Tomato (Lycopersicon esculentum Mill. cv. Puhong 968) seeds were obtained from the Shanghai Academy of Agricultural Sciences, China. Seeds were germinated and grown in plastic nutrition pots filled with growth media (Zhenjiang Peilei Co., Ltd., China). The germinated seedlings were grown under controlled condition (light intensity, 600 μmol m⁻²·s⁻¹; day/night temperature, 25/18°C; light/dark photoperiod, 14 h/10 h; relative humidity, 55–65%) in growth chambers (Ningbo Jiangnan Instrument Factory, Ningbo, China).

After the third true leaf developed, the seedlings were subjected to high-temperature (day/night temperature, 38/28°C; light/dark photoperiod, 14/10 h; relative humidity, 55–65%). The experimental plots included four different treatments: (1) Cont; (2) Spd (1 mM); (3) HT; (4) HT+ Spd (1 mM). The concentration of Spd was selected on the basis of previous experiment (data not shown). One millimole Spd was sprayed to leaves at 17:00 every day, and the control plants were sprayed with distilled water. After 7 days of treatment, the third fully expanded tomato leaves of each treatment were stored at −80°C for physiological and proteomic analysis. The experiment was arranged in a randomized complete block design and biological replicates were independently carried out three times.

The tomato seedlings were washed with sterile distilled water. After wiped with gauze, samples were dried in an oven at 105°C for 15 min followed by 75°C for 72 h, until reaching a constant weight, and then weighed for dry weight. Chlorophyll was extracted with a mixture of acetone, ethanol and water (4.5: 4.5: 1 by volume) and its content was estimated using the method of Arnon (1949). Pn was measured using a portable photosynthesis system (LI-6400, LI-COR Inc, USA).

Protein extraction was performed according to a modified version of the trichloroacetic acid (TCA) acetone precipitation method described by Hurkman and Tanaka (1986). Frozen leaf tissues were ground in liquid nitrogen and suspended in ice-cold extraction buffer (8 M urea, 1% (w/v) dithiothreitol (DTT), 4% (w/v) CHAPS and 40 mM Tris). Then the homogenates were centrifuged at 15,000 × g for 20 min at 4°C, and the supernatants were precipitated overnight with ice-cold acetone containing 10% (w/v) TCA and 0.07% (v/v) β-mercaptoethanol. The resulting protein-containing suspensions were centrifuged at 20,000 × g for 30 min at 4°C, and then the protein pellets were washed three times with cold acetone containing 0.07% (v/v) β-mercaptoethanol. Finally, the protein pellets were air-dried at room temperature and dissolved in rehydration buffer (8 M urea, 1 M thiourea, 2% w/v CHAPS). The protein concentrations were determined by the methods of Bradford (1976) using bovine serum albumin as the standard, and then the protein was stored at −80°C until being subjected to two-dimensional gel electrophoresis (2-DE).

For first dimensional isoelectric focusing (IEF), IPG strips (GE Healthcare, San Francisco, CA, USA, 17 cm, pH 4–7 linear gradient) were used according to the methods of Li et al. (2013). The dry IPG strips were rehydrated at room temperature for 12–16 h in 350 μL rehydration solution [8 M (w/v) urea, 1 M (w/v) thiourea, 2% (w/v) CHAPS, 65 mM DTT, 0.8% (v/v) IPG buffer 4–7, and 1% (w/v) bromophenol blue)]. Following rehydration, the IPG strips were run on an Ettan IPGphor 3 (GE Healthcare, USA) with a gradient of 100 V (1 h), 200 V (1 h), 200 V (1 h), 500 V (1 h), 1000 V (1 h), 4000 V (1 h), and 10,000 V (1 h), finally reaching a value of 75,000 V h. The working temperature was maintained at 20°C with a maximum current of 50 mA per strip. After the first dimension, the IEF strips were equilibrated for 15 min in equilibration solution I [1% (w/v) DTT, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 50 mM Tris–HCl (pH 8.8)], and then in equilibration solution II [2.5% (w/v) iodoacetamide, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 50 mM Tris–HCl (pH 8.8)] for 15 min.

The second dimensional SDS-polyacrylamide gel electrophoresis was performed on running gels (Hoefer SE600 Ruby Standard Vertical System, GE Healthcare; 12.5% polyacrylamide) as described by Laemmli (1970). The strips were embedded on the top of the SDS gel and then sealed with 1% molten agarose solution. Electrophoresis was carried out at 15 mA per gel until the bromophenol blue dye reached the bottom of the gel. After the 2-DE, the gels were stained overnight with Coomassie Brilliant Blue (CBB) R-250 solution (0.1% (w/v) of CBB R-250 in 1:4:5 (v/v) methanol: acetic acid: deionized water) and destained with a 1:1:8 (v/v) methanol: acetic acid: deionized water solution with several changes, until a colorless background was achieved.

The 2-D gels were scanned with an Image Scanner III (GE Healthcare, San Francisco, USA). Spot detection, quantification and matching were performed with Imagemaster™ 2D Platinum software (version 6.0, GE Healthcare, San Francisco, USA). The intensity of each spot on the 2-D gels was quantified based on the volumes percentage (vol. %). Only spots with significant changes (at least 1.5-fold quantitative changes, P < 0.05) were considered to be differentially expressed.

The protein spots were excised from the polyacrylamide gels, and identified using MALDI-TOF/TOF MS by an Ultraflex II mass spectrometer (Applied Biosystems, Foster City, CA, USA). The resulting peptide mass lists were searched in NCBI (http://www.ncbi.nlm.nih.gov) using the software MASCOT version 2.1 (Matrix Science, London, UK). The parameter criteria were as follows: trypsin cleavage, one missed cleavage allowed; carbamidomethyl (C) set as a fixed modification; oxidation of methionines allowed as a variable modification; peptide mass tolerance within 100 ppm; fragment tolerance set to ± 0.4 Da; and minimum ion score confidence interval for MS/MS data set to 95%.

The classification of the identified proteins was performed by searching in the UniProt Knowledgebase (UniProtKB, http://www.uniprot.org).

The hierarchical clustering of the protein expression patterns was performed on the log₂ transformed vol. % of each protein spot using Cluster software (version 3.0). The complete linkage algorithm was enabled, and the results were plotted using Treeview software (version 1.60).

Mapping of the interaction network was performed using the STRING database (http://string.embl.de) based on conformed and predicted interactions.

Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined according to Nakano and Asada (1981) by measuring the rate of ascorbate oxidation at 290 nm (ε = 2.8 mM⁻¹ cm⁻¹). Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity was calculated from the change in absorbance at 265 nm and the extinction coefficient of 14 mM⁻¹ cm⁻¹, as described by Nakano and Asada (1981). Superoxide dismutase (SOD, EC 1.15.1.1) activity was calculated by inhibiting the photochemical reduction of NBT at 560 nm. One unit of SOD activity was defined as the amount of enzyme that caused 50% inhibition of NBT reduction rate (Becana et al., 1986).

The total RNA was extracted from the tomato leaf tissues as described in the TRI reagent protocol (Takara Bio Inc.). The total RNA and cDNA syntheses were performed according to the manufacturer's instructions. The primers were designed according to the NCBI (Supplementary Table 1). qRT-PCR was performed with the SYBR PrimeScript™ RT-PCR Kit (Takara Bio Inc.) according to the manufacturer's instructions. All experiments were repeated three times and the relative gene expression was calculated by the 2^−ΔΔCt method.

All biochemical analyses were conducted at least three times. Data were statistically analyzed with statistical software SPSS 17.0 (SPSS Inc., Chicago, IL, USA) using Duncan's multiple range test at the P < 0.05 level of significance.

After 7 days' treatment with exogenous Spd, no significant differences were observed in the tomato leaves under non-stressful conditions. Phenotypic observations showed that the untreated high-temperature stressed seedlings exhibited chlorosis and yellowing, whereas the Spd-treated seedlings had a better visual appearance (Figure 1A). Under the high-temperature stress, the dry weight, chlorophyll content and net photosynthetic rate (Pn) decreased by 33.0, 16.4, and 58.9%, respectively. However, exogenous Spd application resulted in improvements in these parameters (Figure 1).

To reveal the protective effect of exogenous Spd on the tomato under high-temperature stress, a total of 67 differentially expressed spots were identified using 2-DE and MALDI-TOF-MS (Figure 2, Table 1, Supplementary Figure 1). To better understand which physiological process was regulated by Spd under the high-temperature stress, the identified proteins were grouped into 7 categories based on their biological functions according to Gene Ontology (Figure 3). Among the 67 proteins, the majority were sorted into photosynthesis (27%), followed by cell rescue and defense (24%), protein synthesis, folding, and degradation (22%), energy and metabolism (13%), amino acid metabolism (5%), signal transduction (5%), and unknown (4%).

Compared with the control, there were 33 up-regulated spots and 32 down-regulated spots in response to the high-temperature stress (Figure 4A). For the high-temperature stress induced proteins, the most highly enriched category was cell rescue and defense. However, exogenous Spd up-regulated 35 spots and down-regulated 26 spots compared with the untreated seedlings subjected to high-temperature stress, and of these proteins, the most prevalent category was photosynthesis (Figure 4B).

To obtain a comprehensive overview of the differentially expressed proteins, hierarchical cluster analysis was conducted to categorize the proteins that showed differential expression profiles affected by Spd under the normal and high-temperature stress conditions (Figure 5).

The proteomic results revealed that the abundances of some antioxidant enzymes (spots 25, 29, 35, 36) were changed (Figure 6A), so we further analyzed the associated antioxidant enzyme activities (APX, DHAR, SOD) and related gene expressions (APX 2, APX 6, DHAR 1, DHAR 2, Fe SOD, Cu/Zn SOD). The activities of the enzymes showed significant decreases under high-temperature stress. However, exogenous Spd remarkably increased their activity compared with the high-temperature stress alone (Figure 6B). A similar trend was observed for the expression levels of most of the antioxidant enzyme related genes (Figure 6C).

The proteins act together in the context of networks in cells, rather than performing their functions in an isolated manner (Bian et al., 2015). The STRING database provides a critical assessment and integration of protein–protein interactions, including direct (physical) as well as indirect (functional) associations. A network was used to show the interactions of the identified proteins and revealed the potential information at the protein level (Figure 7). Most energy metabolism related proteins (86.7%) and cell rescue and defense (68.8%) were involved in the protein–protein interaction network. Among the interaction proteins, the energy metabolism related proteins represented the highest proportion (35.1%). More importantly, GAPDH (spot 22) and phosphoglycerate kinase (spot 18) were the important junctions of interacting proteins in the network, suggesting that energy was of the utmost importance for the response to high temperature stress with exogenous Spd treatment.

Photosynthesis is highly sensitive to high-temperature stress and is often inhibited before other cell functions are impaired (Mathur et al., 2014). Importantly, Rubisco and Rubisco activase (RCA) are the primary limiting factors of net photosynthesis under stress (Ahsan et al., 2007; Hu et al., 2015). In this study, we found that proteins related to Rubisco (spots 6, 28, 49) and RCA (spots 16, 64) markedly decreased in response to high-temperature stress, similar to other proteomic studies (Han et al., 2009; Lin K. H. et al., 2015). High temperature can reduce the activation state of Rubisco (Law and Crafts-Brandner, 1999), which is often attributed to the thermolability and loss of activity of RCA under high-temperature stress (Salvucci and Crafts-Brandner, 2004; Sharkey, 2005). However, exogenous Spd had positive effects on Rubisco and RCA in tomato leaves, suggesting that the Calvin cycle and photosynthetic carbon assimilation were maintained at high levels, contributing to the biomass accumulation under high-temperature stress.

Ferredoxin-NADP reductase (spot 23) is the last enzyme in the transfer of electrons during photosynthesis from PS I to NADPH, producing NADPH for CO₂ assimilation (Fukuyama, 2004; Tian et al., 2015). Oxygen-evolving enhancer proteins (spots 32, 39, 57) are also involved in the light reaction of PS II, and are the most heat-susceptible part of the PS II apparatus (Vani et al., 2001). The abundances of ferredoxin-NADP reductase and the oxygen-evolving enhancer (OEE) decreased in response to high-temperature stress, but the expression significantly increased with the application of Spd compared with the stress alone, suggesting that Spd played an active role in the photosynthetic chain, resulted in a higher stability of PS II and an enhancement of oxygen evolving complex capacity, and then subsequently led to an enhancement of the photosynthetic capacity (Shi et al., 2013; Su et al., 2013).

Three spots were identified as proteins implicated in chlorophyll biosynthesis. Glutamate-1-semialdehyde 2,1-aminomutase (spots 11, 13) is an important enzyme to catalyze the formation of 5-aminolevulinic acid (ALA), a vital precursor of chlorophyll (Zhu et al., 2013). Coproporphyrinogen III oxidase 1 (spot 43) catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX in the chlorophyll biosynthesis pathways (Tian et al., 2015). Interestingly, the expression of chlorophyll biosynthesis proteins was increased under high-temperature stress, whereas the chlorophyll content was decreased (Figure 1C). One plausible explanation of this observation is that chlorophyll biosynthesis in plants is very complicated and co-regulated by many factors, but the temperature-related inhibition of the enzyme activity could be an important reason for the inhibition of the chlorophyll biosynthesis.

Plants have evolved a complex sensory mechanism to monitor and adapt to prevailing environmental conditions (Ahsan et al., 2007). Heat shock proteins (HSPs) are typically induced when cells are exposed to high-temperature stress, and are closely related to the acquired thermo-tolerance (Charng et al., 2006). In our study, three forms of HSP70 (spots 3, 4, 5) were identified and significantly up-regulated under high-temperature stress, which is a key part of the high-temperature response (Liao et al., 2014). In addition, two small heat shock proteins (sHSPs, spots 37, 67) were found to be newly induced by high-temperature stress, and were both found to be absent under normal conditions. The sHSPs were further up-regulated by exogenous Spd, suggesting that Spd played a crucial role in maintaining proper folding, facilitating the refolding and preventing the aggregation of the denatured proteins under high-temperature stress (Shi et al., 2013). In this experiment, the stimulation of the heat shock protein with the application of Spd may be relevant to the influence of polyamines on the DNA-binding capacity of heat shock transcriptional factor HSF (Desiderio et al., 1999).

Reactive oxygen species (ROS) metabolism is a universal response to environmental stresses. The stress-induced accumulation of ROS seriously damages the cellular membrane and internal function components, and plants have developed an antioxidant system to regulate the ROS level (Li et al., 2015). In the present study, five proteins were found to have antioxidant-related functions. Among them, Spd increased the abundances of stromal ascorbate peroxidase (APX, spot 25) and dehydroascorbate reductase (DHAR, spot 29) under high-temperature stress (Figure 6A). Further analysis revealed that the activities of APX and DHAR were increased significantly with the application of Spd under high-temperature stress (Figure 6B). The enhanced activities could be largely explained by the up-regulated mRNA levels of APX2, APX6, DHAR1, and DHAR2 (Figure 6C). Interestingly, the expression of superoxide dismutases [Fe] (Fe SOD, spot 35) in the plastid was not in accordance with the superoxide dismutase [Cu-Zn] (Cu/Zn SOD, spot 36) in the chloroplast. Moreover, the protein expression, activities of enzymes and related mRNA levels also showed different change patterns in response to high-temperature and/or Spd treatment. The variance might be due to the post-transcriptional regulation and post-translational modification of SOD through complex mechanisms, which needs further study. Taken together, the exogenous Spd is involved in antioxidant and detoxification defense mechanisms, mitigating oxidative damage and intensifying the resistance to high-temperature stress (Mostofa et al., 2014; Sang et al., 2016).

Generally, abiotic stress causes a transient suppression of de novo protein synthesis (Capriotti et al., 2014). In this study, proteomic analysis identified two spots related to protein synthesis, including elongation factor TuB (spot 17) and mRNA binding protein precursor (spot 66), which were markedly decreased under the high-temperature stress. However, the expression was enhanced after the application of Spd. According to previous data (Li et al., 2012), it can be hypothesized that stimulating the synthesis of specific proteins by exogenous Spd may play important roles in regulating the proteins synthesis and translational machinery, which are important components of the stress response in plants.

Two proteins (spots 38, 46) that induce proper protein folding and/or prevent the aggregation of stress-damaged proteins were preferentially upregulated under high-temperature stress. In agreement with this observation, the upregulation of peptidyl-propyl cis–trans isomerase FKBP 16-3 (spot 38) had been reported in Arabidopsis and rice in response to high-temperature stress (Palmblad et al., 2008; Gammula et al., 2011). The two proteins showed a decreasing pattern under the stress with Spd, indicating that exogenous Spd might regulate protein folding and assembly, participating in the high-temperature stress tolerance.

Proteolysis is a complex process involving many enzymes and pathways in various cellular compartments. Proteases play a central role in metabolism under abiotic stress as they are involved in protein inactivation and the degradation of damaged proteins (Capriotti et al., 2014). In our study, the cysteine proteinase 3-like (spot 52) was up-regulated under high-temperature stress, in agreement with previous studies (Koizumi et al., 1993; Callls, 1995). Interestingly, the expression of ATP-dependent zinc metalloprotease (spot 7) and proteasome subunit alpha type-2-A-like (spot 33) were decreased under high-temperature stress but increased significantly with the application of exogenous Spd. Stimulating the proteolysis of specific proteins by Spd accelerated the degradation of misfolded/damaged proteins, and made tissues more stable by covalently attaching with proteins (Li et al., 2013). Furthermore, the PAs regulated the protein metabolism and may reprogram the proteome in response to abiotic stress (Yuan et al., 2016), which may also account for the resistance to high-temperature stress of the tomato seedlings.

It is well-known that sufficient ATP is necessary in response to abiotic stress in plants (Hu et al., 2014). Three proteins associated with ATP synthase (spots 8, 9, and 53) were significantly upregulated under the high-temperature stress, suggesting a higher energy demand for the degradation and biosynthesis of proteins (Das et al., 2015). However, the ATP synthase proteins were down-regulated by the exogenous application of Spd under high-temperature stress, stabilizing the process of ATP synthesis, and energy metabolism.

ATP is mainly produced by carbohydrate metabolism, such as glycolysis, the tricarboxylic acid cycle and the pentose phosphate pathway (Hu et al., 2015). The first group included 7 proteins involved in glycolysis pathway. Among them, our results showed that fructose-bisphosphate (FBP) aldolase in the cytoplasm (spot 27), and chloroplastic (spot 42) decreased significantly under high temperature. Moreover, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, spot 22), and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (spot 19) also decreased under the stress, which would inhibit the glycolysis pathway and glycolysis associated with intermediate metabolism. However, exogenous Spd up-regulated these proteins, allowing more carbohydrates to enter the glycolic pathway and maintain the normal physiological metabolism of the tomato seedlings, thereby supporting the high-temperature resistance (Shan et al., 2016). The second group included malate dehydrogenase (MDH, spots 21, 65), involved in the tricarboxylic acid cycle. In this study, MDH showed different accumulation patterns in response to high temperature, whereas exogenous Spd sprayed on the leaves maintained the MDH expression at a high level. The third group was protein participating in the pentose phosphate pathway. Under high-temperature stress, the abundance of transketolase (TK, spot 10) and ribose-5-phosphate isomerase (spot 55) decreased. Spd application further improved the abundance of TK, whereas it affected ribose-5-phosphate isomerase unremarkably. Adjusting the EMP-TCA-PPP pathway to produce more energy may be an important mechanism for Spd to alleviate stress induced damage (Li et al., 2015).

Signal transduction pathways play an important role in abiotic stress at the cellular level, leading to changes in metabolic pathways and cellular processes. After the perception of the stress, a signal would be transferred from the cell surface to the nucleus, and then the responsive proteins would be translated (Guo et al., 2013). Within this functional category, we identified calreticulin (spots 1, 2) and harpin binding protein 1 (spot 56). Calreticulin, a major endoplasmic reticulum Ca²⁺-binding chaperone, is involved in a variety of cellular signaling pathways. Calreticulin also plays a crucial role in regulating Ca²⁺ intracellular homeostasis (Nakamura et al., 2001). In our study, calreticulin was significantly up-regulated under the high-temperature stress, but was down-regulated by Spd. These observations suggested that Spd has a relationship with the stress-induced Ca²⁺ signal transduction, probably allowing the release of free Ca²⁺ to relieve stress. Interestingly, harpin binding protein 1 was significantly down-regulated in the tomato leaves under the high-temperature stress, which was concordant with the finding in spring soybean under cold stress (Tian et al., 2015). However, the protein level recovered the controlled level after Spd treatment, and the regulatory mechanism remains unclear.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.