Salt stress may have distinct effects on different organs of plants. As shown in the iTRAQ data, we identified 1966 and 2541 proteins in leaves and roots that possessed more than two unique peptides. Furthermore, 70 and 76 DAPs were identified in leaves and roots, respectively. Among these DAPs, only three were differentially expressed in both roots and leaves, while the remaining 140 DAPs were differentially expressed only in leaves or roots. Functional divergence of the proteins from leaves and roots suggested that leaf and root may have distinct responses to salt stress and may make different contributions to stress-resistance in sugar beet. This result may not represent the entire landscape of protein patterns in leaf and root under stress or control condition owing to the technical limitations of iTRAQ. The possible biological significance of some key DAPS and their relevant metabolic pathways in salt stress adaptation are discussed below.
Analysis of the DAPs response to salt stress in Chloroplast
Chloroplasts, which only exist in leaves, are the most sensitive organelles to salt stress in plants . High salinity will destroy chloroplasts and affect photosynthesis . The experiment showed that chlorophyll content in leaves decreased 0.76-flod under high salt stress in sugar beet (Figure 1a). Unsurprisingly, 22 of the identified DAPs in leaves were related to chloroplasts including 14 up-regulated DAPs and 8 down-regulated DAPs. Three proteins psbQ-like protein 1 (A0A0K9RS47) and Plastocyanin (A0A0J8B4F7) and NAD(P)H quinone oxidoreductase subunit U (A0A0K9R1T8) from the photosynthetic electron transport chain were up-regulated under salt-stress, which may contribute to the maintenance of photosynthesis intensity. In addition, the STRING network analysis showed that the psbQ-like protein 1 was interacted with another up-regulated protein peptidyl-prolyl cis-trans isomerase fkbp16-4 (PPI) (A0A0K9RJJ) (Figure 4a). Other studies have shown the same up-regulation  which imply that plants may respond to salt stress by accumulating PPI to accelerate protein synthesis. Another up-regulated protein A0A0K9QN40 is thioredoxin Y1 that has the ability to regulate the activity of photosynthetic enzymes. Furthermore, DNA repair RAD52-like protein (A0A0K9RXT2) and DNA-damage-repair/toleration protein DRT100-like (A0A0K9S3X5) were also up-regulated proteins in chloroplast, these DAPs may help protect chloroplast DNA from damage under salt stress and enhance salinity tolerance in plant [22, 23].
Glycine betaine is considered to be the best osmotic regulator, which is not only involved in the osmotic regulation of cells, but also plays an important role in stabilizing the structure and functions of biological macromolecules under osmotic conditions, such as protecting the major enzymes and terminal oxidases of TCA (tricarboxylic acid) cycle and stabilizing the peripheral peptides of the light system under salt stress [24-26]. Betaine is an important osmotic regulator, which is produced from choline through two-step oxidation in plants [27-29]. The synthesis of betaine is catalyzed by two enzymes, choline monooxygenase (Q4H1G6) and betaine aldehyde dehydrogenase (Q4H1G7), which are significantly up-regulated under salt-stress . In addition, SEX4 (STARCH-EXCESS 4, also known as Dual specificity protein phosphatase 4, DSP4) (A0A0J8B9Z0) acts as a bridge between light-induced redox changes and protein phosphorylation in the regulation of starch accumulation . The accumulation of SEX4 in this study may suggest that SEX4 may promote the decomposition of transitory starch into soluble sugar to regulate the osmotic pressure in plant cells under salt stress.
Another up-regulated protein LS (6,7-dimethyl-8-ribityllumazine synthase) (A0A0J8E4J4) has been shown to catalyze the penultimate step in the synthesis of riboflavin. In addition to catalyzing riboflavin synthesis and regulating intracellular REDOX reactions, it has been reported that LS plays a role in the JA signaling pathway and participates in plant defense reactions . We observed that ABC transporter B family member 26 (A0A0K9QZ15) was up-regulated under salt stress which may play specific transport role in salt stress response. THI1 (Thiamine thiazole synthase) (A0A0K9Q9I3) was down-regulated in the present study, it has been demonstrated to take part in both guard cell abscisic acid (ABA) signaling and the drought response in Arabidopsis . The abundance of enolase 1 (A0A0J8CFG6) in plastids was down-regulated under salt stress. Previous studies have also documented that the isoenzyme expression of this protein is down-regulated under salinity [33, 34].
Analysis of the DAPs resistant to salt-stress
Osmotic imbalance, ion injury and reactive oxygen species (ROS) coupled with salt stress, which threaten the normal growth and development of plants. Besides betaine, soluble sugar and proline are also essential osmotic regulators. A 3.6-fold increase of proline was detected in leaves (Figure 1b), unfortunately, we did not find differentially accumulated of Proline metabolism-related enzymes (like P5CS) in both leaves and roots. However, we found 2 differentially accumulated sucrose synthase (Q6SJP5 and V7C8M2) in roots. As a widely existing glycosyltransferase in plants, sucrose synthase (SuSy) is a kernel enzyme in sucrose metabolism which can catalyze the decomposition and synthesis of sucrose. The accumulation of SuSy under abiotic stress has been found in many plants, especially in roots [35-37]. It has been reported that SuSy is not only involved in osmotic regulation of plants, but also functions at a branch point to allocation sucrose between cell wall biosynthesis and glycolysis . Thus, choline monooxygenase and betaine aldehyde dehydrogenase may play important roles in osmotic regulation of leaves under salt stress, while SuSy may be pivotal factors in the osmotic regulation of roots in sugar beet.
The damage of NaCl to plants is mainly caused by ion toxicity of sodium ions and chloride ions, as well as the production of ROS induced by stress. The 1.6-flod increase of MDA in leaves reflected the oxidative damage caused by stress (Figure 1c). In plant, excess ingestion of Na+ can affect the absorption of mineral nutrients, such as calcium (Ca2+), magnesium (Mg2+) and potassium (K+) . However, as a salt-tolerant plant, beet can use Na+ replaces of K+ for many functions like osmotic regulation, stomatal regulation and, long-distance transport of anions and so on [40-42]. High levels of chlorine ions in the environment can inhibit the uptake of NO3-, correspondingly, a significantly decrease in the content of high-affinity nitrate transporter (A0A0J8B2J0) under salt stress was observed in roots. In addition, the increase of ROS in plants under salt stress will affect physiological and biochemical processes such as photosynthesis, metabolism and signal transduction. Subjected to salt stress also lead to oxidative burst in plants. However, plants have their own detoxification system which can be divided into three stages: in the first stage, exogenous toxins or cytotoxins are oxidized, reduced or hydrolyzed by enzymes such as cytochrome P450 monooxygenase; then, a coupling reaction of processed products and sugar (or GSH) are catalyzed by enzymes, such as coupling with GSH catalyzed by GST; in the third stage, these conjugates are recognized by ATP coupling transporters in vacuoles or plasma membranes and eventually transported to vacuoles or expelled from the cell[43, 44]. Proteomic studies of roots have shown that two CYP family members (A0A0K9RP46 and A0A0K9RFX0), 3 GST family members (A0A0J8B2G1; A0A0J8CMV9; A0A0J8BAU3) and a F-type H+-transporting ATPase (A0A369ANY6) were differentially expressed. The detection results of root activity showed a 1.5-fold increase of TTC reduction capacity under salt stress (Figure 1d), which is likely to depend on the accumulation of GSTs. These proteins may play an important role in detoxification against salt stress in roots.
Another adaptation of sugar beets to high salt stress results from compartmentalization, excessive salt is selectively distributed to different tissues or organs. Generally, higher salt content is found in petioles and older leaves while lower salt content is found in new leaves, which is conducive to ensuring the function of these functional leaves. Unlike in roots, there was no accumulation of CYP and GST but differential expression of two peroxidase family members (A0A0K9R0G7 and A0A0J8B8Y7) were found in leaves. In addition to the protective enzymes, flavonoids also play an important role in scavenging effect to ROS as non-enzymatic reaction [47, 48]. Chalcone synthase (CHS) and flavanone-3-hydroxylase (F3H) are key enzymes in flavonoid metabolism. An accumulation of CHS (A0A0K9R791) and F3H (A0A0J8CVF6) were detected under salt-stress in leaves. This may be due to the sampling of functional leaves (the third-pair euphylla), and further studies are needed to determine the differences between new leaves and old leaves. These proteins may play an important role in detoxification against salt stress in leave.
Furthermore, non-symbiotic hemoglobin (NsHb) is also an important strategy that plants have evolved to resist stress, which can reduce the damage caused by oxidative stress. Overexpression of NsHb can improve the activity of antioxidant enzyme system in plants [49-51]. Two Non-symbiotic hemoglobin protein (V5QQP3 and V5QR23) and one Non-symbiotic hemoglobin protein (V5QQV5) were increased expression in roots and leaves, respectively. V5QR23, in particular, was upregulated more than two-fold, this intensely induced by salt stress implies it may play an underestimated role in resistant to salt-stress.
Analysis of the DAPs associated with Apoplast and Cell wall
Based on the GO analysis results, a large number of DAPs were associated with apoplast and cell wall in both roots and leaves. However, there were significant differences in response to salt stress between root and leaf cell wall DAPs. The apoplast is the first plant compartment encountering environmental signals. Studies have shown that apoplast protein is not only involved in the response of various environmental signals, but also in the perception and transduction of signals in collaboration with the plasma membrane [53, 54]. The cell wall is the outermost barrier of plant cell, which first senses the stress signal and transmits them into cell to regulate the activity of cell [55, 56]. It is a reticulate structure composed of polysaccharides, enzymes and structural proteins, while changes in composition affect ductility, mechanical support and defense functions of cell wall. In response to stress, cell wall proteins play an important role in cell wall structure, metabolism and signal transduction. It is very interesting that all the DAPs related to apoplast and cell wall were up-regulated in leaves. Specifically, β-galactosidase (A0A0J8B708), β-D-xylosidase 5 (A0A0K9QCY3), endo-1,3;1,4-β-D-glucanase (A0A0J8B9V6), xyloglucan endotransglucosylase/hydrolase protein 24-like (A0A0K9QWM7) were significant accumulation under salt stress in leaves. In higher plants, β-galactosidase is the only enzyme that can inner cleaves β-1,4-galactose to further cleavage of galactose residues from cell wall polysaccharides. Xylan is the main polysaccharide structure in plant cell wall, β-D-xylosidase is a kind of O-glycosyl hydrolases that can hydrolyze Glycosyl bonds between xylans. Endo-1,3(4)-β-D-glucanase has A specific digestive effect on cellulose microfibers and plays an important role in regulating plant cell wall structure . Xyloglucan endotransglucosylase/hydrolases (XTHs) is a family of xyloglucan modifying enzymes that play an essential role in the construction and restructuring of xyloglucan cross-links. In general, the up-regulation of these genes led us to speculate that beet leaves respond to salt stress by maintaining the ductility of cell walls. Leaf cells may increase in volume to compensate for the loss of photosynthetic intensity due to chlorophyll damage, thus ensuring energy supplies.
Diametrically opposed, α-xylosidase 1 (A0A0K9RU87), xyloglucan endotransglucosylase/ hydrolase (A0A0J8CRX9 and A0A0K9QMQ7), β-galactosidase 5 (A0A0K9R1V6), Expansin-like A2 (A0A0K9QJR1), and proline-rich protein 3 (A0A0J8FJ16) were down-regulated under salt stress in root. Expansin is a kind of cell wall relaxation protein, and it has been shown that its accumulation is an important biochemical mechanism for the salt tolerance reaction of wheat varieties . PRP protein (proline-rich protein) is structural protein of plant cell wall that plays an important role in cell wall construction and defense. Overall, the down-regulation of these DAPs suggested that the beet roots resist salt stress by inhibiting cell wall relaxation.
Analysis of DAPs related to Metabolism
The DAPs involved in carbohydrate and energy metabolism are indispensable. Here, NADH-ubiquinone reductase complex 1 MLRQ subunit (Q1H8M8) and Cytochrome c oxidase subunit 5C (A0A0K9Q6K8) participated in energy metabolism were up-accumulated in leaves under salt stress. Both of the two proteins are components of electron transport chain, which are involved in cellular respiration. More widely, 2 EMP components 6-phosphofructokinase (A0A0K9R9W4) and glyceraldehyde-3-phosphate dehydrogenase (A0A0K9R3B7); succinyl-CoA ligase, β subunit (S3HB42) belonging to TCA; ADH (A0A0K9QY28), PDC1 (A0A0K9QG18) and PDC2 (A0A0K9QMI0); ATP synthase α subunits (A0A369AIG8) and ATP synthase β subunits (S3HC31) were all up-accumulated in roots. PDC (Pyruvate decarboxylase) and ADH (Alcohol dehydrogenase) can convert pyruvate metabolism pathway from the synthesis of lactic acid into the synthesis of ethanol. Ethanol is much less toxic to plants than lactic acid or the intermediate acetaldehyde, and spreads easily. These results indicated that sugar beet is able to adapt salt stress by improving energy metabolism.
Phosphatidylcholine (PC) is a crucial metabolite of plant growth and development, and also the main lipid component of plant cell membrane. Besides its structural role, PC is the source of signaling molecules, Serine decarboxylase (SDC: Q4H1G0) can catalyzes the conversion of serine into ethanolamine, which is the first step in PC biosynthesis. Choline /ethanolamine kinase (CEK: A0A0K9RLT3) catalyzes the initial reaction step of choline metabolism that produces phosphoethanolamine . Phosphoethanolamine N-methyltransferase (PEAMTs: Q4H1G5) is a rate-limiting enzyme that catalyzes the phosphoethanolamine to produce choline . The up-accumulated of SDC, CEK and PEAMTs in leaves may not only be involved in the synthesis of cell membranes, but also in the synthesis of betaine and phosphatidic acid (PA) in response to salt stress. Unsurprisingly, the abundance of SDC (Q4H1G0) and GPI ethanolamine phosphate transferase 1 isoform X2 (A0A0J8B1W3) were reduced in roots. Besides, dirigent protein (A0A0K9QD33) involved in yielding lignans was down-regulated in roots after salt stress. These results support our analysis on cell wall related DAPs, which postulates that leaf cells strive to increase volume while root cells maintain it under salt stress.
A similar trend, the abundance of DAPs involved in protein Folding, and degradation were increased in leaves including Tubulin-folding cofactor D (A0A0K9RSC6), Aspartic proteinase nepenthesin-1 (A0A0K9RL53) and Ubiquitin carboxyl-terminal hydrolase 12-like (A0A0K9RR35); while prefoldin subunit 4 isoform X1 (A0A0K9RGH7), DnaJ protein homolog ANJ1(A0A0K9REY5) and Basic 7S globulin 2-like (A0A0K9QE67) were reduced in roots.
Analysis of DAPs involved in transcription and translation processes
Unlike animals, plants have to adapt to environmental changes continuously, they need to adjust their growth and processes of life timely in response to such alterations. Transcription and translation play an irreplaceable role in this adaptation process. A down-regulated accumulation of six DAPs associated with RNA and /or Protein binding were observed in leaves (Figure 4a). Of interest is Glycine-rich RNA-binding protein 2 (GR-RBP2) (A0A0J8FG78), which can affect the expression of genes encoded by mitochondrial genome and thus regulate respiration. Many studies have demonstrated that GR-RBP plays a remarkable role in response to stress [66, 67]. Similarly, a number of DAPs involved in transcription and translation are down-regulated in roots like DNA-directed RNA polymerases II, IV and V subunit 3 (A0A0K9RPV4), DEAD-box ATP-dependent RNA helicase 7 (A0A0J8BDH3), H/ACA ribonucleoprotein complex subunit 2-like protein (A0A0J8BPQ1), and so on. But unlike leaves, the accumulation of 50S ribosomal protein L14 (A0A023ZRD6) and 50S ribosomal protein L22 (A0A369ACK0) during salt stress were higher compared with the control. More remarkable, these two ribosomes are located in mitochondria. Thus, we hypothesize that the global intensity of transcription and translation in beet were decreased during salt stress, while root cells enhanced the synthesis of mitochondrial-related proteins on local level. Such specific regulation may help ensure the proper functioning of mitochondria to obtain sufficient energy against stress.
Analysis of DAPs in regard to Plant hormones
Plant hormones are active substances induced by specific environmental signals and have obvious physiological effects at very low concentrations. Gibberellin regulated protein (A0A0K9RKJ8) was observed to accumulate in the leaves. 1-aminocyclopropane-1-carboxylate oxidase (ACO) is a key enzyme in the ethylene biosynthesis pathway. Long chain acyl-CoA synthetase (LACS) has been reported to have catalytic ability to activate biosynthetic precursors of jasmonic acid (JA) . ACO1 (A0A0J8B2W2), LACS 4-like (A0A0K9RTE2) and auxin-binding protein ABP19a (Q84RC0) were up-accumulated while abscisic acid receptor PYL4 (A0A0J8BHH5) was down-regulated in roots under salt stress. Besides, a significant accumulation of carboxylesterase 1(A0A0J8CQ53), which can demethylate inactive methyl salicylate (MeSA) and methyl jasmonate (MeJA) into active salicylate acid and jasmonic acid, was observed in both leaves and roots under salt stress. These results help us to analyze the potential regulatory functions of diverse hormones in different organs under salt stress.