Membrane Proteomic Profiling of Soybean Leaf and Root Tissues Uncovers Salt-Stress-Responsive Membrane Proteins

Cultivated soybean (Glycine max (L.)), the world’s most important legume crop, has high-to-moderate salt sensitivity. Being the frontier for sensing and controlling solute transport, membrane proteins could be involved in cell signaling, osmoregulation, and stress-sensing mechanisms, but their roles in abiotic stresses are still largely unknown. By analyzing salt-induced membrane proteomic changes in the roots and leaves of salt-sensitive soybean cultivar (C08) seedlings germinated under NaCl, we detected 972 membrane proteins, with those present in both leaves and roots annotated as receptor kinases, calcium-sensing proteins, abscisic acid receptors, cation and anion channel proteins, proton pumps, amide and peptide transporters, and vesicle transport-related proteins etc. Endocytosis, linoleic acid metabolism, and fatty acid biosynthesis pathway-related proteins were enriched in roots whereas phagosome, spliceosome and soluble NSF attachment protein receptor (SNARE) interaction-related proteins were enriched in leaves. Using label-free quantitation, 129 differentially expressed membrane proteins were found in both tissues upon NaCl treatment. Additionally, the 140 NaCl-induced proteins identified in roots and 57 in leaves are vesicle-, mitochondrial-, and chloroplast-associated membrane proteins and those with functions related to ion transport, protein transport, ATP hydrolysis, protein folding, and receptor kinases, etc. Our proteomic results were verified against corresponding gene expression patterns from published C08 RNA-seq data, demonstrating the importance of solute transport and sensing in salt stress responses.


Introduction
Soil salinity is one of the most important environmental factors limiting plant growth and productivity throughout the world [1]. Excessive sodium in the soil inhibits the absorption of mineral nutrients and moisture, leading to the accumulation of toxic ions in plants. Plants employ several strategies to cope with salt stress at the cellular and subcellular levels. Cellular responses in plants to salinity require a new state of cellular homeostasis. These include regulating the expression of specific proteins for the reestablishment of proper cellular ion and osmotic homeostasis with other concomitant processes of repair and detoxification [2]. The salt signal is primarily perceived through roots, which rapidly respond to maintain function and transmit signals to the shoot for appropriate changes in Figure 1. Phenotypes of salt-germinated 7-day-old seedlings of the cultivated soybean genotype C08 under 50 mM and 100 mM NaCl. Treated plants showed an overall stunted growth with decreased root and shoot growth.

Leaf and Root Membrane Proteome under Salt Stress Revealed by Orbitrap
To probe the responses of soybean plasma membrane towards salinity and to search for clues to the mechanisms of these responses, label-free quantitative proteomics, was used to analyze the leaf and root samples from 7-day-old soybean C08 seedlings germinated under salt stress, with three biological replicates for each treatment and tissue. The identified proteins and their relative abundance are listed in Supplementary Tables S1-S4, with an average of 1000-1200 total proteins identified from leaf and root tissues, respectively ( Figure 2). Gene ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the whole-proteome datasets from both tissues were compared based on the molecular functions of the identified proteins between the control and salt-treated samples and categorized by their p-values. Overall, the proteome datasets from both leaf and root tissues were enriched in membrane proteins involved in transporter-related activities such as active ion transmembrane transporters, ATPase-coupled transmembrane transporters, calcium ion binders, GTP binders, channel binders, monovalent transmembrane transporters, proton transmembrane transporters, soluble NSF attachment protein receptors (SNAREs), and proton transporters that are typically present in lower numbers in normal plant proteome datasets ( Figure 3). This suggests our methodology is a good approach to identify transporter-related proteins in plants.
In the root, the 50 mM NaCl treatment induced many proteins, such as transmembrane transporters, pyrophosphatases, ion channels, hydrolases, and anion binding proteins, which were absent in the control dataset ( Figure 3b). In addition, the 100 mM salt treatment also activated proteins such as NADPH dehydrogenases and flavoprotein oxidoreductases, which were missing in both control and 50 mM NaCl-treated root samples. It has been reported earlier that that ion channels and other transmembrane transporters

Leaf and Root Membrane Proteome under Salt Stress Revealed by Orbitrap
To probe the responses of soybean plasma membrane towards salinity and to search for clues to the mechanisms of these responses, label-free quantitative proteomics, was used to analyze the leaf and root samples from 7-day-old soybean C08 seedlings germinated under salt stress, with three biological replicates for each treatment and tissue. The identified proteins and their relative abundance are listed in Supplementary Tables S1-S4, with an average of 1000-1200 total proteins identified from leaf and root tissues, respectively ( Figure 2). Gene ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the whole-proteome datasets from both tissues were compared based on the molecular functions of the identified proteins between the control and salt-treated samples and categorized by their p-values. Overall, the proteome datasets from both leaf and root tissues were enriched in membrane proteins involved in transporter-related activities such as active ion transmembrane transporters, ATPasecoupled transmembrane transporters, calcium ion binders, GTP binders, channel binders, monovalent transmembrane transporters, proton transmembrane transporters, soluble NSF attachment protein receptors (SNAREs), and proton transporters that are typically present in lower numbers in normal plant proteome datasets ( Figure 3). This suggests our methodology is a good approach to identify transporter-related proteins in plants.
In the root, the 50 mM NaCl treatment induced many proteins, such as transmembrane transporters, pyrophosphatases, ion channels, hydrolases, and anion binding proteins, which were absent in the control dataset ( Figure 3b). In addition, the 100 mM salt treatment also activated proteins such as NADPH dehydrogenases and flavoprotein oxidoreductases, which were missing in both control and 50 mM NaCl-treated root samples. It has been reported earlier that that ion channels and other transmembrane transporters are first to be activated upon salt stress, and they continue to function to mitigate more severe or prolonged salt exposure [18]. In the root, SNARE-binding activities were drastically reduced upon salt stress, which warrants further investigation into how roots cope with salt stress in soybean and other plants (Figure 3b).
The leaf proteome data revealed that salt stress induced many proteins such as cation transmembrane transporters, anion transporters, passive membrane transporters, voltage-  KEGG pathway analyses of the leaf and root proteomes provided information on the pathways activated under salt stress. In the root, significant enrichment of proteins involved in linoleic acid metabolism pathway, endocytosis pathway, protein export pathway, and fatty acid biosynthesis pathway was observed in salt-treated samples compared to controls (Figure 3d). Overall, fatty acid metabolism, carbon metabolism, biosynthesis of amino acids, and 2-oxocarboxylic acid metabolism-related proteins were significantly enriched in salt-treated root samples (Figure 3d). In the leaves, salt stress induced different pathways, particularly those related to phagosome, spliceosome, and SNARE interactions in vesicular transport (Figure 3c). A significant enrichment in porphyrin and chlorophyll metabolism was observed in 100 mM salt-treated leaf samples compared to control and 50 mM salttreated samples (Figure 3c).    In the identified proteomes, about 50% of the proteins in both leaf and root tissue were categorized as membrane proteins under cellular component in the UniPro Knowledgebase [19]. All the identified membrane proteins had transmembrane domain (Supplementary Tables S1-S4). In total, we identified 972 membrane proteins from th leaf and root tissues combined, in which 421 proteins were commonly shared by both tissues, 320 membrane proteins were root-specific, and 233 were leaf-specific ( Figure 4a) The 972 membrane proteins were further categorized on the basis of their subcellular lo cations, enzyme activities and the domains they contain (Supplementary Tables S1-S4) Based on enzymatic functions, they can be further sorted into 158 transporter proteins 107 oxidoreductases, 88 primary active transporters, 79 membrane traffic proteins, 41 ATP synthases, 25 SNARE proteins, and 23 chaperone proteins (Figure 4b). According to cel lular component classification, there were 374 proteins identified as integral component of the membrane, 47 membrane proteins, 25 chloroplast thylakoid membrane compo nents, 23 mitochondrial inner membrane components, 21 endoplasmic reticulum mem brane components, and 20 proteins associated to the SNARE complex, photosystem I and chloroplast envelope (Figure 4c). The predominant domains in our membrane proteome were P-loop-containing nucleoside triphosphate hydrolases, AAA + ATPases, smal In the identified proteomes, about 50% of the proteins in both leaf and root tissues were categorized as membrane proteins under cellular component in the UniProt Knowledgebase [19]. All the identified membrane proteins had transmembrane domains (Supplementary Tables S1-S4). In total, we identified 972 membrane proteins from the leaf and root tissues combined, in which 421 proteins were commonly shared by both tissues, 320 membrane proteins were root-specific, and 233 were leaf-specific ( Figure 4a). The 972 membrane proteins were further categorized on the basis of their subcellular locations, enzyme activities and the domains they contain (Supplementary Tables S1-S4). Based on enzymatic functions, they can be further sorted into 158 transporter proteins, 107 oxidoreductases, 88 primary active transporters, 79 membrane traffic proteins, 41 ATP synthases, 25 SNARE proteins, and 23 chaperone proteins (Figure 4b). According to cellular component classification, there were 374 proteins identified as integral components of the membrane, 47 membrane proteins, 25 chloroplast thylakoid membrane components, 23 mitochondrial inner membrane components, 21 endoplasmic reticulum membrane components, and 20 proteins associated to the SNARE complex, photosystem I and chloroplast envelope (Figure 4c). The predominant domains in our membrane proteomes were P-loopcontaining nucleoside triphosphate hydrolases, AAA + ATPases, small GTPase-binding protein domains, chlorophyll a/b binding protein domains, C2 calcium-dependent membrane domains, ABC transporters, Band7 proteins, porin domain, remorins, aquaporins, syntaxins and stomatins (Figure 4d). GTPase-binding protein domains, chlorophyll a/b binding protein domains, C2 calciumdependent membrane domains, ABC transporters, Band7 proteins, porin domain, remorins, aquaporins, syntaxins and stomatins (Figure 4d).

Differentially Expressed Membrane Proteins (DEMPs) in Soybean Leaves and Roots under Salt Stress
Using label-free quantification, we identified 129 differentially expressed membrane proteins (DEMPs) from both tissues and the volcano plots drawn based on a −log10 p-value > 0.05 and absolute fold-change value > 0.5 ( Figure 5 and Table 1). In the leaf, 50 membrane proteins were upregulated and 21 were downregulated, while in the root, 26 were upregulated and 32 downregulated in response to salt stress. GO annotation enrichment analysis of the DEMPs revealed that the membrane proteins upregulated in the leaf were involved in nearly every aspect of transport mechanisms, purine nucleoside biosynthesis and metabolic processes, indicating the important roles of various transporters (e.g., proton transmembrane transporters, cation transporters, energy-coupled transporters, etc.) to combat salt stress in soybean (Supplementary Figure S1a). DEMPs related to membrane and transport were reported to be involved in early events in salt signal transduction [14]. On the other hand, the downregulated membrane proteins in leaf were mostly involved in photosynthetic processes such as light reaction, protein chromophore linkage, response to light stimulus, and generation of precursor metabolites and energy (Supplementary Figure S1b). All the leaf DEMPs are presented in Tables 1 and 2 along with their gene Ensemble IDs, descriptions, Arabidopsis homologs identifier, number of peptides detected, PSMs (peptide spectrum matches), molecular weights and isoelectric points. Among the leaf DEMPs, there were several highly upregulated membrane proteins such as GLYMA_08G224400 (V-type proton ATPase catalytic subunit A), GLYMA_11G120200 (probable NAD[P]H dehydrogenase), GLYMA_11G179300 (NAD[P]H-ubiquinone oxidoreductase C1), GLYMA_02G272600 (RESTRICTED TEV MOVEMENT 2), GLYMA_13G276500 (proton pump-interactor 1), and GLYMA_09G040600 (abscisic acid

Differentially Expressed Membrane Proteins (DEMPs) in Soybean Leaves and Roots under Salt Stress
Using label-free quantification, we identified 129 differentially expressed membrane proteins (DEMPs) from both tissues and the volcano plots drawn based on a −log 10 p-value > 0.05 and absolute fold-change value > 0.5 ( Figure 5 and Table 1). In the leaf, 50 membrane proteins were upregulated and 21 were downregulated, while in the root, 26 were upregulated and 32 downregulated in response to salt stress. GO annotation enrichment analysis of the DEMPs revealed that the membrane proteins upregulated in the leaf were involved in nearly every aspect of transport mechanisms, purine nucleoside biosynthesis and metabolic processes, indicating the important roles of various transporters (e.g., proton transmembrane transporters, cation transporters, energycoupled transporters, etc.) to combat salt stress in soybean (Supplementary Figure S1a). DEMPs related to membrane and transport were reported to be involved in early events in salt signal transduction [14]. On the other hand, the downregulated membrane proteins in leaf were mostly involved in photosynthetic processes such as light reaction, protein chromophore linkage, response to light stimulus, and generation of precursor metabolites and energy (Supplementary Figure S1b). All the leaf DEMPs are presented in Tables 1 and 2 along with their gene Ensemble IDs, descriptions, Arabidopsis homologs identifier, number of peptides detected, PSMs (peptide spectrum matches), molecular weights and isoelectric points. Among the leaf DEMPs, there were several highly upregulated membrane proteins such as GLYMA_08G224400 (V-type proton ATPase catalytic subunit A), GLYMA_11G120200 (probable NAD[P]H dehydrogenase), GLYMA_11G179300 (NAD[P]H-ubiquinone oxidoreductase C1), GLYMA_02G272600 (RESTRICTED TEV MOVE-MENT 2), GLYMA_13G276500 (proton pump-interactor 1), and GLYMA_09G040600 (abscisic acid receptor PYL12) ( Figure 5). On the other hand, the downregulated membrane proteins in leaf contained many chloroplast membrane proteins such as GLYMA_12G059600 (plastid TRANSCRIPTIONALLY ACTIVE 16), GLYMA_05G246900 (PRA1 family protein B5), GLYMA_16G165200 and GLYMA_02G305400 (chlorophyll a/b binding proteins 1 and 1.2), and GLYMA_02G147200 (RAN GTPase-activating protein 1), suggesting a new role of chloroplast membrane proteins in salt stress signaling, or it could simply be a result of the winding down of photosynthetic activities in exchange for ramping up salt stress responses ( Figure 5 and Table 1). receptor PYL12) ( Figure 5). On the other hand, the downregulated membrane proteins in leaf contained many chloroplast membrane proteins such as GLYMA_12G059600 (plastid TRANSCRIPTIONALLY ACTIVE 16), GLYMA_05G246900 (PRA1 family protein B5), GLYMA_16G165200 and GLYMA_02G305400 (chlorophyll a/b binding proteins 1 and 1.2), and GLYMA_02G147200 (RAN GTPase-activating protein 1), suggesting a new role of chloroplast membrane proteins in salt stress signaling, or it could simply be a result of the winding down of photosynthetic activities in exchange for ramping up salt stress responses ( Figure 5 and Table 1).

Salt-Stress-Induced Membrane Proteins from Soybean Leaf and Root Tissues
In both root and leaf proteomes, we found 140 membrane proteins in root and 57 in leaf that were expressed only with salt treatment and absent from the control datasets based on their detection in the form of peptides. These were mainly involved in transport processes inside the cell (Supplementary Figure S2). Salt-stress-induced membrane pro-  [20]. Red bars represent up-regulated gene expressions and blue bars represent down-regulated gene expressions of the corresponding differentially expressed proteins from this study. White bars represent unchanged gene expression levels.

Discussion
To cope with salt stress, soybean plants have evolved complex salt-responsive signaling at the membrane, cellular, organ, and whole-plant levels. Membrane proteins play a variety of roles in crucial biological functions, for example, the transport of biological substances (ions, nutrients, metabolites and signaling molecules), signal transduction, bioenergetic processes, immune response, cell adhesion, and cell-cell interactions. These proteins are challenging to study using biochemical methods because they are only present at low levels and are unstable outside of the lipid bilayers [5]. Hence, they are seldom studied despite their significance in biological systems. Nonetheless, the growing demand for increased quality and quantity of membrane proteomic data has resulted in improved membrane proteomics methodology. High-throughput MS has contributed significantly to the comprehensiveness of proteomics, resulting in the identification of many membrane proteins [4]. Here, we carried out unbiased label-free quantitative proteomic analyses comparing the relative abundance of membrane proteins in salt-treated samples of soybean leaves and roots compared to controls. Among the 972 membrane proteins identified, a total of 233 proteins in leaves and 320 proteins in roots responded to NaCl-stress treatments.
The possible observed pictorial phenotypes of decreased plant height, reduced internode and lateral root number and length were similar to those reported in previous soybean studies where exogenous NaCl applications had a remarkable inhibitory effect on plant growth [21,22] (Figure 1).
Our detected membrane proteome included proteins such as active ion transmembrane transporters [23], ATPase-coupled transmembrane transporters [24], calcium ion binders [25], GTP binders [26], channel proteins [12], monovalent transmembrane transporters [18], transmembrane proton transporters [27], and SNARE receptors [28], confirming their involvement in salt stress tolerance observed in previous studies. In the root, upon 100 mM salt treatment, the activation of NADPH dehydrogenases and flavoprotein oxidoreductases clearly indicated that NADPH regeneration would be essential in the defense mechanism against salt-induced oxidative stress [29].
In leaves, the enrichment of KEGG pathways involved in phagosome, spliceosome and SNARE interactions in vesicular transport strongly suggested that certain splicing factors and autophagy could increase the plant tolerance to salt stress [30,31] (Figure 3c). In the root, the enrichment of KEGG pathways involved in linoleic acid metabolism and endocytosis suggested that salt-stress responses such as lipid metabolism and increased plasma membrane endocytosis could be possible mechanisms for mitigating salt stress in plants [32,33] (Figure 3d). Conserved membrane proteins, including Band7 proteins, porins, remorins, aquaporins, syntaxins and stomatins, could be the major responsive proteins against salt stress in plants because they are crucial in transport, signaling, bioenergetics, and catalysis [34][35][36].
In the leaf, the upregulated proteins, such as abscisic acid receptor PYL12 (GLYMA_09G040600), COP1-interactive protein 1 (GLYMA_11G096400), two cation/H(+) antiporter 8 (GLYMA_09G222500; GLYMA_12G036000), Synaptotagmin-1 (GLYMA_11G107300), V-ATPase subunit A, V-ATPase subunit E3, E1 (GLYMA_08G224400, GLYMA_05G214200, GLYMA_08G020300), and aquaporin NIP2-1 (GLYMA_12G066300), suggest their novel roles in salt-stress responses towards sodium toxicity and ABA-mediated activities inside leaf cells (Table 1 and Figure 4). A soybean sodium/hydrogen exchanger enhanced the salt tolerance through maintaining a higher Na + efflux rate and K + /Na + ratio in Arabidopsis [37]. In rice, a Na + /H + exchanger protein is the sole Na + efflux transporter and its loss-of-function mutant displayed exceptional salt sensitivity [38]. In Arabidopsis, synaptotagmin-1 mutant plants were severely affected by salt stress since this protein is required for the maintenance of plasma membrane integrity [39,40]. During salinity stress, V-ATPases facilitate the sequestration of Na + in the vacuole by establishing an electrochemical proton gradient across the tonoplast [41], and their abundances and activities are usually increased during salinity stress. An adaptive response and induced expression of Nodulin 26-like Intrinsic Protein 2-1 (NIP2-1) were recorded in Arabidopsis under low-oxygen stress [42].
In the root, differentially expressed ion pump proteins, which included vacuolartype proton ATPase subunit E1 (GLYMA_08G020300), ATPase 11, plasma membranetype (GLYMA_04G174800), and calcium-transporting ATPase 4, plasma membrane-type (GLYMA_10G107700), were found to be critical for plant salt tolerance and had key roles in regulating ion transport under salt stress (Table 3 and Figure 5). An improved method using tandem affinity purification tag for the salt-overly sensitive (SOS) pathway indicated that subunits A, B, C, E and G of the peripheral cytoplasmic domain of the vacuolar ATPase were present in an SOS2-containing protein complex to maintain favorable ion ratios in the cytoplasm and for increasing the tolerance of salt stress [9,44]. An overexpressing form of the plasma membrane ATPase that is constitutively active conferred increased salt tolerance [45,46]. It has been reported that the salt-stress-induced elevation in cytosolic Ca 2+ and the new cytosolic Ca 2+ status is regulated by Ca 2+ -ATPases [47]. In the root, the transporter proteins that were highly upregulated by salt stress included ammonium transporter 1 (GLYMA_10G132300) and high affinity nitrate transporter 2.5 (GLYMA_18G141900), suggesting that ammonium transport could alleviate ammonia toxicity caused by salt stress. The overexpression of PutAMT1;1 gene promoted early root growth after seed germination in transgenic Arabidopsis under salt stress [48]. In salt-stressed roots, the upregulation of serine/threonine-protein kinase PBL19 (GLYMA_01G181700), wall-associated receptor kinase-like 6 (GLYMA_05G078000), LRR receptor-like serine/threonine-protein kinase (GLYMA_08G100800), receptor-like protein 4 (GLYMA_11G146900), and calciumdependent protein kinase 2 (GLYMA_15G222300) indicated that they are potential saltstress sensors in the root (Table 3). SlWAK1 is a wall-associated kinase involved in salt tolerance in tomato (Solanum lycopersicum). slwak1 mutants are tolerant to Na + stress, but not to osmotic stress [49]. For example, in Medicago truncatula, the LRR-RLK gene, Srlk, was rapidly induced in roots in response to salt stress [6]. In potato, calcium-dependent protein kinase 2 was identified as a sensor-transducer in the salt stress response in potato plants. Its overexpression promoted ROS scavenging, chlorophyll stability and the induction of stress-responsive genes, thus conferring tolerance to salinity [50].
In the root, the downregulated membrane proteins upon salt stress included aquaporin PIP2-2 (GLYMA_10G211000), proton pump-interactor 1 (GLYMA_20G006500), calnexin homolog (GLYMA_05G199200), (GLYMA_09G243700), and ras-related protein RABF1 (GLYMA_10G208300) ( Table 4 and Figure 5). In Arabidopsis, salt stress is known to localize PIP2 to intracellular compartments, probably to decrease the water permeability of the root [51]. In Arabidopsis, subtilase 6.1 associated with the unfolded protein responded to salt stress through the cleavage of an ER-resident type II membrane protein (bZIP28) [52]. In tobacco, the calreticulin gene from wheat (TaCRT1) improved the salinity tolerance [53]. In Arabidopsis, RabF1 is involved in salt-stress responses through endosomal vesicle transport and may play a crucial role in the recycling and degradation of molecules [54]. We found some chloroplast-related proteins downregulated under salt stress in roots, such as GLYMA_04G095900 (tetratricopeptide repeat domain-containing protein PYG7), GLYMA_05G016100 (CHAPERONE-LIKE PROTEIN OF POR1), GLYMA_14G076000 (outer envelope pore protein 24B), and GLYMA_14G201500 (TIC110), which are essential for chloroplast development, and their suppression plays a role in salt stress responsiveness in chloroplasts. TIC110 is a component of the protein import apparatus in the chloroplast, and its downregulation suggests that salt stress hinders the process of importing nuclear DNA-encoded proteins into chloroplasts. In Arabidopsis, the downregulation of TIC110 expression resulted in the reduced accumulation of a wide variety of plastid proteins [55]. The downregulation of mitochondrial import inner membrane translocase subunit TIM17-2 and mitochondrial import receptor subunit TOM6 indicates that salt stress hinders the import processes in mitochondria in response to stress [56]. The downregulation of mitochondrial ADP, ATP carrier protein 3 (GLYMA_12G205400), means that salt stress disrupts the supply of ATP from the mitochondrial matrix to the cytosol [57].
We also found some proteins in both soybean leaf and root that were present only in the salt-treated samples, suggesting that they were only produced in response to salt. In the root, salt stress induced many transporter proteins, such as ABC transporter C and G family members, membrane magnesium transporter, potassium transporter 4, sodium/hydrogen exchanger 7 (SOS1), and sulfate transporter 1.3 (Tables 5 and 6). Under salinity stress, the ABC transporters have been reported to enhance plant tolerance through the sequestration of sodium salt [58]. In rice, a Mg transporter, OsMGT1, is required for salt tolerance probably by regulating the transport activity of OsHKT1;5, a key transporter for the removal of Na + from the xylem sap at the root maturation zone [59]. In soybean, salt treatment elevated the expressions of SOS1 and AKT1 genes, and reduced the expressions of SKOR and HKT1 genes [37]. A significant accumulation of Na + in the roots of the gmsos1 mutants resulted in the imbalance between Na + and K + , which impaired Na + efflux and increased K + efflux in the roots of soybean under salt stress [60]. Interestingly, the expression of the sulfur transporter AtSULTR3;1 was upregulated in response to drought and salt stress [61].

Soybean Stress Treatment
The surface-sterilized seeds of G. max (accession C08) were germinated in moist vermiculite supplemented with 50 mM or 100 mM NaCl treatments in the greenhouse under normal conditions. After 7 days, whole plant roots and the unifoliate leaves were collected for membrane proteome extraction and quantification. Three independent sets of control and NaCl-treated samples were collected, and each biological replicate represented a pooled sample of three individual plants.

Extraction of Membrane Proteins and Trypsin Digestion
Liquid-nitrogen-frozen leaf and root tissues (about 100 mg) were ground to a fine powder with a pestle and mortar, and membrane proteins were then extracted using a membrane extraction kit according to the manufacturer's protocol using plasma membrane extraction protocols (Cat#SM-005-P; Invent Biotechnologies, Plymouth, MN, USA). The extracted proteins were dissolved in 8 M urea buffer for further digestion and sample preparation.
Digestion of proteins was performed using SMART digest TM trypsin kit (60109-101; Thermo Scientific, Waltham, MA, USA) in solution. Protein reduction and alkylation were achieved with 10 mM dithiothreitol at 56 • C for 30 min followed by 25 mM iodoacetamide at room temperature for 25 min. The digested peptides were purified using Pierce TM C-18 spin columns (Thermo Scientific, Waltham, MA, USA) and finally dissolved in 0.1% formic acid (FA).
The Orbitrap was set up in a data-dependent MS/MS mode under direct control of the Xcalibur software (version 4.1), where a full-scan spectrum (from 375 to 1500 m/z) was followed by tandem mass spectra (MS/MS). The instrument was operated in positive mode with a spray voltage of 2 kV, a capillary temperature of 300 • C, and was calibrated before measurements. Full scans were performed in the Orbitrap with a resolution of 60,000 at 400 m/z, with a precursor ion selection (AGC > 4.0e5) and ion charge >1. Higher energy collisional dissociation, performed at the far side of the C-trap, was chosen as the fragmentation method, by applying a 30% value for normalized collision energy, an isolation window of m/z 1.6, with a maximum injection time of 250 milli seconds (ms) and Orbitrap resolution of 15,000.

Data Analysis and Interpretation
Proteome Discoverer (version 2.3; Thermo Scientific, Waltham, MA, USA), interfaced with an in-house SEQUEST server, was used for data processing, peptide identification, and protein inference, according to the following criteria: Ensembl database of G. max, Enzyme Trypsin, Maximum Missed Cleavage Sites: 2, Precursor Mass Tolerance: 10 ppm, Fragment Mass Tolerance: 0.2 Da, Cysteine Carbamidomethylation as static modification, N-terminal protein acetylation and methionine oxidation as dynamic modifications. The Percolator algorithm was used for peptide validation (peptide confidence: q-value < 0.01, corresponding to false discovery rate (FDR) < 0.01) and only Rank 1 peptides were considered. Peptide and protein grouping were performed using the strict maximum parsimony principle. Label-free quantification analyses using the Minora algorithm were performed with three biological replicates for each treatment throughout the whole proteome analyses. To be considered significantly differentially expressed, a protein had to be quantified with at least three peptides in each biological replicate, a p-value < 0.05, and a fold-change >0.5 for upregulated protein or <−0.5 for down-regulated proteins. The functional annotation of proteins was determined by The Database for Annotation, Visualization and Integrated Discovery (DAVID) and ShinyGo, and then grouped on the basis of their molecular functions, cellular components, and biological processes from Gene Ontology (GO) terms combined with information from the literature [3,26]. Domain analyses were performed using the InterPro website. Volcano plots of differentially expressed proteins were drawn using VolcaNoseR (https://huygens.science.uva.nl/VolcaNoseR/, accessed on 22 February 2022) and by plotting log 10 p-values on the Y-Axis against the log 2 fold-changes on the X-axis. The corresponding RNAseq-based gene expression values of significantly differentially expressed proteins were retrieved from [20] at 24 h and 48 h of salt treatment, and heatmaps were drawn using the heatmapper website.