Effects of combined application of selenium and various plant hormones on the cold stress tolerance of tomato plants

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Here, the effects of Se and various plant hormones on tomato plants under cold stress have been investigated. Methods The biomass, relative electrical conductivity, photosynthetic pigments, malondialdehyde, chlorophyll fluorescence, soluble sugar, proline contents, as well as the regulation of plant hormones were examined. Results Among several plant hormones, the co-application of Se and SA was the most effective in reducing the cold stress of tomato plants. The co-application of Se and SA prevented the oxidative damage caused by cold stress on tomato chloroplasts and preserved the plasma membrane integrity and regulated the osmotic substances under cold stress. The interaction between Se and SA modulated the expression of some cold-induced genes thereby conferring cold tolerance of tomato plants. But the pretreatment with a SA biosynthesis inhibitor (AIP) eliminated the favorable influence of Se on the cold resistance of tomato, indicating that enhancing the cold resistance of plants by regulating the synthesis of SA might be one of the mechanisms by which Se enhanced tomato’s resistance to cold stress. Conclusions Our results clarified the roles of Se and its regulation mechanisms in plant cold stress tolerance and the critical involvement of SA in this process, which might offer a theoretical foundation for using Se fertilizer to increase the production of crops under adversity stresses. cold stress melatonin salicylic acid selenium tomato Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cold stress, including non-freezing chilling stress (0–15℃) and freezing stress (< 0℃) causes severe physiological damage to plants and changes in plant morphology. The symptoms are generally dehydration of plant leaves, gradual wilt, reduced plant growth, and low accumulation of dry matter in plants, resulting in declined yield or quality (Soualiou et al., 2022 ). Moreover, cold stress can affect the activity of key enzymes, impair membrane function and cause cell dehydration, leading to unstable metabolism of plant cells (Guan et al., 2023 ). Some studies reported that plant cells adapted to cold stress via regulating the plasma membrane, intracellular osmotic protective substances, REDOX systems, and photosynthetic rates (Guan et al., 2023 ). Selenium (Se) is a necessary trace element needed by both humans and animals (Rayman, 2012 ). Although Se is not a necessary element for plants, several reports have shown that low levels of Se had positive effects on plants, including stimulating plant growth and development, strengthening plant resistance, and boosting crop quality (Wang et al., 2022 ). Other research revealed that Se could enhance plant resistance to various adverse environments, including high and low temperatures, salinity, drought, heavy metals, etc. (Huang et al., 2018 ; Rady et al., 2020 ). Huang et al. ( 2018 ) discovered that Se could reduce the accumulation of reactive oxygen species and reduce oxidative damage to plants by participating in the redox process in plants and removing excess free radicals in plants. Fan et al. ( 2022 ) reported that Se application improved the photosynthetic efficiency of tomato plants and promoted their growth and development under drought stress. It was found that Se in strawberries induced an antioxidant effect by increasing enzyme compounds (mainly SOD) and non-enzyme compounds (such as ascorbic acid), and increased the photosynthetic rate, chlorophyll content, and stomatal conductivities of strawberry plants in response to cold stress (Huang et al., 2018 ). Many studies have shown that plant hormones play vital roles in plant resistance to a number of environmental stresses (Waadt et al., 2022 ). Some plant hormones effectively regulate plant growth and development at low temperatures, including salicylic acid (SA), melatonin (MT), abscisic acid (ABA), etc. (Tian et al., 2022 ). It was reported that SA (1 and 2 mM) could reduce cold damage in grapevines under low-temperature stress (Li and Wang, 2021 ). SA can inhibit electrolyte leakage, reduce the accumulation of reactive oxygen species, increase the activity of cell protective enzymes, and improve plant photosynthetic efficiency under cold stress (Kang et al., 2004 ). Exogenous melatonin has been demonstrated to improve cold tolerance in a variety of plant species, including Arabidopsis , wheat, and watermelon (Bajwa et al., 2014 ; Turk et al., 2014 ; Li et al., 2017 ). According to Guo et al. ( 2012 ), exogenous ABA administration enhanced pepper seedlings' tolerance to cold-induced oxidative damage primarily through increasing the activity of antioxidant enzymes and the expression of genes associated with cold stress. These reports suggested that plant hormones also played important roles in plant response to cold stress. However, until now, the synergistic effects of Se and a range of plant hormones on the physiological, biochemical, and molecular response of plants under various environmental stresses have been less reported and a thorough investigation is necessary. Tomato ( Solanum lycopersicum L.) is a key vegetable and fruit crop. As one of the main crops cultivated in winter and spring facilities, it is highly vulnerable to cold stress (Han et al., 2020 ). Therefore, low temperature poses a great challenge to the production of its protected land, which seriously affects the growth and development of its adult stage, early yield, and fruit commodity (Zhou et al., 2018 ). Improving cold stress resistance of tomato crops has a certain guiding significance for tomato crop production and optimization of cultivation measures (Haghighi et al., 2014 ). Until now, there are few reports on the impacts of Se on the cold resistance of tomatoes, and few studies on the interaction between Se and exogenous hormones under cold stress, which requires further exploration. This study mainly explored the role of the interaction between Se and exogenous hormones such as SA, MT, and ABA in enhancing tomato seedlings' resistance to cold stress, in order to improve the ability of plants to resist cold stress, so as to provide an important means to improve the yield of vegetables and other crops under cold stress. Our study will provide some theoretical basis for the development and application of compound Se fertilizer. Materials and Methods Plant materials and treatment The tomato seeds ("Ailsa Craig") with full grains and uniform size were selected, sterilized at 55℃ and germinated in a climate chamber. Seeds with consistent germination were sown in the commix matrix for seedling cultivation. The two-leaf tomato seedlings were planted in a tank with 1/4 strength Hogland nutrient solution (pH 5.8). The seedlings were slowed in a climate chamber with the temperature of 25℃/18℃ (day/night) and 75% relative humidity. Na 2 SeO 3 was used as the selenium source, and the hormones were SA, MT, and ABA, respectively. ComCat®, a commercial compound plant growth regulator, was used as positive control by foliar spraying. It dominantly consists of gibberellins, auxins (indole-3-acetic acid), brassinosteroids, amino acids, kitenins, and natural metabolites. The experiments contained ten treatments: (1) CK0: Normal temperature control (25℃/18℃ day and night); (2) CK1: low temperature control (10℃/4℃ day and night); (3) Se: 0.005 mM Na 2 SeO 3 ; (4) SA: 0.1 mM salicylic acid; (5) MT: 0.1 mM melatonin; (6) ABA: 0.05 mM abscisic acid; (7) Se + SA: 0.005 mM Na 2 SeO 3 + 0.1 mM SA; (8) Se + MT: 0.005 mM Na 2 SeO 3 + 0.1 mM MT; (9) Se + ABA: 0.005 mM Na 2 SeO 3 + 0.05 mM ABA; (10) CC: 0.1 g/L ComCat®. After 10 days of cold stress treatment (10℃/4℃ day and night), the fully developed tomato leaves and roots were taken and frozen at -80℃ for use. Plant growth index and electrolyte leakage calculation The plant height (from cotyledon to apex growth point) and stem diameter (1 cm above cotyledon) were measured with a ruler and a vernier caliper. Then the tomato seedlings were washed quickly with deionized water, dried up with tissue, and measured their fresh weight. Then the plants were dried at 105℃ for 12 min then at 80℃ overnight in a drying oven and the dry weight was measured. The relative electrical conductivity (RWC) of the plants was measured with the second fully expanded leaves according to Wu et al. ( 2023 ). The relative water content (REC) was measured according to Fan et al. ( 2022 ). The samples were rinsed thoroughly with deionized water, dried with paper towels, and quickly cut the leaves into strips. A sample of 0.1 g was added with 20 mL of water, shaken gently and the first electrical conductivity (EC1) was tested with a conductivity meter. Then samples were oscillated in a shaker at 100 rpm for 2 h to measure the second electrical conductivity (EC2). Finally, the sample was heated at 100℃ for 20 min, cooled and the conductance value (EC3) was measured. The REC was calculated as follows: REC=(EC2-EC1)/(EC3-EC1)×100%. The photosynthetic pigments content The determination of chlorophyll content was based on Wu et al. ( 2023 ). Tomato leaves were washed with deionized water, dried with paper towels, and quickly cut the leaves into strips. A sample of 0.1g was put into a test tube containing 10 mL of 95% ethanol. The extraction was carried out at room temperature until the leaf tissue was completely turned white. The extract was filled to 25 mL and the absorbance was measured at 470 nm, 649 nm, and 665 nm by an ultraviolet spectrophotometer. The contents of chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Chlx.c) were calculated as described by Arnon ( 1949 ). Measurement of chlorophyll fluorescence parameters Before the determination, the plants were placed in a dark environment for 30 min. Then the chlorophyll fluorescence index Fv/Fm (maximum PSⅡ quantum yield) was determined with the Chlorophyll fluorescence IMAGING system (Image-PAM, Walz, Germany). The sugar contents The soluble sugar contents were determined with the sulphate ketone colorimetry method as described by Fan et al. ( 2022 ). The reducing sugar contents were measured with 3,5-dinitrosalicylic acid (DNS) as described by Deshavath et al. ( 2020 ). The starch contents were measured by anthrone sulfuric acid colorimetry according to de Sousa et al. ( 2022 ). Antioxidant enzymes activity assays 0.3 grams of frozen material were homogenized in 4 mL of sodium phosphate buffer (100 mM, pH 6.8) and centrifuged at 12,000 g for 20 min under 4°C. Then the supernatant was taken and the antioxidant enzymes activity was determined according to Gou et al. ( 2020 ). Reactive oxygen species (ROS) quantitative analysis and histochemical staining According to Han et al. ( 2020 ), the amount of hydrogen peroxide (H 2 O 2 ) was measured using the 2,7-dichlorofluorescin diacetate. The superoxide anion (O 2 •− ) content was assessed as indicated by Shi et al. ( 2014 ). Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) solution were used to visualize the accumulation of H 2 O 2 and O 2 •− according to Zhu et al. ( 2020 ). qRT-PCR analysis Total RNA was isolated using Rn33 Plantpure Universal Plant Total RNA Rapid Extraction Kit (Aidlab, Shenzhen, China), and the first-strand cDNA was generated with High speed-strand cDNA Synthesis Plus Kit (Cofitt, Hongkong, China). The gene expression analysis was performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, USA) using ChamQ Universal SYBR Qpcr Master Mix (Vazyme, Nanjing, China). The β-actin (GenBank: NM_001321306) gene was used as internal control and the primers used were listed in Table S1 . SA biosynthesis inhibitor treatment A salicylic acid biosynthesis inhibitor (2-aminoindan-2-phosphonic acid, AIP) was pre-sprayed on the two-leaf stage seedlings two days prior to the addition of Se and cold treatment. Six treatments were included in this experiment: (1) CK0; (2) CK0 + AIP; (3) CK1 (10℃/4℃ day and night) (4) CK1 + 30 µm AIP; (5) CK1 + 0.005 mM Se; (6) CK1 + 0.005 mM Se + 30 µm AIP; Statistical analysis The test data were processed using Microsoft Excel 2016 software, SPSS24.0, and Sigmaplot14.0 for statistical analysis and plotting. Duncan’s multiple range test (P < 0.05) was applied to test the significant differences. Different letters are used to indicate significant variations between treatment means. Results Effects of Se, various hormones and their interaction on plant growth parameters under cold stress To evaluate the effect of applying sole or combined Se and various plant hormones on plant growth and development under cold stress, tomato plants were treated under low temperature (10℃/4℃, day and night) and exogenous Se and various hormones for 10 d to assess the cold resistance of plants from different treatment groups (Table 1 , Fig. 1 ). According to Fig. 1 A, compared with the normal temperature control group, cold stress resulted in short stature and stunted growth of tomato seedlings. However, after the application of Se and various hormones, the growth of seedlings under cold stress was significantly promoted, and the degree of leaf wilting was not obvious. As shown in Table 1 , application of sole or combined Se and various plant hormones significantly increased the plant height and stem diameter of tomato seedlings compared with the low temperature control group. However, there was no obvious difference between various treatments in promoting plant height and stem diameter. Moreover, application of various exogenous substances significantly increased the fresh weight of tomato seedlings, but there was no significant difference among different treatments (Table 1 ). All the treatments of sole or combined Se and various plant hormones also increased the dry weight of tomato plants under cold stress but the application of Se, SA, MT, and their combined treatments worked best (Table 1 ). Table 1 Influence of exogenous Se on plant growth and root characteristics of tomato seedlings under cold stress Treatment Growth indices Biomass (g/plant) Root morphological traits Plant height (cm) Stem diameter (cm) Total fresh weight Total dry weight Total root length (cm) Total surface area (cm 2 ) Total root Volume (cm3) Average root diameter (mm) CK0 18.67 ± 0.58a 4.97 ± 0.28a 11.2 ± 1.22a 1.15 ± 0.15a 804.75 ± 36.80 a 210.97 ± 32.72a 15.33 ± 5.33a 0.77 ± 0.09d CK1 7 ± 0.82d 3.25 ± 0.3c 2.76 ± 0.5c 0.29 ± 0.07d 130.00 ± 11.11c 47.01 ± 13.83bc 3.58 ± 1.52bc 1.23 ± 0.18ab Se 9 ± 0.71bc 4.23 ± 0.23b 4.93 ± 0.72b 0.44 ± 0.03b 178.85 ± 24.31b 55.63 ± 9.71bc 3.55 ± 0.33bc 1.09 ± 0.07bc SA 9.13 ± 0.48bc 3.99 ± 0.5b 4.53 ± 0.33b 0.4 ± 0.04bc 173.06 ± 13.40b 54.99 ± 12.08bc 3.71 ± 0.83b 1.30 ± 0.06a MT 9.13 ± 0.63bc 4.1 ± 0.21b 4.3 ± 0.49b 0.39 ± 0.04bc 169.42 ± 5.88bc 58.62 ± 3.62bc 3.27 ± 0.54bc 1.02 ± 0.12c ABA 8.25 ± 0.87bc 4.22 ± 0.38b 4.2 ± 0.46b 0.34 ± 0.04cd 132.64 ± 23.06c 50.04 ± 7.32bc 3.01 ± 0.67bc 1.10 ± 0.19bc Se + SA 9.5 ± 0.58b 4.01 ± 0.33b 4.76 ± 0.34b 0.43 ± 0.01b 186.03 ± 12.14b 48.75 ± 8.70bc 3.10 ± 0.50bc 1.03 ± 0.13c Se + MT 9.13 ± 1.31bc 4.18 ± 0.28b 4.73 ± 0.62b 0.42 ± 0.03bc 198.17 ± 40.20b 48.70 ± 12.53bc 2.70 ± 0.95c 0.97 ± 0.12c Se + ABA 8.5 ± 1bc 4 ± 0.1b 4.25 ± 0.27b 0.36 ± 0.04bcd 128.62 ± 7.03c 39.76 ± 8.66c 2.64 ± 0.80bc 1.10 ± 0.16bc BH 8 ± 0.71cd 3.99 ± 0.26b 4.02 ± 0.45b 0.34 ± 0.03cd 190.1 ± 15.02b 62.40 ± 10.07b 4.34 ± 0.57b 1.24 ± 0.16ab Note: Different lowercase letters denote significant differences among different treatments at P < 0.0 Effects of Se, various hormones and their interaction on tomato roots under cold stress Plant root morphology is usually an important evaluation index of plant stress resistance. The total root length, root surface area, and root volume of tomato seedling roots decreased significantly, while the average diameter increased under cold stress (Table 1 ). The application of Se, SA, MT, and their combined treatments had the best effect in promoting root length under cold stress. Effects of Se, various hormones and their interaction on chlorophyll content under cold stress After 10 d of cold stress treatment, the chlorophyll a, chlorophyll b, carotenoid, and total chlorophyll contents in the leaves of tomato seedlings declined significantly due to cold stress compared with control plants (Table 2 ). Compared with the low temperature control, the chlorophyll contents in each treatment group were significantly increased. Among them, the content of chlorophyll b in Se and various hormone combined treatment groups was significantly higher than that in Se and various hormones alone, but the differences between the combined treatment groups were not obvious. Moreover, the content of chlorophyll b in Se and hormone combined treatment was significantly higher than ComCat® treatment group. Table 2 Effects of exogenous selenium, different hormones and their interactions on chlorophyll content of tomato seedlings under cold stress Treatments chlorophyll a (mg g − 1 Fw) chlorophyll b (mg g − 1 Fw) chlorophyll a (mg g − 1 Fw) chlorophyll (a + b) (mg g − 1 Fw) CK0 1.40 ± 0.04a 0.47 ± 0.04a 0.25 ± 0.01a 1.87 ± 0.08a CK1 0.74 ± 0.05d 0.23 ± 0.03e 0.16 ± 0.01f 0.97 ± 0.08d Se 0.89 ± 0.01bc 0.28 ± 0.01d 0.19 ± 0.01bcd 1.17 ± 0.02bc SA 0.91 ± 0.04bc 0.29 ± 0.02cd 0.20 ± 0.01b 1.20 ± 0.06bc MT 0.90 ± 0.02bc 0.30 ± 0.01bcd 0.19 ± 0.00bcd 1.20 ± 0.04bc ABA 0.94 ± 0.09b 0.32 ± 0.04bcd 0.19 ± 0.01bc 1.26 ± 0.12b Se + SA 0.92 ± 0.03bc 0.34 ± 0.02b 0.18 ± 0.01de 1.25 ± 0.05b Se + MT 0.90 ± 0.02bc 0.34 ± 0.02b 0.18 ± 0.01cde 1.24 ± 0.03bc Se + ABA 0.88 ± 0.02c 0.32 ± 0.03bc 0.17 ± 0.01f 1.20 ± 0.04bc BH 0.86 ± 0.01c 0.29 ± 0.01cd 0.17 ± 0.01ef 1.15 ± 0.02c Data are mean ± standard deviation. Different letters in the same column and variety indicate significant differences at the 5% level (Duncan's test). Effects of Se, various hormones and their interaction on the characteristics of chlorophyll fluorescence under cold stress Chlorophyll fluorescence analysis is one way of calculating the plant damage caused by cold stress. Compared with the control plants, cold stress obviously decreased the maximum photochemical quantum yield of photosystem II (Fv/Fm) (Fig. 1 B, C). However, application of sole or combined Se and various plant hormones obviously increased the Fv/Fm value under cold stress. Among them, the combined treatment of Se plus SA or MT significantly increased the Fv/Fm value compared with the single treatments. Effects of Se, various hormones and their interaction on the membrane stability under cold stress As seen in Fig. 2 A, compared with low temperature control, the application of various exogenous substances all significantly reduced the relative electrical conductivity of tomato leaves, with Se, SA, MT, ABA and ComCat® treatments decreased by 26.4%, 30.0%, 29.5%, 24.0% and 17.4%, respectively, and Se + SA, Se + MT and Se + ABA treatment decreased the REC by 36.0%, 33.9%, and 25.3%, respectively. The results showed that Se and SA combined treatment significantly reduced the REC in leaves, and effectively alleviated the damage to cell membrane caused by cold stress. The effect of the combined application of Se and SA was better than that of ComCat®, a commonly used plant growth regulator in the market. Figure 2 A showed the RWC of tomato plants from various treatment groups. Cold stress apparently reduced the water content of tomato leaves, however, the application of sole or combined Se and various plant hormones significantly increased the RWC under cold stress, among which Se plus SA treatment had a better effect. Moreover, ComCat® treatment had no significant effect on plant RWC under cold stress. These results indicated that combined treatment of Se and SA could effectively increase the RWC of tomato leaves under cold stress, and the effect was better than that of ComCat®. As an important index of plant stress resistance, MDA can effectively reflect the damage degree of plant cell membranes. As shown in Fig. 2 B, compared with low temperature control, the MDA contents in tomato leaves and roots were obviously reduced after each treatment. Compared with low temperature control, Se, SA, MT, and ABA treatments decreased the MDA content in tomato leaves by 18.7%, 40.2%, 14.9%, and 12.8%, respectively, while Se + SA, Se + MT, and Se + ABA decreased the MDA content by 48.3%, 27.4%, and 35.0%, respectively. In tomato roots, compared with the control, Se, SA, MT, and ABA treatments decreased the MDA content by 44.3%, 42.6%, 45.1% and 32.9%, and Se + SA, Se + MT, and Se + ABA treatment decreased it by 71.2%, 64.6%, and 57.2%, respectively. Thus, the combined treatment of Se and different hormones had better effects on reducing MDA contents both in tomato leaves and roots than the treatment of Se or hormone alone. Among them, the combined application of Se and SA had the best effect on reducing MDA content in the leaves and roots of tomato seedlings, which was much better than that of the ComCat® treatment group. Accumulation of reactive oxygen species (ROS) As shown in Fig. 3 A, cold stress significantly increased the accumulation of H 2 O 2 content in tomato leaves and roots, however, the application of sole or combined Se and various plant hormones significantly decreased the H 2 O 2 content both in tomato leaves and roots under cold stress. Moreover, cold stress also increased the accumulation of O 2 − . in tomato leaves, which was obviously reduced by most treatments of exogenous substances, and the co-application of Se and SA had a relatively good effect on reducing the O 2 − . accumulation in tomato leaves (Fig. S1 ). Antioxidant enzymes activity in tomato seedlings To study the effect of the treatment of Se and various plant hormones on the antioxidant defense of tomato seedlings under cold stress, we determined the activities of several antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). As shown in Fig. 3 B, cold stress treatment significantly increased the SOD, CAT, and POD activity both in the leaves and roots of tomato seedlings, however, their activity was obviously decreased after the treatment of most exogenous substances. Among them, the application of Se and SA significantly lowered the activity of SOD in tomato leaves and roots and also reduced the CAT activity in tomato roots, while its effect in reducing POD activity did not differ compared to other combined treatments. Measurement of sugar content Based on the previous determination of relevant indexes of tomato seedlings under cold stress, it was found that among the three combined treatments, Se and SA combined treatment had the best effect on alleviating cold stress compared with the ComCat® treatment. Soluble sugars can be used as osmotic regulatory substances to alleviate the damage to plants caused by cold stress. Therefore, in order to explore the effect of Se plus SA treatment on osmoregulatory substances in tomato seedlings under cold stress, the contents of total soluble sugar, reducing sugar, sucrose, and starch in tomato leaves and roots were further determined. As shown in Fig. 4 , cold stress resulted in an apparent increase in the content of various soluble sugars both in tomato leaves and roots. The application of sole or combined Se and SA did not further increase the contents of soluble sugar, reducing sugar, and sucrose compared to low temperature control, but they significantly increased their content in tomato roots. Furthermore, the treatment of sole or combined Se and SA obviously inhibited the starch synthesis both in tomato leaves and roots under cold stress. Expression of cold-induced genes In order to investigate the molecular regulation mechanism of cold stress tolerance mediated by exogenous Se, SA, and their interaction, the transcripts of cold-inducing genes in tomato plants treated with Se and SA under cold stress were detected by qRT-PCR (Fig. 5 ). After cold stress treatment for 10 d, the transcripts of pathogen-related proteins 1 ( PR1 ), Cystatin b ( CYSb ), and Osmotin were significantly up-regulated. Under cold stress treatment, the application of Se induced the expression of CYSb , while SA treatment up-regulated the transcripts of CBF expression 1 ( ICE1 ) and Osmotin genes in tomato leaves. The combination of Se and SA increased the transcripts of a series of cold stress-related genes, including ICE1 , CYSb , and Late embryogenesis abundant protein ( LEA1 ) genes, suggesting that exogenous Se and SA and their interaction could enhance the cold resistance of tomato by modulating the expression of cold stress-related genes under cold stress. Effects of exogenous Se and SA inhibitor (AIP) on tomato seedlings under cold stress Our previous studies showed that among the three combined treatments, the treatment of Se plus SA had the best effect on eliminating the damage of tomato seedlings caused by cold stress compared with the ComCat® treatment group. We further studied the mechanism of exogenous Se and SA and their interaction in enhancing cold resistance of tomato seedlings under cold stress. As revealed in Fig. 6 A and S2, pretreatment with 30 µM AIP (a SA biosynthesis inhibitor) did not change the plant height, stem diameter, fresh or dry weight of tomato plants under normal conditions. In contrast, under cold stress, tomato plants pretreated with AIP had lower biomass and plant height than plants grown without AIP. Se application significantly restored plant growth under cold stress treatment, as shown by a significant increase in plant height and biomass compared with plant seedlings grown under cold stress, however, pre-spraying with AIP diminished the positive effects of Se application (Fig. S2). The chlorophyll fluorescence parameter (Fv/Fm) of tomato seedlings was not affected by pretreatment of AIP under normal condition, but it was reduced under cold stress treatment (Fig. 6 E). Compared with low-temperature control, the Fv/Fm value was increased after Se treatment under cold stress treatment. However, the recovery effect on Fv/Fm of Se was inhibited by AIP pretreatment under cold stress treatment. The trend of Fv/Fm parameter values was consistent with the results of chlorophyll fluorescence imaging (Fig. S3). Under cold stress, Se application obviously reduced the REC and MDA content compared to the plants without Se addition. However, AIP pre-treatment increased the REC and MDA by 24.5% and 20.8% respectively compared with the plants with Se treatment alone under cold stress (Fig. 6 B, C). Finally, Fig. 6 D indicated that pre-treatment of AIP prevented the Se-mediated decrease in H 2 O 2 accumulation under cold stress. Discussions Co-application of Se and various hormones promoted plant photochemical efficiency and growth under cold stress Tomato, as a thermophilic crop, whose biomass, fruit yield, and quality were seriously affected by cold stress (Liu et al., 2019 ). There have been some reports on Se application to alleviate the damage caused by cold stress in plants (Huang et al., 2018 ; Yang et al., 2021 ). However, no studies on the effect of exogenous Se on the cold resistance of tomato plants have been reported, let alone research into the combined application of Se and other plant hormones. It has been reported that treatments with 0.5 and 1.0 mg/kg Se greatly boosted the biomass and chlorophyll content of wheat plants (Chu et al., 2010 ). Moreover, it was found that foliar spraying of 0.5 mM salicylic acid significantly increased the chlorophyll and carotenoid levels in Vitis vinifera in response to frost stress (Jalili et al., 2023 ). Li et al. ( 2021 ) revealed that the application of MT increased the Fv/Fm value and enhanced the cold tolerance of watermelon plants. Yang et al. ( 2021 ) reported that MT contributed to Se-enhanced cold resistance in cucumber plants. Moreover, ABA application promoted the growth of bermudagrass plants and elevated the levels of chlorophyll a fluorescence transient curve in plants (Huang et al., 2017 ). In our study, the combined application of Se and a series of plant hormones significantly increased chlorophyll b content, whose effect was better than that of Se or SA treatment alone (Table 2 ), suggesting an improvement in the plant’s essential metabolic contents. It was reported that Se treatment could improve the growth and development of plants under normal and various environmental stresses, in part because Se could increase the chlorophyll contents in plants, improve the photosynthetic efficiency of plants, and then improve the stress resistance of plants (Diao et al. 2014 ). Fv/Fm was reduced during cold stress, making it a crucial factor in testing for cold tolerance (Baker and Rosenqvist, 2004 ). Our research showed that cold stress decreased Fv/Fm, which prevented plants from effectively using excitation energy and resulted in decreased PSII photochemical efficiency, however, the combined application of Se and a series of plant hormones could enhance the maximum photochemical quantum yield (Fv/Fm) of photosystem II (Fig. 3 – 3 ), which was in accordance with the results of previous research. Co-application of Se and various hormones eliminated the cold-induced oxidative damage Cold stress can lead to the synthesis of excessive reactive oxygen species (ROS), such as H 2 O 2 , O 2− , and O •− , which can damage membranes by causing lipid peroxidation, electrolyte leakage, and membrane degradation (Han et al., 2020 ). SOD, CAT, and POD are efficient antioxidant enzymes that have developed in plants to lower ROS levels in cells and protect them from oxidative stress injuries under a series of abiotic stresses (Fan et al., 2022 ). Huang et al. ( 2018 ) found that treatment of 5 mg/L of Se obviously increased SOD, CAT and POD activities in strawberry leaves under chilling stress. However, in this study, the treatment of Se/SA significantly reduced the activity of SOD in tomato leaves and roots and also reduced the CAT activity in tomato roots, which was consistent with the reports of Saidi et al. ( 2014 ) that lower levels of Se decreased the SOD and POD activity induced by cadmium stress. The co-applications of Se and various hormones decreased H 2 O 2 and O 2 − . Levels and membrane damage of tomato leaves under cold stress (Figs. 4 and 5 ). This result was in accordance with the findings of Liu et al. ( 2021 ) in tea and Huang et al. ( 2018 ) in strawberries. Moreover, in our study, the Se/SA application was the most effective to maintain the cell membrane stability under cold stress, which was manifested by an apparent decrease in relative electrical conductivity and MDA content in tomato leaves (Fig. 2 ). It was worth noting that the effect of co-application of Se and SA was better than the treatment of Se or SA alone, suggesting that there might be synergistic interactive effects between Se and SA in cold stress resistance of tomato plants. Co-application of Se and SA affects osmotic regulation in tomato plants under cold stress Soluble sugars play vital roles in plant osmotic protection and sustaining the biochemical activity of cell membranes in response to cold stress (Fareen et al. 2016 ). To maintain osmotic equilibrium when exposed to cold stress, starch is transformed into soluble sugars (Krasensky and Jonak 2012 ). Other studies have shown that stress-induced regulation of starch metabolism in plants caused by adversity stress can increase cellular sugars or starch accumulation (Dong et al. 2019). Compared with the low temperature control, both Se and SA treatments significantly increased the sugar content in the roots under cold stress, but co-application of Se and SA inhibited the increase of sugar and starch content in the roots (Fig. 4 ), which may indicate that Se combined with SA could effectively reduce the damage caused by cold stress on tomato seedlings, thus indirectly affect the plants' demand for osmotic adjustment substances under cold stress. ComCat® is a natural product that was created to boost plants and increase growth and productivity in various agricultural crops under different abiotic stresses (Workneh et al., 2012 ). In our study, co-application of Se and SA was more effective in alleviating the damage caused by cold stress in tomato plants compared to ComCat®. These results indicate that Se and SA have the potential to be developed as new plant growth regulators. Co-application of Se and SA modulates the transcripts of cold stress-responsive genes Co-application of Se and SA increased the plant’s resistance to cold stress, but the underlying regulatory mechanisms remained unknown. qRT-PCR was conducted to examine the expression of genes associated with cold stress resistance in our study, including PR1 , CYSb , LEA , osmotin, and ICE1 . ICE1 (Inducer of CBF expression 1), an upstream regulator of CBF, positively regulates the expression of CBF. Plants participate in cold stress response through ICE-CBF regulatory pathway (Ashraf et al. 2022). Under cold and other abiotic stresses, CYSb is produced and takes part in the DREB and AREB signal transduction pathways (Zhang et al., 2008 ). LEA (Late embryogenesis abundant) protein is a key protein to enhance plant tolerance to abiotic stress, and it improves biological resistance under high and low temperatures, drought, salinity, and other stresses (Wang et al., 2019 ). Osmotin, a "thaumatin-like protein," protects plant plasma membranes against salt, cold, and heat stressors, which is crucial for the osmotic adaptation of plant cells (Kumar et al. 2015 ). When plants are exposed to low temperatures, osmotin and osmotin-like proteins are implicated to contribute to the accumulation of free proline and ascorbate (Hakim et al. 2018). In our study, both exogenous Se and SA could induce the expression of genes involved in cold stress tolerance, and co-application of Se and SA increased the transcripts of cold stress-responsive genes together (Fig. 5 ). The interaction between them was not a simple superposition effect but through the common coordination to induce the expression of cold stress-related genes to improve the ability of tomato plants to resist cold stress, and then improve the cold resistance of plants. SA contributed to cold stress resistance of tomato plants induced by Se It was reported that SA mediates Se-induced resistance to salt and drought stresses in tomatoes (Fan et al., 2022 ; Wu et al., 2023 ). To further verify the effect of SA on Se-mediated cold stress resistance of tomato plants, the effect of exogenous Se on plant cold stress resistance after inhibiting the SA synthesis was tested by pre-treatment of AIP. AIP inhibits SA synthesis by inhibiting the activity of phenylalanine ammonia-lyase (PAL), the first enzyme in the synthesis of SA through the PAL pathway (Bamneshin et al., 2022 ). Under normal conditions, AIP had no significant effect on plant phenotype, but it appeared to have an inhibitory effect on plant growth and Fv/Fm value when exposed to cold stress (Fig. 5 A), indicating the crucial role of SA in plant resistance to cold stress (Guo et al., 2012 ; Li and Wang, 2021 ). AIP pretreatment eliminated the promoting effect of Se on plant photosynthesis and growth, increased relative conductivity and MDA content, made the accumulation of a large amount of H 2 O 2 and O 2 − ., aggravated the damage caused by oxidative stress, increased the soluble sugar content, and finally eliminated the beneficial effect of Se on plant cold resistance. These findings supported the hypothesis that Se application significantly improved plants' ability to resist cold stress, which was partly caused by the induction of SA synthesis by Se treatment. It was reported that treatment with SeO 3 2− caused the related nonhyperaccumulator Stanleya elata to express its genes for SA synthesis at a constitutively greater rate than the Se hyperaccumulator Stanleya pinnata (Wang et al., 2018 ). Furthermore, Freeman et al. ( 2010 ) discovered that SA was essential for the high Se tolerance in S. pinnata . Li et al. ( 2022 ) reported that it was most effective of 200 mg/L SA to boost the overall uptake of Se, inorganic Se, and organic Se of grapevine to some amount. These results point to intricate interactions between Se and SA in plants, which may vary depending on the Se treatment's concentration, duration, plant type, and capacity for Se accumulation. It is one of the mechanisms for Se to improve the cold resistance of plants by regulating the SA synthesis pathway to enhance the tolerance of plants to cold stress. Therefore, the mechanism of co-application of Se and SA to improve the cold resistance of tomato plants is not a simple superposition and extensive and in-depth research will be conducted on the interactions between Se and SA in response to cold stress treatment. Conclusions In conclusion, our findings suggest that the combined treatment of Se with SA, MT, and ABA had better effects on enhancing the cold stress resistance of tomato plants than the treatment of Se or hormone alone. Among them, the combined application of Se and SA showed the most success in alleviating the cold stress of tomato plants compared with the ComCat® (a commercial compound plant growth regulator) treatment (Fig. 7 ). The co-application of Se and SA also prevented the oxidative damage caused by cold stress on tomato plant chloroplasts and preserved the plasma membrane integrity, as shown by an obvious decrease in MDA, H 2 O 2 , and O 2 − . levels during cold stress. The Se/SA treatment could also reduce the content of reducing sugar, sucrose, total soluble sugar, and starch accumulation caused by cold stress through the regulation of osmotic substances, so as to improve the cold resistance of plants. The interaction between Se and SA modulated the expression of several cold-responsive genes, including PR1 , ICE1 , CYSb , LEA1 , Osmotin , and CBF1 , thereby conferring cold tolerance of tomato plants. It was worth noting that the pretreatment with AIP (a SA biosynthesis inhibitor) eliminated the favorable influence of Se on the cold resistance of tomato plants indicating that enhancing the cold resistance of plants by regulating the synthesis pathway of SA might be one of the mechanisms by which Se enhanced tomato resistance to cold stress. Declarations Funding This work was supported by the National Natural Science Foundation of China (32072561) and the National Agriculture Science and Technology Major Program (No. NK2022090403). 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 Yan Bai and Xin Wang, Qingqing Dai conducted the RT-PCR experiments. Xiangqiang Zhan and Haijun Gong analyzed the original data. The first draft of the manuscript was written by Jia Guo. 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Images of the maximum photochemical efficiency of PSII (Fv/Fm). The image's false-color coding, which can be seen at the bottom, runs from 0 (black) to 1 (red). Cite Share Download PDF Status: Published Journal Publication published 09 Feb, 2024 Read the published version in Plant and Soil → Version 1 posted Editorial decision: Major revisions 12 Sep, 2023 Reviewers agreed at journal 30 Jul, 2023 Reviewers invited by journal 30 Jul, 2023 Editor invited by journal 26 Jul, 2023 Editor assigned by journal 25 Jul, 2023 First submitted to journal 23 Jul, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3176261","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":222713835,"identity":"6a5fd6a8-486a-441a-bb87-83ac7e285af7","order_by":0,"name":"Jia Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYBACAxDxgIFNTl7++MHHYCFm5gbCWhIY2IwNZ/AkG4P5zIxEaWFIbLjBYCYN4RPQYs7e+0wi4RefMePshrTqgoo/0fztQC0/Krbh1GLZc9xMIrGPTY5d5uCx2zPOGOTOOMzYwNhz5jZuh91IY5NI7GEzZmxISLvN22aQ2wDUwszYhkfL/WdgLYkNBxLMikFa5hPUcoONTSLhB1DLjQQzZpCWDYS0WPakMVskNgADuedMsjTPGePcjUAtB/H5xZz9GOOND3+Oycmztx/8zFMhlzvv/OGDD35U4NYCBCwSjG3HUIUO4FMPBMwfGP7UEFAzCkbBKBgFIxoAABKJW2ZZ6COmAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-2943-0656","institution":"Northwest A\u0026F University: Northwest Agriculture and Forestry University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Guo","suffix":""},{"id":222713836,"identity":"0fbc5019-763c-4d21-a168-29dcfd63ab86","order_by":1,"name":"Yan Bai","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Bai","suffix":""},{"id":222713837,"identity":"c37a79cd-abf4-473f-bf4f-c1a351c7332b","order_by":2,"name":"Xin Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Wang","suffix":""},{"id":222713838,"identity":"08ada7e7-9a63-46fd-95e3-88eb0a5a0b4f","order_by":3,"name":"Qingqing Dai","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qingqing","middleName":"","lastName":"Dai","suffix":""},{"id":222713839,"identity":"14959efe-c1eb-460b-adc2-49bc197b3bca","order_by":4,"name":"Xiangqiang Zhan","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiangqiang","middleName":"","lastName":"Zhan","suffix":""},{"id":222713841,"identity":"186b4fda-d735-4b8c-a679-0d6271498905","order_by":5,"name":"Haijun Gong","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haijun","middleName":"","lastName":"Gong","suffix":""}],"badges":[],"createdAt":"2023-07-17 03:01:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3176261/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3176261/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-024-06534-9","type":"published","date":"2024-02-09T15:01:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":41089646,"identity":"84ae476e-4fc5-4763-8e33-744dc4736043","added_by":"auto","created_at":"2023-08-04 17:28:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":296924,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Se, various hormones and their interaction on tomato plant growth under cold stress for 10 d. (A) Observation of plant phenotype of various treatments. (B) Images of the maximum photochemical efficiency of PSII (Fv/Fm). The image's false-color coding, which can be seen at the bottom, runs from 0 (black) to 1 (red). (C) The average values of Fv/Fm of various treatments. CK0: Normal temperature control; CK1: low temperature control; Se: Selenium; SA: salicylic acid; MT: melatonin; ABA: \u003ca href=\"javascript:;\"\u003eabscisic acid\u003c/a\u003e; CC: ComCat\u003csup\u003e®\u003c/sup\u003e. Data are shown as the mean and standard deviation of six biological replicates. Significant differences between various treatments are denoted by different small letters at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-3176261/v1/31317c37dd1b8b941808b881.png"},{"id":41089640,"identity":"d85ecd25-c7a0-45d3-ab97-37275dc5530d","added_by":"auto","created_at":"2023-08-04 17:28:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44796,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Comparison of the relative electrical conductivity (REC) (left) and relative water content (right) of tomato seedlings of various treatments. (B) The malondialdehyde (MDA) content in leaves (left) and roots (right) of tomato plants with various treatments. Data are shown as the mean and standard deviation of six biological replicates. Significant differences between various treatments are denoted by different small letters at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-3176261/v1/a4433247d73a80d056fb2403.png"},{"id":41089644,"identity":"9e8ac91e-3915-488e-b503-7d3da1f2b490","added_by":"auto","created_at":"2023-08-04 17:28:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64278,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Se, various hormones and their interaction on ROS accumulation and activities of antioxidant enzymes in tomato plants of various treatments under cold stress for 10 d. (A) The content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e•-\u003c/sup\u003e in tomato leaves of various treatments. (B) The activity of SOD, CAT, and POD in tomato leaves and roots of various treatments. Data are shown as the mean and standard deviation of six biological replicates. Significant differences between various treatments are denoted by different small letters at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-3176261/v1/cf52777bdb44dbdb0a6fd122.png"},{"id":41089641,"identity":"b7587205-a752-470b-93e0-dca0384211d5","added_by":"auto","created_at":"2023-08-04 17:28:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45173,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Se and SA on sugar and starch contents in the leaves and roots of tomato seedlings of various treatments under cold stress. (A) The soluble sugar contents. (B) The reducing sugar contents. (C) The sucrose contents. (D) The starch contents. Data are shown as the mean and standard deviation of six biological replicates. Significant differences between various treatments are denoted by different small letters at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-3176261/v1/4f84e7dd38d05307d0a1a2d5.png"},{"id":41089645,"identity":"713ebd46-305e-4d7d-8f26-51b4d53d91a2","added_by":"auto","created_at":"2023-08-04 17:28:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":35053,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression profiles of cold-responsive genes in tomato leaves of various treatments under cold stress, including \u003cem\u003ePR1\u003c/em\u003e (A), \u003cem\u003eICE1\u003c/em\u003e (B), \u003cem\u003eCYSb\u003c/em\u003e (C), \u003cem\u003eLEA1\u003c/em\u003e, (D), \u003cem\u003eOsmotin\u003c/em\u003e (E), and CBF1 (F). The \u003cem\u003eβ\u003c/em\u003e-\u003cem\u003eactin \u003c/em\u003egene was used as an internal control to normalize expression. Data are shown as the mean and standard deviation of six biological replicates. Significant differences between various treatments are denoted by different small letters at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-3176261/v1/a1e990efefc27fa35027b2b7.png"},{"id":41090515,"identity":"60206655-8538-49cf-97ce-e3ed93f0777a","added_by":"auto","created_at":"2023-08-04 17:36:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":72513,"visible":true,"origin":"","legend":"\u003cp\u003eThe roles of a SA inhibitor (2-aminoindan-2-phosphonic acid, AIP) in Se-enhanced cold resistance in tomato plants. (A) The fresh (left) and dry weight (right) of plant seedlings. (B) Left: The relative electrical conductivity (REC); Right: the relative water content (RWC). (C) Left: MDA contents; Right: the soluble sugar contents. (D) The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents in tomato leaves. (E) The average values of Fv/Fm of various treatments. CK0: Normal temperature control; CK1: low temperature control; AIP: AIP treatment; Se: selenium. Data are shown as the mean and standard deviation of six biological replicates. Significant differences between various treatments are denoted by different small letters at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-3176261/v1/557d0041cd6cb60c567c655c.png"},{"id":41090883,"identity":"b57b8e81-52f0-4216-8b0a-6e715721dc33","added_by":"auto","created_at":"2023-08-04 17:44:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":101024,"visible":true,"origin":"","legend":"\u003cp\u003eA model for the function of exogenous Se and SA in the cold stress response in tomato plants. When Se and SA were applied to tomato roots, a part of them was transported to the shoots. The interaction of Se and SA promotes the maximum photochemical efficiency of PSII (Fv/Fm), enhances the antioxidant capacity of tomato seedlings, affects osmotic regulation, as well as modulates the expression of cold stress-responsive genes, and finally promoted the cold stress resistance of tomato plants. Moreover, SA plays vital roles in Se‑enhanced cold tolerance in tomato plants. Red arrow indicated an increase, whereas the blue arrow indicates a decrease.\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-3176261/v1/3b83e065c2df133f554550d3.png"},{"id":51005545,"identity":"d5b91fd4-ce00-4519-a74d-ab1b5c870e13","added_by":"auto","created_at":"2024-02-12 15:10:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":944133,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3176261/v1/3dca05c8-0887-4ec0-a0cb-ef6738421ab3.pdf"},{"id":41089643,"identity":"4915bf4a-3fca-4fc6-bd38-7dfe4ba6440d","added_by":"auto","created_at":"2023-08-04 17:28:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13979,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3176261/v1/e67ec708d4c19dc91dca1b5f.docx"},{"id":41089649,"identity":"d68904dc-dbf0-4126-a159-f3b1676050db","added_by":"auto","created_at":"2023-08-04 17:28:54","extension":"ppt","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":390144,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1.\u003c/strong\u003e The Diaminobenzidine (DAB) staining of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) accumulation (upper) and Nitroblue tetrazolium (NBT) staining of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e•-\u003c/sup\u003e (lower) in tomato leaves of various treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S2.\u003c/strong\u003e The plant height and stem diameter of tomato seedlings from various treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S3.\u003c/strong\u003e Images of the maximum photochemical efficiency of PSII (Fv/Fm). The image's false-color coding, which can be seen at the bottom, runs from 0 (black) to 1 (red).\u003c/p\u003e","description":"","filename":"Baisupplementaryfigures.ppt","url":"https://assets-eu.researchsquare.com/files/rs-3176261/v1/2fd8a0f2b8995381d57b3a8e.ppt"}],"financialInterests":"","formattedTitle":"Effects of combined application of selenium and various plant hormones on the cold stress tolerance of tomato plants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCold stress, including non-freezing chilling stress (0\u0026ndash;15℃) and freezing stress (\u0026lt;\u0026thinsp;0℃) causes severe physiological damage to plants and changes in plant morphology. The symptoms are generally dehydration of plant leaves, gradual wilt, reduced plant growth, and low accumulation of dry matter in plants, resulting in declined yield or quality (Soualiou et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, cold stress can affect the activity of key enzymes, impair membrane function and cause cell dehydration, leading to unstable metabolism of plant cells (Guan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Some studies reported that plant cells adapted to cold stress via regulating the plasma membrane, intracellular osmotic protective substances, REDOX systems, and photosynthetic rates (Guan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSelenium (Se) is a necessary trace element needed by both humans and animals (Rayman, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Although Se is not a necessary element for plants, several reports have shown that low levels of Se had positive effects on plants, including stimulating plant growth and development, strengthening plant resistance, and boosting crop quality (Wang et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Other research revealed that Se could enhance plant resistance to various adverse environments, including high and low temperatures, salinity, drought, heavy metals, etc. (Huang et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rady et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Huang et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) discovered that Se could reduce the accumulation of reactive oxygen species and reduce oxidative damage to plants by participating in the redox process in plants and removing excess free radicals in plants. Fan et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that Se application improved the photosynthetic efficiency of tomato plants and promoted their growth and development under drought stress. It was found that Se in strawberries induced an antioxidant effect by increasing enzyme compounds (mainly SOD) and non-enzyme compounds (such as ascorbic acid), and increased the photosynthetic rate, chlorophyll content, and stomatal conductivities of strawberry plants in response to cold stress (Huang et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMany studies have shown that plant hormones play vital roles in plant resistance to a number of environmental stresses (Waadt et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Some plant hormones effectively regulate plant growth and development at low temperatures, including salicylic acid (SA), melatonin (MT), abscisic acid (ABA), etc. (Tian et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It was reported that SA (1 and 2 mM) could reduce cold damage in grapevines under low-temperature stress (Li and Wang, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). SA can inhibit electrolyte leakage, reduce the accumulation of reactive oxygen species, increase the activity of cell protective enzymes, and improve plant photosynthetic efficiency under cold stress (Kang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Exogenous melatonin has been demonstrated to improve cold tolerance in a variety of plant species, including \u003cem\u003eArabidopsis\u003c/em\u003e, wheat, and watermelon (Bajwa et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Turk et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). According to Guo et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), exogenous ABA administration enhanced pepper seedlings' tolerance to cold-induced oxidative damage primarily through increasing the activity of antioxidant enzymes and the expression of genes associated with cold stress. These reports suggested that plant hormones also played important roles in plant response to cold stress. However, until now, the synergistic effects of Se and a range of plant hormones on the physiological, biochemical, and molecular response of plants under various environmental stresses have been less reported and a thorough investigation is necessary.\u003c/p\u003e \u003cp\u003eTomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e L.) is a key vegetable and fruit crop. As one of the main crops cultivated in winter and spring facilities, it is highly vulnerable to cold stress (Han et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, low temperature poses a great challenge to the production of its protected land, which seriously affects the growth and development of its adult stage, early yield, and fruit commodity (Zhou et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Improving cold stress resistance of tomato crops has a certain guiding significance for tomato crop production and optimization of cultivation measures (Haghighi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Until now, there are few reports on the impacts of Se on the cold resistance of tomatoes, and few studies on the interaction between Se and exogenous hormones under cold stress, which requires further exploration. This study mainly explored the role of the interaction between Se and exogenous hormones such as SA, MT, and ABA in enhancing tomato seedlings' resistance to cold stress, in order to improve the ability of plants to resist cold stress, so as to provide an important means to improve the yield of vegetables and other crops under cold stress. Our study will provide some theoretical basis for the development and application of compound Se fertilizer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003ePlant materials and treatment\u003c/p\u003e \u003cp\u003eThe tomato seeds (\"Ailsa Craig\") with full grains and uniform size were selected, sterilized at 55℃ and germinated in a climate chamber. Seeds with consistent germination were sown in the commix matrix for seedling cultivation. The two-leaf tomato seedlings were planted in a tank with 1/4 strength Hogland nutrient solution (pH 5.8). The seedlings were slowed in a climate chamber with the temperature of 25℃/18℃ (day/night) and 75% relative humidity. Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e was used as the selenium source, and the hormones were SA, MT, and ABA, respectively. ComCat\u0026reg;, a commercial compound plant growth regulator, was used as positive control by foliar spraying. It dominantly consists of gibberellins, auxins (indole-3-acetic acid), brassinosteroids, amino acids, kitenins, and natural metabolites. The experiments contained ten treatments: (1) CK0: Normal temperature control (25℃/18℃ day and night); (2) CK1: low temperature control (10℃/4℃ day and night); (3) Se: 0.005 mM Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e; (4) SA: 0.1 mM salicylic acid; (5) MT: 0.1 mM melatonin; (6) ABA: 0.05 mM abscisic acid; (7) Se\u0026thinsp;+\u0026thinsp;SA: 0.005 mM Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.1 mM SA; (8) Se\u0026thinsp;+\u0026thinsp;MT: 0.005 mM Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.1 mM MT; (9) Se\u0026thinsp;+\u0026thinsp;ABA: 0.005 mM Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.05 mM ABA; (10) CC: 0.1 g/L ComCat\u0026reg;. After 10 days of cold stress treatment (10℃/4℃ day and night), the fully developed tomato leaves and roots were taken and frozen at -80℃ for use.\u003c/p\u003e \u003cp\u003ePlant growth index and electrolyte leakage calculation\u003c/p\u003e \u003cp\u003eThe plant height (from cotyledon to apex growth point) and stem diameter (1 cm above cotyledon) were measured with a ruler and a vernier caliper. Then the tomato seedlings were washed quickly with deionized water, dried up with tissue, and measured their fresh weight. Then the plants were dried at 105℃ for 12 min then at 80℃ overnight in a drying oven and the dry weight was measured.\u003c/p\u003e \u003cp\u003e The relative electrical conductivity (RWC) of the plants was measured with the second fully expanded leaves according to Wu et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe relative water content (REC) was measured according to Fan et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The samples were rinsed thoroughly with deionized water, dried with paper towels, and quickly cut the leaves into strips. A sample of 0.1 g was added with 20 mL of water, shaken gently and the first electrical conductivity (EC1) was tested with a conductivity meter. Then samples were oscillated in a shaker at 100 rpm for 2 h to measure the second electrical conductivity (EC2). Finally, the sample was heated at 100℃ for 20 min, cooled and the conductance value (EC3) was measured. The REC was calculated as follows: REC=(EC2-EC1)/(EC3-EC1)\u0026times;100%.\u003c/p\u003e \u003cp\u003eThe photosynthetic pigments content\u003c/p\u003e \u003cp\u003eThe determination of chlorophyll content was based on Wu et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Tomato leaves were washed with deionized water, dried with paper towels, and quickly cut the leaves into strips. A sample of 0.1g was put into a test tube containing 10 mL of 95% ethanol. The extraction was carried out at room temperature until the leaf tissue was completely turned white. The extract was filled to 25 mL and the absorbance was measured at 470 nm, 649 nm, and 665 nm by an ultraviolet spectrophotometer. The contents of chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Chlx.c) were calculated as described by Arnon (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1949\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMeasurement of chlorophyll fluorescence parameters\u003c/p\u003e \u003cp\u003eBefore the determination, the plants were placed in a dark environment for 30 min. Then the chlorophyll fluorescence index Fv/Fm (maximum PSⅡ quantum yield) was determined with the Chlorophyll fluorescence IMAGING system (Image-PAM, Walz, Germany).\u003c/p\u003e \u003cp\u003eThe sugar contents\u003c/p\u003e \u003cp\u003eThe soluble sugar contents were determined with the sulphate ketone colorimetry method as described by Fan et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The reducing sugar contents were measured with 3,5-dinitrosalicylic acid (DNS) as described by Deshavath et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The starch contents were measured by anthrone sulfuric acid colorimetry according to de Sousa et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAntioxidant enzymes activity assays\u003c/p\u003e \u003cp\u003e0.3 grams of frozen material were homogenized in 4 mL of sodium phosphate buffer (100 mM, pH 6.8) and centrifuged at 12,000 g for 20 min under 4\u0026deg;C. Then the supernatant was taken and the antioxidant enzymes activity was determined according to Gou et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReactive oxygen species (ROS) quantitative analysis and histochemical staining\u003c/p\u003e \u003cp\u003eAccording to Han et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), the amount of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was measured using the 2,7-dichlorofluorescin diacetate. The superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) content was assessed as indicated by Shi et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) solution were used to visualize the accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e according to Zhu et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eqRT-PCR analysis\u003c/p\u003e \u003cp\u003eTotal RNA was isolated using Rn33 Plantpure Universal Plant Total RNA Rapid Extraction Kit (Aidlab, Shenzhen, China), and the first-strand cDNA was generated with High speed-strand cDNA Synthesis Plus Kit (Cofitt, Hongkong, China). The gene expression analysis was performed on a StepOnePlus\u0026trade; Real-Time PCR System (Applied Biosystems, USA) using ChamQ Universal SYBR Qpcr Master Mix (Vazyme, Nanjing, China). The \u003cem\u003eβ-actin\u003c/em\u003e (GenBank: NM_001321306) gene was used as internal control and the primers used were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eSA biosynthesis inhibitor treatment\u003c/p\u003e \u003cp\u003eA salicylic acid biosynthesis inhibitor (2-aminoindan-2-phosphonic acid, AIP) was pre-sprayed on the two-leaf stage seedlings two days prior to the addition of Se and cold treatment. Six treatments were included in this experiment: (1) CK0; (2) CK0\u0026thinsp;+\u0026thinsp;AIP; (3) CK1 (10℃/4℃ day and night) (4) CK1\u0026thinsp;+\u0026thinsp;30 \u0026micro;m AIP; (5) CK1\u0026thinsp;+\u0026thinsp;0.005 mM Se; (6) CK1\u0026thinsp;+\u0026thinsp;0.005 mM Se\u0026thinsp;+\u0026thinsp;30 \u0026micro;m AIP;\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe test data were processed using Microsoft Excel 2016 software, SPSS24.0, and Sigmaplot14.0 for statistical analysis and plotting. Duncan\u0026rsquo;s multiple range test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was applied to test the significant differences. Different letters are used to indicate significant variations between treatment means.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eEffects of Se, various hormones and their interaction on plant growth parameters under cold stress\u003c/p\u003e\n\u003cp\u003eTo evaluate the effect of applying sole or combined Se and various plant hormones on plant growth and development under cold stress, tomato plants were treated under low temperature (10℃/4℃, day and night) and exogenous Se and various hormones for 10 d to assess the cold resistance of plants from different treatment groups (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). According to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, compared with the normal temperature control group, cold stress resulted in short stature and stunted growth of tomato seedlings. However, after the application of Se and various hormones, the growth of seedlings under cold stress was significantly promoted, and the degree of leaf wilting was not obvious. As shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, application of sole or combined Se and various plant hormones significantly increased the plant height and stem diameter of tomato seedlings compared with the low temperature control group. However, there was no obvious difference between various treatments in promoting plant height and stem diameter. Moreover, application of various exogenous substances significantly increased the fresh weight of tomato seedlings, but there was no significant difference among different treatments (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). All the treatments of sole or combined Se and various plant hormones also increased the dry weight of tomato plants under cold stress but the application of Se, SA, MT, and their combined treatments worked best (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eInfluence of exogenous Se on plant growth and root characteristics of tomato seedlings under cold stress\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eTreatment\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eGrowth\u003c/p\u003e\n\u003cp\u003eindices\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eBiomass\u003c/p\u003e\n\u003cp\u003e(g/plant)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eRoot morphological traits\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePlant height (cm)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eStem diameter (cm)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTotal\u003c/p\u003e\n\u003cp\u003efresh weight\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTotal\u003c/p\u003e\n\u003cp\u003edry\u003c/p\u003e\n\u003cp\u003eweight\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTotal\u003c/p\u003e\n\u003cp\u003eroot\u003c/p\u003e\n\u003cp\u003elength (cm)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTotal\u003c/p\u003e\n\u003cp\u003esurface\u003c/p\u003e\n\u003cp\u003earea (cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTotal\u003c/p\u003e\n\u003cp\u003eroot Volume (cm3)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAverage root diameter (mm)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCK0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e804.75\u0026thinsp;\u0026plusmn;\u0026thinsp;36.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e210.97\u0026thinsp;\u0026plusmn;\u0026thinsp;32.72a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.33a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09d\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCK1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e130.00\u0026thinsp;\u0026plusmn;\u0026thinsp;11.11c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e47.01\u0026thinsp;\u0026plusmn;\u0026thinsp;13.83bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSe\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e178.85\u0026thinsp;\u0026plusmn;\u0026thinsp;24.31b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e55.63\u0026thinsp;\u0026plusmn;\u0026thinsp;9.71bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07bc\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e173.06\u0026thinsp;\u0026plusmn;\u0026thinsp;13.40b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e54.99\u0026thinsp;\u0026plusmn;\u0026thinsp;12.08bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06a\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e169.42\u0026thinsp;\u0026plusmn;\u0026thinsp;5.88bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e58.62\u0026thinsp;\u0026plusmn;\u0026thinsp;3.62bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eABA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04cd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e132.64\u0026thinsp;\u0026plusmn;\u0026thinsp;23.06c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e50.04\u0026thinsp;\u0026plusmn;\u0026thinsp;7.32bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19bc\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSe\u0026thinsp;+\u0026thinsp;SA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e186.03\u0026thinsp;\u0026plusmn;\u0026thinsp;12.14b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e48.75\u0026thinsp;\u0026plusmn;\u0026thinsp;8.70bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSe\u0026thinsp;+\u0026thinsp;MT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e198.17\u0026thinsp;\u0026plusmn;\u0026thinsp;40.20b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e48.70\u0026thinsp;\u0026plusmn;\u0026thinsp;12.53bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSe\u0026thinsp;+\u0026thinsp;ABA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04bcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e128.62\u0026thinsp;\u0026plusmn;\u0026thinsp;7.03c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e39.76\u0026thinsp;\u0026plusmn;\u0026thinsp;8.66c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16bc\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71cd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03cd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e190.1\u0026thinsp;\u0026plusmn;\u0026thinsp;15.02b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e62.40\u0026thinsp;\u0026plusmn;\u0026thinsp;10.07b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"9\"\u003eNote: Different lowercase letters denote significant differences among different treatments at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEffects of Se, various hormones and their interaction on tomato roots under cold stress\u003c/p\u003e\n\u003cp\u003ePlant root morphology is usually an important evaluation index of plant stress resistance. The total root length, root surface area, and root volume of tomato seedling roots decreased significantly, while the average diameter increased under cold stress (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The application of Se, SA, MT, and their combined treatments had the best effect in promoting root length under cold stress.\u003c/p\u003e\n\u003cp\u003eEffects of Se, various hormones and their interaction on chlorophyll content under cold stress\u003c/p\u003e\n\u003cp\u003eAfter 10 d of cold stress treatment, the chlorophyll a, chlorophyll b, carotenoid, and total chlorophyll contents in the leaves of tomato seedlings declined significantly due to cold stress compared with control plants (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Compared with the low temperature control, the chlorophyll contents in each treatment group were significantly increased. Among them, the content of chlorophyll b in Se and various hormone combined treatment groups was significantly higher than that in Se and various hormones alone, but the differences between the combined treatment groups were not obvious. Moreover, the content of chlorophyll b in Se and hormone combined treatment was significantly higher than ComCat\u0026reg; treatment group.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eEffects of exogenous selenium, different hormones and their interactions on chlorophyll content of tomato seedlings under cold stress\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTreatments\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003echlorophyll a (mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eFw)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003echlorophyll b (mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eFw)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003echlorophyll a (mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eFw)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003echlorophyll (a\u0026thinsp;+\u0026thinsp;b) (mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eFw)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCK0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08a\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCK1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08d\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSe\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01bcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02bc\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02cd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06bc\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01bcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00bcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04bc\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eABA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04bcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSe\u0026thinsp;+\u0026thinsp;SA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01de\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSe\u0026thinsp;+\u0026thinsp;MT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01cde\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03bc\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSe\u0026thinsp;+\u0026thinsp;ABA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04bc\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01cd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01ef\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eData are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Different letters in the same column and variety indicate significant differences at the 5% level (Duncan's test).\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eEffects of Se, various hormones and their interaction on the characteristics of chlorophyll fluorescence under cold stress\u003c/p\u003e\n\u003cp\u003eChlorophyll fluorescence analysis is one way of calculating the plant damage caused by cold stress. Compared with the control plants, cold stress obviously decreased the maximum photochemical quantum yield of photosystem II (Fv/Fm) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). However, application of sole or combined Se and various plant hormones obviously increased the Fv/Fm value under cold stress. Among them, the combined treatment of Se plus SA or MT significantly increased the Fv/Fm value compared with the single treatments.\u003c/p\u003e\n\u003cp\u003eEffects of Se, various hormones and their interaction on the membrane stability under cold stress\u003c/p\u003e\n\u003cp\u003eAs seen in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, compared with low temperature control, the application of various exogenous substances all significantly reduced the relative electrical conductivity of tomato leaves, with Se, SA, MT, ABA and ComCat\u0026reg; treatments decreased by 26.4%, 30.0%, 29.5%, 24.0% and 17.4%, respectively, and Se\u0026thinsp;+\u0026thinsp;SA, Se\u0026thinsp;+\u0026thinsp;MT and Se\u0026thinsp;+\u0026thinsp;ABA treatment decreased the REC by 36.0%, 33.9%, and 25.3%, respectively. The results showed that Se and SA combined treatment significantly reduced the REC in leaves, and effectively alleviated the damage to cell membrane caused by cold stress. The effect of the combined application of Se and SA was better than that of ComCat\u0026reg;, a commonly used plant growth regulator in the market.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA showed the RWC of tomato plants from various treatment groups. Cold stress apparently reduced the water content of tomato leaves, however, the application of sole or combined Se and various plant hormones significantly increased the RWC under cold stress, among which Se plus SA treatment had a better effect. Moreover, ComCat\u0026reg; treatment had no significant effect on plant RWC under cold stress. These results indicated that combined treatment of Se and SA could effectively increase the RWC of tomato leaves under cold stress, and the effect was better than that of ComCat\u0026reg;.\u003c/p\u003e\n\u003cp\u003eAs an important index of plant stress resistance, MDA can effectively reflect the damage degree of plant cell membranes. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, compared with low temperature control, the MDA contents in tomato leaves and roots were obviously reduced after each treatment. Compared with low temperature control, Se, SA, MT, and ABA treatments decreased the MDA content in tomato leaves by 18.7%, 40.2%, 14.9%, and 12.8%, respectively, while Se\u0026thinsp;+\u0026thinsp;SA, Se\u0026thinsp;+\u0026thinsp;MT, and Se\u0026thinsp;+\u0026thinsp;ABA decreased the MDA content by 48.3%, 27.4%, and 35.0%, respectively. In tomato roots, compared with the control, Se, SA, MT, and ABA treatments decreased the MDA content by 44.3%, 42.6%, 45.1% and 32.9%, and Se\u0026thinsp;+\u0026thinsp;SA, Se\u0026thinsp;+\u0026thinsp;MT, and Se\u0026thinsp;+\u0026thinsp;ABA treatment decreased it by 71.2%, 64.6%, and 57.2%, respectively. Thus, the combined treatment of Se and different hormones had better effects on reducing MDA contents both in tomato leaves and roots than the treatment of Se or hormone alone. Among them, the combined application of Se and SA had the best effect on reducing MDA content in the leaves and roots of tomato seedlings, which was much better than that of the ComCat\u0026reg; treatment group.\u003c/p\u003e\n\u003cp\u003eAccumulation of reactive oxygen species (ROS)\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, cold stress significantly increased the accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in tomato leaves and roots, however, the application of sole or combined Se and various plant hormones significantly decreased the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content both in tomato leaves and roots under cold stress. Moreover, cold stress also increased the accumulation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. in tomato leaves, which was obviously reduced by most treatments of exogenous substances, and the co-application of Se and SA had a relatively good effect on reducing the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. accumulation in tomato leaves (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAntioxidant enzymes activity in tomato seedlings\u003c/p\u003e\n\u003cp\u003eTo study the effect of the treatment of Se and various plant hormones on the antioxidant defense of tomato seedlings under cold stress, we determined the activities of several antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, cold stress treatment significantly increased the SOD, CAT, and POD activity both in the leaves and roots of tomato seedlings, however, their activity was obviously decreased after the treatment of most exogenous substances. Among them, the application of Se and SA significantly lowered the activity of SOD in tomato leaves and roots and also reduced the CAT activity in tomato roots, while its effect in reducing POD activity did not differ compared to other combined treatments.\u003c/p\u003e\n\u003cp\u003eMeasurement of sugar content\u003c/p\u003e\n\u003cp\u003eBased on the previous determination of relevant indexes of tomato seedlings under cold stress, it was found that among the three combined treatments, Se and SA combined treatment had the best effect on alleviating cold stress compared with the ComCat\u0026reg; treatment. Soluble sugars can be used as osmotic regulatory substances to alleviate the damage to plants caused by cold stress. Therefore, in order to explore the effect of Se plus SA treatment on osmoregulatory substances in tomato seedlings under cold stress, the contents of total soluble sugar, reducing sugar, sucrose, and starch in tomato leaves and roots were further determined. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, cold stress resulted in an apparent increase in the content of various soluble sugars both in tomato leaves and roots. The application of sole or combined Se and SA did not further increase the contents of soluble sugar, reducing sugar, and sucrose compared to low temperature control, but they significantly increased their content in tomato roots. Furthermore, the treatment of sole or combined Se and SA obviously inhibited the starch synthesis both in tomato leaves and roots under cold stress.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExpression of cold-induced genes\u003c/p\u003e\n\u003cp\u003eIn order to investigate the molecular regulation mechanism of cold stress tolerance mediated by exogenous Se, SA, and their interaction, the transcripts of cold-inducing genes in tomato plants treated with Se and SA under cold stress were detected by qRT-PCR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). After cold stress treatment for 10 d, the transcripts of \u003cem\u003epathogen-related proteins 1\u003c/em\u003e (\u003cem\u003ePR1\u003c/em\u003e), \u003cem\u003eCystatin b\u003c/em\u003e (\u003cem\u003eCYSb\u003c/em\u003e), and \u003cem\u003eOsmotin\u003c/em\u003e were significantly up-regulated. Under cold stress treatment, the application of Se induced the expression of \u003cem\u003eCYSb\u003c/em\u003e, while SA treatment up-regulated the transcripts of \u003cem\u003eCBF expression 1\u003c/em\u003e (\u003cem\u003eICE1\u003c/em\u003e) and \u003cem\u003eOsmotin\u003c/em\u003e genes in tomato leaves. The combination of Se and SA increased the transcripts of a series of cold stress-related genes, including \u003cem\u003eICE1\u003c/em\u003e, \u003cem\u003eCYSb\u003c/em\u003e, and \u003cem\u003eLate embryogenesis abundant protein\u003c/em\u003e (\u003cem\u003eLEA1\u003c/em\u003e) genes, suggesting that exogenous Se and SA and their interaction could enhance the cold resistance of tomato by modulating the expression of cold stress-related genes under cold stress.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEffects of exogenous Se and SA inhibitor (AIP) on tomato seedlings under cold stress\u003c/p\u003e\n\u003cp\u003eOur previous studies showed that among the three combined treatments, the treatment of Se plus SA had the best effect on eliminating the damage of tomato seedlings caused by cold stress compared with the ComCat\u0026reg; treatment group. We further studied the mechanism of exogenous Se and SA and their interaction in enhancing cold resistance of tomato seedlings under cold stress. As revealed in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA and S2, pretreatment with 30 \u0026micro;M AIP (a SA biosynthesis inhibitor) did not change the plant height, stem diameter, fresh or dry weight of tomato plants under normal conditions. In contrast, under cold stress, tomato plants pretreated with AIP had lower biomass and plant height than plants grown without AIP. Se application significantly restored plant growth under cold stress treatment, as shown by a significant increase in plant height and biomass compared with plant seedlings grown under cold stress, however, pre-spraying with AIP diminished the positive effects of Se application (Fig. S2). The chlorophyll fluorescence parameter (Fv/Fm) of tomato seedlings was not affected by pretreatment of AIP under normal condition, but it was reduced under cold stress treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). Compared with low-temperature control, the Fv/Fm value was increased after Se treatment under cold stress treatment. However, the recovery effect on Fv/Fm of Se was inhibited by AIP pretreatment under cold stress treatment. The trend of Fv/Fm parameter values was consistent with the results of chlorophyll fluorescence imaging (Fig. S3). Under cold stress, Se application obviously reduced the REC and MDA content compared to the plants without Se addition. However, AIP pre-treatment increased the REC and MDA by 24.5% and 20.8% respectively compared with the plants with Se treatment alone under cold stress (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). Finally, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD indicated that pre-treatment of AIP prevented the Se-mediated decrease in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation under cold stress.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussions","content":"\u003cp\u003eCo-application of Se and various hormones promoted plant photochemical efficiency and growth under cold stress\u003c/p\u003e \u003cp\u003eTomato, as a thermophilic crop, whose biomass, fruit yield, and quality were seriously affected by cold stress (Liu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). There have been some reports on Se application to alleviate the damage caused by cold stress in plants (Huang et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, no studies on the effect of exogenous Se on the cold resistance of tomato plants have been reported, let alone research into the combined application of Se and other plant hormones. It has been reported that treatments with 0.5 and 1.0 mg/kg Se greatly boosted the biomass and chlorophyll content of wheat plants (Chu et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Moreover, it was found that foliar spraying of 0.5 mM salicylic acid significantly increased the chlorophyll and carotenoid levels in \u003cem\u003eVitis vinifera\u003c/em\u003e in response to frost stress (Jalili et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Li et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) revealed that the application of MT increased the Fv/Fm value and enhanced the cold tolerance of watermelon plants. Yang et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that MT contributed to Se-enhanced cold resistance in cucumber plants. Moreover, ABA application promoted the growth of bermudagrass plants and elevated the levels of chlorophyll a fluorescence transient curve in plants (Huang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In our study, the combined application of Se and a series of plant hormones significantly increased chlorophyll b content, whose effect was better than that of Se or SA treatment alone (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting an improvement in the plant\u0026rsquo;s essential metabolic contents. It was reported that Se treatment could improve the growth and development of plants under normal and various environmental stresses, in part because Se could increase the chlorophyll contents in plants, improve the photosynthetic efficiency of plants, and then improve the stress resistance of plants (Diao et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Fv/Fm was reduced during cold stress, making it a crucial factor in testing for cold tolerance (Baker and Rosenqvist, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Our research showed that cold stress decreased Fv/Fm, which prevented plants from effectively using excitation energy and resulted in decreased PSII photochemical efficiency, however, the combined application of Se and a series of plant hormones could enhance the maximum photochemical quantum yield (Fv/Fm) of photosystem II (Fig.\u0026nbsp;\u0026lt;link rid=\"fig3\"\u0026gt;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u0026lt;/link\u0026gt;\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which was in accordance with the results of previous research.\u003c/p\u003e \u003cp\u003eCo-application of Se and various hormones eliminated the cold-induced oxidative damage\u003c/p\u003e \u003cp\u003eCold stress can lead to the synthesis of excessive reactive oxygen species (ROS), such as H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, O\u003csup\u003e2\u0026minus;\u003c/sup\u003e, and O\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, which can damage membranes by causing lipid peroxidation, electrolyte leakage, and membrane degradation (Han et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). SOD, CAT, and POD are efficient antioxidant enzymes that have developed in plants to lower ROS levels in cells and protect them from oxidative stress injuries under a series of abiotic stresses (Fan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Huang et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) found that treatment of 5 mg/L of Se obviously increased SOD, CAT and POD activities in strawberry leaves under chilling stress. However, in this study, the treatment of Se/SA significantly reduced the activity of SOD in tomato leaves and roots and also reduced the CAT activity in tomato roots, which was consistent with the reports of Saidi et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) that lower levels of Se decreased the SOD and POD activity induced by cadmium stress. The co-applications of Se and various hormones decreased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. Levels and membrane damage of tomato leaves under cold stress (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This result was in accordance with the findings of Liu et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) in tea and Huang et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) in strawberries. Moreover, in our study, the Se/SA application was the most effective to maintain the cell membrane stability under cold stress, which was manifested by an apparent decrease in relative electrical conductivity and MDA content in tomato leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It was worth noting that the effect of co-application of Se and SA was better than the treatment of Se or SA alone, suggesting that there might be synergistic interactive effects between Se and SA in cold stress resistance of tomato plants.\u003c/p\u003e \u003cp\u003eCo-application of Se and SA affects osmotic regulation in tomato plants under cold stress\u003c/p\u003e \u003cp\u003eSoluble sugars play vital roles in plant osmotic protection and sustaining the biochemical activity of cell membranes in response to cold stress (Fareen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). To maintain osmotic equilibrium when exposed to cold stress, starch is transformed into soluble sugars (Krasensky and Jonak \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Other studies have shown that stress-induced regulation of starch metabolism in plants caused by adversity stress can increase cellular sugars or starch accumulation (Dong et al. 2019). Compared with the low temperature control, both Se and SA treatments significantly increased the sugar content in the roots under cold stress, but co-application of Se and SA inhibited the increase of sugar and starch content in the roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which may indicate that Se combined with SA could effectively reduce the damage caused by cold stress on tomato seedlings, thus indirectly affect the plants' demand for osmotic adjustment substances under cold stress. ComCat\u0026reg; is a natural product that was created to boost plants and increase growth and productivity in various agricultural crops under different abiotic stresses (Workneh et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In our study, co-application of Se and SA was more effective in alleviating the damage caused by cold stress in tomato plants compared to ComCat\u0026reg;. These results indicate that Se and SA have the potential to be developed as new plant growth regulators.\u003c/p\u003e \u003cp\u003eCo-application of Se and SA modulates the transcripts of cold stress-responsive genes\u003c/p\u003e \u003cp\u003eCo-application of Se and SA increased the plant\u0026rsquo;s resistance to cold stress, but the underlying regulatory mechanisms remained unknown. qRT-PCR was conducted to examine the expression of genes associated with cold stress resistance in our study, including \u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003eCYSb\u003c/em\u003e, \u003cem\u003eLEA\u003c/em\u003e, osmotin, and \u003cem\u003eICE1\u003c/em\u003e. ICE1 (Inducer of CBF expression 1), an upstream regulator of CBF, positively regulates the expression of CBF. Plants participate in cold stress response through ICE-CBF regulatory pathway (Ashraf et al. 2022). Under cold and other abiotic stresses, \u003cem\u003eCYSb\u003c/em\u003e is produced and takes part in the DREB and AREB signal transduction pathways (Zhang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). LEA (Late embryogenesis abundant) protein is a key protein to enhance plant tolerance to abiotic stress, and it improves biological resistance under high and low temperatures, drought, salinity, and other stresses (Wang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Osmotin, a \"thaumatin-like protein,\" protects plant plasma membranes against salt, cold, and heat stressors, which is crucial for the osmotic adaptation of plant cells (Kumar et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). When plants are exposed to low temperatures, osmotin and osmotin-like proteins are implicated to contribute to the accumulation of free proline and ascorbate (Hakim et al. 2018). In our study, both exogenous Se and SA could induce the expression of genes involved in cold stress tolerance, and co-application of Se and SA increased the transcripts of cold stress-responsive genes together (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The interaction between them was not a simple superposition effect but through the common coordination to induce the expression of cold stress-related genes to improve the ability of tomato plants to resist cold stress, and then improve the cold resistance of plants.\u003c/p\u003e \u003cp\u003eSA contributed to cold stress resistance of tomato plants induced by Se\u003c/p\u003e \u003cp\u003eIt was reported that SA mediates Se-induced resistance to salt and drought stresses in tomatoes (Fan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To further verify the effect of SA on Se-mediated cold stress resistance of tomato plants, the effect of exogenous Se on plant cold stress resistance after inhibiting the SA synthesis was tested by pre-treatment of AIP. AIP inhibits SA synthesis by inhibiting the activity of phenylalanine ammonia-lyase (PAL), the first enzyme in the synthesis of SA through the PAL pathway (Bamneshin et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Under normal conditions, AIP had no significant effect on plant phenotype, but it appeared to have an inhibitory effect on plant growth and Fv/Fm value when exposed to cold stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), indicating the crucial role of SA in plant resistance to cold stress (Guo et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Li and Wang, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). AIP pretreatment eliminated the promoting effect of Se on plant photosynthesis and growth, increased relative conductivity and MDA content, made the accumulation of a large amount of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e., aggravated the damage caused by oxidative stress, increased the soluble sugar content, and finally eliminated the beneficial effect of Se on plant cold resistance. These findings supported the hypothesis that Se application significantly improved plants' ability to resist cold stress, which was partly caused by the induction of SA synthesis by Se treatment. It was reported that treatment with SeO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e caused the related nonhyperaccumulator \u003cem\u003eStanleya elata\u003c/em\u003e to express its genes for SA synthesis at a constitutively greater rate than the Se hyperaccumulator \u003cem\u003eStanleya pinnata\u003c/em\u003e (Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, Freeman et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) discovered that SA was essential for the high Se tolerance in \u003cem\u003eS. pinnata\u003c/em\u003e. Li et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that it was most effective of 200 mg/L SA to boost the overall uptake of Se, inorganic Se, and organic Se of grapevine to some amount. These results point to intricate interactions between Se and SA in plants, which may vary depending on the Se treatment's concentration, duration, plant type, and capacity for Se accumulation. It is one of the mechanisms for Se to improve the cold resistance of plants by regulating the SA synthesis pathway to enhance the tolerance of plants to cold stress. Therefore, the mechanism of co-application of Se and SA to improve the cold resistance of tomato plants is not a simple superposition and extensive and in-depth research will be conducted on the interactions between Se and SA in response to cold stress treatment.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our findings suggest that the combined treatment of Se with SA, MT, and ABA had better effects on enhancing the cold stress resistance of tomato plants than the treatment of Se or hormone alone. Among them, the combined application of Se and SA showed the most success in alleviating the cold stress of tomato plants compared with the ComCat\u0026reg; (a commercial compound plant growth regulator) treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The co-application of Se and SA also prevented the oxidative damage caused by cold stress on tomato plant chloroplasts and preserved the plasma membrane integrity, as shown by an obvious decrease in MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. levels during cold stress. The Se/SA treatment could also reduce the content of reducing sugar, sucrose, total soluble sugar, and starch accumulation caused by cold stress through the regulation of osmotic substances, so as to improve the cold resistance of plants. The interaction between Se and SA modulated the expression of several cold-responsive genes, including \u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003eICE1\u003c/em\u003e, \u003cem\u003eCYSb\u003c/em\u003e, \u003cem\u003eLEA1\u003c/em\u003e, \u003cem\u003eOsmotin\u003c/em\u003e, and \u003cem\u003eCBF1\u003c/em\u003e, thereby conferring cold tolerance of tomato plants. It was worth noting that the pretreatment with AIP (a SA biosynthesis inhibitor) eliminated the favorable influence of Se on the cold resistance of tomato plants indicating that enhancing the cold resistance of plants by regulating the synthesis pathway of SA might be one of the mechanisms by which Se enhanced tomato resistance to cold stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32072561) and the National Agriculture Science and Technology Major Program (No. NK2022090403).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Yan Bai and Xin Wang, Qingqing Dai conducted the RT-PCR experiments. Xiangqiang Zhan and Haijun Gong analyzed the original data. The first draft of the manuscript was written by Jia Guo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArnon DI (1949) Copper enzymes in isolated chloroplasts polyphenoloxidase in \u003cem\u003eBeta vulgris\u003c/em\u003e. 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Plant Physiol Biochem 156:209\u0026ndash;220. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.plaphy.2020.09.014\u003c/span\u003e\u003cspan address=\"10.1016/j.plaphy.2020.09.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cold stress, melatonin, salicylic acid, selenium, tomato","lastPublishedDoi":"10.21203/rs.3.rs-3176261/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3176261/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eThe roles of selenium (Se) in relieving the harmful effect of cold stress have been reported, but there are few studies on the interaction between Se and various plant hormones in plants in response to cold stress. Here, the effects of Se and various plant hormones on tomato plants under cold stress have been investigated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe biomass, relative electrical conductivity, photosynthetic pigments, malondialdehyde, chlorophyll fluorescence, soluble sugar, proline contents, as well as the regulation of plant hormones were examined.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAmong several plant hormones, the co-application of Se and SA was the most effective in reducing the cold stress of tomato plants. The co-application of Se and SA prevented the oxidative damage caused by cold stress on tomato chloroplasts and preserved the plasma membrane integrity and regulated the osmotic substances under cold stress. The interaction between Se and SA modulated the expression of some cold-induced genes thereby conferring cold tolerance of tomato plants. But the pretreatment with a SA biosynthesis inhibitor (AIP) eliminated the favorable influence of Se on the cold resistance of tomato, indicating that enhancing the cold resistance of plants by regulating the synthesis of SA might be one of the mechanisms by which Se enhanced tomato\u0026rsquo;s resistance to cold stress.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results clarified the roles of Se and its regulation mechanisms in plant cold stress tolerance and the critical involvement of SA in this process, which might offer a theoretical foundation for using Se fertilizer to increase the production of crops under adversity stresses.\u003c/p\u003e","manuscriptTitle":"Effects of combined application of selenium and various plant hormones on the cold stress tolerance of tomato plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-04 17:28:49","doi":"10.21203/rs.3.rs-3176261/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2023-09-12T08:00:42+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-07-31T03:04:18+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-07-31T01:43:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2023-07-26T04:07:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-07-26T01:46:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2023-07-23T22:12:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"eba68741-fb2e-42b8-a0c4-09a5dc41bb15","owner":[],"postedDate":"August 4th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-02-12T15:04:12+00:00","versionOfRecord":{"articleIdentity":"rs-3176261","link":"https://doi.org/10.1007/s11104-024-06534-9","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2024-02-09 15:01:06","publishedOnDateReadable":"February 9th, 2024"},"versionCreatedAt":"2023-08-04 17:28:49","video":"","vorDoi":"10.1007/s11104-024-06534-9","vorDoiUrl":"https://doi.org/10.1007/s11104-024-06534-9","workflowStages":[]},"version":"v1","identity":"rs-3176261","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3176261","identity":"rs-3176261","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}