iTRAQ protein profile analysis of leaves and roots of sugar beet (Beta vulgaris) differing in response to salt stress

Salinity is one of the most serious threat to agriculture worldwide. Sugar beet is an important sugar-yielding crop and has a certain tolerance to salt. However, the molecular mechanism of salt tolerance in beta vulgaris are poorly understood. Proteomics can provide a new perspective and deeper understanding for the research of beet salt-tolerant. Here, leaves and roots were used to identify the differentially abundant protein species between salt-stress and control conditions in beta vulgaris. As a result, 70 and 76 DAPs were identified in leaves and roots, respectively. The functions were determined for the classification of the DAPs, mainly involved in cellular processes, environmental information processing, genetic information processing and metabolism. These processes can work cooperatively to reconstruct the favorable equilibrium of physiological and cellular homeostasis under salt stress. Some candidate DAPs are closely related to salt resistance such as choline monooxygenase, betaine aldehyde dehydrogenase, glutathione S-transferase (GST) and F-type H+-transporting ATPase. The expressional pattern of 10 DAPs encoding genes were consistent with the iTRAQ data. and have distinct This study provided some into the underlying the of higher plant identified some Interacts with Ca2+-Dependent Protein CPK33 and Modulates the S-Type Anion Channels


Background
Salinity is one of the most serious threat to agriculture worldwide. Sugar beet is an important sugar-yielding crop and has a certain tolerance to salt. However, the molecular mechanism of salt tolerance in beta vulgaris are poorly understood. Proteomics can provide a new perspective and deeper understanding for the research of beet salttolerant.

Results
Here, leaves and roots were used to identify the differentially abundant protein species between salt-stress and control conditions in beta vulgaris. As a result, 70 and 76 DAPs were identified in leaves and roots, respectively. The functions were determined for the classification of the DAPs, mainly involved in cellular processes, environmental information processing, genetic information processing and metabolism. These processes can work cooperatively to reconstruct the favorable equilibrium of physiological and cellular homeostasis under salt stress. Some candidate DAPs are closely related to salt resistance such as choline monooxygenase, betaine aldehyde dehydrogenase, glutathione S-transferase (GST) and F-type H+-transporting ATPase. The expressional pattern of 10 DAPs encoding genes were consistent with the iTRAQ data.

Conclusions
Our results demonstrated that during adaptation of beet to salt stress, leaves and roots have distinct mechanisms of molecular metabolism regulation. This study provided some significative insights into the molecular mechanism underlying the response of higher plant to salt stress, and identified some candidate proteins against salt stress. Background 3 Salinity is one of the most severe abiotic threats that affects the growth and development of crops [1,2] . Soil-salinization is a growing problem for agriculture that may negatively decrease the quality and yield of crops. The common effect of soil salinity on plants comes from the inhibition of growth by Na + and Clˉ accumulation [3] . Unlike other abiotic stresses, salinity causes both osmotic stress and ion toxicity in plants [4] . Plant growth may rapidly impaired by osmotic stress in a first phase and then specific ion toxicity primarily from Na + and Clˉ accumulation may cause membrane disorganization, the generation of reactive oxygen species, metabolic toxicity, inhibition of photosynthesis, and the attenuation of nutrient acquisition in a second phase of salt stress [5,6] . Although the growth of most crops is adversely affected by soil salinity, some cultivars are able to adapt to saline conditions to achieve good harvests.
Sugar beet (Beta vulgaris ssp. vulgaris or B. vulgaris) is one of the most important sugaryielding crops in the world. As recently domesticated crop, cultivated beets inherited certain salt-tolerance traits from its wild ancestor Beta vulgaris ssp. maritima ( B. maritima or 'sea beet') [7] . Cultivar 'O68' is an excellent parent used in traditional crossbreeding with strong salt tolerance. Our previous study showed that under 300 m mol·L − 1 NaCl treatment, the relative germination rate of this cultivar was more than 70% and the seedling can grow normally [8] . In addition, it has a strong regeneration capability of petiole explants which is ideally suited for use in molecular breeding. Therefore, O68 is a good choice for studying the mechanism of salt-stress response in B. vulgaris.
Analysis of the proteome responses to stress provides more direct insights into the molecular phenotype, since proteome is a better reflection of organism's actual response to environmental changes than the transcriptome. Isobaric tags for relative and absolute quantitation (iTRAQ) is one of the most reliable labeling techniques available for proteome quantification [9][10][11] . Li et al. analyzed the changes of membrane proteins under salt stress using iTRAQ technology in sugar beet monosomic addition line M14 [12] . Yu et al. analyzed the changes in proteome and phosphoproteome of M14 leaves induced by shortterm salt stress (30 min and 1 hour) [13] . Wu et al. studied changes in the proteome of beet seedlings treated with 50 mm NaCl for 72 h and 30 and 105 differentially expressed proteins were identified in the shoots and roots, respectively [14] . However, how plants respond to salinity depends on the organ, intensity and duration of the stress, which may lead to various changes at the proteome level [15,16] . In addition, as suggested by Shavrukov [17] , salt treatments can be divided into two types: salt stress (gradual exposure to rising salt levels) or shock (immediate exposure to a high-salt environment).
Due to the difference in response to salt-stress between the two approaches [7] , the method of salinity application should be carefully considered with respect to the interpretation of results. As the increase of salt concentration in nature usually occurs gradually, a method of gradual adding NaCl is used for treatment.
In the present study, iTRAQ-based quantitative proteomic analysis was employed to identify differentially abundant protein species (DAPs) in leaves and roots of cultivar beet 'O68', respectively. The final concentration of treatment was set as 300 mM, and the treatment time was 24 hours after reaching the final concentration. A total of 70 and 76 DAPs were identified in leaves and roots, respectively. Then Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups of proteins (COG) and STRING were used to elucidate the function of the DAPs in response to salt stress. These results will provide some insights into the underlying molecular mechanisms of stress responses and improve the understanding of the salt-stress response of B. vulgaris.

Results
The purpose of this study was to compare the protein levels changes associated with physiological and biochemical mechanisms involved in salt response between leaves and roots of B. vulgaris. To achieve this, we performed iTRAQ analysis of leaves sampled and roots sampled from plants treated with gradually increasing NaCl levels.

Effects of salinity on physiological indexes of B. vulgaris
Acetone extraction is used to determine the content of chlorophyll, a 0.76-fold decrease in chlorophyll content was detected in leaves under salt stress compared to control plants

Primary Data Analysis and Protein Identification Information by iTRAQ
To investigate the mechanism of sugar beet against to salt stress, the iTRAQ-based comparative proteome analysis at 36 hour after NaCl treatment of leaf and root was performed. A total of 31,438 and 39,522 MS/MS counts were generated from leaves and roots of B.vulgaris, respectively. In leaf, 10121 unique peptides and 3,175 proteins were identified against the UniProt database, 61.6% proteins (1,966) of which had at least two unique peptides. Also in root, 13248 unique peptides and 3,935 proteins were identified, of which 64.6% proteins (2,541) had at least two unique peptides. The length and number distribution of the peptides are provided in Figure. S1, statistical analyses showed that most peptides have 8-15 amino acids. The peptide number distribution of proteins indicated that 90% of identified proteins contain less than 8 segments (Figure. S2). The mass of the identified proteins suggested that 60 and 70 low molecular weight proteins (Mr < 10 kDa), 300 and 417 high molecular weight proteins (Mr > 100 kDa) were identified using the iTRAQ strategy ( Figure S3), respectively. The distribution of protein coverage showed that coverage with less than 10 %, 10 % -30 %, and 30 % 100% accounted for 57.6 %, 33.9 % and 8.4% in leaf ( Figure S4a), and 55.6 %, 30.6 % and 13.8 % in root ( Figure S4b).

Identification of Differential Abundance Protein Species (DAPS)
Proteins with at least two unique peptides in this study were used to screen DAPS. The  Table S1 and Table S2.

Transcriptional Analyses of the Corresponding Genes Encoding DAPS
To know the correlation between the abundance of DAPs and the transcript level of their corresponding genes, twelve DAPs (six from leaves and six from roots with one intersection) were selected for qRT-PCR analyses. The results showed that ten of the twelve selected DAPs keep the same express trend between transcript and protein level, the other two DAPs showed no significantly changed at transcript level (Table 1). This discrepancy may be due to temporal differences between the transcriptional level and the protein level of the salt stress response.

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 B. vulgaris ( Figure 1a). Changes must be implemented in response to this reduction.
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 upregulated under salt-stress, which may contribute to the maintenance of photosynthesis intensity. In addition, protein-protein interaction analysis showed that the psbQ-like protein 1 was interacted with another up-regulated protein PPI (A0A0K9RJJ3 |peptidylprolyl cis-trans isomerase fkbp16-4) (Figure 4a). The same up-regulation has been shown in other studies [19] 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 [20] .
Furthermore, DNA repair RAD52-like protein (A0A0K9RXT2) and DNA-damagerepair/toleration protein DRT100-like (A0A0K9S3X5) were also up-regulated proteins in chloroplastic, these DAPs may help protect chloroplast DNA from high salt and enhance salt tolerance in plant [21,22] .
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. Betaine is an important osmotic regulator, which is produced from choline through two-step oxidation in plants [23][24][25] . 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 (A0A0J8B9Z0 |STARCH-EXCESS 4, also known as Dual specificity protein phosphatase 4, DSP4) acts as a bridge between light-induced redox changes and protein phosphorylation in the regulation of starch accumulation [26] , 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 was LS (A0A0J8E4J4 |6,7-dimethyl-8-ribityllumazine synthase), which catalyzes 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 [27] . 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 (A0A0K9Q9I3 |Thiamine thiazole synthase) 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 [28] . 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 [29,30] .

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 [31][32][33] . 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 [34] . 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 B. vulgaris.
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 (Ca 2+ ), magnesium (Mg 2+ ) and potassium (K + ) [35] . 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 [36][37][38]  Another adaptation of sugar beets to high salt stress results from compartmentalization [41] , 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 [42] . 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 [43,44] . Chalcone Furthermore, non-symbiotic hemoglobin (NsHb) is also an important strategies 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 [45][46][47] . Two Non-symbiotic hemoglobin protein (V5QQP3 and V5QR23) and one Nonsymbiotic 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 saltstress.

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 [48] . 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 [49,50] . 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 [51,52] . 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 [53] . 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 [54] . 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 [55] . 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 [56] . 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), Expansinlike 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 [57] . 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-regulated of these DAPs illuminates that the beet roots resist salt stress by inhibiting cell wall relaxation.

Analysis of DAPs related to Metabolism
The conversion of serine into ethanolamine, which is the first step in PC biosynthesis [58] .
Choline /ethanolamine kinase (CEK: A0A0K9RLT3) catalyzes the initial reaction step of choline metabolism that produces phosphoethanolamine [59] . Phosphoethanolamine Nmethyltransferase (PEAMTs: Q4H1G5) is a rate-limiting enzyme that catalyzes the phosphoethanolamine to produce choline [60] . 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.

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 downregulated accumulation of six DAPs associated with RNA and /or Protein binding were observed in leaves (Figure 4a). Of concern 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 [61] . Many studies have demonstrated that GR-RBP plays a remarkable role in response to stress [62,63] . 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 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 surmised 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.

Plant Materials and Treatments
The cultivar sugar beet O68 were from our own laboratory (Heilongjiang, China). Let the seeds soaked in water for 10h, then sterilized in 0.1% (v/v) HgCl 2 for 10 min, washed repeatedly with distilled water, and germinated on wet filter paper in germination box at 26 °C for 2 days. After germination, seedlings were selected, transferred to plastic pots

Physiologic Indexes Detection
A total of 0.1g, 0.5g and 1g fresh leaves from the third pair of euphylla were used to detect chlorophyll content (acetone extraction), proline content (Ninhydrin colorimetry method), and malondialdehyde content (Thiobarbital acid method) following Gao [65] , respectively. 0.5g Fresh roots were collected for the determination of root activity by TTC reduction method base on Gao [65] . These parameters were analyzed by a UV-2100PC ultraviolet-visible spectrophotometer (UNICO. LTD), and each treatment was repeated three times.

Protein Extraction, Protein Digestion and iTRAQ Labeling
ITRAQ analysis was carried out in LC Sciences (Hangzhou, China). The leaf or root tissue from every 10 plants was pooled as one biological replicate, and three biological replicates were conducted for iTRAQ-based comparative proteomics analysis. The total proteins of the leaves and roots from each sample were extracted, according to a previous report [66] .

Nano-LC-ESI-MS/MS Analysis
The labeled peptides powder was dissolved with 52 μl (10 mM ammonium formate, pH 10) and then fractionated into fractions by Waters E2695 liquid chromatography system using

Protein Identification and Quantification
A MaxQuant (version 1.5.5.1) was used for iTRAQ protein identification and quantification [67,68] . For protein identification, the beet (B. vulgaris) protein database of UniProt (https://www.uniprot.org/uniprot/? query=beta%20vulgaris&fil=reviewed%3Ano&sort=score) was used with the criterion of a false discovery rate (FDR) < 0.01. The parameters of library searching were as following: fixed modifications include Carbamidomethyl on cysteine residues, iTRAQ 8 plex (N-term) and iTRAQ 8 plex (K); oxidative modification on methionine was set as a variable modification. The peptide mass tolerance was set as ±10 ppm and the fragment mass tolerance was 0.2 Da.

Bioinformatics Analysis
The biological and functional properties of proteins were analyzed with GO

RNA Extraction and qRT-PCR
Total RNA was extracted from leaves and roots by MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan). Approximately 2 μg of total RNA was reverse-transcribed using High-Capacity cDNA Reverse Transcription Kits in a 20 μl of reaction volume (ThermoFisher Scientific, US). The reactions were incubated for 10 min at 25 °C, followed by 37 °C for 120 min, and finally the reactions were terminated at 85 °C for 5 min. All the primers were listed in Table S3. PP2A+ UBQ5 and PP2A+25S were used as endogenous control in leaves and roots, respectively. For qRT-PCR, the gene-specific primers were designed using Primer-BLAST online (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi). The qRT-PCR reactions were performed using iTaq Universal SYBR® Green Supermix (BIO-RAD, Hercules, CA) on the CFX Real-time PCR system (BIO-RAD, CA). To avoid non-specific amplification, melting curve was carried out for each PCR product. The expression level of the miRNAs in different samples were calculated by comparative 2 − △△CT method.

Data Treatment and Statistical Analysis
For the data of the physiological parameters and qPCR analysis, the mean and SD were calculated from three repeats of each treatment, and the differences were analyzed by Duncan's multiple range test (p < 0.05) and an independent-samples t-test (p < 0.05).
Additional Files Figure S1. Distribution of peptide length in leaf (a) and root (b) of B. vulgaris.

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download.  Figure S1~S4.pdf Table S1 Detailed information of DPAs in leaves.xlsx