Integration of transcriptomic and proteomic analyses of Rhododendron chrysanthum Pall. in response to cold stress in the Changbai Mountains

Cold stress is one of the abiotic stresses that affect plant growth and development, as well as life and geographical distribution important. For researching how plants react to low temperature stress, Rhododendron chrysanthum Pall. (R. chrysanthum) growing in Changbai Mountains of China is an essential study subject. R. chrysanthum was cold-treated at 4 °C for 12 h (cold-stress group-CS, and controls-CK), combined with transcriptomics (RNA-seq) and proteomics (iTRAQ) techniques, to investigate the response mechanisms of R. chrysanthum response to cold stress. Cold stress resulted in the discovery of 12,261 differentially expressed genes (DEGs) and 360 differentially expressed proteins (DEPs). Correlation of proteomic and transcriptome data, proteome regulation of distinct subcellular localization, and gene/protein functional groupings are all part of the investigation. The combined analysis showed that 6378 DEPs matched the corresponding DEGs when the control was compared with the cold-treated samples (CK vs CS). The analysis identified 54 DEGs–DEPs associated with cold stress. cold-tolerant DEGs–DEPs were enriched with hydrolase activity, acting on glycosyl bonds, carbon–oxygen lyase activity and ferric iron binding. Seven potential DEGs–DEPs with significant involvement in the cold stress response were identified by co-expression network analysis. These findings identify the synergistic effect of DEGs–DEPs as the key to improve the cold tolerance of R. chrysanthum and provide a theoretical basis for further studies on its cold resistance subsequently.


Background
Rhododendron chrysanthum Pall. (R. chrysanthum), which grows in the Changbai Mountains of China, is an endemic and endangered species with medicinal and ornamental value that grows in cold, high-altitude environments, making it a good material for studying abiotic stresses on plants [1]. It is one of the only shrubs that can grow above 1000 m in the Changbai Mountains and is one of the ground covers of the Changbai Mountain birch forests and alpine tundra areas [2]. The harsh growing conditions and poor soils of the Changbai summit are a serious challenge to the plant [3]. The R. chrysanthum has evolved resistance to low temperatures and other abiotic stresses over a long period of flexible evolution [4][5][6].
Low temperatures widely affect plant growth and development as well as life and geographic distribution [7]. Plant hardiness refers to a plant's capacity to withstand cold temperatures (0-15 °C) without injury or damage [8]. Comprehensive analysis of maize after cold stress using transcriptome and hormone revealed its adaptive mechanisms to cold stress [9], and a comprehensive physiological and transcriptomic analysis of wild rice after cold stress revealed the adaptive mechanisms of cold tolerance [10]. However, few studies on R. chrysanthum cold stress resistance have been published. Abiotic stress-induced physiological changes in R. chrysanthum plants, including chlorophyll content and antioxidant enzyme activities, have been extensively studied [3,4]. Furthermore, there are few reports on the molecular mechanism of R. chrysanthum resistance to low temperature stress, which might be attributed to the unknown proteome and genome of R. chrysanthum. The results indicated photosynthesis was inhibited under cold stress. At present, our studies on the effects of cold stress in R. chrysanthum have focused on morphological, physiological and molecular changes.
Transcriptome analysis is a powerful tool for identifying genes that may help plants resist abiotic stress [11]. Furthermore, plants have a variety of highly regulated protein networks in growth and development that play an important role in low temperature stress resistance [12]. A combined transcriptomic and proteomic approach was used to provide new insights into the molecular and cellular mechanisms of plant responses to cold stress [13]. Transcriptomic and proteomic analysis of selenate-treated Cardamine violifolia to investigate mechanisms of selenium accumulation and tolerance in Cardamine violifolia [14], phosphorylation level, heat-induced calcium-mediated signaling, ROS homeostasis and endomembrane trafficking, were found to be differentially enriched in spinach following infection with heat stress [15]. Our analysis first examines of protein and transcript abundance, before exploring the primary effects of cold stress on the proteomes and genes. Individual pathways and proteins that are highly affected are studied and discussed. The integration of various technologies enables a better understanding of and response to environmental pressures and the enabling strategies of the mechanisms involved [16]. The present study assessed the interaction and expression relationships between these DEPs and DEGs, and a total of 54 DEGs-DEPs were expressed at different levels during cold stress. This will enable us to understand the molecular mechanisms by which R. chrysanthum responds to cold stress.

Plant materials and treatment
On Changbai Mountain at altitudes between 1300 and 2650 m, we collected Rhododendron chrysanthum Pall. For the experiment, we selected 8-month-old seedlings of essentially uniform growth as the test material [2]. These seedlings are exposed to cold stress treatment for 12 h at 4 °C (CS), which in the normal temperature 12 h at the same time as the control group (CK) (n = 3).

Transcriptomic analysis
Using a TransZol kit, total RNA extraction from finger millet leaf tissue was carried out (TransGenBiotech, Inc., Beijing, China). RNA Library Prep Kit for Illumina1 (NEBNex-t1UltraTM) was used to create sequencing libraries (NEB, Ipswich, MA, USA). In the CK and CS group, there were three biological replicates. cDNA libraries were constructed using the Illumina HiSeq™ X-Ten platform (BGI, Shenzhen, China). Constructed libraries were quality checked with an Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System and sequenced after passing. The clean reads were assembled using Trimmomatic filtering using Tirinity. Fragments per kilobase million were used to quantify the results (FPKM). DEseq2 was used to identify differentially expressed genes (DEGs) based on an absolute fold change value |log 2 FC|≥ 1 and an adjusted P value ≤ 0.01.

Proteome analysis
A high intensity ultrasonic processor (Scientz) was used to sonicate R. chrysanthum leaf tissue samples three times on ice in lysis buffer (8 M urea, 100 mM TEAB, pH 8.0). Three biological replicas from each group were obtained, and the protein content in the supernatant was calculated using a two-dimensional quantification kit (GE Healthcare, USA). Following the TMT kit/iTRAQ kit protocols, the peptides were processed after being reconstituted with 0.5 M TEAB ([10 plex] Ther-moFisher Scientific 90406 Waltham USA). By using tandem mass spectrometry (MS/MS) and an electrospray voltage of 2.0 kV, the resultant peptides were examined. The entire scan was conducted in the 350-1800 m/z range, and an Orbitrap 70,000 resolution was used to identify the intact peptides. By using tandem mass spectrometry (MS/MS) and an electrospray voltage of 2.0 kV, the resultant peptides were examined. The intact peptides were found using an Orbitrap 70,000 resolution during the complete scan, which covered the m/z range of 350 to 1800. The Maxquant search engine was used to process the final MS/MS data. Significantly different proteins were defined as having a fold change > 1.3 and an FDR-corrected p-value 0.05 in comparison to the control.

Data processing and statistical analysis
The agriGO online tool's single enrichment analysis was used to conduct Gene Ontology (GO) enrichment analysis. To look into the high-level uses and functions of the biological system, the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was used. The subcellular localization of proteins was predicted using the online Wolfpsort subcellular localization prediction software. The STRING database was used to retrieve all differentially expressed protein database information or sequences to construct a network of DEGs-DEPs interactions. Cytoscape 3.9.0 was chosen to analyze and visualize the reciprocal network. SPSS 20 (NY, USA) was used for statistical analysis of the data.

Differential photosynthetic performance under cold stress
The photosynthetic performance of Fv/Fm, qP, Fm, and Y(II), were compared to understand how cold stress affected R. chrysanthum. The fluorescence of chlorophyll was measured with an IMAGING PAM (Fig. 1A). Under cold stress, Fv/Fm, Fm, and Y(II) were all shown to be significantly lower(Supple. Table 1). Cold stress reduced the Fv/Fm and Fm content in CS by 19.20% and 4.68%, respectively ( Fig. 1B, C), respectively. Cold stress decreased plant photosynthetic performance, according to these findings.

Transcriptomic analysis of R. chrysanthum leaves under cold stress
Transcriptome analysis of CK and CS group R. chrysanthum leaves using the Illumina HiSeq platform. RNA-seq was performed on different treatment conditions of R. chrysanthum and a total of 206,321 transcripts were received. Transcriptome was compared three times to determine the gene difference (DEG) between CS and CK ((|log2FC|≥ 1, p ≤ 0.01). Among them, there were 6811 and 5450 up-and down-regulated DEGs ( Fig. 2A). After 12 h of cold stress, the transcriptome of R. chrysanthum revealed a total of 5179 GO words, 2056 of which were highly enriched to. The majority of DEGs and DEPs were involved in metabolic processes in response to cold stress by Gene Ontology (GO) enrichment analysis. In the cellular component analysis, the majority of DEGs were assigned to membrane part, organelle and macromolecular, and 45 DEPs were assigned to cell (Supple. Figure 1A, B). For the molecular function analysis, the majority of DEGs were annotated to transporter activity and catalytic activity, and up to a total of 127 DEPs were enriched in catalytic activity (Supple. Figure 1C). Between the comparisons of CS and CK, KEGG pathway enrichment analysis of DEGs revealed 131 similar pathways, and we screened the top 20 enriched KEGG pathways. The darker blue colour indicates a more significant QValue (Fig. 2C). Thus, Plant-pathgen interaction, MAPK signaling pathwayplant and Plant hormone signal transduction deserve to be focused on, as these pathways are significantly enriched and relatively abundant in terms of DEGs, which may be related to the response of R. chrysanthum to cold stress in response to cold stress.

Differential expression of proteins and proteomic analysis of R. chrysanthum leaves under cold stresss
The first TMT-based study to determine differentially expressed proteins in cold-stressed (CS) and control (CK) R. chrysanthum leaves. PCA analysis was carried out on all samples at the proteomic level. While the second OPLS component (PC2) described 22.9% of the data set's variation, the first component (PC1) explained 54.2% of the total variation (Fig. 5c). It showed that the protein expression of R. chrysanthum leaves differed under the two different treatments. At the proteomic level, 175 and 185 DEPs in CS vs CK (Fig. 2B), which could be associated with cold tolerance of R. chrysanthum. CS and CK were subjected to proteomic analysis. DEPs were found in the category of molecular function. The results of subcellular localization study revealed that up-regulation proteins were primarily found in chloroplast (31%) while down-regulation proteins were primarily found in chloroplast (48%) (Fig. 3A, B). KEGG pathway analysis revealed that the Circadian rhythmplant, Nitrogen metabolism, and Ribosome under cold stress DEPs in the CK vs CS group were considerably enriched (Fig. 3D).

Transcriptomic and proteomic interaction analysis to explore the response of R. chrysanthum to cold stress
Comparing the quantitative correlations between the two histologies provides a rapid understanding of the potential regulatory relationships between proteins and transcripts. There were also 360 DEPs and 12,261 DEGs between CK and CS. There are a total of 175 DEPs that are up-regulated and 185 that are down-regulated when CK is compared to CS in Fig. 6a. There are 5450 downregulated DEGs and 6811 upregulated DEGs. With a positive association between DEPs and DEGs in the CK vs CS group, the Pearson correlation between CK vs CS transcripts at both histological levels and their one-to-one corresponding protein expression was examined (r = 0.042) (Fig. 4B).The DEGs and DEPs in CS vs CK were cross-referenced according to each other, comparing the two groups at the academic level. Comparative analysis allowed the classification of proteins or transcripts into eight different types of expression profiles, each of which could correspond to a regulatory relationship (Fig. 4D). To further understand the biological processes in which the proteins or transcripts (The DEGs and DEPs that overlap for correlation analysis are named DEGs-DEPs) with different regulatory relationships are involved, we performed GO and KEGG enrichment analysis on these proteins or transcripts and further analysed them by clustering heat maps (Fig. 4E). GO analysis revealed that DEGs-DEPs in the CK vs CS group were associated with a number of biological processes, including cell wall macromolecule metabolic process, cellular polysaccharide metabolic process, cellular glucan metabolic process and cellular ion homeostasis (Fig. 4C). The enriched cellular components In the KEGG clustering analysis, samples with significantly up-regulated proteins and transcripts after cold stress were significantly enriched in Linoleic acid metabolism, suggesting that R. chrysanthum may respond to cold stress damage through this pathway (Fig. 4E). The overlapping samples with both DEPs and DEGs down-regulated were significantly enriched in Nitrogen metabolism, suggesting that cold stress significantly affected plant nitrogen metabolism, which is consistent with the significant decrease in photosynthetic rate of R. chrysanthum detected by cold stress. Cold stress may alter the ability of R. chrysanthum to absorb and utilize different nitrogen sources, thus causing changes in this metabolic pathway. Interestingly, nine samples with overlapping DEPs up-regulated with DEGs down-regulated and eleven samples with overlapping DEPs down-regulated with DEGs up-regulated were not significantly enriched (Fig. 4E).

Analysis of DEPs and DEGs and PPI networks associated with cold stress tolerance in R. chrysanthum
To better understand the mechanism of cold tolerance in R. chrysanthum, the DEGs and DEPs with the most significant changes were further assessed. A examination of GO and KEGG enrichment analysis revealed 54 DEGs-DEPs to be connected to cold tolerance (Fig. 5A). The protein interaction network of DEGs-DEPs linked to cold stress was predicted using the STRING database, and the co-regulatory interactions between them were shown using Cytoscape 3.9.0. 43 nodes and 46 edges made up the resulting co-regulatory network for cold tolerance (Fig. 5B). Under cold stress, the majority of the nodal genes were strongly expressed, and KEGG in Ribosome enriched the DEPs-DEGs clusters in the dashed circles.

Discussion
Transcriptomic and proteomic analysis have been widely employed in plant studies to identify the molecular pathways of response to abiotic stress [17,18]. The proteomic and transcriptomic approaches were integrated with a biochemical method in our current study to analyze R. chrysanthum at the gene, protein, and molecular levels. These regulated genes and proteins covered various biological functions and were localized in multiple subcellular structures. These data are the first report of an integrated transcriptome and proteome analysis of R. chrysanthum. Low temperature is the main factor restricting the distribution of plants in high latitude and high altitude [7]. However, there are few reports on cold stress research of R.chrysanthum. Abiotic stress causes an excessive buildup of reactive oxygen species (ROS) in alpine tundra [2].
Cold stress causes a variety of plant responses, including signal transduction, substrate metabolism, protein ubiquitination [19]. The data also provide an opportunity to compare protein expression with their mRNA transcripts. Therefore, it is necessary to comprehensively analyze the transcriptome and proteome to determine the mechanism of cold stress response. By comparing CS and CK, we discovered certain genes that may be connected to cold tolerance.In the present work, cold stress significantly reduced the photochemical efficiency of R. chrysanthum leaves, as evidenced by a significant decrease in Fv/Fm versus Fm in CS. In the mean photochemical quenching qP, there was no statistical difference (Fig. 1B, C; Table S1). However, the qP change after cold stress was not significant, which may imply that R. chrysanthum has some strategies to mitigate the damage of photosystem II (PSII) by low  temperature. This result, in agreement with the findings related to PSII inhibition by cold stress [3,20]. The considerable decrease in Fm found in our study suggests that PSII cooperativity was inhibited and that a major portion of the PSII reaction centers was depleted. However, this is the same as the effect of tropical tree species on PSII under cold stress [21]. The functional annotation of transcripts from R. chrysanthum leaf tissue before and after cold stress is thoroughly analyzed in this paper, along with data on DEGs involved in many aspects of the response to cold stress as well as information on KEGG and GO enrichment analysis. Under cold stress, enrichment pathways were mainly associated with Plant hormone signal transduction, photosynthesis, and MAPK signaling pathway (Fig. 2C). In previous studies, similar pathways were found to be present in plants in response to cold stress [19,22]. Analysis of proteomic data identified a number of key protein families involved in the regulation of cold stress response, including the Ferritin-like superfamily and the Glycoside hydrolase superfamily, which enhance cold and stress tolerance in plants by stimulating cell membranes. When the environment changes, the cell membrane is the first to sense the stimulus and its fluidity and stability are both affected [23]. In studies related to the response of rice to low temperatures, the altered distribution of sugars in the cytoplasm under cold stress may have a role in slowing down cell membrane The number of proteins interacting with a specific gene or protein is shown by the size of the circles coagulation, which may be a protective mechanism for cold tolerance in plants [24,25].Sugar signal are often used in conjunction with phytohormone signal in response to abiotic stresses, such as abscisic acid signals in conjunction with sucrose signals in response to cold stress [26]. Studies have shown that glycoside hydrolases can alter membrane integrity by affecting the glycosylation of the OmpA family-like protein PXO 02523 [27].These proteins improve the ability of plants to tolerate cold by stimulating the cell membrane. In addition, proteins associated with cold stress are enriched in Nitrogen metabolism, Ribosome, Circadian rhythm-plant, and cold stress in R. chrysanthum may be associated with the response of these pathways (Fig. 4E). Circadian rhythm regulation helps plants to better photosynthesize nutrients for growth and promotes the opening and closing of leaf stomata, allowing leaves to curl at night to prevent water loss [28].
Co-regulation analysis was carried out on the identified DEGs and DEPs in order to comprehend the relationship between DEGs-DEPs. In the cold stress response pathway, the cold stress-related protein interaction network found 54 DEGs-DEPs, and seven of them were enriched in ribosome processes with subcellular localization in the chloroplast stroma (Fig. 5). In conclusion, we provided transcriptomic and proteomic data on the response of R. chrysanthum to cold stress and performed an integrative analysis. Under cold stress, more DEGs were up-regulated than down-regulated in association with cold stress. Comparison of the transcriptome with the proteome showed that DEGs-DEPs were enriched in linoleic acid metabolism, dioxygenase activity and cell wall macromolecule metabolic process after cold stress. The majority of DEGs-DEPs were significantly upregulated, suggesting that R. chrysanthum may grow at high altitude and high latitude in the Changbai Mountains through these pathways. The response mechanism of R. chrysanthum to cold stress is thus linked to these key transcriptional regulators and proteins. Through further studies, these key cold stress response genes could serve as an important reference for functional analysis and mechanisms of resistance to abiotic stress in plants such as R. chrysanthum.

Conclusions
Combined analysis of transcriptomic and proteomic data showed that 6378 DEPs matched the corresponding DEGs when control was compared with samples treated with cold stress for 12 h (CK vs CS). A total of 54 DEGs-DEPs co-regulated by analysis were identified to be involved in R. chrysanthum response to cold stress. The DEGs-DEPs associated with cold stress tolerance were involved in important pathways such as Circadian rhythm and Nitrogen metabolism. Co-expression network analysis revealed seven key DEGs-DEPs that were found to play important roles in cold stress response. These results suggest that the synergistic action of DEGs-DEPs is crucial for improving cold tolerance in plants.