Roles of salicylic acid in selenium-enhanced salt tolerance in tomato plants

doi:10.21203/rs.3.rs-1857198/v1

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Research Article

Roles of salicylic acid in selenium-enhanced salt tolerance in tomato plants

https://doi.org/10.21203/rs.3.rs-1857198/v1

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Aims

Selenium (Se) has been reported to mitigate the harmful effect of salt stress on plants, however the internal mechanisms are still unknown. Here, the effects of Se supplementation on tomato under salt stress were investigated.

Methods

The biomass, relative electrical conductivity (REC), relative water content (RWC), malondialdehyde (MDA), soluble sugar and proline content, and the regulation of plant hormones of Se application in tomato were investigated after exposure to Se and salt stress treatments.

Results

Exogenous Se application improved photosynthesis and the water use efficiency (WUE) of tomato plants under salt stress, thereby promoting the growth of tomato plants under salt stress. Se supplementation also reduced the REC and MDA, decreased the content of H₂O₂ and O₂^•−, and mitigated the oxidative damage caused by salt stress. Exogenous Se increased the salicylic acid (SA) content in tomato leaves and roots via up-regulating the PAL or ICS pathways of SA biosynthesis. After pretreatment with the SA inhibitor (ABT), the photosynthetic efficiency of tomato plants decreased, the growth was weakened, and the relative electrical conductivity was increased, indicating that the alleviating role of Se on salt stress was abolished.

Conclusions

Our results clarified the roles of Se and its regulation mechanisms in plant salt stress tolerance, as well as the critical involvement of SA in this process. The study of Se in plant abiotic stress tolerance will give more theoretical foundation for the usage of exogenous Se in agricultural production to enhance the crop growth and yield under adversity stresses.

SA biosynthesis inhibitor

salicylic acid

salt stress

selenium

tomato

Soil salinity is one of the major abiotic stresses which seriously restrict plant growth and crop yield (Soltabayeva et al., 2021; Shrivastava and Kumar, 2015), which has been exacerbated by poor irrigation practices, rising population, and industrial pollution (Yu et al., 2020). Currently, 20% of arable land of the world and 50% of the irrigated areas are grievously affected by extreme salt stress (Wichern et al., 2020). Salt stress simultaneously leads to ionic toxicity, osmotic and oxidative stresses, which affects many plant physiological and biochemical processes, including photosynthesis, energy metabolism, and water and nutrient uptake of plants, etc. (Yang et al., 2020). Plants have developed various strategies to survive in saline environments such as ion homeostasis and compartmentalization, biosynthesis of osmoprotectants, activation of antioxidant compounds, and regulation of a number of stress-responsive genes (Gupta and Huang, 2014). On the other side, the application of exogenous substances (melatonin, silicon, brassinosteroids, polyamine, and selenium, etc) may be a promising strategy to improve plant salt tolerance effectively, which also saves both time and effort (Rady et al., 2020).

Selenium (Se) is a trace element that is required by both humans and animals (Gupta and Gupta, 2017). Adequate supplementation of Se per day plays a variety of important physiological functions in human body, such as preventing cardiovascular diseases and type-2 diabetes, regulating human immunity, and reducing the risk of cancer (Rayman et al., 2012). Since plants are the primary source of dietary Se, studies on Se compounds in plants are therefore necessary (Zhu et al., 2018). Selenate (SeO₄²⁻) and selenite (SeO₃²⁻) are the main forms of Se available for plants (Gupta and Gupta, 2017). Although there is no direct evidence that Se is essential for higher plants, studies in recent years have shown that Se at low concentrations could influence a series of plant growth and physiological processes, such as enhancing plant photosynthesis (Borbély et al., 2021), enhancing root and shoot growth (Diao et al., 2014), promoting the germination and nitrogen assimilation, increasing starch accumulation, and delaying plant senescence (Hajiboland et al., 2019). In addition, a series of studies have reported that Se could mitigate the adverse impacts on plants of different environmental stresses, e.g., salt, drought, high temperature and heavy metals (Elkelish et al., 2019; Rady et al., 2020). It was reported that Se could enhance the plant growth index of maize under salt stress (Jiang et al., 2017). Several studied have shown that Se could protect plants against severe stresses via improving antioxidant capacity, increasing the photosynthetic indices, and producing secondary metabolism (Elkelish et al. 2019; Zsiros et al., 2019). However, a lot of researches remain to be done to explore the impacts of exogenous Se supplementation on plants in response to salinity.

Previous studies have showed that some phytohormones played important roles in Se-enhanced tolerance of abiotic stresses, such as abscisic acid (Zahedi et al., 2019), auxin (Jia et al., 2018), jasmonic acid and ethylene (Tamaoki et al., 2008). However, whether salicylic acid (SA) is involved in Se-induced salt tolerance of plant has not been reported. SA is an endogenous plant hormone, which is crucial in the regulation of a variety of physiological and developmental processes in plants, including plant growth, maturity, senescence, plant photosynthesis, and transpiration, etc. (Koo et al. 2020). It also serves as a signaling molecule, triggering allergic reactions and induce stress resistance of plants to salt, drought, heat, heat, cold, and heavy metals (La et al., 2019; Wassie et al. 2020). Khalvandi et al. (2021) found that 0.5 mM SA treatment could improve photosynthetic performance, maintain membrane permeability, induce stress-response related proteins and increase antioxidant enzyme activity, effectively alleviating the negative impacts caused by drought stress on the winter wheat growth. Ardebili et al. (2014) found that using a combination of Se and SA to mitigate the effects of salt stress on soybean was more successful than using either Se or SA respectively. Although Se or SA can improve salt stress tolerance on their own, the interactions between Se and SA during salt stress are unknown. The mechanisms of Se-enhanced plant tolerance under salt stress, as well as the impact of plant hormone like SA, should be investigated.

Tomato (Solanum lycopersicum L.), one of the most frequently cultivated and consumed horticultural crops, has long been considered a model plant for the study of plant resistance to various adverse stresses, whose growth and yield are severely impaired when it is exposed to soil salinity (Fan et al. 2021). In addition, compared to other vegetables such as leafy vegetables and legumes, tomato accumulates far less Se, which makes it an excellent model for exploring the biochemical and molecular mechanisms of Se-enhanced stress resistance in plants (Puccinelli et al., 2019). The goal of this work was to learn more about Se’s involvement and regulatory mechanism under salt stress through investigating the plant photosynthesis, plant growth indicators, antioxidant system of the chloroplasts, and the regulation of plant hormones of Se application. This study will help to clarify the inner mechanism of Se in enhancing tomato’s salt resistance, and provide some theoretical support for the usage of Se fertilizer in agricultural production in salinity areas.

Plant materials and treatment

The experimental materials were tomato (Solanum lycopersicum L.) cultivar "Ailsa Craig”. Plump and uniform seeds were sterilized by soaking in deionized water for 15 min at 55°C, then germinated for 2 days on wet filter paper in a growth chamber without light at 28℃. The germinated seeds were planted in the medium in the greenhouse with the day/night temperature of 25℃/18℃. At the three-leaf stage, the seedlings were moved to the half-strength modified Hoagland solution (Gou et al., 2020a), which was aerated for 10 min every 3 h. The culture solution pH was adjusted to 6.2 and changed every 3 days. Five days later, the seedlings were subjected to Se or salt stress treatment via adding 0.25 mM sodium selenite (Na₂SeO₃) or 150 mM sodium chloride (NaCl) to the nutrient solution. Among the different Se doses tested, supplementation with 0.025 mM Se had the most noticeable effect in relieving salt stress damage in our early pre-experiment (data not shown). These experiments include four different treatments, including: (1) CK: no Se and no NaCl; (2) Se: 0.025 mM Na₂SeO₃; (3) NaCl: 150 mM NaCl; (4) NaCl + Se: 150 mM NaCl + 0.025 mM Na₂SeO₃. After 15 days of salt stress, the fully expanded leaves and roots of tomato seedlings were wrapped in tin foil and preserved at -80℃ after being frozen in liquid nitrogen.

The biomass, relative electrical conductivity (REC), and relative water content (RWC)

A straightedge and a vernier caliper were used to measure the plant height and stem diameter of six randomly picked plants. The REC of tomato leaves was measured as follows: the fully expanded third leaves were taken and cut into pieces. Weigh 0.1 g sample and put into a centrifuge tube containing 20 ml of deionized water, shaken for 2 h and its electrical conductivity was measured (EC1). The sample was then boiled for 10 min, cooled and measured for electrical conductivity (EC2). The REC was calculated with the formula: REC (%) = EC1⁄EC2*100. The RWC of leaves was measured as described by Fan et al (2022).

Photosynthetic Parameters Measurement

The second fully expanded leaves of tomato were used to determine the photosynthetic parameters using the Li-6800 portable photosynthesis system (LI-COR, Lincoln, USA). The formula: WUE = Pn/Tr was used to calculate the instantaneous water use efficiency (WUE).

Se content determination

Plant tissues were dried for 3 days under 80°C in an oven and ground into powder. 0.2 g sample was weighted and placed in a digestion tube containing 5 mL extracting solution (nitric acid: perchloric acid = 4:1). After incubation overnight, the solution was first digested at 120°C in the metal bath for 2 h, and then warmed up to 170°C. The solution was heated until the remaining volume is about 2 mL, cooled, and diluted to 30 m. 5 mL of the solution was taken out, added the same volume of 6 mol/L hydrochloric acid, reaction at 100°C for 30 min. Then it was mixed with 5 mL of 6 mol/L hydrochloric acid, incubated for 30 min in a boiling water bath. After making the blank control and selenium standard curve, an atomic fluorescence spectrometer (LC-AFS-8530, China) was applied to determine the Se content.

The malondialdehyde (MDA), soluble sugar and proline content

The MDA content was measured using thiobarbituric acid reaction according to Gou et al (2020a). According to Fan et al (2022), the soluble sugars were determined with the sulphate ketone colorimetry method.

The proline was measured as described by Shi et al. (2016). 0.1 g sample was added with 3 mL of 3% sulfosalicylic acid, incubated for 10 min in a boiling water bath, then cooled down and centrifuged at 6,000 g for 10 min. After that, 1 ml of the supernatant was collected and added with 4 mL extraction buffer (distilled water: glacial acetic acid: acidic ninhydrin = 1:1:2). The mixture solution was boiled for 1 h and cooled down. After that, 2 mL toluene was added and stratification was carried out for 2 h. After full extraction, the red toluene phase was taken for colorimetric analysis at 520 nm. The proline content was calculated based on a standard curve.

Quantification and visualization of reactive oxygen species (ROS)

The hydrogen peroxide (H₂O₂) content was assayed with a fluorescent dye 2,7-dichlorofluorescin diacetate as described by Han et al. (2020). The superoxide anion (O2^•−) content was determined using the sulphanilic acid and α -naphthylamine solution as described by Shi et al. (2014). The visualization of H₂O₂ and O^2•− accumulation was performed with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) solution as reported by Zhu et al (2020).

Antioxidant enzymes activity assays

The weight of 0.3 g of frozen material was ground into homogenate with 4 mL ice-cold sodium phosphate buffer (100 mM, pH 6.8). The homogenate was centrifuged under 12,000 g at 4°C for 20 min, and the supernatant was collected to determine the antioxidant enzymes activity (Gou et al., 2022b).

Plant hormone content

The weight of the material was ground into powder with liquid nitrogen, then mixed thoroughly with 2 mL of the extraction buffer (isopropanol: sterile water: HCl = 2:1:0.002) and incubated at 4℃ overnight. The supernatant was obtained by centrifuging the solution at 7000 g for 12 min at 4°C. The supernatant was collected, added with 2 mL of dichloromethane and shaked in an oscillation incubator at 4°C for 1 h, and centrifuged at 7000 rpm for 10 min at 4°C. Then, 2 mL of the lower fraction was taken, blew dry with nitrogen, added with 100 µL methanol and filtered through a 0.22 µm microporous membrane. The liquid chromatography (QTRAP5500, AB, USA) was applied to determine the content of various phytohormones.

Analysis of gene expression

Total RNA was isolated and purified using Trizol Reagent (Omega, Norcross, USA), and first-strand cDNA was generated with HiScript® Ⅱ Q RT SuperMix for qPCR kit (Vazyme, Nanjing, China). The gene expression analysis was performed on a QuantStudio®5 Real-Time PCR System (ABI, Carlsbad, CA) using SYBR Premix Ex Taq™ kit (Takara). The specific primers were listed in Table S1. The GAPDH (GenBank: NM_001321306) gene was selected as internal control.

SA biosynthesis inhibitor treatment

Two days in advance, the four-leaf stage seedings were pre-sprayed with a salicylic acid biosynthesis inhibitor ABT (1-aminobenzotriazole, Yang et al., 2018), followed by the addition of Se and NaCl. The experiment included four treatments: (1) CK + 100 µM ABT; (2) 150 mM NaCl; (3) 150 mM NaCl + 0.025 mM Se; and (4) 150 mM NaCl + 0.025 mM Se + 100 µM ABT. The PH of culture solution was adjusted to 6.2 and changed every 3 days.

Statistical analysis

The data was analyzed using SPSS 19.0 software (IBM, Armonk, NY). Data are means ± SD (n = 3) calculated with three biological replicate samples. One-way analysis of variance (ANOVA) and Duncan’s multiple range test were performed for comparison of significant differences between treatments (P < 0.05). All the experiments were repeated at least three times.

Effects of Se on plant growth parameters under salt stress

In order to investigate the function of Se on plant growth and development under salt stress, tomato seedlings were applied with 150 mM NaCl and exogenous Se to evaluate the salt resistance of various treatment groups (Fig. 1). Under normal condition, Se supplementation significantly increased plant height, but had no effect on stem diameter of tomato seedlings (Fig. 1B). After salt stress treatment for 15 d, both the seedlings with and without Se treatment showed shorter plant height and smaller stem diameter compared with the control. Moreover, the tomato seedlings without Se treatment showed obvious wilting, petiole drooping, while the Se-treated plants did not exhibit obvious symptoms of salt damage (Fig. 1A). In addition, compared with the seedlings without Se application, the plant height and stem diameter of tomato seedlings under Se treatment significantly increased by 17.8 and 23.0% respectively (Fig. 1B).

The relative electrical conductivity (REC) is a key parameter of stress damage, which was investigated. In addition, the relative water content (RWC) of tomato seedlings from different treatment groups was also tested (Fig. 1C). Exogenous Se treatment did not change the REC and relative water content of tomato plants compared with the control under non-stress condition. However, compared to the seedling under salt stress without Se treatment, the REC of tomato leaves treated with Se decreased by 16.2%, and the RWC of leaves with Se treatment increased by 9.8%.

Exogenous Se promoted photosynthesis under salt stress

To study the impact of exogenous Se on the plant photosynthesis under salt stress, the photosynthetic parameters of various treatment groups were investigated, such as the net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO₂ concentration (Ci) (Table 1). Under normal condition, Se addition had no effect on the photosynthetic characteristics of tomato plants compared to control plant. Salt stress treatment dramatically reduced the Pn, Tr, Gs, and Ci in tomato seedlings. But compared to the seedling treated with salt stress without Se addition, Se supplementation significantly increased the Pn, Tr, Gs, and Ci of tomato leaves by 27.9%, 11.0%, 73.3%, and 10.7%, respectively. Under normal and stress conditions, Se treatment did not increase water use efficiency (WUE) of tomato leaves.

Table 1

Influence of Se on the photosynthetic characteristics in tomato plants under salt stress
Treatments	Net photosynthetic rate Pn (µmol CO₂ m^− 2s^− 1)	Transpiration rate Tr (mmol H₂O m^− 2s^− 1)	Stomatal conductance Gs (mmol H₂O m^− 2s^− 1)	Intercellular CO₂ concentration Ci (µmol CO₂ mol^− 1 air)	Water use efficiency WUE (mmol CO₂ mol^− 1 H₂O)
CK	4.95 ± 0.2a	3.86 ± 0.87a	111.74 ± 5.6a	329.49 ± 34.09a	1.28 ± 0.26c
Se	4.73 ± 0.25a	3.71 ± 0.28a	95.13 ± 7.3a	303.08 ± 22.13ab	1.27 ± 0.13c
NaCl	2.72 ± 0.2c	1.73 ± 0.08c	40.68 ± 3.34c	260.5 ± 8.85c	1.58 ± 0.17b
NaCl + Se	3.48 ± 0.37b	1.92 ± 0.06b	70.52 ± 15.2b	288.2 ± 10.92b	1.81 ± 0.16a
Note: Different lowercase letters denote significant differences among different treatments at P < 0.05.

Se contents in different parts of tomato plants

We measured the Se content in tomato roots, stems, and leaves with exogenous Se addition under normal and salt stress (Table 2). The results showed that adding Se to the culture solution obviously increased the Se content in different tomato tissues.

Table 2

Influence of exogenous Se on Se content in various parts of tomato under salt stress
Treatment	Root (µg/g)	Stem (µg/g)	Leave (µg/g)
CK	0.37 ± 0.2c	ND	ND
Se	15.07 ± 5.12a	0.7 ± 0.09	0.52 ± 0.23
NaCl	0.16 ± 0.06c	ND	ND
NaCl + Se	8.85 ± 1.45b	0.42 ± 0.17	0.75 ± 0.05
Note: Different lowercase letters denote significant differences between treatments (P < 0.05).

As shown in Table 2, applying Se to the culture solution obviously raised the Se content in tomato leaves, stems, and roots under both non-stress and salt stress conditions, demonstrating that making the plants absorb Se by their roots is a convenient and effective strategy to enhance Se content in different tomato tissues.

Effects of Se on lipid peroxidation and osmoprotectants under salt stress

As shown in Fig. 2, under normal condition, the MDA, soluble sugar and proline content was not changed by exogenous Se application, either in tomato leaves or roots. Their contents were significantly induced by salt treatment for 15 d. However, compared to the seedling under salt stress without Se treatment, Se application remarkably decreased the MDA and soluble sugar content of leaves under salt stress by 30.1% and 37.6% respectively (Fig. 2A, B). Se also reduced their contents in tomato roots in response to salt stress by 44.7% and 29.8%, respectively. In addition, added Se slightly decreased the proline content in leaves but did not change its content in roots under salt stress (Fig. 2C).

H₂O₂ and O₂^•- accumulation and antioxidant defense

The histochemical staining experiment showed that Se application did not affect the H₂O₂ and O₂^•− accumulation in tomato leaves under non-stress environment (Fig. 3A). Salt stress obviously increased the ROS accumulation in tomato leaves, which was significantly decreased by Se application (Fig. 3A upper). Se supplementation remarkably decreased the contents of H₂O₂ and O₂^•− in tomato leaves under salt stress treatment by 22.2% and 47.2% respectively (Fig. 3A lower).

In order to investigate the influence of exogenous Se on the antioxidant defense of tomato plants in response to salt stress, several antioxidant enzymes activity was investigated furtherly, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) (Fig. 3B). Se treatment had no effect on the activity of SOD and POD either in the leaves or roots of tomato plants under non-stress conditions, however, it did diminish the activity of CAT in tomato leaves but had no effect on that of in tomato roots. Salt stress treatment remarkedly increased the activity of SOD, POD and CAT, while the added Se obviously decreased their activity by 19.2%, 39.6% and 34.1% in leaves, and 21.7%, 15.1% and 34.1% in roots compared to seedlings in the absence of Se treatment.

Phytohormones contents

To investigate the influence of Se addition on the contents of phytohormones under salt stress treatment, we measured the level of several phytohormones in different tomato tissues after salt stress treatment for 5 days with or without Se addition, including salicylic acid, abscisic acid (ABA), melatonin (MT), and jasmonic acid (JA) (Fig. 4). As shown in Fig. 4A, exogenous Se application obviously raised the SA content in tomato leaves and roots both under non-stress and salt stress environments. In salt stress conditions, the SA contents in leaves and roots of Se-treated tomatoes were 88.8% and 81.4% higher respectively compared with the tomato seedlings without Se supplementation. No significant difference in ABA content was tested among the tomato leaves of different treatment groups. The ABA content in se-treated tomato roots was lower than that of the control plant (Fig. 4B). The MT content in different tomato tissues was not changed significantly by Se addition both under non-stress and salt stress conditions (Fig. 4C). Under normal growth conditions, Se application did not change the JA content in tomato leaves. Salt treatment significantly reduced the JA content in tomato leaves regardless of Se addition or not. Both under non-stress and stress conditions, the JA content in tomato roots was decreased by Se treatment (Fig. 4D).

The expression of genes associated with SA biosynthesis and transformation

Furthermore, qRT-PCR was applied to analyze the transcripts of some genes involved in SA biosynthesis and transformation (Fig. 4E). The transcripts of PAL1 were up-regulated in tomato leaves in response to Se application both under normal and salt stress conditions for 5 d. However, added Se induced the expression of PAL1 under salt stress treatment but did not change its expression in tomato roots in normal condition. Under normal condition, Se application did not change the transcripts of ICS in tomato leaves, but down-regulated its expression in the roots. In addition, exogenous Se treatment especially increased the expression of ICS gene in tomato leaves in response to salt stress. Under normal and stress conditions, the expression of SAMT gene in tomato leaves was not changed by Se treatment. However, added Se up-regulated the PAL1 expression in tomato roots under normal condition but did not change its expression under salt stress condition. Se application down-regulated the expression of SABP2 in different tomato tissues under the normal condition, however added Se did not change its expression under salt stress condition.

Roles of SA in Se-induced salt stress tolerance in tomato seedlings

As shown in Fig. 5A, the tomato pre-treated with 100 µM ABT (a SA biosynthesis inhibitor) still maintained high plant height and stem diameter, indicating that ABT had no obvious inhibition effect on plant growth. The plant height and stem diameter of tomato seedlings were significantly reduced by salt stress treatment, while these two growth indexes were higher in Se-treated plant seedlings (Fig. 5A). However, pre-sprayed with ABT weakened the promoting effect of Se on stem diameter of tomato seedlings. Then the effects of ABT on plant photosynthesis after selenium application in response to salt stress were studied (Fig. 5B). The Pn, Gs, Ci and WUE were significantly reduced under salt stress treatment. However, these parameters were significantly recovered after selenium treatment, while ABT pre-treatment attenuated the promotion effects of Se treatment on plant photosynthesis, as manifested by a significant decline of Pn, Gs, Ci and WUE by 53.0%, 79.5%, 41.7%, and 29.8%. In salt stress condition, ABT pre-treatment also obviously raised the REC by 12.9% compared to the plants with Se treatment alone but did not change the relative water content (Fig. 5C). Se treatment obviously mitigated the wilting status of tomato plants induced by salt stress, but the plants became wilting again after pretreatment with ABT (Fig. 5D). The H₂O₂ visualization in leaves with ABT and Se addition under salt stress condition was investigated by histochemical staining experiment as well (Fig. 5E). Se-mediated decrease in H₂O₂ accumulation under salt stress was obviously abolished by ABT pre-treatment.

Exogenous Se promoted photosynthesis and growth of plants under salt stress

Excess salinity stress leads to osmotic stress and ion toxicity, which increases the Na⁺ content in plant tissue, aggravates the cell membrane peroxidation, causes oxidant damage, and suppresses plant photosynthesis, thus leading to nutrient deficiency, retarded plant growth and biomass reduction (Gupta and Huang, 2014). In our study, we analyzed the impact of Se application in enhancing tomato plant growth through its involvement in the regulation of key physiological and biochemical parameters. Our study showed that the Se supplementation boosted plant development under salt stress treatment, as evidenced by an obvious increase in plant height and stem diameter. Moreover, Se also relieved the wilting state of tomato plants caused by salt stress (Fig. 1). Furthermore, Previous studies showed that Se could improve plant growth and development in many plants exposed to various abiotic stresses, which were partly attributed to its promoting effect on photosynthetic efficiency of plants (Fan et al., 2022; Diao et al., 2014), which was also supported by our results. The decrease of plant photosynthesis under salinity was probably due to the lower stomatal conductance (Gs) and the net photosynthetic rate (Pn) caused by salt stress (Jiang et al., 2017). In our study, added Se increased the Pn and Gs under salt stress, thus protecting the photochemical and increasing Tr, and further enhancing photosynthesis and promoting the plant growth compared to the tomato seedlings in the absence of Se (Fig. 1, Table 1). Under normal condition, exogenous Se application raised the plant height of tomato seedlings, since Se had been found to play important roles in the production and signaling of some phytohormones involved in plant internode elongation, such as cytokinin and gibberellin (Shan et al., 2021; Jiang et al., 2019).

Exogenous Se promoted the accumulation of osmoprotectants of plants under salt stress

Soluble sugars (sucrose, glucose and fructose), a kind of important osmoprotectants, play crucial roles in regulating osmotic adjustment, maintaining the water balance of plants, scavenging excess ROS, and protecting cell membranes under various adverse environmental stresses (Dien et al., 2019). In addition, soluble sugars could act as a kind of chelating agent to entrap Na⁺ within starch granules, which helps in neutralizing the Na⁺ toxicity of plants under salt stress (Kanai et al., 2007). Proline is an important amino acid and provide several benefits to plants in response to salinity (El Moukhtari et al., 2020). It not only acts as an osmoprotectant to regulate the water balance of plant cells, but also plays important roles in ROS scavenging and stabilizing membranes to prevent electrolyte leakage (Shamsul et al., 2012; Naliwajski et al., 2021). Previous studies reported that the soluble sugar and proline content was evidently enriched when plants are subjected to salt stress, which might contribute to the osmotic and salt stress tolerance of plants (Boriboonkaset et al., 2013; El-Katony et al., 2021), while other studies reported opposite or inconsistent results. For example, Watanabe et al. (2000) reported that the total soluble sugar content increased in young Populus euphratica leaves but decreased in the mature leaves after salt stress treatment. It was also reported that the soluble sugar content in stress-resistant rice decreased in response to drought stress (Xu et al., 2015). Naliwajski et al. (2021) reported that the acclimated cucumber plants accumulated lower level of proline under salt stress compared to the non-acclimated plants, both under normal and stress conditions. These studies showed that the change of soluble sugars and proline content in plants in response to salt stress varied among different plant species, different varieties and different plant growth stage (Xu et al., 2015; Naliwajski et al., 2021). Furthermore, in our study, the content of soluble sugars and proline of Se-treated tomato seedlings under salt treatment for 15 d was lower compared to the tomato plants without Se supplementation (Fig. 2B, C), which suggested that the duration of Se application in plants under salt stress might also be one of the factors affecting the accumulation of soluble sugars and proline, since the toxicity and damage to plants caused by salt stress was basically mitigated by Se addition for 15 d (Fig. 1A) and it was not necessary for plants to keep producing high level of osmotic protective substance, which indirectly indicated the promoting effect of selenium on plant salt stress.

Exogenous Se alleviated the oxidative damage caused by salt stress treatment

Salt stress is often accompanied with the secondary oxidative damage for plants, since high salinity leads to the accumulation of excessive ROS, such as H2O2, O^2-, and O^•−, which potentially causes cell membrane lipid peroxidation and even cell death (Zhang et al., 2016). Moreover, MDA levels and relative electrolyte leakage in plants can represent the integrity of their cellular membranes in response to salt stress (Gou et al., 2020b). Plants have evolved an efficient antioxidant system, including enzymes such as SOD, POD and CAT to scavenge ROS to avoid oxidative damage caused by various environmental stresses (Gill and Tuteja, 2010). It was reported that exogenous Se could regulate antioxidant defense system via upregulating the activities of several antioxidant enzymes, reflected by decreased H₂O₂ and MDA content in the cells of different plant species under various environmental stresses (Ashraf et al., 2018; Kaur and Nayyar, 2015). In this research, Se supplementation clearly mitigated the oxidative damage produced by salt stress (Fig. 3A), whereas the activity of various antioxidant enzymes was lower in Se-treated tomato seedlings under salt stress than in tomato plants in the absence of Se addition (Fig. 3B), which was probably due to the oxidative damage induced by salt stress treatment was basically eased by Se supplementation for 15 d, thereby indirectly decreasing the plant demand for antioxidant enzymes to eliminate ROS. Furthermore, it has been reported that Se was an integral part of the enzymes like glutathione peroxidase (GPx) and other seleno-chemical compounds, all of which played vital roles in reducing cell peroxidation and oxidative damage (Puccinelli et al., 2017), suggesting Se might regulate other antioxidant mechanisms under salt stress.

SA was involved in Se-induced salt tolerance in tomato plants

Previous studies reported that SA could regulate various plant metabolic processes, including regulating the stomatal and water potential, enhancing plant nutrient uptake and photosynthesis, and promoting plant growth and development, which were some of the mechanisms by which SA regulated the plant stress resistance (Khan et al., 2015). PAL and ICS encode phenylalanine ammonia-lyase and isochorismate synthase respectively, which are the two key enzymes of the two SA bio synthesis pathways (Ding and Ding, 2020). It has been observed that the transcripts of PAL1 were remarkedly up-regulated in tomato leaves and roots under salt stress whereas the expression of ICS was only induced by Se addition in tomato leaves (Fig. 4E). Moreover, the SA content was obviously induced in different tomato tissues (Fig. 4A). These results suggested that the accumulation of SA in tomato leaves under salt stress was probably attributed to the up-regulation of PAL pathway by Se application, while its accumulation in roots might only be related to the up-regulation of ICS pathway. As described by Park et al. (2007), SAMT1 catalyzes SA to become methylated SA (MeSA), which was hydrolysed by SABP2 and turned back into SA again. The expression of SAMT1 and SABP2 was not changed by exogenous Se treatment, indicating Se did not regulate the mutual transformation between SA and MeSA in response to salt stress (Fig. 4E). Interestingly, pre-sprayed with a SA biosynthesis inhibitor (ABT) reduced the stimulating effect of Se on plant growth and plant photosynthesis (Fig. 5), worsen the damage caused by oxidative stress, and eventually abolished the beneficial effect of Se application on the plant salt resistance, which further proved the important function of SA in the Se-enhanced plant salt resistance.

Exogenous Se application improved the photosynthetic rate of tomato, thereby promoting plant growth and development. Se addition also reduced the relative electrical conductivity and MDA, decreased the content of H₂O₂ and O₂^•−, and mitigated the oxidative damage caused by salt stress. Exogenous Se increased SA content both in tomato leaves and roots via up-regulating of PAL pathway of SA biosynthesis. After pre-sprayed with a SA inhibitor ABT, the photosynthetic efficiency of tomato plants decreased, the growth was weakened, and the relative electrical conductivity was increased, indicating that the alleviating effect of Se on salt stress was abolished. Our results clarified the roles and its regulation mechanisms in plant salt stress tolerance, as well as the critical involvement of SA in this process (Fig. 6). The study of Se under abiotic stress tolerance will give more theoretical foundation for the usage of exogenous Se in agricultural production to enhance the crop growth and yield under salt stresses.

ABT, 1-aminobenzotriazole; DAB, diaminobenzidine; H₂O₂, hydrogen peroxide; MDA, malondialdehyde; NBT, nitroblue tetra-zolium; O₂^•-, superoxide anion; qRT-PCR, quantitative real-time PCR; ROS, reactive oxygen species; SA, salicylic acid; Se, selenium

Funding

This work was supported by National Natural Science Foundation of China (32072561).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Hong Wu and Shuya Fan. HG analyzed the original data. The first draft of the manuscript was written by Jia Guo.

Data availability

The data generated during the current study are available from the corresponding author on reasonable request.

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Roles of salicylic acid in selenium-enhanced salt tolerance in tomato plants

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