–20 cm) with a sandy loam texture, a bulk density of 1.40 g·cm⁻³, a field capacity of 23% (mass water content), and alkaline hydrolysable nitrogen, available phosphorus, and available potassium content of 52.5, 19.5, and 196.1 mg·kg⁻¹, respectively. The soil had a pH of 8.6, electrical conductivity (EC) of 0.31 dS·m⁻¹, and organic matter content of 12.2 g·kg⁻¹.

2.2. Agricultural Cultivation Measures

The experimental pots had dimensions of 30 cm in diameter and 50 cm in height. The soil, air-dried and sieved through a 2 mm mesh, was packed into the pots in three layers. Approximately 15 cm of soil was compacted once during filling. The basal fertilizer was evenly blended into the third layer, filling the pots up to a depth of 3–5 cm below the rim. Each pot contained an air-dried soil mass of 43.4 kg, ensuring a bulk density of 1.40 g·cm³. The application rates of nitrogen, phosphorus, and potassium fertilizers were uniform across all treatments, with application rates of 0.13, 0.08, and 0.13 g·kg⁻¹ dry soil, respectively. During potting, all phosphorus fertilizers and 30% of the total nitrogen and potassium fertilizers were applied as basal fertilizers. The remaining 70% of nitrogen and potash fertilizer were applied with water at the onset of fruit expansion in each spike. The tomato variety used for the test was ‘Jingfan 404’. Each pot contained one plant, which was transplanted when it had five leaves and one heart. Three spikes of fruiting were topped with three terminal leaves.

2.3. Experimental Design

This experiment had two factors: different irrigation levels (W) with a control water volume range of ±5%, and different foliar sprayed exogenous selenium (Na₂SeO₃) concentrations (S) with a control concentration range of ±0.5 mg·L⁻¹. Four concentrations of foliar sprayed exogenous selenium (Na₂SeO₃) were used as follows: Se0, Se2.5, Se5, and Se10, which correspond to 0 mg·L⁻¹, 2.5 mg·L⁻¹, 5 mg·L⁻¹, and 10 mg·L⁻¹ respectively. During the flowering and fruiting period, the leaves of the plants were sprayed twice at 20-day intervals with four different concentrations of selenium. Each spray was applied until water dripped from the leaf surface, and S0 used water as the control treatment. Throughout the exogenous selenium spraying process, a waterproof plastic sheet was laid over the soil surface to hinder any dripping of Na₂SeO₃ solution into the soil, thus safeguarding the integrity of the test results. Each exogenous selenium concentration was paired with three irrigation control levels. The lower irrigation limits were designated at 50%, 65%, and 80% of the field’s water-holding capacity, denoted as W1, W2, and W3, respectively. The irrigation quota was maintained at 2.0 L. A total of 12 treatments were established, encompassing complete combinations of exogenous selenium concentrations and irrigation control levels (

Table 1. Experimental treatments.

Treatments	Exogenous Selenium Se Concentration (mg·L−1)	Water Content (%)
W1Se0	0	50% field capacity
W1Se2.5	2.5
W1Se5	5
W1Se10	10
W2Se0	0	65% field capacity
W2Se2.5	2.5
W2Se5	5
W2Se10	10
W3Se0	0	80% field capacity
W3Se2.5	2.5
W3Se5	5
W3Se10	10

). To accommodate the requirement for destructive sampling, 20 pots were allocated to each treatment, resulting in a total of 240 pots per treatment. For precise control over irrigation volume in each treatment, a drip irrigation system was employed. From 7:30 a.m. to 8:30 a.m. each day, three chosen representative pots from each treatment were weighed, and the soil moisture content was calculated for irrigation control. To prevent overgrowth and to produce robust plants, tomatoes were uniformly irrigated to 90% of the field holding capacity after fixation, and when the soil moisture content dropped to 55–60% of the field holding capacity, all treatments were again irrigated to 90% of the field holding capacity, after which the treatments were irrigated in accordance with the lower limit of the designed irrigation control.

2.4. Observation Items and Methods

2.4.1. Plant Height and Stem Diameter

Three representative plants from each treatment, exhibiting comparable overall growth, were selected and labeled for the assessment of tomato plant height, leaf area, and stem thickness. Measurements were performed every 10 days, commencing on the 30th day after transplanting. Stem thickness was measured at 2 cm above the soil surface utilizing a caliper and employing the crisscross method.

2.4.2. Biomass

At the flowering and fruiting stages and at the ripening and harvesting stages, three plants of equal size were randomly selected for each treatment, separated by stems, leaves, and fruits, and their fresh weights were measured separately, after which they were placed in an oven at 105 °C for 30 min, and then dried at 75 °C to constant weight, and their dry weights were measured separately.

2.4.3. Measurement of Photosynthesis Indices

We employed a portable photosynthesis meter (LI-COR Inc., Lincoln, NE, USA) to gauge various photosynthetic indices, encompassing the net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO₂ concentration (Ci), and transpiration rate (Tr), of the fully expanded leaves of tomato, counting the third leaf from the tip downwards (inverted trifoliate), at 9:00–11:00 a.m. The SPAD values of tomato functional leaves were measured using a SPAD analyzer (SPAD-502 Plus, Konica Minolta, TKY, Japan). In every treatment, three tomato plants with analogous growth conditions were designated, each plant bearing a singularly marked leaf. Subsequently, three points on each leaf were measured and averaged.

2.4.4. The Levels of Peroxide and Osmotic Regulatory Substances in Tomato Leaves

Between 8:00 and 10:00 a.m., on the 10th day following the application of exogenous selenium treatment, functional leaves ranging from the 5th to the 8th positions from the apex of the tomato plants were harvested. The leaves were rinsed with deionized water to eliminate impurities, swiftly dried, enveloped in aluminum foil, and promptly frozen in liquid nitrogen. Following transportation to the laboratory, the samples were stored in an ultra-low temperature refrigerator set at −80 °C. The content of proline was determined using the acid ninhydrin method [27]; the content of soluble sugars was determined using the anthrone method [28]; the content of malondialdehyde (MDA) was determined using the thiobarbituric acid (TBA) colorimetric method [29]; the content of H₂O₂ was determined using the titanium sulfate colorimetric method [29]; and the content of O₂⁻ was determined using the hydroxylamine hydrochloride oxidation method [29].

2.4.5. Selenium Content of Aboveground Organs of Tomatoes

For each treatment, the aboveground parts of the plants were selected at the fruit ripening and harvesting stage, and classified into leaves and fruits, which were washed and swabbed dry with distilled water, and then the organ samples were placed in an oven, first dried at 105 °C for 30 min and then dried at 75 °C to a constant mass, and then the Se content in the leaves and fruits was determined by inductively coupled plasma mass spectrometry (ICP-MS) [30].

2.5. Statistical Analysis

Analysis of variance (ANOVA) was performed using SPSS 20.0 (IBM Corp., Armonk, NY, USA). Two-way ANOVA was applied to test water content (W), selenium treatment (Se), and the interaction of W × Se. All experimental data were expressed as means ± standard deviation. All treatment means (n = 3) were compared for any significant differences using Duncan’s multiple range tests at p < 0.05. Graphical presentation was carried out in Graph Pad Prism 8.0.2 software for Windows (La Jolla, CA, USA).

3. Results

3.1. Growth Status of Tomato

Table 2. Effects of foliar selenium application and soil moisture on tomato growth.

Treatments	Height (cm)	Stem Diameter (mm)
W1Se0	98.6 ± 2.7 ef	8.1 ± 0.1 ef
W1Se2.5	99.8 ± 1.7 def	8.5 ± 0.3 def
W1Se5	95.7 ± 2.1 f	8.0 ± 0.1 f
W1Se10	97.5 ± 6.8 f	8.0 ± 0.2 f
W2Se0	107.2 ± 1.9 bcd	8.7 ± 0.3 cde
W2Se2.5	105.8 ± 6.3 cde	8.8 ± 0.7 cd
W2Se5	106.2 ± 6.8 cde	9.4 ± 0.3 bc
W2Se10	111.3 ± 5.0 bc	8.5 ± 0.4 def
W3Se0	115.2 ± 2.7 ab	9.2 ± 0.1 bc
W3Se2.5	120.7 ± 2.0 a	10.0 ± 0.2 a
W3Se5	115.3 ± 5.7 ab	9.5 ± 0.7 ab
W3Se10	108.7 ± 4.9 bc	8.4 ± 0.2 def
W	**	**
Se	ns	**
W × Se	ns	**

Note: “ns” indicates means not significant (p > 0.05), and ** indicates significance at p < 0.01. Means were separated according to Duncan’s multiple range test. The means with the same small case letters are statistically non-significant.

shows that the interaction between water content and selenium treatment concentration (W × Se) had a significant effect on stem diameter but not on height. Both plant height and stem thickness were significantly affected by water content. Selenium treatment had a significant impact on stem diameter but not on height.

Under the same foliar selenium concentration, plant height and stem diameter increased with increasing soil moisture content. Compared to W3, the average plant height and stem diameter decreased by 14.9% and 12.4%, respectively, in W1. Under the same soil moisture condition, the average stem diameter initially showed an increasing trend and later followed a decreasing trend with increasing foliar selenium concentration, reaching a maximum at Se2.5, with an increase of 5.0% compared to Se0. These results suggest that exogenous selenium at low concentrations may mitigate the adverse effects of drought stress on plants and enhance plant growth.

3.2. Aboveground Dry Matter of Tomato Plants

The dry matter of various organs of tomato plants and the total aboveground dry matter were significantly influenced by water content (W), selenium treatment (Se), and the interaction of W × Se.

The dry matter of fruits, leaves, and stems accounted for 31.3% to 41.8%, 29.9% to 37.8%, and 26.9% to 35.1% of the total aboveground dry matter, respectively, with the proportion of fruits higher than the proportion of stems and leaves (Figure 1). Under the same foliar selenium concentration, the average dry matter of fruits, leaves, stems, and total aboveground dry matter significantly increased with increasing soil moisture content (p < 0.001). Compared to the W3 treatment, the dry matter of fruits, leaves, stems, and total aboveground dry matter in the W1 treatment decreased by 29.6%, 40.8%, 36.0%, and 35.5%, respectively. Under the same soil moisture condition, the average dry matter of fruits, leaves, stems, and total aboveground dry matter initially showed a significant increasing trend followed by a decrease with increasing foliar selenium concentration (p < 0.05). Compared to the Se0 treatment, the dry matter of fruits, leaves, stems, and total aboveground dry matter increased by 26.3%, 26.4%, 35.2%, and 29.0%, respectively, in the Se2.5 treatment. Foliar spraying of low-concentration selenium under drought stress and normal irrigation conditions significantly increased the dry matter of tomato plants.

3.3. Physiological Characteristics of Tomato Plants

The study investigated the effects of varying selenium spray concentrations and soil moisture conditions on the Pn, Gs, Ci, Tr, and SPAD of tomato leaves. The Pn, Gs, Ci, Tr, and SPAD of tomato plants were significantly influenced by water content (W), selenium treatment (Se), and the interaction of W × Se. (

Table 3. Influence of foliar selenium application and soil moisture on physiological characteristics of tomato.

Treatments	Pn (μmol CO2 m−2 s−1)	Gs (mol H2O m−2 s−1)	Ci (μmol mol−1)	Tr (mol H2O m−2 s−1)	SPAD
W1Se0	4.82 ± 0.47 f	0.05 ± 0.00 g	208.47 ± 8.30 g	1.87 ± 0.15 f	62.03 ± 0.49 f
W1Se2.5	14.70 ± 1.63 bc	0.14 ± 0.02 de	228.99 ± 8.18 f	4.75 ± 0.29 d	66.2 ± 0.61 d
W1Se5	14.31 ± 0.56 bc	0.21 ± 0.04 bc	275.54 ± 1.66 abc	7.12 ± 0.22 bc	64.57 ± 0.49 e
W1Se10	11.96 ± 1.66 d	0.16 ± 0.01 de	250.44 ± 4.35 de	6.54 ± 0.38 c	64.00 ± 0.61 e
W2Se0	6.95 ± 1.60 e	0.08 ± 0.00 fg	228.32 ± 5.62 f	3.26 ± 0.88 e	68.07 ± 0.50 bc
W2Se2.5	16.44 ± 1.63 ab	0.21 ± 0.02 bc	242.00 ± 5.21 ef	8.50 ± 0.59 b	70.83 ± 0.49 a
W2Se5	14.04 ± 1.95 c	0.18 ± 0.03 cd	262.28 ± 13.72 cd	8.06 ± 1.22 b	68.83 ± 0.21 b
W2Se10	10.69 ± 1.08 d	0.17 ± 0.03 cd	262.77 ± 17.12 cd	7.54 ± 1.02 bc	66.30 ± 1.06 d
W3Se0	11.45 ± 0.53 d	0.12 ± 0.01 ef	270.76 ± 8.11 bcd	4.46 ± 0.32 de	66.47 ± 0.93 d
W3Se2.5	18.03 ± 0.65 a	0.38 ± 0.06 a	291.57 ± 22.93 a	13.21 ± 0.26 a	67.93 ± 0.61 bc
W3Se5	16.10 ± 0.26 abc	0.36 ± 0.01 a	284.17 ± 10.16 ab	11.87 ± 1.24 a	67.03 ± 0.25 cd
W3Se10	16.31 ± 0.39 ab	0.25 ± 0.01 b	269.13 ± 7.66 bcd	11.91 ± 1.40 a	66.47 ± 0.86 d
W	**	**	**	**	**
Se	**	**	**	**	**
W × Se	**	**	**	**	**

Note: ** indicates significance at p < 0.01. Pn: net photosynthetic rate; Gs: stomatal conductance; Ci: intercellular CO₂ concentration; Tr: transpiration rate; and means were separated according to Duncan’s multiple range test. The means with the same small case letters are statistically non-significant.

). At the same sprayed selenium concentration, Pn, Gs, Ci, and Tr of tomato leaves decreased significantly as the soil moisture content decreased. The SPAD value initially tended to increase and decrease with increasing soil water content. It reached its maximum value in W2. Pn, Gs, Ci, and Tr in W1 were reduced by 26.0%, 49.5%, 13.6%, and 51.1%, respectively, compared to W3. Additionally, SPAD increased by 6.7% in W2 compared to W3. Under the same soil moisture condition, the Pn, Gs, Ci, Tr, and SPAD of tomato leaves showed a significant trend of increasing and then decreasing with the increase in selenium spraying concentration, which was not significant or even decreased when the selenium concentration was more than 2.5 mg·L⁻¹. Pn, Gs, Ci, Tr, and SPAD of Se2.5 increased by 111.8%, 166.7%, 7.8%, 175.6%, and 4.3%, respectively, compared to Se0. Under drought stress, spraying low and medium concentrations of selenium can effectively enhance crop physiological performance.

3.4. Antioxidant Capacity and Osmoregulation Substances of Tomato Leaves

The study revealed that the MDA, H₂O₂, O₂⁻, proline, and soluble sugars in tomato leaves were significantly influenced by water content (W), selenium treatment (Se), and the interaction of W × Se (Figure 2 and Figure 3). Under the same selenium concentration, the levels of MDA, H₂O₂, O₂⁻, proline, and soluble sugars increased gradually with decreasing soil water content. Compared to W3, the content of MDA, H₂O₂, O₂⁻, proline, and soluble sugars in W1 increased by 21.2%, 42.3%, 29.9%, 110.0%, and 23.2%, respectively. Under the same soil moisture condition, the average activity of proline and soluble sugars tended to increase and then decrease with increasing selenium concentration, while the content of MDA, H₂O₂, and O₂⁻ first showed a decreasing trend and later increased with increasing selenium concentration. Compared to Se0, Se2.5 increased the proline and soluble sugars by 96.7% and 52.7%, respectively, while decreasing MDA, H₂O₂, and O₂⁻ by 23.5%, 25.1%, and 13.3%, respectively. Different water treatments also resulted in variations in the ability and effectiveness of selenium to scavenge free radicals. Compared to the well-watered treatment, selenium showed better free radical scavenging effects under drought stress. Under the Se2.5 treatment, the H₂O₂ decreased by 34.9%, and MDA decreased by 34.1% in the W1 treatment, whereas in the W3 treatment, H₂O₂ and MDA only decreased by 18.0% and 16.3%, respectively.

3.5. Content of Selenium in the Different Organs of the Tomato

Applying exogenous selenium through foliar spraying significantly increased the selenium content in both leaves and tomato fruits. The interaction between foliar spraying of exogenous selenium and soil moisture had a significant effect on the selenium content in leaves (p < 0.001), but no significant effect on the selenium content in fruits was observed (Figure 4). Under the same soil moisture treatment, the average selenium content in leaves significantly increased by 532.0%, 749.3%, and 1853.3% in S2.5, S5, and S10, respectively, compared to S0. Similarly, the average selenium content in fruits significantly increased by 254.5%, 536.4%, and 918.2% in the S2.5, S5, and S10 treatments, respectively.

4. Discussion

Drought stress may accumulate reactive oxygen species ROS in excess in plants, resulting in damage to cellular architecture. Selenium plays a vital role in restraining the excessive production of ROS triggered by drought stress [31]. The results indicate that as soil moisture decreased, malondialdehyde, hydrogen peroxide, and superoxide anion levels in tomato leaves increased (Figure 2). However, after selenium treatment, low concentrations of selenium were found to reduce the levels of hydrogen peroxide, superoxide anion, and malondialdehyde in leaves. Plants can synthesize two amino acids, namely selenocysteine (SeCys) and selenomethionine (SeMet) through the biosynthesis of selenium via the sulfur assimilation pathway [32]. Selenocysteine serves as a constituent of glutathione (GSH), which is crucial for plant responses to various stresses. Glutathione peroxidase (GSH-Px) is regarded as a pivotal antioxidant enzyme that can be stimulated by selenium exposure under various abiotic stresses. GSH-Px, with the assistance of GSH, acts as a potent scavenger of hydroperoxides [12]. Additionally, research by Hartikainen and Hussain has shown that the addition of exogenous selenium can increase antioxidant enzyme activity in plants, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), assisting in the removal of ROS [33,34]. Nevertheless, with the escalation of selenium spray concentration, there was a gradual rise in the level of hydrogen peroxide, superoxide anion, and malondialdehyde in the leaves (Figure 2). This indicates that high selenium concentration not only fails to protect the organism but also exacerbates the degree of damage and makes the destructive effects produced by stress more severe [35]. High selenium concentration fosters the generation of free radicals and peroxidation [36].

Under the conditions of drought stress, plants typically increase proline synthesis to act as an osmoregulator, aiding in the maintenance of intracellular water balance. Selenium may impact the synthesis and accumulation of proline and its related metabolites [25]. With the gradual decrease in soil moisture, proline and soluble sugars gradually increased in tomato leaves (Figure 3). Accumulation of osmoregulatory substances serves as an efficient defense mechanism for plants against various environmental stresses, notably drought stress [29]. Several reports suggest that environmental stress can cause plants to overproduce proline, which maintains cell osmotic balance, stabilizes membranes, and prevents electrolyte leakage to increase plant stress tolerance. This could keep ROS levels within normal limits, helping to prevent oxidative damage to the plant [37,38]. Low selenium concentrations increased leaf proline and soluble sugar in this study. In Khan’s study [39], by upregulating the level of proline synthase (glutamyl kinase) activity, selenium application increased proline levels. Also, Se treatment enhanced soluble sugar synthesis. Nawaz additionally illustrated that the external application of selenium upheld intracellular osmotic equilibrium by accumulating both organic osmoregulatory substances and inorganic ion content. This action further elevated leaf relative water content (RWC) within plant tissues [40]. Based on this foundation, the application of selenium emerges as an efficacious approach for sustaining osmotic pressure equilibrium in response to drought stress. In Regni’s study, it was concluded that sodium selenate acted by stabilizing the proline content in olive leaves as well as decreasing the release of proline from the roots under salt stress conditions [41].

Amidst the challenge of drought, the photosynthetic system of plants is disrupted, resulting in an inability to photosynthesize properly [42]. As shown in Table 3, Pn, Gs, Ci, Tr, and SPAD all decrease with lower soil moisture. Under drought stress conditions, plant stomata close, preventing CO₂ from entering the plant and leading to a decrease in photosynthesis. Simultaneously, due to the unavailability of external CO₂, photoinhibition occurs in the leaves, resulting in sustained damage or irreversible destruction of the chloroplasts’ ultrastructure [43]. As a consequence, chlorophyll degradation occurs alongside fragmentation, leading to the inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and subsequent stomatal closure. These factors reduce the photochemical efficiency of photosystem II (PSII). Drought stress reduces the photosynthetic rate as well as inhibits the processes of primary light energy conversion, electron transfer, photosynthetic phosphorylation, and photosynthetic dark reaction in the photosynthetic light reaction. This ultimately leads to a decline in photosynthesis [44]. The reduction in gas exchange properties induced by drought has been well documented in many crops, such as wheat [31], maize [45], and tobacco [46].

In contrast, the application of low selenium concentrations led to enhanced Pn, Gs, Ci, Tr, and SPAD (Table 3). This suggests that the damage caused by stress to the leaves was reduced and their photosynthetic performance was restored. The increase in the net photosynthetic rate at low Se levels may be related to Gs because the increasing trend of Gs was essentially consistent with that of the net photosynthetic rate [47]. In addition, since stomata are the only pathway for transpiration, the transpiration rate is closely related to Gs. The chlorophyll content and photosynthetic parameters were significantly increased after low Se treatment, indicating that the photosynthesis of tomato was strengthened. The results of this study were consistent with those of previous studies. The study of Li shows that low Se increased the chlorophyll content and photosynthetic parameters of lettuce, thereby enhancing photosynthesis and promoting the growth of lettuce [48]. Additionally, exogenous selenium was found to attenuate drought stress-induced stomatal restriction. This enabled the leaves to have enough photosynthetic substrates, thereby reducing the limitation of photosynthetic rate caused by drought stress [49]. This finding is consistent with Zhang’s research [50], that selenium exerts a positive influence in alleviating the adverse effects of drought stress on plants by restoring the light and system to restore plant photosynthesis, growth, and yield. The appropriate concentration of Se treatment could not only improve the content and total amount of various photosynthetic pigments in leaves but also optimize the ratio of chlorophyll. This is because part of chlorophyll a and all chlorophyll b mainly function as antenna pigments. The decrease in chlorophyll a/b value also means that the leaf’s ability to capture and transfer light energy has improved and up-regulated the photosynthetic electron transport chain in plants [51]. Additionally, Cysteine residues play a pivotal role in the structure and function of proteins within the protein chain, facilitating processes such as disulfide bond formation, enzyme catalysis, and the formation of metal binding sites. SeCys, which is larger than cysteine, more reactive, and more prone to deprotonation, is harmful to protein structure and function when it replaces cysteine [52]. For instance, iron–sulfur (Fe-S) cluster proteins are integral components of electron transport chains of chloroplasts and mitochondria and they are susceptible to substitution by SeCys, leading to impaired organelle function [53]; a decline in photosynthetic performance after excessive selenium application may be attributed to selenium’s interference with normal protein synthesis, resulting in the production of malformed selenoproteins, which affects normal plant growth [54].

Under non-drought conditions, spray application of selenium improved photosynthetic performance (Table 3). This implies that exogenous selenium impacts not just stomatal regulation but also modulates the photosynthetic system through the regulation of non-stomatal factors like PSII photochemical activity and carbon assimilation efficiency. Valkama’s study supports this idea and suggests that selenium may contribute to the net photosynthetic rate [55].

Drought stress is a severe abiotic stressor for plants, which restricts their growth and biomass production [56]. The experiment showed that decreasing soil moisture resulted in a decrease in tomato plant height, stem thickness, and aboveground dry matter mass. This implies that the growth of plants is notably impacted by drought stress, primarily due to oxidative harm and photoinhibition. Photosynthesis is a fundamental physiological process in plants, providing a crucial energy source for growth, development, and metabolic processes. Damage to the photosynthetic system can result in a temporary or permanent decrease in the plant’s photosynthetic efficiency [57]. In general, plant biomass is most directly related to plant photosynthesis. Photosynthesis, occurring predominantly in leaves, drives the synthesis of organic compounds essential for biomass accumulation. Efficient photosynthetic activity directly influences leaf biomass, while the products of photosynthesis serve as substrates for fruit development, thereby contributing to fruit biomass [58]. During the experiment, it was noted that the dry matter of W2Se2.5 exhibited a considerable increase in comparison to W2Se0. Additionally, the MDA levels between W2Se2.5 and W3Se0 showed no statistically significant variance, whereas a notable enhancement in the photosynthetic rate was evident in W2Se2.5 compared to W3Se0. These findings indicate that selenium efficiently alleviated the oxidative harm induced by drought stress. Bocchini’s experiment showed the same result, where selenium biofortification under drought stress significantly increased the dry matter mass of the aboveground portion of maize by 59% [18]. After spraying a high concentration of selenium, the decline in dry matter accumulation could potentially be attributed to the progressive rise in reactive oxygen species (ROS), depletion of osmoregulatory substances, and impaired photosynthetic efficiency induced by selenium stress. This stress is produced by the spraying of high concentrations of selenium, which subsequently leads to high absorption of selenium into the plant’s body. According to the text, selenium acts on plants in two ways, depending on how concentrated it is, as reported by Hartikainen [33]. During pot experiments, lower doses of selenium were found to enhance the growth of ryegrass seedlings, whereas higher doses displayed a pro-oxidant impact, leading to reduced yields and the onset of metabolic disorders. Hawrylak’s research further confirmed this discovery in cucumber [59], where the biomass of the roots and aboveground parts was negatively correlated with selenium concentration.

Moreover, given selenium’s dual role in humans, which is both essential and potentially toxic, and its narrow range of dietary requirements (40–400 μg·d⁻¹) [15], it is important to examine the concentration of selenium in tomato fruit. According to the food safety standards in China (GH/T 1135-2017) [60], the selenium content of fruit with spraying 2.5 mg·L⁻¹ of sodium selenite meets the requirements of GH/T 1135-2017 for selenium-rich vegetables (0.1–1 mg/kg). This means that the concentration of selenium in tomato fruit of Se2.5 treatments is lower than the toxic concentration for humans and that the recommended daily intake would be safely met.

Figure 4 shows that selenium levels in various aboveground organs of tomato plants are greatly increased by applying foliar selenium, including organic and inorganic selenium. The concentration of selenium also increases with the selenium application concentration. These results suggest that foliar selenium application is an effective method for enriching plants with selenium. The absorption of selenium by plant leaves involves two steps: penetration of selenium through the protective tissues of the leaf into the internal tissues on the leaf surface, followed by contact with mesophyll cells and entry into the mesophyll cells through the corresponding absorption pathways. Studies have shown that nutrients on the foliar surface first enter the mesophyll cells by diffusion and are then absorbed and utilized [61]. It is generally believed that the epidermal cell wall has little effect on the entry of nutrients into the cell, and the transport mechanism of nutrients into the cytoplasm through the epidermal cell wall is no different from that of root cells [62]. Due to the difficulty of extracting intact leaf cells, there are few studies on the mechanism of nutrient uptake by leaves at home and abroad, and it is difficult to simulate the actual situation. The mechanism of nutrient uptake by leaves has been recognized as similar to that of the root system, mesophyll cells and root hair cells transport external nutrients across membranes [63].

In our study, selenium application occurred during the flowering and fruit setting stage, different from prevailing practices that predominantly target the seedling or seed stages for selenium supplementation to alleviate stress. This departure has unveiled varying optimal selenium concentrations, with the application of 2.5 mg·L⁻¹ of sodium selenite (31.7 μM Se) in our experimental findings. In contrast, Haghighi observed 2.5 μM [23], while Saleem reported 10 mM [26]. It reflects the differential tolerances of tomatoes at different growth stages to exogenous selenium. For instance, there might be specific growth stages where processes such as Se absorption, translocation, or accumulation are more active, leading to varying degrees of Se demand or responsiveness. Moreover, our study has found an additional facet: the promotional effect of exogenous selenium on tomatoes under sufficient irrigation. The results showed that lower doses of selenium were found to enhance the growth, biomass, and photosynthetic parameters. This is in contrast with some existing research findings [22,64]. Because tomato is a non-Se-accumulator species, quantities greater than 25 μg of Se per gram of dry weight of roots and leaves, in general, are toxic to the species. We attribute this disparity to differences in the timing of selenium application, and the form in which Se was supplied may also influence the results [65].

Our research holds paramount significance as it contributes novel insights into the optimal timing of selenium application, shedding light on its efficacy across the flowering and fruit-setting stages of tomatoes. By expanding the scope beyond traditional application practices, we underscore the necessity of considering the nuanced interactions between selenium supplementation, growth stage, and irrigation conditions. These findings not only deepen our understanding of selenium-mediated stress alleviation but also offer practical implications for optimizing agricultural practices to enhance crop productivity and resilience. This study represents a step towards exploring selenium application strategies with the potential to contribute to the development of more refined and advancing sustainable agricultural methodologies in the face of evolving environmental challenges.

Although selenium significantly influenced tomato growth and physiological parameters in this experiment, it only applies selenium to tomatoes during the flowering and fruiting stages. Additionally, it fails to comprehensively monitor the dynamic changes in various indicators of plants after selenium application. To improve the study, it is recommended to monitor the plants continuously after selenium application and to include a control group for comparison. Therefore, to further understand selenium’s role in plant antioxidant systems, molecular analyses are required, including a genomic perspective. The dynamic changes in antioxidant systems after selenium application are currently unclear.

5. Conclusions

In this study, we investigated the way selenium enhances the tolerance of tomatoes to drought. The findings suggest that oxidative stress in plants can be caused by drought stress, which can lead to damage to cell membranes. Low levels of selenium can be beneficial to tomatoes through the maintenance of a balance between clearance systems and ROS production and through the improvement of photosynthetic properties. But tomatoes can be harmed by excessive concentrations of selenium, regardless of whether they are under drought stress or unstressed conditions. Based on the physiological status of plants, the results suggest that spraying 2.5 mg·L⁻¹ of sodium selenite under drought conditions (W2Se2.5) can efficiently decrease the levels of ROS and MDA in tomato leaves, thereby reducing oxidative damage to the plants. Spraying 2.5 mg·L⁻¹ of sodium selenite under well-watered conditions (W3Se2.5) can also increase the photosynthetic capacity and improve the growth of tomatoes. Future experiments could study the dynamic effects of applying selenium to tomatoes to determine the optimal timing and frequency of selenium spraying. This may establish a new theoretical foundation for producing selenium-enriched tomatoes and providing technical assistance.