Correlation Analysis of Response of Sorbus pohuashanensis Leaves to High-temperature Stress Based on Label Free-based Quantitative Proteomic and Transcriptomic

Abstract

Background: High-temperature stress is the main abiotic stress of Sorbus pohuashanensis (Hance) Hedl. introduced from the mountains to plain areas. To investigate the mechanisms of heat response in S. pohuashanensis, we compared the transcriptome and proteome after high temperature stress at 43 °C for 8 h.

Results: Proteomic sequencing yielded 92 (51 upregulated and 41 downregulated) DAPs, involving heat-killed proteins, molecular chaperones, energy metabolism, signal transduction, cellular autophagy, and reactive oxygen species scavenging systems. TEM results indicated that high-temperature stress led to disruption of the structural integrity of chloroplasts in mature chloroplast cells, reduction of chloroplast vesicle-like lamellae, accumulation of lipophilic granules, and an increase in the number of vesicles. Quantitative combined transcriptomic and proteomic analyses yielded 34 pairs of DAPs/DEG-associated genes. The functional enrichment of these DAPs/DEGs indicated that their functions were related to chaperones, energy metabolism, ROS scavenging system, and cell wall modification.

Conclusion: 92 DAPs were identified in the proteomic analysis of S. pohuashanensis under high temperature stress, and co-regulatory analysis revealed 34 DAPs/DEGs in the comparison group, with 24 genes having the same protein and mRNA expression trends and 10 genes with opposite protein and mRNA expression trends. These genes are collectively involved in response to high temperature stress by encoding proteins related to molecular chaperones, energy metabolism, ROS scavenging systems and cell wall modifications. These results provide a basis for the complex HSF–Hsp regulatory network contributes to the heat tolerance of S. pohuashanensis. 

1. Background

Sorbus pohuashanensis (Hance) Hedl. is widely distributed in north, north-west, and north-east China. It grows on mountain slopes or in valley forests at altitudes of 900-2500 m and is resistant to low temperatures [1]. S. pohuashanensis is of very high ornamental value in the landscape and as a food source for some wild birds [2]. However, the vertical introduction of native tree species from high altitude mountains to low altitude areas within the same climatic zone is still difficult [3]. Seeds from higher altitudes tend to grow poorly at lower altitudes because they have developed different growth rhythms over a long period of time as they adapt to light and heat factors at different altitudes [4]. The introduction of alpine species to lower altitudes is characterised by the most drastic changes in habitat temperature [5]. Sunburn occurs on the leaves of plants when they are exposed to the sun during the hot summer months [6]. Sunburn is one of the most serious problems faced when applying horticultural plants such as S. pohuashanensis, Purus communis [7], Eriobotrya japonica and Malus domestica [8]. The occurrence of sunburn in plants appears to be related to climatic factors involving light intensity, temperature and air humidity, yet little is known about the causes of sunburn in summer in S. pohuashanensis [8].

Current research on the molecular biology of S. pohuashanensis is relatively focused on molecular pharmacology, molecular markers, gene cloning and functional genomics sequencing [9-12]. The mechanisms of the species' response to heat stress are still being explored, and some progress has been made. Peng et al. found that S. pohuashanensis is heat-sensitive plant [13]. On the molecular mechanism of the response of S. pohuashanensis to high temperature, Liu et al. sequenced the transcriptome of S. pohuashanensis, cloned SpHsp70-1 from unigenes and investigated its response mechanism and expression pattern to high temperature [11, 14]. Zhang et al. cloned SpHsp17.3 and SpHsp23.8 and investigated their tissue-specific expression and response to abiotic stress [15, 16]. Pei et al. sequenced the transcriptome of S. pohuashanensis leaves treated continuously at 43°C for 8 h. A total of 1221 DEGs were obtained, with candidate genes involved in calcium signaling pathways, phytohormone signaling, heat shock factors (HSFs), molecular chaperones, protein ubiquitination degradation, cell wall modification, cellular autophagy, reactive oxygen species (ROS) scavenging enzymes [17]. However, studies at the transcriptional level alone are insufficient to elucidate the molecular mechanisms underlying the response of S. pohuashanensis to high temperature stress, and regulation at the translational level is not yet known.

Previous studies have shown that high temperature stress causes structural changes in the chloroplast capsular membrane of plants, which in turn affects the enzymatic activities of photosystem I (PS-Ⅰ) and photosystem II (PS-Ⅱ) reducing the photosynthetic rate of plants [18, 19]. In addition, high-temperature stress reduces the photosynthetic efficiency of plants by affecting the carbon assimilation of photosynthesis [20, 21]. High-temperature stress can induce changes in the expression of many proteins in terms of quantity and species [22]. Under high-temperature stress, plants can regulate the expression of members of the heat stress protein family including HSP70, HSP90, sHSP, heat shock homolog 70 (HSC70) and HSC80 to ensure normal protein folding or to enhance the stability of some unfolded proteins [23-25]. The abundance of ROS scavrnging enzymes including catalase (CAT), peroxiredoxin/thioredoxin (Prx/Trx), ascorbicacid-glutathione (AsA-GSH), glutathione S-transferase (GST) pathway and peroxidase (POD) pathway are all involved in the scavenging of H₂O₂ produced in excess under high-temperature stress in plants [24, 26, 27]. Glucose and energy metabolic processes (such as, tricarboxylic acid cycle glycolytic pathway) play an important role in plant stress response [18, 27-29]. The expression pattern of glycolysis-related enzymes is also affected by high-temperature stress[18, 20, 21, 23, 30-32]. Further research on the molecular mechanisms of tree responses to high temperature stress is an important guide to the discovery of tree resources, determination of forest tree resistance, and selection of heat-tolerant tree species [33, 34]. Due to variable shearing and post-translational modifications in the translation process, the expression level of mRNA in cells cannot fully represent the expression level of proteins. Therefore, it is necessary to study the changes in protein expression to understand the network synergistic response mechanism of plants to high temperature at the systems biology level [35]. 

In this study, molecular regulatory networks at the translational level in response to high temperature stress were constructed using label-free quantitative proteomics and TEM, and combined with previous high temperature transcriptomics to reveal the molecular mechanism system of S. pohuashanensis in response to high-temperature [17]. The results of the correlation analysis will help to further explore the key mechanisms by which S. pohuashanensis cope with high-temperature stress and provide an information base for breeding heat-tolerant S. pohuashanensis varieties.

2. Methods

2.1. Plant materials and treatment

S. pohuashanensis was provided by the forest germplasm resources nursery of the National Forest Genetic Resources Platform (NFGR), Beijing University of Agriculture. Beijing, China, and one-year-old grafted clones of three genotypes from Mount Tai (Shandong, China), Mount Lao (Shandong, China), and Pingquan (Hebei, China) were used in the experiment. Jian Zheng undertook the formal identification of the plant material used in this study. Voucher specimens of this material have not yet been deposited in a publicly available herbarium. 

Treatment samples were obtained from the same grafted S. pohuashanensis as the previously sequenced transcriptome [17]. The high-temperature treatment group (HT) and a control group (CK) were pretreated for 3 days in an artificial climate chamber (BIC-400; Bosun, Shanghai, China) under 16 h/25°C day, 8 h/18°C night, 70% relative humidity and a light intensity of 180 μmol·m^-2·s^-1. After pretreatment, the HT group was subjected to high-temperature stress at 43 ℃ for 8 h under light conditions (other conditions unchanged). In contrast, the conditions of the CK group remained unchanged. At the end of treatment, all leaves of the HT and CK were collected, frozen in liquid nitrogen, and stored at -80 ℃ for protein extraction. In addition, the leaves of HT and CK groups used for transmission electron microscopy were selected and high-temperature treatment was performed at 0, 2, 4, 6 and 8 h, and recovery for 24 h.

2.2. Protein preparation

After 43 ℃ 8h treatment, six leaves samples from HT and CK groups were extracted by phenol extraction, and an appropriate amount of the sample was grounded thoroughly in liquid nitrogen. Thereafter, pre-cooled BPP buffer (Sinopharm, Shanghai, China) was added to three volumes as plant homogenate, and the samples were vortexed at 4 ℃ for 10 min. An equal volume of Tris-saturated phenol (pH≥7.8) (T0250, Solarbio, Shanghai, China) was added, followed by vortexing at 4 ℃ for 10 min and then centrifuged for 20 min (12,000×g, 4 ℃). Added 5 volumes of pre-cooled ammonium acetate (Sigma, Santa Clara, CA, USA) methanol (Sinopharm, Shanghai, China) solution and precipitate the protein overnight at -20 °C; the next day, centrifuged at 4 °C for 20 min at 12000×g and discard the supernatant; added 90 % pre-cooled acetone to the precipitate and mixed well, centrifuged and discarded the supernatant, repeat twice; dissolved the precipitate in Halt Protease Inhibitor Cocktail (ThermoFisher Scientific, Wilmington, DE, USA); ultrasound on ice for 2 min; centrifuged at 4 °C for 12000× g 20 min and remove the protein supernatant. Standard samples were prepared according to the instructions provided with the Pierce BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Wilmington, DE, USA) and the absorbance of each sample was measured at 562 nm using a Multiskan MK3 ELISA (Thermo, Waltham, MA, USA). The protein concentration of the samples was calculated from the standard curve and the volume of sample used.

2.3. Protein digestion and labeling

TEAB (Sigma, Santa Clara, CA, USA) was added to a final concentration of 100 mM, followed by Bond-Breaker TCEP Solution (Thermo Fisher Scientific, Wilmington, DE, USA) to a final concentration of 10 mM at 37 ℃ for 60 min and added iodoacetamide (≥99 %, chromatographically pure crystalline, Sigma, Santa Clara, CA, USA) to a final concentration of 40 mM at 25 ℃ for 40 min, protected from light. Thereafter it was centrifuge at 10,000×g for 20 min and then the precipitate was removed. The sample was dissolved in 150 µL of 100 mM TEAB, and trypsin (Promega, Madison, WI, USA) was added at an enzyme: protein ratio of = 1:50 and digested overnight at 37 ℃. After trypsin digestion, the peptides were dried using vacuum pump. The enzymatically dried peptides were dissolved in 0.1 % TFA (Thermo Fisher Scientific, Wilmington, DE, USA). The peptides were desalted by HLB (Thermo Fisher Scientific, Wilmington, DE, USA), and each sample was divided into two portions and dried using a vacuum concentrator. The peptides were quantified using a Pierce quantitative colorimetric peptide assay (23275, Thermo Fisher Scientific, Wilmington, DE, USA).

2.4. Liquid chromatography-tandem mass spectrometry 

The peptides were quantified by mass spectrometry using mass spectrometry loading buffer at an equivalent concentration of 0.5 μg/μL. Liquid-phase tandem mass spectrometry was performed using EASY-nLC 1200 (ThermoFisher Scientific, Wilmington, DE, USA) with Q-Exactive (ThermoFisher Scientific, Wilmington, DE, USA). A 75 μm × 25 cm C18 column reversed-phase column (ThermoFisher Scientific, Wilmington, DE, USA) was selected and using high-performance liquid chromatography system (Thermo Xcalibur, Version 3.0, https://www.thermofisher.com/order/catalog/product/OPTON-30487?SID=srch-srp-OPTON-30487 ). Separation was performed using buffer A solution of 0.1 % formic acid water and buffer B solution of 0.08 % formic acid acetonitrile solution (80 % acetonitrile). The chromatographic column was balanced with 95 % A. Samples were loaded by an automatic sampler first to a mass spectrometry C18 trap column and then separated using an analytical C18 column with a flow rate of 300 nL/min. The relevant liquid phase gradient was performed as follows: 0 min, A: B = 100:0 (v/v); 1 min, A:B = 95:5 (v/v); 63 min, A:B = 77:23 (v/v); 77 min, A:B = 71:29 (v/v); 86 min, A:B = 62:38 (v/v); 88 min, A:B = 52:48 (v/v); 89 min, A:B = 0:100 (v/v); 95 min, A:B = 0:100 (v/v); 96 min, A:B = 100:0 (v/v); and 120 min, stop. Each sample was separated by capillary high-performance liquid chromatography and then analyzed using Q-Exactive Mass Spectrometers.

2.5. Label-free protein identification and quantification

Mass spectrometry raw data were analyzed qualitatively and quantitatively using Proteome Discoverer (Version 2.2) and Xcalibur Qual Browser Version 3.0. The database used in this study was UniGene_ORF.fa.transdecoder.pep. The data were derived from  transcriptome sequencing data [17]. The database search parameters were as follows: fixed iodoacetamide of Cys alkylation, fixed modifications of oxidation (M), acetyl (Protein N-term), modifications of carbamidomethyl (C), fixed trypsin of enzyme name (Full), trypsin fragment with up to 2 missed cleavages, peptide tolerance was set at 10 ppm, and peptide false discovery rate analysis ≤ 0.01. Identification of the peptides BLAST Nr database ( https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/ ) to obtain annotation information. DAPs were screened using a threshold of fold change (FC). FC ≥ 1.2 was considered up-abundant, FC ≤ 0.83 was considered down-abundant, and 0.83<FC<1.2 was considered to have no significant change in expression and visualization of DAPs using TBtools. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org ) via the iProX partner repository with the dataset identifier PXD027218 [36]. ‍ 

2.6. Bioinformatic analysis of proteins 

Functional annotations of GO (Gene Ontology, http://www.geneontology.org ) describe three aspects of molecular function, cellular component and biological process. AgriGO (Version 2.0; http://systemsbiology.cau.edu.cn/agriGOv2/index.php ) was used to identify GO terms that were significantly enriched by DAPs after high-temperature stress. KEGG is a key public database associated with pathways (http://www.kegg.jp/kegg/pathway. html )[37]. GO and KEGG functional annotation analysis were performed for the differentially identified proteins. In addition, the main biological functions of DAPs and the major biochemical metabolic and transduction pathways involved was determined using GO and pathway functional significance enrichment analysis.

2.7. Proteomic and transcriptomic correlation analysis

The mRNA and protein encoded by the same gene were linked when the protein encoded by this gene was identified in the DAPs in this study and the gene was a DEG in the previous transcriptome [17]. The proteins and associated genes were counted in terms of both quantifiable and significantly different components. Once the association information for the gene and protein was obtained, the association results were annotated for GO function. At GO level_2, the profiles of differential transcripts and differential protein functional annotations were compared. KEGG pathway annotation was performed on the association results to compare the KEGG pathways involved in the differential transcripts and differential proteins. Quantitative association analysis of transcriptomics and proteomics-linked genes based on the annotation of DEGs and DAPs to determine their expression at the transcriptional and translational levels.

2.8. Quantitative RT-PCR determination

qRT-PCR was performed using a Bio-Rad CFX96 real-time PCR detection system. Fifteen primers specific for DAP corresponding to DEGs were designed using Primer Premier Version 6.24 (Supplementary Table S1). qRT-PCR was performed using SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biology, Hunan, China) according to the manufacturer's instructions. SpActinQ was used as an internal reference gene. Differences in quantitative results were evaluated using 2^-ΔΔCt method^[38]. Three technical replicates of qRT-PCR were performed for each gene.

2.9. Transmission electron microscope observation

The washed leaves were placed on wax dishes; avoiding the veins, they were cut into long strips of 1-1.5 mm width and 2-3 mm length. The cut leaves were immersed in 2.5 % glutaraldehyde fixative (111-30-8, SPI, USA) at 4 ℃ for more than 8 h. Thereafter the samples were fixed overnight at 4℃ under light-proof conditions using 1% osmium fixative (Ted Pella Inc., CA, USA). The dehydrated samples were embedded in Epon812 (4.70 g Epon812, 2.56 g MNA, 2.62 g DDSA, 0.12 gDMP-30) embedding agent and the material was placed in a 45 ℃ thermostat for 12 h before being transferred to a 60 ℃ thermostat for 24 h to complete polymerization. Samples were sectioned using an EM UC7 frozen ultrathin sectioning machine (Leica, Wetzlar, Germany) and an Ultra 45° diamond sectioning knife (Daitome, Switzerland). Sections that produced a yellowish color were selected for slicing interference, and slices were retrieved using a 150 mesh Formvar Film copper mesh. The copper mesh was stained with 2 % uranyl acetate saturated with ethanol for 8 min, followed by 2.6 % lead citrate solution protected from carbon dioxide for 8 min, and vacuumed for 30 min on a 7700 transmission electron microscope (Hitachi, Tokyo, Japan). CCD conversion was performed to obtain electronic images.

2.10. Statistical analysis

The t-test in R Version 3.5.0 was used to calculate the p-value for significant differences between samples. 

The g plot in the R package was used to cluster the significant DAPs using the distance calculation algorithm: Spearman between samples, Pearson between genes, and h cluster (complete algorithm).

GO functional and KEGG pathway significance enrichment analysis of DAPs was performed using a hypergeometric distribution and the test was Fisher's exact test. 

3. Results

3.1. Quantitative identification of proteins

In this study, after high temperature treatment, leaves of S. pohuashanensis were collected and subjected to label-free proteomic analysis. After merging data from three replicates, a total of 10,362 peptides and 3,801 proteins were identified (Table 1). The distribution of the peptide numbers is shown in Figure 1. In addition 115 protein fragment-contained 6 peptides, 3,629 fragments had 7–11 peptides, and 6,618 peptide fragments were more than 12 in length. Protein masses were normally distributed with 2,438, 304, and 21 proteins 1–50, 51–100, and above 101 kDa in size, respectively (Figure. 2).

Table 1 Mass Spectrometry Primary Analysis of Protein Samples.

3.2. Quantitative identification of proteins and DAPs

Full-spectrum annotation of the identified proteins showed that 2,531 of the 3,801 proteins were annotated in Nr, accounting for 66.59% (Table 2).

Table 2 Statistics of annotation results for S.ohuashanensis leaves proteins.

The 3,801 proteins in the raw data obtained using the label-free quantification technique had at least one significant peptide sequence. The p-value of the difference between samples was calculated using the t.test function in R. In this study, 92 DAPs, of which 51 had increased protein abundance and 41 had decreased protein abundance were obtained, and the screening criteria for DAPs were p < 0.05, FC < 0.83 or FC > 1.20 (Table 3). DAPs were visualized using TBtools (Supplementary Figure S1). The 92 DAPs were classified according to their annotation information. The predominant category was heat shock proteins, with a total of 17 Hsps within 7 Hsp70, 2 Hsp90, 4 Hsp100, and 4 sHsp in increased abundance. In addition, five energy metabolism-related proteins with increased abundance, two ROS scavenging-related proteins with increased abundance, two intracellular signalling-related proteins with increased abundance, and two intracellular signaling-related proteins with decreased abundance were also identified.

Table 3. DAPs under high temperature stress in S.ohuashanensis proteome.

3.3. Functional annotation of DAPs

DAPs were significantly enriched in 36 GO terms (q-value ≤ 0.05). Among the biological process categories, intracellular processes (GO: 0009987) (n=91) and metabolic processes (GO: 0008152) (n=89) were the most enriched. Within the cellular formation category, cellular parts (GO: 0044464) (n=88) and organelles (GO: 0043226) (n=51) were the most abundant subcategories. Among the molecular functional categories, catalytic activity (GO: 0003824) (n=108) and binding (GO: 0005488) (n=96) were the enriched subcategories (Figure 3).

Further studies identified KEGG biological pathways involved in DAPs under high temperature stress in S. pohuashanensis, with a total of 148 KEGG pathways, which are mainly involved in protein processing in the endoplasmic reticulum (ko04141), antigen processing and presentation (ko04612), longevity regulating pathway-multispecies (ko04213), endocytosis (ko04144), spliceosomes (ko03040), MAPK signaling pathways (ko04010), and plant-pathogen interactions (ko04626) (Figure 4).

3.4. DEGs and DAPs correlation analysis of S. pohuashanensis

The genes and proteins correlated to S. pohuashanensis were counted at both quantifiable and significantly different levels (Table 4 and Supplementary Table S2). Differential ploidy obtained using RNA-Seq and Protein-Seq was used as data points and correlated by a nine-quadrant plot (Figure 5).

Table 4. Statistical table of transcriptome and proteome data association.

The genes in quadrants ①, ③, ⑦ and ⑨ in Figure 5 are sorted out. That is, the genes associated with transcriptome and proteome results. A total of 34 associated genes were obtained, with 25 of the 92 identified DAPs showing the same pattern of abundance (increase or decrease) as the corresponding DEG expression levels (up- or down-regulation) (Tables 5 and 6).

Table 5. Genes with increased abundance of DAPs with corresponding upregulated expression of DEGs.

Table 6. Genes with reduced abundance of DAPs and corresponding downregulated expression of DEGs.

Nine additional DAPs showed patterns of abundance (increased or decreased) that did not coincide with the corresponding DEG expression levels (up- or down-regulated) (Tables 7 and 8).

Table 7. Genes with increased abundance of DAPs with corresponding downregulated expression of DEGs.

Table 8. Genes with reduced abundance of DAPs with corresponding upregulated expression of DEGs.

3.5. DEGs and DAPs correlation functional analysis of S. pohuashanensis

After obtaining the association information of genes and proteins, GO functional annotation of the association results is required. At GO level 2 terms, the profiles of differential transcripts and differential protein functional annotations were compared. In the DEGs and DAPs, metabolic processes (GO: 0008152), intracellular processes (GO: 0009987) and monomeric processes (GO: 0044699) were the most abundant subcategories within the biological processes category. In the cell-forming category, cells (GO: 0005623) and cell parts (GO: 0044464) were the richest subcategories. Among the molecular functional categories, catalytic activity (GO: 0003824) and binding (GO: 0005488) were the enriched subcategories (Figure 6). In particular, membranes (GO: 0016020) and organelles (GO: 0043226) were also the most enriched subcategories (Figure 6).

After obtaining the association information of genes and proteins, the association results were further annotated with KEGG pathways to compare the KEGG pathways involved in the differential transcripts and differential proteins. In DEGs and DAPs, these pathways were mainly involved in carbohydrate metabolism, amino acid metabolism, and energy metabolism (Figure 7). The difference is that coenzyme and vitamin metabolism, and other amino acid metabolism are enriched annotated pathways in DEGs. Furthermore, the synthesis of other secondary metabolites, lipid metabolism, and transport and degradation are enriched annotated pathways in DAPs (Figure 7).

3.6. Validation of quantitative RT-PCR of genes under transcript and translation level

Fifteen genes were randomly selected for qRT-PCR to validate the results of the combined analysis. The results showed that the relative expression patterns of these genes in the HT and CK groups (Figure 8a) were generally consistent with the transcriptome sequencing of the genes (Figure 8b and Supplementary Table S2).

3.7. The characteristics of leaf flesh cells of S. pohuashanensis under high temperature

To better understand the changes in S. pohuashanensis cells under high temperature stress, we observed leaf flesh cells during 8 h of stress at 43 ℃ and after recovery for 24 h. The ultrastructure of cell walls, cell membranes, vesicles, chloroplasts, mitochondria, nuclei and other organelles of S. pohuashanensis were all altered in response to high temperature stress (Figure 9). The structural integrity of the chloroplasts was disrupted as the cell membrane, nuclear membrane, cytoplasmic vesicles and other components of the membrane system of mature leaf flesh cells of S. pohuashanensis wrinkled to varying degrees (Figure 9f); chloroplasts gradually expanded from shuttle-shaped, and the lamellae of vesicle-like basal granules within the chloroplasts disappeared. Their distribution was disrupted (Figure 9e); starch granules within the chloroplasts were blurred, lipophilic granules accumulated in large numbers, and the number of vesicles increased (Figure 9g). These results indicated that high-temperature stress caused damage to the structure and function of the chloroplasts, affecting normal photosynthetic activity. However, chloroplasts that recovered for 24 h after high-temperature treatment re-produced new starch granules, indicating that the short duration of high temperature stress did not cause irreversible damage to the structure and function of chloroplasts (Figure 9i).

4. Discussion

Based on label-free quantitative technology and TEM, this study obtained high-quality proteomic data and conducted cellular microstructural changes in the leaves of S. pohuashanensis in response to high-temperature stress. A total of 92 DAPs (51 increased abundance and 41 decreased abundance) were obtained, including heat kinases, molecular chaperones, energy metabolism, signalling, cellular autophagy and reactive oxygen species scavenging systems. Thirty-four pairs of DAPs/DEG-associated genes were obtained, and gene function enrichment annotation indicated that the functions of these associated DAPs/DEGs were involved in molecular chaperones, energy metabolism, ROS scavenging system and cell wall modifications. High temperature stress resulted in disruption of chloroplast structural integrity, reduction of chloroplast vesicle-like lamellae, accumulation of lipophilic granules and an increase in the number of vesicles in mature chloroplasts.

4.1 Proteomics analysis of S. pohuashanensis under high temperature stress

The DAPs were mainly focused on heat stress proteins, molecular chaperones, energy metabolism, cell wall and membrane systems, calcium signaling and ROS, indicating that high temperature stress has a multifaceted effect on the vital activities of S. pohuashanensis. 

Chaperones are proteins that help other proteins fold, assemble, translocate and mediate the degradation of misfolded proteins [39]. Thus, chaperones can effectively regulate the production of abnormally folded proteins in plant cells, avoiding the formation of aggregates and reducing cell damage caused by stress [40]. Many chaperones have been identified and are divided into three main groups, the CPN family, the HSP family, and other small protein molecules such as nucleoporins, DnaJs, SecBs and CSPs. Hsp70s are an important class of chaperones with significant roles in biotic and abiotic stresses, and their activity is regulated by DnaJs [39]. The interaction between Hsp70s and unfolded proteins (their substrates) is regulated by molecular co-chaperones, including sHsps [41]. SlHsp70s regulate SlHsfA1 function and inhibit SlHsfA1 activity through direct interactions [42]. The HSF-Hsp regulatory network may be involved in the regulation of high-temperature stress response [42, 43]. Activation of HSFs in response to heat stress is released by association with and inhibition of Hsp70s and Hsp90s chaperones, as chaperone molecules bind to misfolded proteins caused by high temperature stress [44]. In addition, Hsps, including sHsp, Hsp90, and Hsp70, and their auxiliary chaperones play a role in preventing protein denaturation and maintaining protein homeostasis [45]. In particular, we have identified chaperone proteins ClpB1, ClpB3, and ClpB4, which belong to the Hsp100 family, and ClpBs function as molecular chaperones to enhance their resistance to various stresses [46]. ClpB3 is involved in protein folding during high-temperature stress and pigment body variation to regulate the formation of vesicle membranes in chloroplasts and enhance the heat resistance of chloroplasts [47]. ClpB1 is not essential for plant germination and development under normal conditions; however, it is important under heat stress. AtClpB3 also acts in response to heat stress and chloroplasts by binding to signal peptides and promoting chloroplast development [48]. In the results of our correlation analysis, DJ1B-like was upregulation and BIP5-like was upregulated at the protein level (Tables 5 and 8). DJ-1 protein in plant and mammalian cells, DJ-1 directly affects SOD activity in a highly conserved manner, thereby preventing cell death [49]. BIP, a molecular chaperone of the Hsp70 family, is localized in the ER and plays a key role in protein translocation, protein folding and quality control in the ER [50]. S. pohuashanensis regulates their homeostasis and reduces the damage they suffer when exposed to heat damage by reorganizing various proteins denatured by heat stress through molecular chaperones. Furthermore, the plant BAG family plays a broad role in plant programmed cell death (PCD) processes and is involved in the co-chaperone regulation of cellular pathways [51]. For example, the AtBAG protein is a nucleotide exchange factor for Arabidopsis Hsp70/Hsc70, and its mechanism for regulating protein folding is conserved in plants [51]. These results indicate that proteins with a chaperone role regulate their homeostasis by reconstituting various proteins denatured under high-temperature stress when S. pohuashanensis were subjected to high-temperature damage, reducing the damage it suffered.

At the cellular level, the effects of high-temperature stress on S. pohuashanensis were mainly manifested in the disintegration of cyst-like structures and increased vesicles. Transmission electron microscopy showed that after 8 h of high-temperature stress. The membrane system components, such as the cell membrane, nuclear membrane, and cytoplasmic membrane vesicles of mature leaf flesh cells of S. pohuashanensis, showed different degrees of wrinkling and the structural integrity of chloroplasts was disrupted (Fig. 9e). The content of starch decreased, the number of lipophilic granules, and vesicles increased (Fig. 9g). This suggests that high temperature stress caused a certain degree of damage to the structure and function of the chloroplasts, affecting the normal conduct of photosynthesis and that stromal vesicles may have played a very important role in response to the high-temperature stress. The results showed that the short duration of heat stress did not cause irreversible damage to the chloroplast structure and function. As a biological function of cell quality control, autophagy removes loss-of-function proteins and damaged cellular components, a homeostatic pathway that is important for the energy balance of plant cells. Recent studies have shown that autophagy is involved in the response of plants to high temperature stress, and impaired autophagy in Arabidopsis and Solanum lycopersicum leads to the accumulation of aggregated proteins, resulting in reduced heat tolerance [52, 53]. The water potential of cells increases under high temperature stress, with water loss from vesicles and wilting of leaves, whereas the number of liposomes increased and autophagy was activated. The main intracellular The main organelle, the chloroplast, was deprived of its structure and photosynthesis was affected.

4.2 Correlation analysis of S. pohuashanensis under high temperature stress.

The transcriptomic results reflect the expression of genes in the samples, whereas the proteomic results show the translation of genes after expression. To understand the relationship between DEGs/DAPs further and detect possible key pathways, co-regulatory analyses of genes/proteins found in S. pohuashanensis were performed. In the co-response network for high temperature stress, more genes were switched on than switched off after exposure to 8 h of high-temperature stress [19]. The results of the co-analysis showed that the pattern of abundance (increase or decrease) of 34 of the 92 identified DAPs correlated with the expression levels (up- or down-regulated) of the corresponding DEGs. Chaperones, stress responses, cell wall modifications and photosynthesis were involved.

The correlation analysis revealed downregulated expression of FKBP53-like and the pan-stress protein gene usp-like in the transcriptome (Supplementary Table S2), increased abundance of FKBP62 and uspA-like and decreased abundance of BAG1 in the proteome (Table 3). PPIase accelerates protein folding. It catalyzes the cis-trans isomerization of the proline imino peptide bond in oligopeptides. It positively regulates thermotolerant co-chaperones by interacting with HSP90.1 and increasing HSFA2-mediated accumulation of small HSP family chaperones [54]. As a histone chaperone, AtFKBP53 binds to 18S rRNA chromatin and negatively regulates its expression level, playing a role in chromatin remodelling and transcriptional regulation [55]. Under high temperature stress, most enzyme activities would decrease, which directly affected the normal metabolism of plant cells, particularly photosynthesis. UGE1 and AKR4C9-like were upregulated at both transcriptional and translational levels, whereas ndhO and AT47H were downregulated at both the transcriptional and translational levels (Tables 5 and 6). UGE1 catalyzes the interconversion between UDP-glucose and UDP-galactose and the interconversion between UDP-arabinose and UDP-xylose [56]. AKR4C9 is an aldehyde reductase that acts on various substrates, reduces ketosteroids, aromatic aldehydes, ketones, sugars and other aliphatic aldehydes, and oxidizes hydroxysteroids, acting as a detoxifying enzyme to reduce the range of toxic aldehydes and ketones produced during stress [57]. ndhO transfers electrons from NAD(P)H via FMN and iron-sulfur centers to quinones in the photosynthetic chain and possibly to quinones in the respiratory chain of the chloroplast. The direct electron acceptor of this enzyme is considered to be a proton quinone, combining redox reactions with proton translocation, and conserving the redox energy in the proton gradient [58]. AT74H is a phosphoglycerate-mutase like protein that lacks PGM activity and may play a role in carbohydrate metabolism [59]. These results indicate that high-temperature stress affects normal photosynthesis in the chloroplasts of S. pohuashanensis.

Toxic effects can occur if ROS are produced too rapidly and exceed the plant's own antioxidant scavenging capacity [60, 61]（尹永强, 2007 #54）. Under high temperature stress, ROS, such as H₂O₂, O₂^—, and ¹O₂, are produced in the chloroplasts and mitochondria of plants, which acquire heat tolerance by activating ROS scavenging systems [62, 63]. Previous studies found a decrease in POD, SOD, and APX enzyme activities in leaves after 8 h of 43℃ stress in S. pohuashanensis; data from DEGs of high temperature stress showed that most of the genes related to ROS scavenging enzymes were down-regulated in expression: for example GSTU17-like, peroxidase P7-like, peroxidase 47, PER47-like , MDAR5, PMI1-like and L-ascorbate oxidase-like; whereas the increased abundance of GSTU17-like in the proteomic results suggests that the ROS scavenging system of S. pohuashanensis is severely affected by high temperature stress when it occurs (Supplementary Table S2 and Table 3). GSTU belongs to the group of GSTs involved in integrating the photosensitive pigment PHYA-mediated photomorphogenesis and various phytohormone signals that regulate various aspects of plant development by affecting the glutathione sink [64]. Due to the effects of high temperature stress, the activity of enzymes such as APXs, PODs, and GSHs in S. pohuashanensis cells is reduced and the ability to scavenge reactive oxygen species is inhibited, leading to an excessive accumulation of reactive oxygen species in the cells, but glutathione may play an exclusive role in the scavenging of ROS in S. pohuashanensis. In addition to the ROS scavenging system, detoxification also plays an important role in the response of S. pohuashanensis to high-temperature stress. Upregulation of CYP71AV8-like expression and reduced abundance of CYP71A1-like in the proteome were found in the high-temperature stress transcriptome of S. pohuashanensis (Supplementary Table S2 and Table 3). Cytochrome P450 is a class of heme-containing multifunctional single-chain proteins with an absorption peak at 450 nm when bound to CO in its reduced state [65]. It is the largest family of enzymatic proteins in plants and plays an important role in plants [66]. The evolution of P450s has been linked to the metabolism of many secondary products with defensive functions in higher plants. When exposed to environmental stress, P450s exert their detoxifying effects while participating in the synthesis of sterols, flavonoids, alkaloids and terpenoids to enhance plant protection [67].

Genes involved in cell wall synthesis, cytoskeleton construction, and regulating of proteins that regulate life activity are all affected when S. pohuashanensis is subjected to high temperature stress. The cell wall plays an important role in cell shape, cell stability and development, and cell protection. It acts as a powerful dynamic barrier that can respond to changes triggered by external stresses [68, 69]. In this study, correlation analysis revealed that cell wall synthesis-related genes TBL1, TBL6, TBL19, TBL38, XTH23 and GRP-like were down-regulated in transcriptome results, and cell wall modification-related genes PME35, PME1-like and CER26 were downregulated; TBL38 abundance decreased and PEM35 abundance increased in proteome results, indicating that the cell wall of S. pohuashanensis was affected by high temperature stress (Supplementary Table S2, Tables 6 and 8). The protein encoding the pectin synthesis process-related gene TBL contributes to the synthesis and deposition of secondary wall cellulose by acting as a bridging protein that binds pectin and other cell wall polysaccharides [70]. The plant cell wall is accompanied by various metabolic and structural adaptations including the formation of cell wall modifications (e.g. cuticle above ground and suberin widely distributed in roots) and possesses a specific phenolic ester-based protection system [71]. PME modifies the cell wall through demethylation of cell wall pectin and may be an important gene involved in early plant developmental events [72]. These results suggest that pectin synthesis in the cell wall of S. pohuashanensis is affected by high-temperature stress, affecting the regeneration of the cell wall. The changes in cell wall modifications have positive implications for the resistance of S. pohuashanensis to high temperatures.

5. Conclusion

In this study, 92 DAPs were identified in the proteomic analysis of S. pohuashanensis under high temperature stress, and co-regulatory analysis revealed 34 DAPs/DEGs in the comparison group, with 24 genes having the same protein and mRNA expression trends and 10 genes with opposite protein and mRNA expression trends. These genes are collectively involved in response to high temperature stress by encoding proteins related to molecular chaperones, energy metabolism, ROS scavenging systems and cell wall modifications.

Abbreviations

AsA-GSH: Ascorbicacid-glutathione; CAT: Abundance of catalase; DAPs: Differential abundant proteins; DEGs: Differential expression genes; FC: Fold change; GO: Gene Ontology; GST: Glutathione S-transferase; HSC: Heat shock cognate protein; HSFs: heat shock factors; PCD: programmed cell death; POD: Peroxidase; Prx/Trx: Peroxiredoxin/Thioredoxin; PS-Ⅰ: photosystem I; PS-Ⅱ: photosystem II; ROS: Reactive oxygen species.

Declarations

Ethics approval and consent to participate

This study including sample collection was conducted according to China's Biodiversity Conservation Strategy and Action Plan (2011-2030) (Index number: 000014672/2010-00714) and Seed Law of the People's Republic of China (2015 Revised Version), which permits use of biological resources to Chinese for scientific research purpose.

Consent to publish

We agree to publish the manuscript in this journal.

Availability of data and material (data transparency)

The proteome data sets used and analyzed during the study are available from the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD027218. The transcriptome data sets used and analyzed during the study are available from the SRA (https://www.ncbi.nlm.nih.gov/sra), and the accession number of this project is PRJNA699178.

Competing interests

The authors declare no conflict of interest.

Funding

National Natural Science Foundation of China (grant number 31770369).

Authors' Contributions

X.P. performed the experiments and bioinformatics analysis, and drafted the manuscript; L.Z. and Y.L. planted the sampling plants and analyzed the data; X.G. guided and analyzed the observation results of TEM. R.Z., Y.Z., and D.D. evaluated the protocol and data. J.Z. critically evaluated the protocol and data, and revised the final version of the manuscript. All of authors read and approved the final manuscript for publication.

Acknowledgements

We would like to thank the National Forest Genetic Resources Platform (NFGRP) for providing the S. pohuashanensis plant resources and the National Natural Science Foundation of China (grant number 31770369) for funding. And we would like to thank Editage (www.editage.cn) for English language editing.

Authors’ Information

¹ School of Landscape Architecture, Beijing University of Agriculture, Beijing, 102206, People’s Republic of China.

² Shandong Provincial Center of Forest and Grass Germplasm Resources, Shandong Province, 250102, People’s Republic of China.

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