Phenotype and physiological responses of HT and HS under heat stress
After 48 h of heat stress, HS seedlings showed more wilting than HT, suggesting that the effect of heat stress on pepper seedlings was slighter in HT compared with HS (Fig. 1A). After 48 h of heat stress, the contents of proline and soluble sugar were increased in the two pepper genotypes, while their contents in the HT genotype were higher compared with the HS genotype (Fig. 1B, C). Compared with the control samples, the content of MDA was significantly increased both in HT and HS after 48 h of heat stress. However, HS showed a stronger up-regulation (Fig. 1D). RWC values after 48 h of heat stress were decreased in leaves, and such reduction was greater in HS compared with HT (Fig. 1E).
iTRAQ data analysis and responses of DAPs to heat stress
Based on the iTRAQ experiment, a total of 1,628,623 spectra were identified from pepper seedlings. Moreover, 23,706 peptides, 21,335 unique peptides, and 5,098 proteins were detected from 193,696 known spectra (Fig. S1A). The MS data were deposited into the identifier PXD019556. Fig. S1B shows the mass distribution of proteins, and the majority of them were 10-70 kDa in size. In addition, sequence coverage of most proteins was<35% (Fig. S1C). The coefficient of variation of replicates was calculated to evaluate the reliability of proteomic data. The result showed that proteins with 20% coefficient of variation were more than 80% of identified proteins (Fig. 1D), suggesting that the data were reliable. In a PCA model based on 24 samples, control (0 h) and heat-treatment samples (3, 28 and 48 h) were clearly separated (Fig. S2). PCA1 accounted for 40.30% of the variability, while the PCA2 accounted for 38.30% of the variability (Fig. S2).
Compared with the control samples, a total of 1,591 DAPs were identified under heat stress in HT and HS. Among these DAPs, 157/156, 589/301, and 976/1,025 were significantly accumulated in HT/HS under heat stress at 3, 28, and 48 h, respectively (Fig. 2A). In short, there were more DAPs identified in HT compared with HS (Fig. 2A). Moreover, 457 and 394 specific DAPs were identified in HT and HS, respectively, while 740 common DAPs were found both in HT and HS (Fig. 2B).
Functional analysis of DAPs in response to heat stress
Of the 1,591 DAPs, 997 DAPs were categorized into 24 categories using COGs database. The largest group was general function prediction only, followed by post-translational modification, protein turnover, chaperones, translation, ribosomal structure and biogenesis, energy production and conversion, carbohydrate transport and metabolism, and amino acid transport and metabolism (Fig. 3A and Table S1).
To further characterize all heat-responsive proteins, these DAPs identified in HT and HS were subjected to GO analysis. Of the 1,591 DAPs identified in HT and HS under heat stress, 1,453 proteins were annotated into three groups as cellular component (CC), molecular function (MF), and biological process (BP). GO analysis showed that cell part, cell, and organelle were the major CC terms; catalytic activity, binding, structural molecule activity and transporter activity were the dominant MF terms; and cellular process, signaling, metabolic process, single-organism process and response to stimulus were the most dominant BP terms (Fig. 4).
To further investigate the major metabolic pathways responding to heat stress, KEGG enrichment analysis was carried out with heat-responsive proteins from HT and HS. The result showed that DAPs were matched to 99 KEGG pathways in HT and 106 KEGG pathways in HS. Heat stress could affect protein processing in photosynthesis, endoplasmic reticulum, terpenoid backbone biosynthesis, porphyrin and chlorophyll metabolism, carbon fixation in photosynthetic organisms, and pentose phosphate pathways in both two genotypes (Fig. 5). Furthermore, KEGG metabolic pathways, including pyrimidine metabolism, galactose metabolism and amino sugar and nucleotide sugar metabolism, were highly enriched in HT, whereas other pathways, such as glyoxylate and dicarboxylate metabolism, phenylpropanoid biosynthesis, carbon metabolism, nitrogen metabolism, biosynthesis of amino acids, glutathione metabolism, phenylalanine metabolism, and glycine, serine and threonine metabolism, were considerably enriched in HS (P<0.05, Table S2 and S3).
Antioxidants in response to heat stress
In response to heat stress, antioxidant enzymes (GST, POD, SOD, DHAR, APX and CAT) were differentially accumulated according to proteomic data. Under heat stress, most GSTs were significantly increased with the increase of treatment time (Fig. 6A). Some of them were accumulated in both two genotypes, while three of them were suppressed in response to heat stress. Among six identified PODs, POD 12-like and 3-like were significantly increased in HT, while POD p7-like, 17-lke, and 51-like were decreased in HT and HS (Fig. 6A). Under heat stress, two SODs were identified in response to heat stress, and both of them were greatly increased in HT, while there were no obvious changes in HS (Fig. 6A). One DHAR and one CAT were significantly increased in HT, while they were slightly increased in HS (Fig. 6A). In addition, most APXs were increased in HT, while they were only there APXs were increased in HS.
Furthermore, in order to further compare the differences of antioxidant capacity between HT and HS, the content of GSH and the activities of GST, POD, SOD, APX, and CAT were determined at 0 and 48 h after the heat treatment. Fig. 7A shows that the content of GSH was increased in both two genotypes under heat stress. However, its content was higher in HT compared with HS. The activities of GST and POD were higher in HT compared with HS after 48 h of heat stress (Fig. 7B, C). Compared with the control samples, the activities of SOD, APX, and CAT were significantly increased in HT. However, there were no obvious changes in HS (Fig. 7D, E, F).
Specific DAPs of HT in response to heat stress
To further investigate the mechanism underlying the higher heat tolerance in HT, the functions of 457 specific DAPs were studied based on GO analysis (Table S4). In short, these DAPs were mainly enriched in protein autophosphorylation, hexokinase-dependent signaling, and regulation of ROS metabolic process. Among these proteins, 95 proteins were up-regulated at each treatment time point under heat stress, including glutamate decarboxylase (Capana00g003786), 26s protease regulatory subunit 8 homolog a (Capana01g002147), sHSP (18.2 kDa class I heat shock protein), universal stress protein a-like protein, and so on (Table S4). Moreover, some DAPs related to signal transduction were also identified in HT, such as calcium-dependent protein kinase (Capana04g002552), serine threonine-protein kinase ht1 (Capana07g001488), and atp-dependent 6-phosphofructokinase 6 (Capana07g001111).