TMT-Based Proteomic Proling Reveals the Molecular Mechanism in the Cold Tolerance of Rhododendron Aureum Georgi.

Background: Cold stress is one of the important factors that restrict plant growth and regional distribution. Under cold acclimation conditions, plants growing in temperate zones reprogram the gene expression in the cells, making the plants better cope with the coming cold stress. However, under natural environmental conditions, the climate is complex and changeable. The sudden large temperature drop will bring serious disasters to plants. Rhododendron aureum Georgi, as an evergreen plant growing in high altitude areas of Changbai Mountain, the harsh ecological environment may endow it with different cold tolerance characteristics. Results: In this study, the proteomic difference between samples under control and cold stress were compared pairwise. A total of 360 DAPs were identied, of which 175 were down-regulated and 185 were up-regulated when comparing these two sets of data. The high cold tolerance of Rhododendron plants can be attributed to: accumulation of chaperone proteins; the up-regulation of components related to translation; enhancement of catabolism and reduction in anabolism, provide energy for plants and ght against cold stress; enhanced cellular antioxidant capacity; modication of components in cell wall, membrane, and cytoskeleton. Conclusion: These results provide an in-depth understanding of the cold tolerance mechanism of Rhododendron. The identied genes and metabolic pathways provide a certain reference for the genetic improvement of plant cold tolerance.

phosphorylation.). An equal volume of Tris-saturated phenol (pH 8.0) was added. Then, the mixture was further vortexed for 5 min. After centrifugation (4 °C, 10 min, 5 000g), the upper phenol phase was transferred to a new centrifuge tube. Proteins were precipitated by adding at least four volumes of ammonium sulfate-saturated methanol and incubated at -20 °C for at least 12 h. After centrifugation at 4 °C for 10 min, the supernatant was discarded. The remaining precipitate was washed with ice-cold methanol, followed by ice-cold acetone for three times. The protein was redissolved in 8 M urea and the protein concentration was determined with BCA kit according to the manufacturer's instructions[6].

Trypsin Digestion, TMT labeling, and HPLC fraction
Dithiothreitol was added to each protein sample to reach a nal concentration of 5 mM, and the reduction reaction was performed under conditions of 56 °C for 30 minutes. Then adding iodoacetamide to make the nal concentration of 11 mM, and incubate for 15 minutes at room temperature in darkness. The concentration of urea in protein sample was diluted to less than 2 M by adding 100 mM TEAB for digestion with trypsin. Protein sample was digested with trypsin at a ratio of 1:50 overnight at 37 °C and then adding new trypsin at a ratio of 1:100 to continue enzymatic hydrolysis for another 4 hours. The peptide mixture was desalted by Strata X C18 SPE column (Phenomenex) and labelled with TMT reagent. The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length). Brie y, peptides were rst separated with a gradient of 8 % to 32 % acetonitrile (pH 9.0) over 60 minutes into 60 fractions. Then, the peptides were combined into 6 fractions and dried by vacuum centrifuging [7].

LC-MS/MS Analysis
The tryptic peptides were dissolved in buffer A (0.1 % formic acid and 2 % acetonitrile), directly loaded onto a reversed-phase analytical column and separated with a linear gradient of buffer B (0.1 % formic acid and 90 % acetonitrile) at a flow rate of 500 nL/min on an EASY-nLC 1000 UPLC system: held in 6-23 % buffer B for 26 minutes, 23-35 % buffer B for 8 minutes, 35-80 % buffer B for 3 minutes and 80 % buffer B for 3 minutes.
The peptides are separated by the ultra-high performance liquid system and injected into the NSI ion source for ionization and then analyzed by Q Exactive mass spectrometry. The ion source voltage was set to 2.0 kV, and the peptide precursor ions and their secondary fragments were detected and analyzed by highresolution Orbitrap. The scanning range of the primary mass spectrum is set to 350-1800 m/z, and the scanning resolution is set to 70,000; the scanning range of the secondary mass spectrum is set to a xed starting point of 100 m/z, and the secondary scanning resolution is set to 17,500. The data acquisition mode uses the data-dependent scanning (DDA) program, that is, the rst 20 peptide precursor ions with the highest signal intensity are selected to enter the HCD collision cell sequentially using 28 % and 31 % of the fragmentation energy for fragmentation[8].

Database search
The raw data were searched against Rhododendron_lapponicum_313330 protein database (45945 sequences) using Maxquant (v1.5.2.8). The parameter setting method of protein identi cation is in accordance with Zhou's method [5].

Identi cation of DAPs
To get an overview of the protein changes of Rhododendron under low temperature, we conducted a proteomic analysis of Rhododendron leaves before and after cold stress. DAPs are the key players that we are most concerned about that can be used to clarify the cold-resistant performance of Rhododendron plants. When the fold change ratio of the protein species before and after the cold treatment is greater than 1.2 or less than 0.833 and the p value is less than 0.05, we de ne such proteins as up-regulated proteins or down-regulated proteins. Using this cut-off point standard, a total of 360 DAPs were identi ed, of which 175 were down-regulated and 185 were up-regulated under cold stress versus room temperature growth.

Classi cation of DAPs
Gene Ontology (GO) is an important bioinformatics analysis method used to characterize various attributes of genes and gene products. GO annotations are divided into 3 categories: Biological Process, Cellular Component and Molecular Function, explaining the biological role of proteins from different perspectives.
We have made statistics on the distribution of DAPs in GO secondary annotations. A total of 179 DAPs are enriched in biological processes, where metabolic processes account for the largest proportion, followed by single-organism process categories and cellular process categories. Among the cell components, the membrane ranks rst in terms of the number of DAPs, the other components identi ed correspond to cells, macromolecular protein complexes, organelles, and extracellular regions. Catalytic activity is the most representative category of molecular functions, accounting for 50 % of this category. The results of GO data suggest that cold exposure mainly affects the metabolic activities, intracellular material transport and signal transduction in Rhododendron plants. 360 DAPs are assigned to 21 categories according to the Clusters of Orthologous Groups (COG) database. The main functional categories include General function prediction only (17.24%), posttranslational modi cation, protein turnover, chaperones (10.78 %), Energy production and conversion (9.91 %), Translation, ribosomal structure and biogenesis (8.62 %), Carbohydrate transport and metabolism (8.19 %), Inorganic ion transport and metabolism (6.9 %), Intracellular tra cking, secretion, and vesicular transport (6.03 %), Signal transduction mechanisms (6.03 %), and amino acid transport and metabolism (5.17 %) (Fig. 1).

Correlation analysis of DAPs and DEGs
Proteomics provides some clues on translation and post-translational levels in response to cold stress. However, the integration of transcriptome and proteome data can show a global picture of gene expression regulation in Rhododendron plants. In this study, we performed a correlation analysis on the results of the transcriptome and proteome. To identify those DEGs and DAPs that respond to cold stress, we used the following criteria as a threshold for screening (|log 2 FC|>1, p value <0.001 for DEGs; Proteins with FC value >1.2 or <0.83 and P value <0.05 are regarded as signi cant DAPs). A total of 6378 genes had corresponding proteins in the transcriptome, we have got a very poor positive correlation between the changes in the transcript level and the protein level for DAPs (Pearson correlation coe cient r = 0.042). More DEGs (n=587) and DAPs (n=306) are not correlated. 54 genes are shared between the transcriptome and proteome, among which 34 genes have similar expression trend, and the remaining 20 genes have opposite expression trends, which implies that complicated regulation process exist from transcription to translation when Rhododendron plants suffer from cold stress (Fig. 2). For those genes whose transcription is down-regulated but the translation level is up-regulated, it means that these genes may enter the translation machinery more e ciently to synthesize protein products during periods of cold stress. To obtain the metabolic pathways most closely related to cold stress, we performed KEGG enrichment analysis on the data in the correlation analysis, and the results are shown in Fig. 3 We observed that these DAPs were enriched in 9 pathways, including circadian rhythm; oxidative phosphorylation; cyanoamino acid metabolism; ascorbate and aldarate metabolism; SNARE interactions in vesicular transport; nitrogen metabolism; amino sugar and nucleotide sugar metabolism; avonoid biosynthesis; phenylpropanoid biosynthesis. These results indicate that the proteins involved in various metabolic pathways, photoperiod and vesicle transport respond to cold stress.

Discussion
DAPs involved in Posttranslational modi cation, protein turnover, chaperones Low temperature stress increases the risk of protein unfolding, misfolding, degradation and oxidation. The accumulation of these abnormal proteins in the cell can have harmful consequences for the cell. Therefore, increasing the abundance of proteins with protective functions is necessary to protect proteins from damage [9]. Correspondingly, in our proteomics experimental results, we observed that the abundance of some proteins such as molecular chaperones and heat shock proteins increased under low temperature stress. The obvious feature of chloroplast photosystem II is that it is particularly susceptible to photooxidative damage during cold stress. DnaJ acts as molecular chaperone that plays essential role in contributing to maintenance of photosystem II. In our current research, the up-regulated levels of DnaJ may provide protection for the photosystem II under cold stress environments. DnaJ has been reported to have an interaction with HSP70. Consistent with the up-regulation of DnaJ, the abundance of HSP70 also appeared to accumulate after Rhododendron was exposed to cold stress [10]. Therefore, here DnaJ and HSP70 may form a complex to work together. This interaction will prompt HSP70 to hydrolyze ATP to recruit other client proteins to perform functional diversi cation. Obviously, this combined machine will be more e cient and have broader functions. In addition to HSP70, other chaperone-related proteins such as Chaperonin 60 and 10 kDa chaperonin 1 (CPN10-1) have also been shown to accumulate in response to cold stress [11]. These two proteins together with Cpn20 that form a complex are required for the fold of the RuBisCo small subunit after they translocate from cytoplasm to chloroplast. The cpn60α1 knockdown mutant exhibits yellow leaf phenotypes, dwar ng, and chloroplast collapse that disrupt both photosynthesis and photorespiration [12]. In addition to being recognized as aiding the folding of proteins in the chloroplast, other roles of Chaperonin 60 and its co-molecular chaperones in cold stress still need to be further explored. Protein degradation mediated by protease and ubiquitin modi cation system plays an important role in the process of plants coping with cold stress. In our study, the abundance of 26S proteasome non-ATPase regulatory subunit 13 (RPN9B), serine protease EDA2, and Aspartic proteinase A1 identi ed as being involved in protein degradation were all down-regulated under cold stress. It has been revealed that Aspartic proteinase A1 is involved in biotic and abiotic stress response, reproductive development and chloroplast metabolism, while, to our best knowledge the report on the functional analysis of the other two proteins in plant abiotic stress has not been discovered. As we know, similar to drought stress, cold stress can also bring about the imbalance of plant water status. As an important hub gene for ubiquitin mediated proteolysis pathway, SUMO-activating enzyme subunit 2 (SEA2) plays an important role in avoiding excessive water loss during drought stress [13]. In our study, cold stress also induced the accumulation of SEA2, and the up-regulation of this protein may play a positive role in the adaptation of Rhododendron to severe cold (Table 1).

DAPs involved in translation, ribosomal structure and biogenesis
In eukaryotic cells, the nucleus and ribosomes are considered to be the central hub for integrating stress responses. The protein synthesis process includes four stages: initiation, extension, termination, and ribosomal cycle, among which translation initiation is the main regulatory step of protein synthesis [14]. So far, there is not much direct evidence that ribosomal proteins (RP) are involved in cold stress. Tronchoni et al. found that the improvement of translation e ciency is an important means for S. kudriavzevii strain to adapt to low temperature environment [15]. Rogalski  soybeans found that overexpression of sense and antisense ribosomal protein L34-like gene in transgenic plants showed characteristics of cold sensitivity and cold resistance, respectively, indicating that SOL34 plays a negative regulatory role in the metabolic process that adapts to low temperature during seed imbibition [17]. However, most evidence pointed to the up-regulation of RP accumulation under cold condition. In our study, we also noticed that 6 plastic ribosomal proteins (RPS) and 7 RP were up-regulated after cold stress ( Fig. 4) (Table 1)

DAPs involved in intracellular tra cking
For eukaryotes, vesicle transport not only plays a role in plant growth and development by maintaining the speci city and integrity of the compartment, but also plays a role in the response to abiotic and biotic stress, although the latter studies are relatively small, but it has been supported by increasing evidence. The main internal membrane transport pathways in plant cells include secretory and endocytic pathways [19]. The transport of protein cargo from one organelle to another is mediated by vesicle transport, it has been demonstrated that cold stress affects the intracellular protein transport pathway to varying degrees. The SYP51/SYP52 of the SYP5 family is located on PVC or tonoplast, and forms a complex with other SNARE proteins, including VAMP722, SYP22, and VTI11, which is critical for protein transport and PSV formation [20]. Our proteomics results showed that the abundance of SYP51, SYP52, and SYP22 were downregulated, which implies that the tra cking destined to the vacuole may be affected. Exposure to high salt or high osmotic pressure conditions causes excessive accumulation of intracellular ROS, which is carried by vesicles into vacuoles. Suppressing the expression of AtVAMP7C gene makes these vesicles unable to fuse with the tonoplast and stay in the cytoplasm, which can maintain the function of the vacuole [21]. In our research, cold acclimation may lead to an increase in ROS accumulated in cells and a decrease in the abundance of SNARE protein, which reminds us whether Rhododendron plants alleviate oxidative stress in the same way to preserve the function of tonoplast. FAB1A/B function as PtdIns 3,5-kinase to catalyze PtdIns 3-P to produce PtdIns (3,5) P2, which is necessary for maintaining endomembrane homeostasis [22]. Increased FAB1A/B abundance was detected in the cold acclimation of Rhododendron. The above results let us speculate that Rhododendron actively preserve the homeostasis of the endometrial system during cold acclimation. Annexins are an evolutionarily conserved multi-gene family that relies on Ca 2+ to bind the negatively charged phospholipids on the membrane. In addition to Ca 2+ binding properties, other protein domains also endow it with various other functions including membrane tra c, cytoskeletal responses, ion transport, and stress responses [23]. Regarding the performance of Annexins in stress response, the existing conclusions are not completely consistent, and many of their functions are inferred from their expression in stress. In Arabidopsis, the two genes encoding Annexins (AtANN1 and AtANN4) are regulated by abiotic stress [24]. Single mutant annAt1 and annAt4 plants both show drought and salt tolerance, while their overexpression plants show Stress-sensitive characteristics [24]. In wheat, TaANN3 was induced by cold stress for 1h. In a time-course experiment, TaANN3 decline to a level lower than the initial expression 24h after treatment with cold stress [25]. However, Li et al. discovered that OsANN3 was induced by drought and ABA in rice, and overexpression plants showed better drought resistance [26]. In our research, the abundance of Annexin3 has been down-regulated (Table 1). Therefore, the exact role of Annexins in plant cold stress response should be further investigated according to plant species and cold stress treatment conditions.

DAPs involved in carbohydrate transport and metabolism
In higher plants, NADPH is mainly synthesized through two pathways. One is produced by photosynthetic machinery through the photosynthetic electron transport chain. The other is produced by the pentose phosphate pathway. Glucose 6-phosphate dehydrogenase is the key enzyme that oxidizes glucose 6phosphate to NADPH. But under light, this pathway in photosynthetic tissue is inhibited by the reduction of F-type thiooxidized protein [27]. In our study, we noticed that the abundance of glucose 6 phosphate dehydrogenase increases during cold stress. In addition, the abundance of F-type thioredoxin also increased. The glucose 6 phosphate dehydrogenase of Chlorella was transferred to Saccharomyces cerevisiae. Although the increase in the abundance of glucose 6 phosphate dehydrogenase did not increase the enzyme activity, it could effectively alleviate the oxidative stress caused by freezing damage [28]. The second enzyme of the pentose phosphate pathway, 6-phosphogluconolactonase, also increased its protein abundance when subjected to cold stress, which indicated that the pentose phosphate pathway of Rhododendron plants was activated after being induced by cold stress. The pentose phosphate pathway not only provides the reducing equivalent of NADPH and the necessary carbon skeleton for plants, but is also related to the assimilation of nitrogen [29]. The pentose phosphate pathway provides NADPH for NIR. In our study, the expression of NIR was also up-regulated after being induced by cold stress. Reduced carbon ows into the pentose phosphate pathway and leaves assimilate nitrite ions to produce organic molecules, which help plants maintain vitality. Glucose-6-phosphate, as the interface carbon source of glycolysis and pentose phosphate pathway, can be catalyzed by glucose 6-phosphate dehydrogenase to generate 6phosphogluconic acid to enter the pentose phosphate pathway, or it can be converted to Fructose 1,6bisphosphate under the action of phosphohexose isomerase and phosphofructokinases to enters the glycolytic pathway [30]. The accumulation of glucose 6-phosphate dehydrogenase and the decrease in abundance of phosphofructokinase in our study suggest that the carbon ow shifts from glycolysis in the catabolic pathway to the anabolic pentose phosphate pathway. Adversity environments often compromise photosynthesis of plants, and the sugar concentration will be reduced to the level of "sugar de cit", which triggers a sugar starvation response in plants. If the duration of the stress is prolonged, the plant will be depleted of sugar. Therefore, in most cases, people generally accept the conclusion that cold stress triggers starch degradation [31]. In our research, we found that the abundance of Alpha-amylase related to starch hydrolysis increased under cold stress (Table 1). This implies that the cold stress environment may make Rhododendron plants in a state of sugar de cit, requiring the conversion of starch to sugar to respond adaptively to stress.

DAPs involved in amino acid transport and metabolism
In addition to being a building block for protein synthesis, amino acids are also involved in plant physiological processes [32]. Some differentially expressed proteins related to amino acid metabolism were identi ed after Rhododendron was exposed to cold stress. Branched-chain amino acid aminotransferase 2 and Methionine gamma-lyase showed a tendency of up-regulation after cold induction. This result indicates that the anabolism of branched amino acids is positively regulated in response to cold signals compared to controls. The precursor molecule of isoleucine synthesis is α-ketobutyric acid which can be generated either from the decomposition of methionine catalyzed by methionine gamma-lyase or the deamination of threonine. The last four steps of branched amino acid (isoleucine and valine) synthesis share the same enzymes, and Branched-chain amino acid aminotransferase 2 is responsible for catalyzing the nal step of this pathway [33]. Therefore, the up-regulation of these two enzymes implies an increase in the accumulation of branched amino acids following cold treatment. It is reported that the branch amino acid content in potato leaves increases more than the proline content after drought stress. Isoleucine and valine can also be served as osmotic regulators to deal with osmotic stress, although they are not as familiar as proline [34]. In addition, it has been proposed that the accumulation of free branched amino acids can be used as a substrate for stress-induced proteins or as a signal molecule to regulate gene expression. Therefore, we suggested that the two enzymes related to branch amino acid synthesis induced by low temperature stress play a positive regulatory role in the process of Rhododendron cold resistance. The reactive oxygen species (ROS) brought about by oxidative stress is the main factors affecting plant tness and reproduction when plants encounter cold stress. Plants evolved a powerful defense system including antioxidant enzymes and antioxidant compounds to reduce or eliminate ROS. Tocopherols and avonoids are such antioxidants. Tocopherol is a class of lipophilic compounds mainly found in plastids, and its main function is to protect the chloroplast membrane from oxidative stress. Overexpression of 4-Hydroxyphenylpyruvate dioxygenase (HPPD) in Arabidopsis resulted in an up-regulation of the tocopherol content in the leaves by 37% [35]. In our study, the abundance of HPPD also showed a signi cant upregulation after the cold stress treatment. As an important category of avonoids, anthocyanins not only provide colorful colors to plants, but are also important antioxidants. The supply of phenylalanine is one of the factors affecting the biosynthesis of anthocyanins. Arogenate dehydratases are the key enzymes that catalyze the formation of phenylalanine from arogenate [36]. Genetic analysis showed that arogenate dehydratases2 (ADT2) contributed the most to the promotion of anthocyanin synthesis among the six isoenzymes [36]. Moreover, we noticed that the abundance of 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHD-SDH), a key enzyme in the synthetic pathway of aromatic amino acids, also increased signi cantly after the Rhododendron was exposed to cold stress. The up-regulated expression of these three enzymes means that Rhododendron shifts the metabolic ux from the synthesis of aromatic amino acids to the production of antioxidant substances under cold stress. We cannot determine the role of up-regulated expression of HISN2 in cold stress. Energetically, it has been proved that the synthesis of histidine requires a high metabolic cost, which is obviously unsuitable for Rhododendron under cold stress. More importantly, the overexpression of HISN2 has no effect on the level of histidine [37,38]. Therefore, we speculate that the elevated accumulation of HISN2 is not to increase the abundance of histidine, but to synthesize metabolic intermediates and divert the metabolic ow to the corresponding pathway. It is known that asparagine plays an important role in nitrogen storage and long-distance transportation of nitrogen. The level of asparagine synthase is closely related to the amount of free asparagine [39]. In our study, we observed that asparagine synthase accumulates to high levels during cold stress. The up-regulation of asparagine synthase may be related to the turnover of protein during cold stress, which produces more ammonia that is toxic to living cells (Table 1). In addition to the detoxi cation of ammonia and the carrier of nitrogen, the relationship between increased abundance of asparagine synthase and cold stress still needs further exploration.

DAPs involved in energy production and conversion
It is reported that the components of complex I, II and III in the respiratory chain are signi cantly downregulated under cold stress [40]. In our study, we also observed decrease in the abundance of Cytochrome c1, which led us to speculate that this may be related to the disruption of the metabolism or the reduction of the reducing equivalents during cold stress. In addition, we found that Iron-sulfur cluster assembly protein 1 (ISU1) is also sensitive to cold stress, and its abundance is down-regulated compared to the control. However, the abundance of Cytochrome c has been up-regulated, which may be related to the functional diversi cation of this protein. In addition to the classic role as electron transport carrier, Cytochrome c also participates in the synthesis of ascorbic acid and the stabilization of the electron transport chain. Moreover, it is also the target of cyanide attacking the electron transport chain, and electrons cannot be transferred to molecular oxygen via complex III. Under normal conditions, cyanide is combined into non-toxic glycosides and stored in vacuoles, while glycosidases are localized in the cytoplasm. The vacuole membrane under environmental stress is damaged; glycosidase directly hydrolyzes the glycosidic bond and releases hydrogen cyanide, causing damage to plants. β-cyanoalanine synthase is directly involved in the detoxi cation of cyanide, concentrating cyanide and cysteine to synthesize cyanoalanine [41]. In our study, we observed increased abundance of β-cyanoalanine synthase under cold stress. The fuel required for oxidative phosphorylation comes from the reducing equivalent produced by metabolic pathways such as the tricarboxylic acid (TCA) cycle and β-oxidation pathway [42]. Glyoxylate cycle involves the concentration of acetyl-CoA and oxaloacetate catalyzed by citrate synthase to produce citric acid and coenzyme A. The up-regulation of citrate synthase 2, a key enzyme in glyoxylate cycle, indicates that more acetyl groups may enter TCA cycle from peroxisome. The substrate of the glyoxylate cycle comes from the beta oxidation of fatty acids in the peroxisome. Evidence suggests that acetyl-CoA oxidase 3 is responsible for catalyzing the rst step of β-oxidation of medium-length fatty acids in peroxisome [43]. In our study, the enhanced accumulation of acetyl-CoA oxidase 3 implies that the mobilized lipids may be oxidized to meet the substrate requirements of the glyoxylate cycle. Furthermore, the abundance of acylcarnitine carriers (BOU) related to acetyl or acyl transport also increased after being induced by cold stress. This indicates that in addition to the glyoxylate pathway, alternative pathway, the BOU pathway, which also participates in providing metabolic substrates to the TCA cycle. 2-oxoglutarate dehydrogenase complex is responsible for catalyzing the second oxidative decarboxylation reaction in the TCA cycle. In our study, the level of dihydrolipoyl succinyl transferase, E2 subunit of 2-oxoglutarate dehydrogenase (OGDHC) complex1, was down-regulated, suggesting that this enzyme is sensitive to cold stress ( Table 1). The combined effect of the instability of OGDHC and the increased in ux of acetyl groups into the TCA cycle may cause the accumulation of 2-oxoglutarate. As an important organic molecule in the cell, 2-oxoglutarate plays regulatory role at least in three aspects: (i) carbon skeleton for nitrogen assimilation; (ii) modulation in amino acid metabolic network; (iii) regulation in carbon-nitrogen interaction. The inhibition of 2-OGDHC in potato, via chemical inhibitors, culminated with reduction of the intermediate of the TCA cycle except for succinic acid and down-regulation of amino acids related to nitrogen assimilation. Our results may differ from the above conclusions: (i) The acetyl group derived from β oxidation feeds the TCA cycle, allowing metabolism to ow from citrate synthase to isocitrate dehydrogenase; (ii) although not signi cant, the down-regulated glutamate decarboxylase (required for the GABA pathway) may not be able to supplement succinate. The lack of concerted up-regulation of glutamine synthase and glutamate synthase makes us uncertain that the carbon skeleton of 2-oxoglutarate will switch to nitrogen assimilation. Therefore, we cannot determine which metabolic pathway the accumulated 2-oxoglutarate will lead to with our existing data.

DAPs involved in antioxidation, inorganic ion transport and metabolism
Superoxide dismutase (SOD) converts O 2into H 2 O 2 and O 2 through disproportionation. It is the rst line of defense for antioxidant enzymes against reactive oxygen species. According to the metal cofactors in the active center, SOD in plants is divided into three categories: Copper/zinc SOD (CuZnSOD), manganese SOD (MnSOD), and iron SOD (FeSOD). CuZnSODs are localized in cytoplasm, chloroplast and peroxisome. MnSODs are found in the chloroplast. FeSODs are mainly located in chloroplasts, and parts of them are presented in peroxisomes and apoplasts. Proteomic results revealed that CuZnSODs showed increased abundance after cold stress treatment in wheat leaves, while opposite conclusion was obtained in rice under such treatment. In our study, among the three types of SOD, only the abundance of CuZnSODs changed signi cantly and showed up-regulation under cold stress. CuZnSOD is a homodimer enzyme containing copper and zinc. Copper is a key cofactor necessary for enzymes to perform catalytic functions [44]. Copper chaperones are involved in the tra cing of coppers and release them to copper-containing proteins. Those proteins that insert coppers into CuZnSODs are called Cu chaperone of SOD (CCS) [45]. In our study, we observed a concerted up-regulation of CCS after cold stress treatment. In addition to copper ions, another metal ion, iron, is also interesting because it participates in the electron transport chain. Ferritin is an iron storage protein synthesized in the cytoplasm and transported to mitochondria or chloroplasts. Mutants devoid of ferritin did not show obvious growth and development defects under normal conditions, but is sensitive to oxidative stress caused by methylviologen. Transgenic mutants overexpressing alfalfa ferredoxin showed resistance to photoinhibition induced by low temperature stress [46]. In our study, we observed increased abundance of Ferritin-1 and Ferritin-3 after plants were exposed to cold stress (Table 1).
This indicates that ferritin is required to relieve the oxidative stress caused by cold stress to maintain the redox homeostasis in Rhododendron plants. As the main soluble antioxidant substance in plants, Ascorbic acid (ASA) plays a vital role in detoxifying ROS produced by photosynthesis, respiration and abiotic stress. In the process of ASA biosynthesis, D-galacturonate reductase catalyzes the reduction of D-galacturonic acid to L-galactonic acid. L-galactonic acid is then converted to L-galactono-1,4-lactone, and is further oxidized to produce ASA. It has been shown that heterologous overexpression of strawberry GalUR, the content of ASA increases to the original 2 to 3 times in Arabidopsis [47]. In tomato overexpression lines, although the content of ASA only increased moderately, the transgenic lines showed the characteristics of photoprotection against photooxidation [47]. Therefore, we believe that D-galacturonate reductase plays a positive role in enhancing the cold tolerance of Rhododendrons.

DAPs involved in cytoskeleton
It is well known that when plants encounter cold stress, they are often accompanied by osmotic stress. Actin-depolymerizing factor 2 was identi ed as an up-regulation of abundance after cold exposure, indicating that this protein may be required in response to osmotic stress. In fact, the correlation between the regulation of potassium channels in guard cells and osmotic stress has long been reported. During the period of cold stress, the actin laments depolymerization caused by osmotic stress further strengthens the in ux of potassium ions in the guard cells [48]. Therefore, the up-regulation of Actin-depolymerizing factor 2 abundance here can be regarded as sensors of osmotic stress protect cells from excessive water loss. Another protein (Tubulin β-2) related to the cytoskeleton also accumulated after being induced by cold stress. This accumulation may come from the biosynthesis of the protein or the disassembly of microtubule brils. The assumption of microtubule depolymerization is consistent with the consensus that cold stress induces a decrease in membrane uidity, accompanied by calcium ion in ux and microtubule depolymerization [49]. In addition to microtubules and micro laments, phospholipase D is also an important part of the cold signaling [50]. In our study, the abundance of PLD-α1 was down-regulated when Rhododendron was exposed to cold stress ( Table 1). As we know that PLD-α1 can aggravate the damage of freezing stress to plants, the reduction of PLD-α1 abundance can be seen as a manifestation of Rhododendron plants actively responding to cold stress and reducing freezing damage.

DAPs involved in cell wall, aquaporins, and H + -ATPase
The cell wall protects plants from environmental stress, provides structural support and acts as a barrier to diffusion. UDP-xylose is a direct donor for the synthesis of cell wall polysaccharides xylose and xylan [51].
Our results indicate that the enzymes UDP-glucuronic acid decarboxylase (UXS), UXS2 and UXS6, involved in the synthesis of UDP-xylose, were down-regulated after the Rhododendron plants were subjected to cold stress. However, in view of the irregular xylose structure in the uxs3 xus5 uxs6 triple mutants and no obvious phenotype in all 6 single uxs, we cannot determine the effect of down-regulation of UXS2 and UXS6 on the xylose content in the cell wall [52]. Ferulic acid extensively dimerizes, facilitating formation of cross-links between cell wall polysaccharides, and contributes to the recalcitrance of cell wall [53]. In this study, the abundance of Aldehyde dehydrogenase family 2 member C4 (ALDH2C4), the enzyme responsible for oxidizing coniferaldehyde to ferulic acid, was down-regulated. Another protein involved in the regulation of cell wall polymer is Secretory Carrier-Associated Membrane Proteins (SCAMP), which may in uence the composition of the cell wall by nely regulating the level of cell wall precursors and the secretion of proteins participated in cell wall synthesis and transport. It has been reported that PttSCAMP3 knockdown mutants increase the accumulation of carbohydrates and phenolics on the secondary cell wall [54]. Therefore, the down-accumulation of SCAMP3 in this study may play an important role in the cold tolerance of Rhododendron plants by changing the composition of the cell wall. We assume that the altered cell wall composition may be an adjustment to cold stress, and perception of cold stress triggers the cold response of plants. The cell membrane plays a very important role in the interaction between the cell and the environment. For example, it can act as thermo sensors, permeable barrier, and the boundary of a cell. Many functions of the cell membrane are completed with the participation of membrane-bound proteins [55].
Aquaporin and plasma membrane H + -ATPase, as important proteins on the plasma membrane, play an important role in regulating the entry and exit of various materials. In addition to transporting water, aquaporins can also transport neutral solutes and ammonia [56]. The response of aquaporin to abiotic stress has been widely reported in the literature. For example, transgenic plants overexpressing wheat TaAQP7 (PIP2) show cold tolerance [57]. However, opposite pattern was revealed in our research. All the DEPs, PIP1-2, PIP1-3, PIP2-4, PIP2-1, and PIP2-8 related to the water channel were down-regulated after being subjected to cold stress. We speculate that this is related to the growth environment of the Rhododendron. Cold memory allows plants to reduce water into the apoplast to avoid the formation of ice crystals in the apoplast when freezing stress comes, causing further dehydration of the plant and physical damage to the plasma membrane. The plasma membrane H + -ATPase is responsible for the active transport of cations. This process is accompanied by the hydrolysis of ATP and the e ux of protons. In addition, H + -ATPase is also involved in other physiological processes such as salt resistance and pH adjustment. The results of the time course experiment showed that the amount of H + -ATPase increased by the induction of cold stress [58]. However, in our study, the abundance of plasma membrane ATPase 4 and 10 both decreased after being subjected to cold stress (Table 1). For the opposite result that appeared in the experiment, we cannot give further explanation for the time being.

Conclusion
In this study, we made a comparative analysis of Rhododendron plants under normal temperature and cold stress according to their proteomic response. Our results indicate that Rhododendron plants have divergent cold stress tolerance mechanisms, which can be summarized as follows: (a) Accumulation of chaperone proteins like DnaJ, HSP70, and Chaperonin 60 in order to stabilize the protein and facilitate protein folding, and protect the protein from cold stress-induced denaturation; (b) The up-regulation of components related to translation, such as ribosomal proteins, Translation initiation factor, and translation initiation factor, may be crucial to the cold tolerance of rhododendrons; (c) Enhancement of carbohydrate and lipid metabolism and reduction in anabolism, provide energy for plants and ght against cold stress; (d) Change the metabolic pathway of aromatic amino acids towards the synthesis of antioxidants, such as tocopherol, and synthesize other antioxidants-related substances such as ascorbic acid and SOD to detoxify the attack of ROS; (e) Modi cation of components in cell wall, membrane, and cytoskeleton, contributing to the transmission of stress signals and adjusts the water state in the cell. According to the proteomics data, the response of Rhododendron to cold stress can be summarized in Fig. 5 and Fig. 6. These results provide an in-depth understanding of the cold tolerance mechanism of Rhododendron.

Abbreviations
TMTs: Tandem mass tags; FC: Fold change; DEGs: Differentially expressed genes; GalUR: D-galacturonate reductase; GABA: g-aminobutyric acid; TGN: trans-Golgi network; PVC: prevacuolar compartment Declarations Ethics approval and consent to participate We obtained permission for our eld study from Changbai Mountain Planning and Natural Resources Bureau.

Not Applicable
Availability of data and material The data sets supporting the results of this article are included in this article.

Competing interests
The authors declare that they have no conflict of interest.     DAPs involved in translation, ribosomal structure and biogenesis. The green boxes represented up-regulated proteins under cold stress.