DAPs involved in Posttranslational modification, 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 photo-oxidative 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 diversification. Obviously, this combined machine will be more efficient 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, dwarfing, 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 modification 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 identified 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 efficiency is an important means for S. kudriavzevii strain to adapt to low temperature environment[15]. Rogalski et al. reported Rpl33 knockout plants exhibited a compromised recovery when transferred from chilling stress condition to standard growth conditions, indicating Rpl33 is required for efficient translation under cold stress[16]. However, Cheng et al.'s research in 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). Therefore, the experimental results of the up-regulation of RP abundance are consistent with previous studies on model organisms such as Arabidopsis. Regarding the explanation of this phenomenon, here we adopt the viewpoint of Molina et al.: Under cold acclimation, the Rhododendron needs to change the composition of ribosomes to meet the proper translation rate of protein[18].
DAPs involved in intracellular trafficking
For eukaryotes, vesicle transport not only plays a role in plant growth and development by maintaining the specificity 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 down-regulated, which implies that the trafficking 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 Ca2+ to bind the negatively charged phospholipids on the membrane. In addition to Ca2+ binding properties, other protein domains also endow it with various other functions including membrane traffic, 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 6-phosphate 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 flows 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 6-phosphogluconic acid to enter the pentose phosphate pathway, or it can be converted to Fructose 1,6-bisphosphate 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 flow 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 deficit”, 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 deficit, 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 identified 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 final 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 fitness 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 flavonoids 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 significant up-regulation after the cold stress treatment. As an important category of flavonoids, 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 significantly after the Rhododendron was exposed to cold stress. The up-regulated expression of these three enzymes means that Rhododendron shifts the metabolic flux 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 flow 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 detoxification 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 significantly down-regulated 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 diversification 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 detoxification 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 first 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 influx 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 flow from citrate synthase to isocitrate dehydrogenase; (ii) although not significant, 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 O2- into H2O2 and O2 through disproportionation. It is the first 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 significantly 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 trafficing 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 identified 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 filaments depolymerization caused by osmotic stress further strengthens the influx 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 fibrils. The assumption of microtubule depolymerization is consistent with the consensus that cold stress induces a decrease in membrane fluidity, accompanied by calcium ion influx and microtubule depolymerization[49]. In addition to microtubules and microfilaments, 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 influence the composition of the cell wall by finely 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 efflux 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.