Journal of Proteomics & Bioinformatics
Open Access

Membrane Proteomic Responses in Sugar Beet Roots to Salt Stress

Jianing Kang^1,2,3, Peng Liao^1,2, Bing Yu^1,2, Sixue Chen^1,3, Haiying Li^1,2 and Chunquan Ma^{1,2* *}Correspondence: Chunquan Ma, Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin 150080, China, Email:

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Introduction

Soil salinity has become one of the most serious environmental constraints to crop production [1,2]. Plant response and tolerance to salt stress is a hot topic in current plant biology [3-6]. Salt stress reflects a combination of other stresses, including ionic stress, osmotic stress, and oxidative stress [7-10]. Although different strategies to enhance plant tolerance have been explored, including creating transgenic plants with a limited number of candidate genes, the outcomes have not met expectation and most crops could not grow well on saline soil. Therefore, a comprehensive study of a salt-tolerant crop (e.g., a sugar beet monosomic addition line M14) using omics technologies is important. Proteomics has shown utility in unraveling the molecular mechanisms of plant response to salt stress [11], and the systemic knowledge will greatly facilitate rational engineering and/or breeding for salt tolerance.

The sugar beet line M14 is a unique germline, which was obtained by crossing sugar beet Beta vulgaris L. with B. corolliflora Zoss. The M14 has the entire 18 chromosomes from B. vulgaris L. genome with the addition of chromosome 9 from B. corolliflora Z. It exhibits characteristics of apomixes and tolerance to drought, cold and salt stress [12]. Our previous studies have shown that the M14 seedlings can grow under 500 mM NaCl treatment for 7 days without losing viability, making it an interesting material for studying salt stress tolerance [13]. To date, we have made progress in the study of the M14 responses to salt stress at the transcriptomic and proteomic levels [13-16]. Especially, we have studied the changes in leaf membrane proteome of the M14 plants in response to salt stress (0, 200, 400 mM NaCl) and found 40 membrane proteins increased and 10 membrane decreased under salt stress [14]. However, analysis of the leaf membrane proteome of the M14 plants in response to salt stress is inadequate, since we missed the roots, which are the first organ exposed to salt stress [17,18]. Hence, studying root membrane proteome of the M14 plants in response to salt stress is a natural progression toward characterizing root membrane proteins in the M14 tolerance to salt stress.

Plant cell membrane system and many different proteins in or associated with the membranes play important roles in cell functions [19-21]. Especially, the plasma membrane, which separates the environment from the intracellular reactions, is essential for information exchange (signal transduction), material transport, and adaptation to stress conditions [22]. Membrane proteins are certainly important in these processes. Due to the hydrophobicity and low abundance of membrane proteins, it is difficult to analyze the membrane proteome using traditional technologies such as 2D gel electrophoresis [22,24]. In this study, we employed a modern iTRAQ LC-MS/MS technology to analyze differentially expressed root membrane proteins of the M14 seedlings in response to salt stress. A total of 115 differentially expressed proteins were identified, with 96 increased and 19 decreased in levels. The proteins were mainly involved in transport, signaling, stress and defense, energy, protein degradation, and transcription. The results have improved understanding of how the M14 plants tolerate salt stress. The resources of the salt stress-related genes and proteins may be applied to improving crop stress tolerance and yield.

Materials and Methods

Plant materials and NaCl treatment

Sugar beet monosomic additional line M14 seeds were sown and grown on vermiculite, and then the seedlings were transferred to Hoagland solution. They were grown in a growth chamber under a 14 h/10 h light/dark photoperiod (450 μmol m-2s-1 PAR) at 25°C/20°C (day/night) and 65% relative humidity. Five-week-old plants were divided into three groups, i.e., control group (without NaCl), 200 mM NaCl treatment group, and 400 mM NaCl treatment group. The plants were treated for 7 days. The NaCl concentrations were chosen according to our previous report showing that the M14 plants can tolerate up to 500 mM NaCl [13]. Plant materials were harvested directly into liquid nitrogen and stored in -80°C freezer. Three biological replicates were collected for each sample [25-33].

Physiological and biochemical analyses of control and salt-stressed roots

Root relative water content (RWC). After different concentrations of salt treatment, fresh weight was measured immediately after roots were harvested, and dry weight was obtained after the samples were dried at 75°C for 48 h. Turgor weight was determined by subjecting leaves to rehydration for 2 h after their dry weight was measured. The RWC was determined according to a previous method: RWC (%) = (fresh weight-dry weight)/(turgor weight-dry weight) × 100.

Membrane permeability: Membrane permeability was assessed by electrolyte leakage (EL). Root samples from different treatments (0, 200 mM, 400 mM NaCl) were rinsed three times with deionized water and placed in a tube containing 10 mL of deionized water. The tubes were placed in a vacuum chamber until the roots became wilted. The initial electrical conductivity of the solution (EC1) was determined. After autoclaving the tubes at 120℃ for 30 min, the final electrical conductivity (EC2) was obtained. EL was calculated as a percentage of EC1/EC2.

SOD activity: One-gram root material was ground in 2 mL 50 mM phosphate buffer (pH 7.8) with 1 × protease inhibitor cocktail. After centrifugation at 4°C 12000 rpm for 10 min, the supernatant was used for SOD activity assay. Three biological replicates were prepared for each sample. The SOD activity was assayed by measuring the inhibition of photochemical reduction of nitro blue tetrazolium (NBT) using a spectrophotometer at 560 nm as previously described. The 3 mL reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 μM methionine, 63 μM NBT, 1.3 μM riboflavin and 100 μL enzyme extract. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of NBT reduction.

Microsomal membrane protein extraction

A total of 50 g fresh roots were harvested from control, 200 mM and 400 mM NaCl treated samples. Each sample was ground into a fine powder in liquid nitrogen, followed by suspension and grinding in 15 ml of homogenization buffer (10 mM Tris-HCl pH7.4, 10 mM KCl, 1.5 mM MgCl₂, 10 mM DTT, 0.5 M Sucrose, and 1 mM PMSF). The resulting slurry was filtered through four layers of cheesecloth, and the filtered homogenate was centrifuged at 4℃, 800 g for 10 min to remove debris. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 4℃, 100,000 g for 1.5 h. The microsome pellet was washed with cold 100 mM Na₂CO₃ and homogenized with a Dounce homogenizer. The homogenate was centrifuged again at 4℃, 100,000 g for 1.5 h. The microsome pellet was collected with 500 µL resuspension buffer (100 mM HEPES pH7.0, 0.05% Triton X-100 and 0.5 M Sucrose) and centrifuged at 4℃, 800g for 10 min. The supernatant microsome was carefully transferred to a new eppendorf tube and kept at -80℃. The protein concentration was estimated using a Bradford Protein Assay Kit according to the manufacturer's instructions (Sigma, USA).

SDS-PAGE and western blot analysis

Protein samples of 15 μg microsomal protein each were loaded onto SDS polyacrylamide gels. The gel was blotted to PVDF membrane (146 mA, 2.5 h) and probed with primary antibodies against aquaporin (1:1000 dilution) and actin (1:5000 dilution) (Agrisera Inc., Sweden) for 2 h at room temperature. The blot was washed four times with a TBST solution (200 mM Tris-HCl pH 7.6, 137 mM NaCl and 0.05%Tween 20, v/v), and then incubated with a secondary antibody (goat-anti-rabbit IgG horse radish peroxidase conjugated, at 1:5000 dilutions, Zhong Shan Jin Qiao Inc., China) for 1 h at room temperature. The blot was finally washed with the TBST and a TBS solution (200 mM Tris-HCl pH 7.6 and 137 mM NaCl), and then developed using a DAB Kit (Zhong Shan Jin Qiao Inc., China). A replicate protein gel was stained with Coomassie Brilliant Blue R-250 for equal loading control.

iTRAQ labeling and LC-MS/MS identification

The acetone pellets of the microsome preparations from control, 200 mM NaCl treated and 400 mM NaCl treated samples (each of 100 μg protein) were collected by centrifugation at 20,000g, 4°C and dissolved in 1% SDS, 100 mM triethylammonium bicarbonate, pH 8.5. Reduction, alkylation, trypsin digestion and peptide labeling were conducted using the iTRAQ 8-plex reagent kit following the manufacturer's instructions (AB Sciex Inc., Framingham, MA, USA). The control replicates were labeled with iTRAQ tags 113, 114 and 115; the 200 mM NaCl replicates with tags 116 and 117; and the 400 mM NaCl replicates with tags 118, 119, and 121. The labeled microsomal peptides were desalted by C18-solid phase extraction and dissolved in strong cation exchange (SCX) solvent A (25% (v/v) acetonitrile, 10 mM ammonium formate, and 0.1% (v/v) formic acid, pH 2.8). The peptides were fractionated using an Agilent high-performance liquid chromatographer (HPLC) 1260 with a polysulfethyl A column (2.1 × 100 mm², 5 μm, 300 A; PolyLC, Columbia, MD, USA). Peptides were eluted with a linear gradient of 0−20% solvent B (25% (v/v) acetonitrile and 500 mM ammonium formate, pH 6.8) over 60 min followed by ramping up to 100% solvent B in 5 min. The absorbance at 280 nm was monitored, and a total of 16 fractions were collected. The fractions were lyophilized and resuspended in LC solvent A (0.1% formic acid in 97% water (v/v), 3% acetonitrile (v/v)). A quadrupole Orbitrap (Q Exactive) mass spectrometry (MS) system (Thermo Fisher Scientific, Bremen, Germany) is used to identify the labeled peptides. The instrument was run in a data-dependent mode with a full MS (400−2000 m/z) resolution of 70 000 and five MS/MS experiments using high energy collision dissociation at a normalized collision energy of 28%. The isolation width was 3 Th, and the AGC target was 1e6. Each sample fraction was loaded onto an Easy-nLC 1000 system (Thermo Fisher Scientific, Bremen, Germany), which flows at 300 nL/min during a linear gradient from solvent A (0.1% formic acid (v/v)) to 30% solvent B (0.1% formic acid (v/v) for 80 min and to 99.9% acetonitrile (v/v)) for an additional 10 min.

Analysis of differentially expressed proteins

The iTRAQ MS/MS data were processed by a thorough database searching considering biological modification and amino acid substitution against a non-redundant B. vulgaris database (53,260 entries) with Proteome Discoverer v1.4 (Thermo Fisher Scientific, Bremen, Germany). A differentially expressed protein must have at least three confident MS/MS spectra (1% FDR) and a p-value<0.05 with fold change thresholds (<0.5 or >2.0). Functions and known subcellular localizations of the identified proteins were inferred from the UniProt (http://www.ebi.uniprot.org/index) and literature.

Unknown subcellular localization was predicted using Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/). Prediction of protein transmembrane helices was conducted using the TMHMM Server v. 2.0 (http://genome.cbs.dtu.dk/services/TMHMM-2.0/). Pathways related to the differentially expressed proteins were obtained from KEGG pathway database (http://www.kegg.jp/).

Quantitative real-time PCR (qRT-PCR) analysis

Total RNA was isolated from the frozen samples using a TRIZOL reagent (Invitrogen Inc., USA). After removing genomic DNA using DNase I, cDNA was synthesized using a PrimeScript RT Reagent Kit (TakaRa, Shiga. Japan). Gene specific primers of selected proteins were designed using online Primer3 Plus. Quantitative RT-PCR was performed in a 20 μL mixture containing 10 μL 2 × SYBR^® Premix Ex Taq™ (TaKaRa, Shiga, Japan), 2 μL cDNA, 0.3 μL gene-specific primers, and 7.7 μL double distilled H₂O. The PCR conditions were: 95°C for 3 min; 95°C for 15 s, 61°C for 30 s, 40 cycles, on a CFX96 Real-Time PCR Detection System (Bio-Rad Inc., USA). Three biological replicates were used for each sample. All the data were analyzed using CFX Manager software (Bio-Rad Inc.) and the delta CT method was used for quantification.

Metabolite measurement

Cellular glucose was assayed using a Glucose (GO) Assay Kit following the manufacturer's instructions (Sigma-aldrich, USA). Briefly, glucose is oxidized to gluconic acid and hydrogen peroxide by glucose oxidase. Hydrogen peroxide reacts with o-dianisidine in the presence of peroxidase to form a colored product, which reacts with sulfuric acid to form a more stable colored product. The intensity of the pink color was measured at 540 nm, which is proportional to the original glucose concentration. The content of reduced glutathione (GSH) was measured based on the increase of absorbance at 412 nm when 5,5'-dithiobis-(2-nitrobenzoic acid) was reduced by GSH using the published methods.

Results and Discussion

Morphological, physiological, and biochemical responses to salt stress

To determine how salt stress affects the M14 seedlings, we inspected their morphological responses to different NaCl concentrations (0, 200 mM and 400 mM NaCl). As shown in Figure 1A, the M14 seedlings under 200 mM and 400 mM NaCl treatment grew slowly, and the fully expanded leaves appeared slightly chlorotic as compared with the control plants. The growth inhibition under the 400 mM NaCl treatment was marked, but the seedlings could survive the 400 mM NaCl treatment for seven days. The root length was also decreased with the increasing salt concentrations (Figure 1B). These results are consistent with our previous observation [15].

Figure 1: Morphological changes of sugar beet M14 seedlings under 0, 200 mM, 400 mM NaCl treatments A. Phenotype of sugar beet M14 seedlings. B. Root length of sugar beet M14 seedlings.

In addition to morphological changes, relative water content (RWC) and membrane permeability of the M14 roots were affected by the salt stress. Compared with the control, RWC of the M14 roots under 200 mM and 400 mM NaCl treatment decreased by 12.37% and 28.46%, respectively (Figure 2A). Salt stress caused membrane permeability to increase and then intracellular electrolyte to leak. As shown in Figure 2B, electrolyte leakage (EL) under 200 mM and 400mM NaCl treatments increased by 8.86% and 20.88%, respectively, compared with the control. As part of the salt stress response, SOD activity increased after the salt treatments (Figure 2C).

Figure 2: Effects of salt stress on RWC, membrane permeability and SOD activity of sugar beet M14 roots under 0, 200, 400mM NaCl treatments A) RWC of sugar beet M14 roots. B) Membrane permeability. C) SOD activity. a, b, c indicates control and salt-stressed samples are significantly different (p-value<0.05). Values represent mean ± standard deviation of three biological replicates.

Evaluation of microsomal membrane purity

Western blotting was used to evaluate the purity of the microsomal membrane preparations. PIP proteins are aquaporins that regulate the transport of water and small neutral molecules across cell membrane [33]. Actin is a family of globular proteins that form microfilaments in the cytoplasm [34]. The antibodies against PIP1 and actin were used to evaluate the relative purity of the microsomal membrane preparations and soluble protein extracts. As shown in Figure 3A, there was an obvious band of PIP1 (^~ 28 kDa) in the different membrane preparations, but not detectable in the soluble protein extracts. As to actin, we detected the signal in the microsomal membrane preparations, but the level is significantly lower than in the soluble protein extract (Figure 3B). Although the microsomes were prepared using ultrahigh- speed centrifugation and washed repeatedly with Na₂CO₃, it is possible that small number of soluble proteins was trapped in the vesicles. Based on these results, the microsomal membrane preparations were highly enriched for the membrane proteomics experiments.

Figure 3: Purity assessment of the microsomal membrane preparations using Western blotting. A) Western blotting of microsomal membranes and total soluble proteins of sugar beet M14 with anti-PIP1 antibody. B) Western blotting of microsomal membranes and total soluble proteins of sugar beet M14 with anti-Actin antibody. C) Coomassie blue staining of the membrane and soluble proteins to ensure equal loading.

Differentially expressed membrane proteins of M14 roots in response to salt stress

Using the iTRAQ labeling and two-dimensional LC-MS/ MS approach, a total of 343 differentially expressed proteins were identified at a 99% confidence level. These proteins were significantly increased or decreased (a fold change >2.0 or <0.5, p<0.05) in response to the NaCl treatments. Prediction of transmembrane helices in the identified proteins showed that 115 of the 343 proteins had one or more transmembrane domains (TMDs) (Table 1, Supplemental Table 1). This result suggests that many proteins in our membrane preparations may be peripheral membrane proteins or membrane associated proteins in spite of extensive washing steps with sodium bicarbonate.

We predicted the subcellular location of the 115 differentially expressed membrane proteins. These proteins are mainly located in nucleus, vacuole and plasma membrane (Figure 4A). Based on their function categories in Uniprot and literature (Figure 4B), these proteins fell into nine groups, including metabolism, transport, cellular structure, degradation, energy, signaling, stress and defense, transcription related and unknown. The majority of the proteins (32%) were transport-related. The other differentially expressed membrane proteins were classified into metabolism (28%), cellular structure (8%), signaling (7%), stress and defense (6%), energy (4%), degradation (4%), transcription related (4%) and unknown (7%).

Figure 4: Subcellular localization and functional classification of 74 differentially expressed membrane proteins in sugar beet M14 root. A) Subcellular distribution. B) Functional classification.

Comparison of the proteome data with transcriptome and metabolite data

To determine whether gene transcription level changes correlated with the protein level changes, we selected 10 genes encoding the M14 differentially expressed membrane proteins and analyzed their transcriptional changes using real-time PCR (Figure 5). Overall, seven genes showed a positive correlation between transcriptional and protein levels. Three genes showed different patterns between transcriptional and protein levels, i.e., glutamate receptor protein, glucose-6-phosphate translocator and sucrose transporter 4 showed opposite trends in transcript and protein levels (Table 2).

Figure 5: Real-time PCR analysis of the genes encoding the membrane proteins in sugar beet M14 root after salt treatments.

We measured the contents of glucose and glutathione (GSH) in the samples. With the increase of salt concentration, glucose content in the M14 roots gradually decreased. Strikingly, glucose content dropped more than 70% under 400 mM salt treatment compared with control (Figure 6). In the metabolic pathway (Figure 7), beta- D-glucan exohydrolase (gi|566232333) increased under 200 mM salt treatment. However, there is no direct correlation between this enzyme and glucose content, indicating other pathways may affect the glucose content. GSH is an important antioxidant in plant cells, preventing damage to important cellular components caused by excessive production of reactive oxygen species [35]. With the increased of salt concentration, GSH concentration increased, e.g., by 25% in the 400mM salt treatment (Figure 6). Glutamate receptor protein (gi|590571602) also increased in the 200 mM and 400 mM NaCl treatments with similar trend, indicating that the glutamate receptor protein might contribute to the increase of GSH under the salt treatments (Figure 7). Interestingly, in Arabidopsis innate immunity response, a glutamate receptor was found to mediate GSH trigger calcium transients and transcriptional changes [36]. The functions of the M14 glutamate receptor and GSH in salt stress response are to be investigated in future studies.

Figure 6: Related metabolite contents in sugar beet M14. A) Content of glucose. B) Content of GSH. a, b, c indicates control and salt-stressed samples are significantly different (p-value<0.05). Values represent mean ± standard deviation of three biological replicates.

Figure 7: Schematic presentation of systematic salt tolerance mechanisms in M14 roots. The red and green highlighted proteins indicate increased and decreased proteins, respectively.

Discussion

Using iTRAQ LC-MS/MS, we have identified and quantified many proteins from the sugar beet M14 plants under control and salt stress conditions. Many of the proteins are membrane proteins with diverse functions, such as transport, metabolism, signaling and energy. Here we focus on discussing membrane protein changes toward understanding the salt stress mechanisms of the M14 roots.

Transport and energy related proteins

Root is the initial organ to sense salt stress in soil. Since the main function of roots is to transport of water and nutrients, it is reasonable that the largest functional category of the membrane protein is transport-related. In this work, we identified 37 differentially expressed membrane proteins associated with the transport, with 24 increased and 13 decreased compared to the control (Table 1). It is known that Na⁺ in the roots is transported out by plasma membrane Na⁺/H⁺ antiporter or is transported into vacuoles through vacuolar Na⁺/H⁺- antiporter. Interestingly, we identified a Na⁺/H⁺ antiporter with two-fold increase under 400 mM NaCl treatment (Table 1). However, it appears to be targeted to chloroplast thylakoid membrane. It is not known how this Na⁺/H⁺ antiporter contributes to salt tolerance. Another transporter that displayed a 2.8 fold increase under 400 mM NaCl is a sugar transport protein 13 (gi|590657945). Sugar can function as a signaling molecule and/or provides energy to enhance plant stress tolerance [37]. In addition, an ABCG/PDR subfamily ATP binding cassette (ABC) protein (gi|375273925) showed a 3.2 fold increase under salt stress. The ABC transporter superfamily proteins form a large and ubiquitous protein family. In previous studies, the functions of different members were found to be redundant [38,39]. In Arabidopsis, there are more than one hundred ABC transporters [40]. The genome size of rice is about 3.5 times that of Arabidopsis, but it has almost the same number of ABC transporter genes [41]. The original function of this transporter was associated with detoxification [42]. The ABC transporters have been found to involve in many biological processes, such as hormone transport, stomatal movement, metabolite transport and response to environmental stresses [43]. It was reported that ABC proteins can form homo-dimers to mediate abscisic acid (ABA) transport across the membrane under abiotic stresses [44]. Nevertheless, its function in glycophytes is mainly focused on glutathione conjugate and chlorophyll catabolite transport, heme transport and helC homologue [45-47]. In salt stress proteomics of Arabidopsis [48,49] and rice [50,51], members of the ABC transporters were not found. Interestingly, in halophytes (e.g., Suaeda salsa), ABC transporter 1 is increased in both salt and heat shock stress [52]. Although ABC transporters appear to be important for halophytes, this protein was not found in leaf membrane proteomics of sugar beet M14 [14], but it was identified in the roots here. This evidence suggests that the ABC transporters may have different functions in glycophytes and halophytes. For, ABC transporters may play an important role in salt stress tolerance in a tissue-specific manner.

Table 1: Proteins identified and quantified in the microsomal membrane preparations from the control and NaCl treated sugar beet M14 using iTRAQ LC-MS/MS.

In addition to ABC transporter that use ATP directly for transport, here we identified five differentially expressed membrane proteins associated with energy and transport, four increased and one decreased in response to the salt stress treatments (Table 1). AAA-type ATPase family protein (gi|566206055) can hydrolyze ATP and transfer energy to target proteins, resulting in extensive conformational changes. In addition, a variety of specific adapter proteins can simultaneously control the conformation of AAA proteins and transfer energy to their target proteins [53]. V-ATPases (gi|733214298) in vacuolar membrane hydrolyze ATP to produce transmembrane electrochemical gradient, which drives a variety of cations (e.g., Na⁺) to accumulate into the vacuoles in order to maintain cell turgor, ion balance and promote plant growth under salt stress [54].

As to decreased transporters, three aquaporins, PIP1 (gi|1402833), MipI (gi|4884866) and TIP1,3 (gi|590563830), showed decreases in the M14 roots under salt stress. They transport water, and are involved in plant osmotic stresses (e.g., drought and high salt) [55]. The decreases of these proteins may reflect the capability of the M14 roots in maintaining water in the root cells [56,57].

Metabolism and signaling related proteins

Under salt stress, 32 differentially expressed membrane proteins associated with metabolism, including thirty increased and two decreased proteins (Table 1). First, NADPH-cytochrome P450 reductase provides electrons for cytochrome P450s in response to salt stress, and cytochrome P450s are involved in a variety of pathways involving oxidative metabolisms (e.g., synthesis of defensive metabolites) [58]. Second, beta-D-glucan exohydrolase family protein (gi|566232333) can hydrolyze glycosidic bonds between two or more carbohydrates and between carbohydrates and non-carbohydrate molecules [59]. Third, beta-glucosidase 8 (gi|350534796) can hydrolyze biologically inactive ABA-glucose ester to produce active ABA. It cooperates with ABA de novo synthesis to regulate cellular ABA levels in the course of plant growth and response to environmental stresses [60]. Fourth, cholesterol acyltransferase (gi|566161131) is one of the major enzymes involved in lipid metabolism, and may catalyze the transfer of acyl groups from lecithin to sterol, stabilizing the number of free sterols in the membrane [61]. Phytosterols are important components of plant plasma membrane. The increased cholesterol acyltransferase may enhance membrane stability, thereby enabling the M14 salt stress tolerance. Fifth, (gi|11386885), which acts on hexose phosphorylation, is one of the key enzymes in the process of plant respiration and metabolism, and has been found to play a vital role in plant sugar signaling [62,63]. Most of the hexokinase-1 activity is associated with intracellular membranes. Such specific localization may be favorable for their involvement in compartmentalized metabolism or signal transduction [64,65].

Signal transduction plays an important role in plant salt stress response and tolerance [66]. Here we identified eight differentially expressed membrane proteins associated with the signaling, and they were increased under salt stress compared with control plants (Table 1). One of the proteins is calcium-binding EF hand family protein (gi|566182885), which increased 2.5 times. Calcium-binding EF hand family protein is involved in plant stress response through calcium signaling. As a calmodulin, it has 2-6 EF-hands motifs, the EF-hand motifs bind to Ca²⁺ through helix-loop-helix [67]. Another signaling protein is a lectin receptor kinase (gi|914726339). This group of kinases plays important roles in saccharide signaling, stress perception and plant innate immunity [68]. Its function in plant salt stress response has not been reported.

Correlation between transcript, protein and metabolite levels

Out of the 10 genes encoding differentially expressed membrane proteins in different functional categories, seven showed positive correlations between transcript and protein levels, and three showed opposite trends of changes (Table 2). The opposite trends could be attributed to differential regulation at transcriptional, posttranscriptional and posttranslational levels, as well as differential life-time and stability of the transcripts and proteins [69].

Table 2: Comparison of the transcriptional level and protein level of the differentially expressed root membrane proteins.

To test whether the changes of the differentially expressed proteins correlate with related metabolites, we measured the contents of glucose and GSH under 0 mM, 200 mM and 400 mM NaCl (Figure 6). GSH is not only a pivotal component of the glutathione-ascorbate cycle, but also the precursor of phytochelatins and glutathione oligomers that chelate heavy metals such as cadmium. In addition, small oxidoreductases such as glutaredoxins use GSH as a substrate [70,71]. Interesting, both GSH and a glutamate receptor protein (gi|590571602) were increased under the 200 mM and 400 mM NaCl treatments. The glutamate receptor protein may be likely to regulate GSH levels under salt stress in the M14 plants [36]. As to glucose, its decreased levels could be attributed to decreased photosynthesis and increased energy consumption under salt stress, e.g., for metabolism and membrane transport activities (see previous sections). The increase of beta-D-glucan exohydrolase found in proteomics did not contribute to the changes of glucose, highlighting the importance of molecular analyses at different levels.

Overview of membrane protein functions in M14 salt stress tolerance

A previous leaf membrane proteomics work of the M14 plants under salt stress (200 mM and 400 mM NaCl) revealed 30 increased and 10 decreased proteins compared to control, and the proteins were mainly localized in chloroplast and mitochondria [14]. While here the differentially expressed membrane proteins in the M14 roots are mainly localized in the nucleus, vacuole and mitochondria. The differences in the distribution may reflect the functions and relative abundances of membrane proteins in roots and leaves (Figure 7). Interestingly, a differentially expressed membrane protein dolichyl-diphospho oligosaccharide-protein glycosyltransferase (gi|590703969) was observed in both roots and leaves of the M14. It had about two-fold increases in the two tissues of M14 under salt treatments, suggesting protein glycosylation may constitute an important posttranslational modification (PTM) in the M14 salt stress tolerance. Table 1 summarizes root salt-responsive membrane proteins in different organelles including nucleus, vacuoles and mitochondria. For example, the increase of apyrase (gi|117622284) involved in purine and pyrimidine metabolism in the extracellular matrix is really interesting [72]. In addition to energy metabolism, the extracellular nucleotides (e.g., adenosine, ADP and ATP) can regulate ROS production, membrane channel activity and auxin transport [73-75]. The decrease of root growth under salt treatment (Figure 1) may be attributed to the inhibition of polar auxin transport caused by extracellular ATP and apyrase activity [75], which pose a negative regulatory role in the growth of plant roots. The functions of many interesting proteins discovered in this study deserve further investigation.

Conclusion

The iTRAQ LC-MS/MS based quantitative proteomics identified 343 unique membrane proteins in the roots of sugar beet M14 line. A total of 115 proteins exhibited significant changes in response to salt stress, with 96 increased and 19 decreased in protein levels (fold change >2 or <0.5, p<0.05). The salt responsive membrane proteins were mainly involved in transport, signaling, stress and defense, and energy, and most of them were located in the vacuolar membrane, nuclear membrane, plasma membrane and mitochondrial membrane. Many interesting salt-stress responsive membrane proteins were discovered in the M14 roots, e.g., aquaporins, ABC transporter, Ca2⁺-binding protein, lectin receptor kinase, glutamate receptor and apyrase. Compared with glycophytes, sugar beet M14 ABC transporters from different subfamilies may play important roles in salt stress tolerance. The increase of a dolichyl-diphospho oligosaccharide glycosyltransferase in both root and leaf membrane proteomes highlights the importance of protein glycosylation in plant salt stress tolerance. Future studies on protein PTMs in the M14 will enhance understanding of the regulatory mechanisms important for plant salt stress signaling and tolerance.

Acknowledgment

This research was supported by the National Science Foundation of China (Project 31471552: The response of antioxidant enzymes to salt stress in sugar beet M14, Project 31671751: Regulatory mechanisms of BvM14-glyoxalase I transcription factor involved in the response to salt stress, Project 31501359: Study on phosphorylation sites of a Ser/Thr protein kinase from sugar beet M14 line in response to salt stress and Project 31401441: Identification of root variation related proteins in sugar beet (Beta vulgaris L.) monosomic addition line M14 using LC-MS/MS analysis) and the National Science Foundation of Heilongjiang Province (Project C2015026), and the Common College Science and Technology Innovation Team of Heilongjiang Province (Project 2014TD004). The authors thank Daniel Chen from Honors College, Biomedical Sciences Program, University of South Florida for critical reading and editing of the manuscript.

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