Tomato (Solanum lycopersicum L. cultivar “Money Maker”) seeds were soaked in water at 55°C for 25 min and then sown in a tray containing a vermiculite matrix. The seedlings with three leaves were transferred to a 1/2-strength Yamasaki tomato nutrient solution (Li C. et al., 2016) for 7 days. Subsequently, the seedlings were transferred to fresh 1/2-strength Yamasaki tomato nutrient solution (Stanleygroup, Shandong, China) without (control) or with (i) foliar spraying with 24-epibrassinolide (BR, 0.1 μmol/L; 24-Epicastasterone, Yuanye Biotechnology Co., Shanghai, China), (ii) root exposure to Ca(NO₃)₂ solution (Ca, 100 mmol/L), (iii) foliar spraying with 0.1 μmol/L BR plus root exposure to 100 mmol/L Ca(NO₃)₂ solution (Ca+BR) for continued cultivation for 5 days. The concentration used for spraying BR (0.1 μmol/L) was selected based on previous studies (Nie et al., 2019; Li et al., 2020). The entire foliar portion was sprayed with 0.1 μmol/L BR or distilled water once every 2 days, and salt stress treatment was carried out on the day of the second spray. The osmotic potential of 100 mmol/L Ca(NO₃)₂ was –0.97 MPa at 25°C, which was determined using a WESCOR Vapro 5600 osmometer (WESCOR, ELITechGroup, Inc, Stoneham, MA, United States). Leaf samples were taken from three randomly selected tomato seedlings with uniform growth for each treatment at different time points. Briefly, all leaflets from the second and third fully expanded leaves of the selected tomato seedlings were collected 1, 3, and 5 days after the salt stress treatment, and mixed abundantly for physiological analysis and protein extraction. The harvested leaf samples were immediately frozen in liquid nitrogen before storing in ultra-low temperature refrigerators to determine the H₂O₂, malondialdehyde (MDA), ascorbic acid (AsA), SOD, POD, CAT, and other indicators. The plant material taken 5 days after the salt stress treatment was divided into aboveground and underground parts. They were put in the oven at 105°C for 15 min and kept at 60°C until a constant weight was attained. The second leaf from the top was dried for the amino acid analysis.

The fresh weight (FW) of the leaves was measured by a rapid weighing method as described by Zhang et al. (2018). The chlorophylls of the second fully expanded leaves were extracted with acetone (80%) and the chlorophyll content was determined by monitoring the absorbance at 645 and 663 nm (Ahmad et al., 2018). The protein content and proline content were determined as described by Bradford (1976) and Bates et al. (1973), respectively. Three samples were used for the determination of the chlorophyll and proline content. Photosynthetic measurements were performed on fully expanded leaves (the second leaf from the top) on 5 days of salinity treatment at 10:00–11:00 a.m. A standard leaf chamber (2 × 3 cm²) fitted on a portable photosynthesis system (6400XT, Li-Cor Inc., Lincoln, NY, United States) was used at ambient relative humidity: 50–60%; carbon dioxide (CO₂); 400 μmol^⋅mol^–1; flow rate: 500 μmol s^–1; vapor pressure deficit < 2, and photosynthetically active radiation: 800 μmol^⋅m^–2⋅s^–1. Each leaf was allowed sufficient time for equilibration in the chamber until constant readings were obtained. The photosynthetic parameters such as the net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO₂ concentration (Ci), transpiration rate (Tr), the instantaneous water use efficiency (WUE), calculated through Pn/Tr, and the stomatal limitation (Ls) defined as 1-Ci/Ca (Ca denotes the atmospheric CO₂ concentration) were measured according to Hao et al. (2019). The microstructure of the leaf was prepared by the paraffin sectioning method as described by Li et al. (2020). The leaf sections were observed and photographed under an optical microscope.

Amino acid extraction and determination were carried out as described by Ohtsuki et al. (2016). Approximately 0.1 g of the oven-dried sample was soaked in 2% 5-sulfosalicylic acid (1.5 ml) for 24 h. After the centrifugation at 13,681 × g for 10 min, the supernatant (800 μl) was diluted to 5 mL with 0.02 mmol/L hydrochloric acid (HCl) and then the diluted supernatant was filtered through a 0.45 μm filter membrane (Linghanglab, Tianjin, China) and placed in a 1.5 ml injection bottle (Linghanglab, Tianjin, China). An amino acid analyzer (L-8800, HITACHI, Chiyoda City, Tokyo, Japan) was used to determine the content of different amino acids. Each treatment was determined with three biological replicates and three technical replications.

The H₂O₂ and MDA levels were measured as described by Ahmad et al. (2018). Briefly, the fresh leaf tissue (0.3 g) was homogenized with 0.1% trichloroacetic acid (TCA) and centrifuged at 12,000 × g for 10 min. Approximately, an equal volume of supernatant, 100 mM potassium phosphate buffer (pH 7.0), and 1M potassium iodide were mixed and the absorbance was noted at 390 nm with a spectrophotometer (Shimadzu UV-2450, Japan). The H₂O₂ content was expressed as nmol^⋅g^–1 FW. The MDA content was measured on the basis of thiobarbituric acid reaction (Bailly et al., 1996). In brief, a 0.5 g sample was macerated in 8 ml of 0.1% (w/v) TCA and centrifuged at 4,830 × g for 10 min at 4°C. The supernatants were obtained and mixed with 0.5% (w/v) of TCA made in 5% (w/v) TCA. The reaction mixture was heated at 100°C on a water bath for 20 min; afterward, the mixture was put on ice to stop the reaction. After cooling, a step of centrifugation at 7,888 × g for 10 min was done and the absorbance was taken at 450, 532, and 600 nm.

Enzyme extraction has been improved with reference to the Gong et al. (2005) method. One gram of leaf sample was ground into a homogenate, using 8 ml of a pre-cooled sodium phosphate buffer (0.05M, pH7.0). The homogenate was transferred into a 15 ml tube and centrifuged at 4°C 9,661 × g for 20 min. The supernatant obtained was used for the determination of the activity of enzymes, such as SOD (EC 1.15.1.1), POD (EC 1.11.1.7), and CAT (EC 1.11.1.6). The SOD activity was analyzed using nitroblue tetrazolium (NBT) as described by Gong et al. (2005). The SOD activity was expressed as units (U) of SOD mg^–1 of protein, in which one SOD unit is defined as the amount of enzyme that inhibits 50% of the reduction rate of NBT. The determinations of the POD and CAT activity were according to the method of Yang with slight modifications (Yang et al., 2010). The POD and CAT activities were expressed as 1U with a change of 1 per minute in OD₄₇₀ and OD₂₄₀, respectively, and expressed as U mg^–1 protein. The non-enzymatic antioxidant AsA contents were measured using a V_C (vitamin C, ascorbic acid) Assay Kit (A009, Jiancheng, Nanjing, China) by noting the absorbance at 536 nm (Quan et al., 2015).

The total protein was extracted from the fully expanded second and third leaves of the tomato seedlings from top to bottom. Briefly, 1 g of the leaf sample from every biological replicate were finely crushed in liquid nitrogen and mixed with 10% (w/v) TCA/acetone solution having 65 mM dithiothreitol (DTT) for 1 h (−20°C). Afterward, the extracted sample was centrifuged for 45 min at 10,000 × g and the obtained pellet was vacuum-dried and solubilized in 1/10 volume of SDT buffer (4% sodium dodecyl sulfate (SDS), 100 mM dithiothreitol (DTT), and 150 mM Tris–HCl, pH 8). After being incubated for 3 min, the suspended solution was ultrasonicated (80 w, 10 s ultrasonication at a time, every 15 s, and 10 times), and re-incubated at 100°C for 3 min followed by a step of centrifugation at 13,000 × g at 25°C for 10 min. The protein content in each sample was calculated using a Bicinchoninic acid Protein Assay Reagent (Promega, Madison, WI, United States) and the samples were stored at −80°C until use. Protein digestion was performed according to the FASP procedure described by Wiśniewski et al. (2009) and the resulting peptide mixture was marked according to the instructions of the manufacturer (AB SCIEX, Framingham, MA, United States) with 8-plex isobaric tags for relative and absolute quantification (iTRAQ). The detailed description related to the sample preparation, digestion, and analysis is provided in the Supplementary Methods.

The LC-MS/MS experiment was performed on a Q Exactive Mass Spectrometer coupled to an Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific Waltham, MA, United States). A volume of 10 μl of each fraction was injected for the LC-MS/MS analysis. The instrument was run with peptide recognition mode enabled and the detailed information related to the sample preparation and instrument conditioning is provided in the Supplementary Methods. The LC-MS/MS analysis was carried out at HooGen Biotech, Shanghai, China. The MS/MS spectra were searched using the Mascot search engine (Matrix Science, London, United Kingdom; version 2.2) embedded in Proteome Discoverer 1.3 (Thermo Electron, San Jose, CA, United States) against the UniProt S. lycopersicum database (35,921 sequences, download at 20180118) and the decoy database. The search parameters are provided in the Supplementary Methods. Differentially modulated proteins were identified as proteins with fold change (FC) ratio > 1.20 or <0.83 (P < 0.05; Li G. et al., 2017; Zhong et al., 2017).

The Gene Ontology (GO) program Blast2GO¹ was adopted to annotate differential expression proteins (DEPs) to create histograms of the GO annotation based on their role in the biological process, molecular function, and cell components. For the DEPs pathway analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, using the KEGG automatic annotation server (KAAS) program² was used. The GO enrichment analysis of each module was performed using Cytoscape v3.7.2.

Additionally, the protein accumulation determined by the iTRAQ analysis was further quantified and analyzed through LC-Parallel reaction monitoring (PRM) MS. Complete information related to the LC_PRM/MS analysis is appended in the Supplementary Methods. The analysis of the raw data was carried out via the Skyline 3.5.0 software (MacCoss Lab, University of Washington, United States; Peterson et al., 2012), where the intensity of every signal given specific peptide sequence can be measured for each sample after the normalization of each protein with the reference standard.

All physiological data were checked for statistical significance using ANOVA and presented as the mean ± SD of three biological replicates. Duncan’s multiple range test was applied to compare the means at the P < 0.05 level in SPSS (version-21.0). The proteomic experiments were also repeated with three independent biological replicates. The 95% confidence (Z score = 1.96) was set to pick the proteins whose distribution was removed from the main distribution. The cut-off values for the up-regulated or down-regulated proteins were taken as 1.2- or 0.83-fold, respectively (Li M. et al., 2017; Zhong et al., 2017).

To elucidate the effects of Ca(NO₃)₂-induced salt stress, the growth of tomato seedlings in response to Ca(NO₃)₂ treatment was investigated. As shown in Figure 1, Ca(NO₃)₂ stress repressed shoot biomass. However, the foliar application of BR alleviated the Ca(NO₃)₂-induced shoot growth inhibition in tomato seedlings (Figures 1A,B).

Observations of the microstructure of the tomato leaves showed that Ca(NO₃)₂ stress disrupted the arrangement of the epidermis, palisade mesophyll, and spongy mesophyll in tomato leaves (Figure 1C). The intercellular space enlarged, the palisade mesophyll and spongy mesophyll deformed, and the boundaries became blurred after Ca(NO₃)₂ stress. Foliar spraying with BR also affected the mesophyll structure in the tomato leaves. Compared with the control, the palisade mesophyll elongated, and the spongy mesophyll arranged chaotically, while the boundaries between the spongy mesophyll, the palisade mesophyll, and the epidermal cells became clear and neatly arranged in the BR-treated tomato leaves. Compared with Ca(NO₃)₂ stress alone, the thickness of the upper epidermal cells increased, and the cells were tightly arranged in the Ca+BR treatment. Moreover, the palisade mesophyll was arranged regularly, and an increased number of spongy mesophyll cells with clear cell boundaries were observed in the leaves of Ca+BR-treated tomato seedlings.

To elucidate the molecular mechanisms underlying BR-alleviated Ca(NO₃)₂ stress, we performed proteomics analysis and revealed the differentially changed proteins (DCPs) using the iTRAQ technique (Figure 2A). A total number of 419,939 secondary mass spectrums were obtained in the Ca(NO₃)₂ stress and/or BR-treated seedlings. Among these spectra, 117,757 spectra were matched to the 25,486 identified peptides. Finally, a total number of 25,486 unique peptides and 5,670 proteins were determined [P < 0.05, FC > 1.2] (Supplementary Figure 1A and Supplementary Tables 1, 2). There were 63.47% of the proteins that included at least two peptides (Supplementary Figure 1B) and the most enriched protein masses are 20–30 and 30–40 kDa, followed by 40–50, 50–60, and 10–20 kDa proteins (Supplementary Figure 1C).

Compared with the control, 469 proteins showed significantly changed accumulation (244 with increased accumulation and 225 with decreased accumulation) in the leaves of Ca(NO₃)₂-treated seedlings (Figure 2B). Moreover, a total of 172 (79 with increased accumulation and 93 with decreased accumulation) and 442 (251 with increased accumulation and 191 with decreased accumulation) proteins with significantly different accumulation were identified in the BR/control and Ca+BR/Ca comparison groups, respectively (Figure 2B).

Functional annotations of the 172, 469, and 442 DCPs belong to the BR/control, Ca/control, and Ca+BR/Ca groups, respectively, (Figure 2B; Supplementary Figure 2; and Supplementary Tables 4–7) showed that the DCPs in the Ca/control were annotated into 47 functional terms, including 11 molecular function terms, 16 cellular component terms, and 20 biological process terms (Supplementary Figure 2A). Additionally, 442 DEPs in the Ca+BR/Ca were annotated to 47 functional groups, including 18, 17, and 12 terms in biological process, cellular component, and molecular function, respectively (Supplementary Figure 2B). The proteins were selected based on different GO terms shown in Figure 2C.

The DCPs were then blasted KEGG genes to retrieve their KEGG ortholog (KOs) and were subsequently mapped to the pathways in KEGG (Supplementary Figure 3). In the Ca(NO₃)₂-treated seedlings, the KEGG pathways were enriched in carbohydrate metabolism including pyruvate metabolism, glycolysis, glyoxylate, and dicarboxylate metabolism, and carbon fixation in the photosynthetic organisms involved in energy metabolism (Supplementary Figure 3A). In the Ca+BR-treated seedlings, the KEGG pathways were enriched in photosynthesis, carbon metabolism, propionate metabolism, pyruvate metabolism, and glycolysis (Supplementary Figure 3C). These results collectively indicated that compared with Ca(NO₃)₂ stress, foliar spraying with BR largely affected carbohydrate and energy metabolism pathways.

The iTRAQ data were subsequently confirmed using Skyline software. The detailed data of the 25 target peptide fragments are shown in Supplementary Table 8. The accumulation of several antioxidant-related proteins, such as P30264, Q6X1D0, K4ASJ5, and K4ASJ6, and the light-harvesting complex chlorophyll A-B binding protein (P27524) elevated in the leaves of the Ca(NO₃)₂-treated seedlings. As shown in Supplementary Tables 4, 6, 8, the iTRAQ validation showed that Ca(NO₃)₂ stress increased the accumulation of Chlorophyll a-b binding protein (P27524, P27489, K4CXU8, K4C768) and ferredoxin (K4D1V7) and decreased the accumulation of cytochrome P450-type monooxygenase 97A29 (D2CV80). These results were consistent with the iTRAQ results, which indicated that Ca(NO₃)₂ stress affected photosynthesis and antioxidant defense in the leaves of the tomato seedlings.

Foliar spraying with BR increased the accumulation of antioxidant-related proteins such as POD (K4ASJ6, K4ASJ5) and cysteine proteinase 3 (Q40143), the proteins involved in the KEGG pathways of biosynthesis of amino acids (sly01230), photosynthesis-antenna proteins (sly00196), and protein processing in the endoplasmic reticulum (sly04141; Supplementary Tables 4, 5, 8).

Exogenous BR also affected the photosynthesis-related proteins under Ca(NO₃)₂ stress. Compared with Ca(NO₃)₂ stress, the accumulation of chlorophyll a-b binding protein (K4DC08) down-regulated, while that of PSBR (Q40163), PsbQ 543931 (Q672Q6), psbH (A0A0C5CEE1), and ferredoxin-1 (Q43517) up-regulated in the Ca+BR treatment (Supplementary Tables 4, 7, 8). Moreover, the changes in protein accumulation detected by the PRM assay were consistent with the iTRAQ results, indicating that the iTRAQ results were sufficiently valid.

Proteomics analysis revealed that exogenous BR affected the accumulation of proteins involved in chlorophyll metabolism and photosynthesis in tomatoes under Ca(NO₃)₂ stress (Figure 3A). The key enzyme in the metabolism of porphyrin and chlorophyll is nicotinamide adenine dinucleotide phosphate (NADPH)-protochlorophyllide oxidoreductase (K4DCQ6). Calcium nitrate stress down-regulated the accumulation of K4DCQ6 in the tomato leaves. Moreover, in the pathway of porphyrin and chlorophyll metabolism, the significant increase of red chlorophyll catabolite reductase (Q1ELT8) under Ca(NO₃)₂ stress enhanced the electron transport in chlorophyll decomposition (Figure 3A). Foliar spraying with BR had no significant effect on the red chlorophyll catabolite reductase (Fragment; Q1ELT8) under salt stress, but K4DCQ6 was up-regulated in the “Ca+BR/Ca” group, and thereby modulating chlorophyll levels in the Ca+BR treatment (Figure 3A). Meanwhile, Ca(NO₃)₂ stress increased the chlorophyll contents in the tomato leaves; however, foliar spraying with BR did not affect the chlorophyll contents in the Ca(NO₃)₂-treated tomato seedlings (Figures 3B,C). Altogether, these results indicated that BR alleviated Ca(NO₃)₂ stress by improving the function and stability of the photosynthetic system in tomato leaves. Next, we evaluated the effects of BR and Ca(NO₃)₂ on the photosynthetic parameters. Foliar spraying with BR decreased the WUE and Ls in the tomato leaves (Figure 3D and Table 1). Calcium nitrate stress inhibited the Pn and Tr, and decreased the Gs and Ci, but increased the Ls and WUE in the tomato leaves (Figure 3D and Table 1). However, foliar spraying with BR improved the Pn, Tr, Gs, and Ci, but decreased the WUE and Ls in the Ca(NO₃)₂-treated seedlings (Figure 3D and Table 1).

The overproduction of ROS and subsequent oxidative damage commonly occur under salt stress (Alexander et al., 2020; Yang et al., 2020). Proteomics analysis showed that Ca(NO₃)₂ stress affected the accumulation of proteins involved in stress responses and antioxidant defense, and exogenous BR increased the accumulation of antioxidant enzymes and proteins involved in the responses to osmotic stress in tomatoes under Ca(NO₃)₂ stress (Figure 4A). To further confirm the proteomics results, we investigated the levels of H₂O₂ and oxidative damage in the Ca(NO₃)₂-treated tomato plants. Calcium nitrate stress significantly increased the content of H₂O₂ and MDA at 3 and 5 days after the treatment, respectively. However, foliar spraying with BR significantly reduced the H₂O₂ accumulation and MDA content in the leaves of Ca(NO₃)₂-treated tomato seedlings after 3 and 5 days of treatment, respectively (Figures 4B,C).

The time-course of the antioxidant enzyme activity showed the differential effects of Ca(NO₃)₂ and BR. For instance, Ca(NO₃)₂ significantly increased the activity of SOD after 1 and 5 days, but not after 3 days of treatment. Unlike this trend, Ca(NO₃)₂ stress significantly increased the activity of POD and CAT 1 day after treatment but it reduced the activity of POD and CAT in the tomato leaves after 3 and 5 days compared with the control (Figures 5B,C). However, compared with the seedlings only exposed to Ca(NO₃)₂ stress, the activity of these three enzymes was significantly increased by BR treatment after 1 day of Ca(NO₃)₂ stress. Foliar spraying with BR reduced Ca(NO₃)₂-induced SOD activity (Figure 5A) and improved the CAT activity in the leaves of Ca(NO₃)₂-treated tomato seedlings after 3 and 5 days of treatment (Figure 5C). Meanwhile, foliar spraying with BR further reduced POD activity after 3 days in the Ca(NO₃)₂-treated tomato seedlings (Figure 5B). However, the activity of POD in the Ca+BR-treated tomato seedlings was not significantly different from that in the only Ca(NO₃)₂-treated tomato seedlings after 5 days of treatment.

Calcium nitrate stress markedly increased the content of proline (Pro) in the tomato leaves throughout the experimental period. However, exogenous BR significantly reduced the Ca(NO₃)₂-induced increases in the Pro content after 3 and 5 days of Ca(NO₃)₂ stress, and the effect of BR was more profound with the prolongation of the treatment time (Figure 6A). Meanwhile, the AsA content differentially fluctuated with time in the Ca(NO₃)₂-treated tomato seedlings (Figures 6A,B). Compared with the control, the AsA content decreased after 1 day, however, it eventually increased after 5 days of Ca(NO₃)₂ stress (Figure 6B). Calcium nitrate alone or combined with BR did not alter the AsA accumulation at 3 days of the Ca(NO₃)₂ treatment. Also, the BR treatment on the Ca(NO₃)₂-treated tomato seedlings did not affect the AsA content compared with the only Ca(NO₃)₂-treated tomato seedlings after 3 and 5 days of the Ca(NO₃)₂ treatment. These results collectively supported the proteomics data and indicated that exogenous BR alleviated Ca(NO₃)₂-induced growth inhibition by increasing the antioxidant enzyme activity, thus, reducing the level of membrane lipid peroxidation, finally improving Ca(NO₃)₂ stress tolerance in tomatoes.

The proteomic analysis showed that Ca(NO₃)₂ stress disrupted the amino acid metabolism in the tomato leaves (Figure 7A). The phenylalanine (Phe) content increased under Ca(NO₃)₂ stress, while BR decreased the Phe content of the salt-stressed tomato seedlings. This change was negatively correlated with the decrease of PAL (K4CQI0) accumulation in the Ca/control and the increase of K4CQI0 accumulation in the Ca+BR/Ca (Figures 7, 8 and Supplementary Table 7). Moreover, the levels of amino acids such as threonine (Thr), glutamate (Glu), alanine (Ala), valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu), histidine (His) and proline (Pro) increased in the Ca(NO₃)₂-treated tomato seedlings, thereby reprogramming the primary metabolism of plants to salt stresses, while after foliar spraying with BR, all the amino acid content decreased in the Ca(NO₃)₂-treated tomato seedlings (Figures 7, 8). All of the amino acids tested, except glycine (Gly), cysteine (Cys), and tyrosine (Tyr), showed markedly increased content in the leaves of the Ca(NO₃)₂-treated seedlings. Foliar spraying with BR decreased the contents of amino acids which were otherwise induced in the Ca(NO₃)₂ treatment (Figure 7B and Supplementary Table 9).

Soluble sugars and soluble proteins, as intracellular osmoregulatory substances, play important roles in modulating plant responses to salt stress. The KEGG pathway analysis involved in carbohydrate metabolism indicated that five pathways were containing 20 identified DCPs that were enriched in the Ca(NO₃)₂-treated plants (viz.) glycolysis/gluconeogenesis, glyoxylate and dicarboxylate metabolism, propanoate metabolism, pyruvate metabolism, citrate cycle (TCA cycle). The Ca(NO₃)₂ treatment also up-regulated the accumulation of malate dehydrogenase (K4B6N4, K4DCV3, K4CW40), thereby promoting the TCA cycle; in contrast, they down-regulated the accumulation of pyruvate dehydrogenase E1 component subunit beta (K4CJJ4) in the tomato seedlings (Supplementary Table 10). The proteomic analysis also showed that BR modulated the sugar and protein metabolism pathways in the Ca(NO₃)₂-treated tomato seedlings (Supplementary Table 10). We thus speculated if BR alleviated salt-induced stress by increasing the contents of osmoregulatory substances. To address this question, we measured the levels of soluble sugars and soluble proteins in the tomato seedlings. Calcium nitrate stress increased the content of soluble sugar throughout the study period. Although foliar spraying with BR decreased the soluble sugar content after 1 day of salt stress, BR further increased the content of leaf soluble sugars after 3 and 5 days of salt stress compared with the tomato seedlings only treated with Ca(NO₃)₂. The soluble protein content more or less increased in the Ca(NO₃)₂-treated tomato seedlings throughout the study period, whereas foliar spraying with BR increased the content of the soluble proteins significantly at 3 days after the Ca(NO₃)₂ treatment (Figures 9A,B). Notably, the positive regulatory effect of BR was more profound on the soluble sugars than soluble proteins under Ca(NO₃)₂ stress after 5 days.