Salt stress may have distinct effects on different organs of plants. Herein, we have identified 1,966 and 2,541 proteins in leaves and roots that possessed more than two unique peptides. Furthermore, 70 and 76 DAPs were identified in leaves and roots, respectively, of which only three were differentially expressed in both of them. Functional divergence of the proteins in leaves and roots suggested distinct responses to salt stress and different contributions to stress-resistance in sugar beet. While we are aware that owing to the technical limitations of iTRAQ these results may not represent the entire landscape of protein patterns in leaves and roots under salt stress adaptation, the possible biological significance of some key DAPS and their relevant metabolic pathways are discussed below.
Analysis of the DAPs response to salt stress in Chloroplast
Chloroplasts only exist in leaves and are the most sensitive organelles to salt stress in plants [18]. We have shown that chlorophyll content in leaves decreased 0.76-fold under high salt stress, consistent with the fact that high salinity destroys chloroplasts and affects photosynthesis [19]. Unsurprisingly, 22 of the identified DAPs in leaves were related to chloroplasts, 14 of which were up-regulated and 8 down-regulated. Three proteins, psbQ-like protein 1 (A0A0K9RS47), 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 represent efforts to maintain photosynthesis. In addition, the STRING network analysis showed that psbQ-like protein 1 interacted with another up-regulated protein, peptidyl-prolyl cis-trans isomerase fkbp16-4 (PPI) (A0A0K9RJJ) (Fig. 4a), consistent with previous studies [20]. This suggests that plants respond to salt stress by increasing PPIs in order to accelerate protein synthesis. Another up-regulated protein is thioredoxin Y1 (A0A0K9QN40), which regulates the activity of photosynthetic enzymes [21]. Furthermore, DNA repair RAD52-like protein (A0A0K9RXT2) and DNA-damage-repair/toleration protein DRT100-like (A0A0K9S3X5) were also up-regulated proteins in the chloroplast. These DAPs may help protect the chloroplast DNA from damage under salt stress and enhance salinity tolerance [22, 23].
Glycine betaine, considered to be the best osmotic regulator, is not only involved in osmotic regulation of cells but also in the stabilization of macromolecules. For example, it protects the major enzymes and terminal oxidases of the TCA (tricarboxylic acid) cycle and stabilizes the peripheral peptides of the light system [24-26]. In plants, betaine is produced from choline via two oxidation steps and catalyzed by two enzymes that are significantly up-regulated under salt-stress: choline monooxygenase (Q4H1G6) and betaine aldehyde dehydrogenase (Q4H1G7) [27-29]. 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 [30]. SEX4 may help promote the decomposition of transitory starch into soluble sugar, in order to regulate osmotic pressure in plant cells.
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 and regulates intracellular REDOX reactions. In addition, LS plays a role in the JA signaling pathway and participates in plant defense reactions [31]. The ABC transporter B family member 26 (A0A0K9QZ15) was also up-regulated, which may be related to specific transport functions. THI1 (Thiamine thiazole synthase) (A0A0K9Q9I3) was down-regulated. This protein takes part in both guard cell abscisic acid (ABA) signaling and drought response in Arabidopsis [32]. Finally, enolase 1 (A0A0J8CFG6) in plastids was down-regulated, consistent with previous reports [33, 34].
Analysis of salt stress resistant DAPs
Salt stress affects the normal development of plants in the form of osmotic imbalances, ion injury and reactive oxygen species (ROS) formation. Soluble sugar and proline, like betaine, are also essential osmotic regulators. A 3.6-fold increase in proline was detected in leaves, although proline metabolism-related enzymes (like P5CS) did not accumulate in either leaves or roots. We did find two differentially accumulated sucrose synthases (Q6SJP5 and V7C8M2) in roots. Sucrose synthase (SuSy) is a widely distributed glycosyltransferase in plants that catalyzes the decomposition and synthesis of sucrose. Accumulation of SuSy under abiotic stress has been found in several plants, especially in roots [35-37]. SuSy is not only involved in osmotic regulation of plants, but also functions at a branch point to allocate sucrose to either cell wall biosynthesis or glycolysis [38]. Thus, in sugar beets, choline monooxygenase and betaine aldehyde dehydrogenase may play a role in osmotic regulation of leaves, whereas SuSy may be important in the osmotic regulation of roots.
The toxicity of NaCl to plants is mainly caused by sodium and chloride ions, as well as ROS production. In leaves, the observed 1.6-fold increase in MDA reflects the oxidative damage caused by stress. In plants, excess ingestion of Na+ can affect the absorption of mineral nutrients such as Ca2+, Mg2+ and K+ [39]. However, being a salt-tolerant plant, sugar beet can use Na+ instead of K+ for osmotic regulation, stomatal regulation and long distance transport of anions [40-42]. Consistent with the fact that high levels of chloride can inhibit the uptake of NO3-, a significant decrease in high affinity nitrate transporter (A0A0J8B2J0) was observed in roots. Increase of ROS and oxidative bursts can affect photosynthesis, metabolism and signal transduction. However, plants have their own detoxification system. First, exogenous toxins or cytotoxins are metabolized by enzymes such as cytochrome P450 monooxygenase. Second, enzymes like GST catalyze coupling reactions between processed products and sugar (or GSH). Third, these conjugates are recognized by ATP coupling transporters and are transported to vacuoles or secreted [43, 44]. Two CYP family members (A0A0K9RP46 and A0A0K9RFX0), 3 GST family members (A0A0J8B2G1; A0A0J8CMV9; A0A0J8BAU3) and an F-type H+-transporting ATPase (A0A369ANY6) were differentially expressed. Root activity results showed a 1.5-fold increase in TTC reduction capacity, likely due to accumulation of GSTs. Thus, these proteins may play an important role in detoxification against salt stress in roots.
Sugar beets respond to salt stress by compartmentalization [45]. In general, a higher salt content is found in petioles and older leaves whereas lower salt is found in new leaves, which ensures their correct function [46]. Unlike what was found in roots, in leaves there was no accumulation of CYP and GST, but differential expressions of two peroxidase family members (A0A0K9R0G7 and A0A0J8B8Y7). Chalcone synthase (CHS) (A0A0K9R791) and flavanone-3-hydroxylase (F3H) (A0A0J8CVF6) were detected in leaves. These are key enzymes in the metabolism of flavonoids, which play an important role in non-enzymatic scavenging of ROS [47, 48], therefore these proteins may be involved in detoxification. This finding may be due to the sampling of functional leaves (the third-pair euphylla). Further studies are needed to determine the differences between new and old leaves.
Plants also use overexpression of non-symbiotic hemoglobin (NsHb) as a strategy to reduce the damage caused by oxidative stress, by improving the activity of the antioxidant enzyme system [49-51]. One non-symbiotic hemoglobin protein, V5QQV5, was up-regulated in leaves, whereas two, V5QQP3 and V5QR23, were found in roots. The latter was upregulated more than two-fold, suggesting an underestimated role in the resistance against salt stress.
Analysis of the DAPs associated with Apoplast and Cell wall
GO analysis results showed that, in both roots and leaves, a large number of DAPs associate with apoplast and cell wall. However, cell wall DAPs in root and leave responded differently to salt stress. The apoplast is the first plant compartment encountering environmental signals [52], and apoplast proteins are involved in the response to these signals and in the perception and transduction of signals together with the plasma membrane [53, 54]. Stress signals are first detected by the cell wall, which transmits them into cells to regulate their activity [55, 56]. Interestingly, 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), and xyloglucan endotransglucosylase/ hydrolase protein 24-like (A0A0K9QWM7) were significantly accumulated. In higher plants, β-galactosidase is the only enzyme that can cleave β-1,4-galactosan internally and removes galactose residues from cell wall polysaccharides [57]. Xylan is the main polysaccharide in plant cell walls, and β-D-xylosidase is an O-glycosyl hydrolases that hydrolyzes glycosyl bonds in xylans [58]. 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 [59]. Xyloglucan endotransglucosylase/hydrolases (XTHs) play an essential role in the formation of xyloglucan cross-links [60]. Up-regulation of these genes in sugar beet leaves suggests a response to salt stress that results in maintaining the ductility of cell walls. Leaf cells may increase in volume to compensate for chlorophyll damage, thus ensuring energy supply.
In contrast, down-regulation of the following DAPs suggest that sugar beet roots resist salt stress by inhibiting cell wall relaxation. Indeed, α-xylosidase 1 (A0A0K9RU87), xyloglucan endotransglucosylase/ hydrolase (A0A0J8CRX9 and A0A0K9QMQ7), β-galactosidase 5 (A0A0K9R1V6), Expansin-like A2 (A0A0K9QJR1) and proline-rich protein (PRP) 3 (A0A0J8FJ16) were down-regulated. Expansin is a cell wall relaxation protein and its accumulation is a biochemical mechanism for salt tolerance in wheat varieties [61], whereas PRP is a structural protein involved in cell wall construction and defense.
Analysis of DAPs related to Metabolism
Consistent with the fact that DAPs involved in carbohydrate and energy metabolism are indispensable, we found up-regulation of NADH-ubiquinone reductase complex 1 MLRQ subunit (Q1H8M8) and Cytochrome c oxidase subunit 5C (A0A0K9Q6K8) in leaves. In roots, up-regulation was found for 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 (A0A369AIG8) and β subunits (S3HC31). PDC (Pyruvate decarboxylase) and ADH (Alcohol dehydrogenase) can switch production of lactic acid to ethanol, which is much less toxic to plants, or to intermediate acetaldehyde. These results indicate that sugar beet adapts to salt stress by improving energy metabolism.
Phosphatidylcholine (PC) has not only a structural role in membranes, but is the source of signaling molecules. Serine decarboxylase (SDC: Q4H1G0) catalyzes the first step in PC biosynthesis: the conversion of serine into ethanolamine [62]. Choline /ethanolamine kinase (CEK: A0A0K9RLT3) catalyzes the initial reaction step of choline metabolism to produce phosphoethanolamine [63]. Phosphoethanolamine N-methyltransferase (PEAMTs: Q4H1G5) is a rate-limiting enzyme that catalyzes the production of choline from phosphoethanolamine [64]. In leaves, up-regulation of SDC, CEK and PEAMTs may be related to both cell membrane synthesis and to the synthesis of betaine and phosphatidic acid (PA). Unsurprisingly, SDC (Q4H1G0) and GPI ethanolamine phosphate transferase 1 isoform X2 (A0A0J8B1W3) were down-regulated in roots as well as dirigent protein (A0A0K9QD33), involved in yielding lignans. These results are consistent with our analysis of cell wall-related DAPs in that, under salt stress, leaf cells strive to increase volume while root cells maintain it.
Similarly, DAPs involved in protein folding and degradation were increased in leaves, e.g., Tubulin-folding cofactor D (A0A0K9RSC6), Aspartic proteinase nepenthesin-1 (A0A0K9RL53) and Ubiquitin carboxyl-terminal hydrolase 12-like (A0A0K9RR35). In contrast, prefoldin subunit 4 isoform X1 (A0A0K9RGH7), DnaJ protein homolog ANJ1(A0A0K9REY5) and Basic 7S globulin 2-like (A0A0K9QE67) were down-regulated in roots.
Analysis of DAPs involved in transcription and translation processes
Plants require continuous adaptation to the environment, and one mechanism is by regulating transcription and translation. In leaves, we observed down-regulation of six DAPs associated with RNA and /or Protein binding, especially Glycine-rich RNA-binding protein 2 (GR-RBP2) (A0A0J8FG78), which can affect the expression of genes encoded by mitochondrial genome and thus regulate respiration [65]. GR-RBP plays a remarkable role in the response to stress [66, 67]. In roots, a number of DAPs involved in transcription and translation were down-regulated, like DNA-directed RNA polymerases II, IV and V subunit 3 (A0A0K9RPV4), DEAD-box ATP-dependent RNA helicase 7 (A0A0J8BDH3) or H/ACA ribonucleoprotein complex subunit 2-like protein (A0A0J8BPQ1). Also, unlike in leaves, 50S ribosomal protein L14 (A0A023ZRD6) and 50S ribosomal protein L22 (A0A369ACK0) were higher than in the control. Since these two ribosomes are located in the mitochondria, we hypothesize that global transcription and translation in sugar beet is reduced during salt stress, whereas root cells locally enhance the synthesis of mitochondrial-related proteins. Such specific regulation may help ensure the proper functioning of mitochondria to obtain sufficient energy.
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. In roots, the enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) is involved in the ethylene biosynthesis pathway, whereas long chain acyl-CoA synthetase (LACS) can activate the biosynthetic precursors of jasmonic acid (JA) [68]. ACO1 (A0A0J8B2W2), LACS 4-like (A0A0K9RTE2) and auxin-binding protein ABP19a (Q84RC0) were up-regulated, whereas abscisic acid receptor PYL4 (A0A0J8BHH5) was down-regulated. Both leaves and roots showed increased carboxylesterase 1(A0A0J8CQ53), which can demethylate inactive methyl salicylate (MeSA) and methyl jasmonate (MeJA) into active salicylate acid and jasmonic acid.