Physiological and Proteomic Analyses Reveal Adaptive Mechanisms of Ryegrass (Annual vs. Perennial) Seedlings to Salt Stress

Ryegrass has a relatively high salt tolerance and is considered to be a promising species for both foraging and turf purposes in salt-affected soils in China. While annual ryegrass and perennial ryegrass are two different species, they have similar genomes. However, little is known about their physiological and molecular response mechanisms to salinity stress. Here, biomass, chlorophyll fluorescence, and inorganic ion and organic solute content were measured. 2-DE-based proteomic technology was then used to identify the differentially expressed proteins in the salt-treated seedlings. The results showed that salt stress reduced growth and photosynthesis in the seedlings of both species, but much more so in annual ryegrass. With increasing salinity, the Na+ concentration increased while the K+ concentration decreased in both species, and the sugars and proline increased as the primary organic solutes used to cope with osmotic stress. Additionally, proteomic analysis revealed 33 and 37 differentially expressed proteins in annual and perennial ryegrass, respectively. Most of the identified proteins were involved in carbohydrate and energy metabolism, photosynthesis, genetic information processes, amino acid metabolism, stress defense, and protein synthesis and folding. The results suggest that the two-ryegrass species had different physiological and proteomic responses. These findings can provide new insights into physiological mechanisms by which ryegrass species respond to salt stress.


Introduction
Annual ryegrass (Lolium multiflorum Lam.) and perennial ryegrass (Lolium perenne L.) are widely distributed in Europe, North Africa, and temperate Asia [1]. These two species have very similar genomes but have great differences in morphological characteristics and uses. For example, annual ryegrass can be grown together with other grass species to improve pasture quality [2,3]. Perennial ryegrass is also valued for high yield potential as forage grass, but this species can also be used for a variety of turf applications (e.g., home lawns, fairways, athletic fields, and golf courses) [4,5]. In addition, both ryegrasses have good salt tolerance and are considered promising in the bioremediation of salt-affected soil in northeastern China.
Hoagland nutrient solution was applied to the seedlings. The nutrient solution containing 5.00 mM Ca 2+ , 2.00 mM Mg 2+ , 6.04 mM K + , 22 [27]. When the seedlings were grown to 60 d, they were treated with stress treatments, and three salt concentrations of 0 mM (CK), 100 mM NaCl (S1), and 200 mM NaCl (S2) were applied, and the stress treatment was carried out for 7 d. The seedlings of annual ryegrass and perennial ryegrass were then harvested.

Fresh Weight and Dry Weight of the Seedlings
The harvest seedlings of ryegrass were washed with distilled water three times. The shoots and roots were separated, and the shoots were placed in an oven at 105 • C and then dried at 80 • C to reach a constant weight, and the fresh and dry weights of the seedlings were measured for each plant [28].

Chlorophyll Fluorescence
Chlorophyll fluorescence imaging of the seedlings in each treatment was measured with a pulse amplitude modulation chlorophyll fluorescence imaging system (Open FluorCam FC 800-O, Photon System Instruments, Brno, Czech Republic). The minimum fluorescence (F 0 ), maximum fluorescence (Fm), maximum photochemical efficiency (Fv/Fm), photosynthetic electron transport rate (ETR), actual photochemical efficiency of PS II (Φ PSII ), photochemical quenching coefficient (q p , and non-photochemical quenching (NPQ) were evaluated. These parameters were calculated as follows: where PPFD was the actinic light intensity, 0.5 was the proportion of light energy assigned to PS II, and 0.84 meant that 84% of the incident light was absorbed by seedlings [29]. All the treatments have three replicates.

Inorganic Ion Contents
Dry samples of plant material (25 mg) were treated with 10 mL deionized water at 100 • C for 1 h and the extract was taken to determine the inorganic ions contents. The extract was centrifuged and passed through a microporous membrane. Na + and K + contents were determined by an atomic absorption spectrometer (TAS-990, Purkinje General, Beijing, China) in three independent replicates.

Proline and Total Soluble Sugar Contents
Proline was extracted with 3% sulfosalicylic acid for 1 h, and the absorbance was measured with ninhydrin at 520 nm in three independent replicates per sample [30]. Total soluble sugars were extracted with absolute ethanol and freshly prepared anthrone reagent for 15 min, after which the absorbance was measured at 620 nm also in three independent replicates per sample [31].

Protein Extraction
The proteins were extracted using the phenol method [23]. Proteins were extracted from aliquots of 1 g FW samples taken from the control and 200 mM NaCl treated ryegrass seedlings. Frozen leaf tissue was ground in liquid nitrogen to a fine powder and incubated in ice-cold extraction buffer (from 0.1 M pH 8.0 Tris-HCL, 0.9 M sucrose, 10 mM EDTA, and 0.4% β-mercaptoethanol) overnight at −20 • C. After centrifugation, the precipitate was washed with ice-cold 80% acetone. Total protein was extracted in a lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT, 4% proteinase inhibitor cocktail, and 2% (v/v) pH 4-7 ampholytes). The subsequent supernatant was collected. Protein concentrations were determined with a 2D-Quant-Kit (GE Healthcare, Princeton, NJ, USA). The precipitate protein was stored at −80 • C for further analysis.

Two-Dimensional Electrophoresis
A total of 1300 µg of proteins in each sample was loaded on 24 cm IPG strip by rehydration bromophenol blue) loading at room temperature. Rehydration solution contains 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 20 mM DTT, 0.5% (v/v) pH 4-7 IPG Buffer, and 0.002% (v/v) bromophenol. After rehydration, isoelectric focusing was conducted in the following steps: 30 V for 8 h, 50 V for 4 h, 100 V for 1 h, 300 V for 1 h, 500 V for 1 h, 1000 V for 1 h, and 8000 V for 12 h. After IEF, the strips were incubated in equilibration buffer containing 0.05 M Tris-HCl pH 6.8, 2.5% SDS, 10% (v/v) glycerol, and 2% DTT, and shaken for 15 min, and then for another 15 min with the iodoacetamide replaced DTT equilibration buffer [32]. The second-dimension separation of proteins was performed on SDS-PAGE gel (12.5% polyacrylamide) using an Ettan™ Daltsix apparatus (GE Healthcare, Waukesha, WI, USA).

Gel Image Scanning and Protein Expression Abundance Analysis
The decolorized SDS-PAGE gel was placed on an Image Scanner III gel sweeper (GE Healthcare Waukesha, WI, USA) to complete the gel image scan. Image analysis was performed with ImageMaster 2D Platinum Software Version 7.0 (GE Healthcare, Waukesha, WI, USA). The molecular weight, relative abundance, and isoelectric point of each protein spot were obtained, and the differentially expressed protein between the control and stress treatment was analyzed for subsequent mass spectrometry analysis.

In-Gel Digestion and MALDI-TOF/TOF Analysis
Selected spots were excised from 2D gels, washed with sterile deionized water, and digested with trypsin as described previously [33]. Analysis was performed using MALDI TOF/TOF mass spectrometry (Applied Biosystems). By matching the parameters and setting the results, the information about the protein abundance change was obtained, and the peptide and family information were saved and integrated. After mass spectrometry identification, the obtained mass spectrometry results were searched by MASCOT Dameon software in the NCBI protein database. For the proteins identified by MASCOT, their accession numbers were directly searched against the NCBInr database (http://blast.ncbi. nlm.nih.gov/) and UniProt database (http://www.ebi.uniprot.org/) to obtain the proteins corresponding blast information. Non-commercial databases, KEGG (http://www.genome.jp/kegg/pathway.html), were utilized to search for protein functional classification. By matching the parameters and setting the results, the information about the protein abundance change is obtained, and the peptide and family information are saved and integrated.

Statistical Analysis
The experimental data processing was performed using SPSS software (Version 13.0, SPSS Inc., Chicago, IL, USA). One-way ANOVA was performed to test the differences between control and NaCl-treated seedlings of annual and perennial ryegrass by Tukey post hoc test. The differences were considered significant when p < 0.05.

Physiological Changes of Annual Ryegrass and Perennial Ryegrass under Salinity
With increasing salinity, both the fresh and dry weights of annual ryegrass and perennial ryegrass decreased (Table 1); however, the magnitude of the decreases were different. For example, the dry weight of annual ryegrass at 200 mM salinity decreased by 52.6% compared with the control group. However, the dry weight of perennial ryegrass only decreased by only 38.3% at the same salt concentration. With increasing salinity, the Fv/Fm, ΦPS II, NPQ, and q p values of annual ryegrass and perennial ryegrass seedlings generally decreased, but the decreases differed based on salinity and ryegrass species (Figure 1). For example, the Fv/Fm in the seedlings of annual ryegrass at the highest salinity (200 mM) decreased by 30% compared with the control group (p < 0.05). However, no significant change was found in the seedlings of perennial ryegrass at the 200 mM salt concentration ( Figure 1A,B). In addition, while the responses to changing salinities were similar, the ΦP SII and q p values in the seedlings of perennial ryegrass were higher than those in annual ryegrass under both control and high salinity conditions ( Figure 1C,D,G,H).
Logically, the Na + concentrations in seedlings of annual ryegrass and perennial ryegrass increased with the increasing salinity and were highest in the 200 mM salinity conditions (p < 0.05; Figure 2A,B). However, the K + concentrations decreased with increasing salinity. The K + concentrations decreased by 15.3% and 31.8% at the 100 and 200 mM treatments, respectively, for annual ryegrass, while they decreased by 20.8% and 21.1% in the same treatments for perennial ryegrass ( Figure 2C,D).
The seedlings of the two-species accumulated large amounts of organic solutes in the high salinity conditions. For example, proline content increased 15.2-fold and 21.3-fold at 200 mM salinity for annual and perennial ryegrass, respectively (p < 0.05; Figure 2E,F). The total soluble sugar content in annual ryegrass increased from 227.6 µmol/g to 478.7 µmol/g and increased from 250.0 µmol/g to 637.2 µmol/g in perennial ryegrass at 200 mM salinity (p < 0.05; Figure 2G,H).

Proteomic Analysis of Annual Ryegrass and Perennial Ryegrass under Salinity
To investigate the changes of proteome profiles in the seedlings of annual ryegrass and perennial ryegrass under salinity stress, we measured changes in expression of salt-responsive proteins using 2-DE gels. Differences were by comparing between the control (0 mM NaCl) and high salinity (200 mM NaCl) treatments in the seedlings of annual and perennial ryegrass. When a protein spot had an average fold change > 1.5 and a p-value < 0.05, it was considered to be differentially expressed. In this analysis, more than 709 protein spots in annual ryegrass and 854 protein spots in perennial ryegrass seedlings were reproducibly detected on gels using the Melanie 7.0 software. The results showed that 33 protein spots in the seedlings of annual ryegrass were differentially accumulated, while 37 proteins in the seedlings of perennial ryegrass were differentially expressed (Tables 2 and 3, respectively).
The 70 differentially expressed protein spots were submitted for identification by MALDI-TOF/TOF. The information on the mass spectrometry analysis was showed in Supplementary Material 1. Through peptide matching in the MASCOT database ( Figure 3; Figures S1 and S2). Compared with the control group, of the 33 differentially accumulated proteins in annual ryegrass seedlings, 5 proteins were up-regulated and 28 proteins were down-regulated (Table 2). For the 37 differentially accumulated proteins of perennial ryegrass seedlings, 6 proteins were up-regulated under high salinity conditions and 31 proteins were down-regulated (Table 3).         Figure 4A. b Database access ion numbers according to NCBInr. c The plant species that matched the peptides. d The oretical mass (kDa) and pI of identified proteins. The oretical values were retrieved from the protein database. e Mascot score reported after searching against the NCBInr database. f The amino acid sequence coverage for the identified proteins. g The number of unique peptides identified for each protein.   Figure 4C. b Database access ion numbers according to NCBInr. c The plant species that matched the peptides. d The oretical mass (kDa) and pI of identified proteins. The oretical values were retrieved from the protein database. e Mascot score reported after searching against the NCBInr database. f The amino acid sequence coverage for the identified proteins. g The number of unique peptides identified for each protein.
carbohydrate energy metabolism (10), photosynthesis (5), genetic information processes (2), amino acid metabolism (6), stress defense (7), protein synthesis and folding (4), signal transduction (1), and other pathways (2) ( Figure 4B). The results showed that there were six proteins shared in common between annual and perennial ryegrass, three of which had opposite accumulation patterns in the two species ( Figure 4C), which indicated that annual ryegrass and perennial ryegrass have different mechanisms by which they respond to salt stress.  The characters of these proteins were determined based on BLAST analyses using the NCBInr database. The differentially accumulated proteins from the annual ryegrass seedlings were divided into seven functional groups based on their roles in different metabolic pathways. The proteins were grouped into nine categories based upon their biochemical functions, including carbohydrate energy metabolism (9), photosynthesis (11), genetic information processes (3), amino acid metabolism (1), stress defense (5), protein synthesis and folding (3), and others (1) ( Figure 4A). The same grouping classifications were used for perennial ryegrass, and the categories included carbohydrate energy metabolism (10), photosynthesis (5), genetic information processes (2), amino acid metabolism (6), stress defense (7), protein synthesis and folding (4), signal transduction (1), and other pathways (2) ( Figure 4B). The results showed that there were six proteins shared in common between annual and perennial ryegrass, three of which had opposite accumulation patterns in the two species ( Figure 4C), which indicated that annual ryegrass and perennial ryegrass have different mechanisms by which they respond to salt stress.

Physiological Response of Annual and Perennial Ryegrass under Salinity Stress
The seedling growth period is one of the most critical periods in the life cycle of plants and can be especially sensitive to stressful conditions [18]. Salinity in the soil can cause low water potential, destroy plant tissues and structures, and strongly affect seedling growth [34]. In the present study, the seedling weights in both species decreased with increasing salinity, but the decrease was greater in the dry weight of annual ryegrass at 200 mM salinity (Table 1), which indicated that salt tolerance of the two ryegrasses were different. Photosynthesis is considered to be one of the clearest indicators that plants are experiencing physiological stress [35]. In addition, the chlorophyll fluorescence parameters are particularly useful as they reflect the photosynthetic characteristics of plants [36]. In our fluorescence experiments the Fv/Fm value did not change much at the highest salinity (200 mM) in perennial ryegrass, but significantly decreased in annual ryegrass ( Figure 1A,B), suggesting that the photosynthetic electron transport of annual ryegrass was more inhibited than perennial ryegrass under salt stress. Moreover, compared with annual ryegrass, the perennial ryegrass also had higher ΦPSII and q p values under high salinity conditions. Plants living in high salinity conditions can accumulate toxic amounts of Na + , while at the same time they are prevented from absorbing K + , which can induce metabolic disorders in plants [37]. In this study, both species accumulated high levels of Na + , while the K + concentration decreased ( Figure 2). Similar results were found in wheat by Li et al. [38] and barley by Yang et al. [39]. Under such conditions, most plants can accumulate proline and soluble sugars in the protoplasm to maintain their osmotic balance. In this study, the annual ryegrass and perennial ryegrass were also able to accumulate osmotic adjustment solutes, including proline and soluble sugars, to compensate for the osmotic imbalance, which may be related to the excess Na + . Notably, the proline and soluble sugar content in perennial ryegrass seedlings were much higher than those in annual ryegrass seedlings. These results indicated that the perennial ryegrass was able to respond more positively to salt stress than annual ryegrass.

Carbohydrate and Energy Metabolism
In this study three metabolic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, spot 112), fructose bisphosphate aldolase (FBA, spot 337), and phosphoglycerate mutase (PGM, spot 186), involved in the glycolysis pathway, were differentially accumulated in annual ryegrass seedlings ( Table 2). GAPDH can catalyze the conversion of glyceraldehyde-3-phosphate to 1, 3-diphosphoglycerate, which plays an important role in the plant sugar metabolism pathway [17]. PGM plays a significant role in glycolysis, which catalyzes phosphate from 3-phosphoglyceric acid to a second carbon to form 2-phosphoglyceric acid. The results clearly showed that the abundance of these two proteins (GAPDH and PGM) increased in the annual ryegrass under salt stress, indicating that this grass species was able to provide more intermediate metabolites through glycolysis in response to salinity stress.
For perennial ryegrass, the metabolic enzymes fructose bisphosphate aldolase (FBA, spot 515), pyruvate dehydrogenase (PDH, spot 517), phosphoglycerate kinase (PGK, spot 532), and sucrose synthase (SS, spot 567), involved in glycolysis pathway, were all down-regulated under salt stress (Table 3). FBA can catalyze the conversion of fructose-1, 6-diphosphate to dihydroxyacetone phosphate. In the present study, the isoform of aldolase is cytosolic. PGK mainly catalyzes the formation of glycerophosphates from 1, 3-diphosphoglycerate, key enzymes in the first two stages of the glycolysis pathway. The reduced abundance of these proteins indicated that the glycolysis pathway of perennial ryegrass was inhibited by salinity stress. In addition, PDH catalyzes the oxidative decarboxylation of pyruvate to produce acetyl-CoA, NADH, and CO 2 . SS can catalyze the reversible reaction of sucrose metabolism and is also a key enzyme that promotes sucrose into various metabolic pathways, such as glycolysis. The reduced abundance of these proteins indicated that perennial ryegrass had insufficient material for glycolysis and was inhibited from utilizing carbohydrates to meet energy demands.
Plants require large amounts of ATP to provide sufficient energy for their growth and development under high salinity conditions [40]. In this study, we found that the accumulations of ATP synthase in annual (spot 338) and perennial ryegrass (spot 479 and 553) were reduced under salinity stress (Tables 2 and 3). This result indicated that ATP synthesis in both annual and perennial ryegrass was inhibited by salt stress.
The TCA cycle provides essential precursors for amino acid biosynthesis and energy metabolism, and also plays a key role in response to abiotic stressors [41]. Succinate dehydrogenase (SDH), involved in mitochondrial electron transport, is responsible for transferring electrons from succinic acid to coenzyme Q. This enzyme plays a vital role in the TCA cycle pathway and is also capable of integrating multi-subunit enzymes on the inner mitochondrial membrane [42]. It can provide electrons and oxygen to the respiratory chain of cell production, which is a part of carbohydrate metabolism. Here, we found that the abundance of SDH under salt stress in annual ryegrass (spot 349) was reduced ( Table 2) but showed no change in perennial ryegrass (Table 3). This result suggested that TCA cycle activity was decreased in annual ryegrass, but unchanged in perennial ryegrass. Similarly, some other studies have reported that decreased TCA cycle activity was related to reduce consumption of organic acids, which might be a strategy to save carbon structures for the synthesis of compounds required to endure stressful conditions [43]. Therefore, the reduction of TCA cycle activity in annual ryegrass might benefit the synthesis of substances used for osmotic regulation in order to cope with salt stress.
Some proteases related to the pentose phosphate pathway are capable of maintaining cellular homeostasis. They can provide amino acids and nucleotide biosynthesis prerequisites for plants, and also reduce molecules against oxidative stress [44]. We found that ribulose-phosphate 3-epimerase (RPE, spot 327), ribose-5-phosphate isomerase 3 (RPI3, spot 328), and S-adenosylmethionine synthetase (SAMS, spot 341) were reduced in abundance in annual ryegrass under salt stress (Table 2). RPE and RPI3 catalyze the reversible epimerization of D-ribulose 5-phosphate to D-xylulose 5-phosphate, which are key members of the pentose phosphate pathway. SAMS can catalyze the reaction of ATP and methionine to form S-adenosylmethionine, which is an important enzyme in both plant metabolism, and responses to stress [45]. The abundance of these proteins was reduced in annual ryegrass, which indicated that salt stress destroyed cell homeostasis of annual ryegrass and caused oxidative damage ( Figure 5). In addition, we also found that the abundance of trans ketolase (TK, spot 324 and 562) was reduced in perennial ryegrass (Table 3). TK is a key enzyme in the non-oxidative branch of the pentose phosphate pathway. Its accumulation promotes the ability of non-oxidative branches to produce ribonucleic acids and amino acids via NADPH production [46]. This result indicated that salt stress disrupted the redox balance of perennial ryegrass. Figure 5. Schematic view of differentially accumulated proteins involved in key metabolic pathways in annual and perennial ryegrass seedlings under salinity stress. Each protein is represented by an ellipse, the protein with increased abundance is red, and the protein with reduced abundance is green. FBA, fructose-bisphosphate aldolase; PKG, phosphoglycerate kinase; OEC, oxygen-evolving complex; OEE, oxygen-evolving enhancer protein; CA, carbonic anhydrase; RuBisCo, ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate; GADPH, glyceraldehyde3-phosphatedehydrogenase; EF-Tu, elongation factor Tu; TCA cycle, tricarboxylic acid cycle; SDH, succinate dehydrogenase; GST, glutathione S-transferase.

Photosynthesis
One previous study has shown that photosynthesis of plants was greatly inhibited by salt stress as chlorophyll biosynthesis was reduced along with photosystem II efficiency [47]. Our proteomic analysis results showed that three photosynthetic proteins were differentially accumulated in annual ryegrass under salt stress. Chlorophyll a-b binding protein (spot, 324) and photosystem II extrinsic subunit (spot 325) were decreased in annual ryegrass under salt stress (Table 2). Chlorophyll a-b binding protein acts as a light-harvesting complex (LHC) that captures and excites energy transfer to the optical system, which helps PS II convert more light energy and reduce energy loss caused by reductions in PS II efficiency [48]. The photosystem II extrinsic subunit is involved in stabilizing the structure of the photosystem II oxygen-evolving complex (OEC), the ion environment of oxygen evolution, and protects the OEC against heat-induced inactivation. The reductions in protein abundances indicated that the activity of the photosystem II was inhibited in annual ryegrass under salinity stress. In addition, we found that the photosynthesis NDH subunit complex B1 (spot 345), associated with the electron transport chain, was also reduced in annual ryegrass under salt stress ( Table 2). The reduced abundance of this protein may mean that photosynthetic electron transport was inhibited in annual ryegrass, potentially due to the salt stress destroying a part of the photosystem in this grass, leading to impaired molecular function in some proteins. However, the expression of these proteins associated with the photosystem II and electron transport chain showed no change in perennial ryegrass ( Figure 5). These results are consistent with the Fv/Fm values of the chlorophyll fluorescence parameters in this study, which indicated that the photosynthetic electron transport in annual ryegrass was inhibited more than in perennial ryegrass under salt stress.
Moreover, we found that the accumulation of proteins associated with the Calvin cycle in annual ryegrass all decreased under salt stress, including ribulose bisphosphate carboxylase/oxygenase activase (RuBisCO activase, spot 340), ribulose 1, 5-bisphosphate carboxylase/oxygenase small chain (spot 315 and 316), ribulose 1, 5-bisphosphate carboxylase/oxygenase large chain (spot 317 and 330), RuBisCO large subunit-binding protein subunit beta (spot 308), and carbonic anhydrase (spot 326, Table 2). RuBisCO activase is a molecular chaperone that controls the switching of Rubisco from the inactive to active conformation and is also a key enzyme that affects photosynthetic rate [49]. Carbonic anhydrase (CA) plays an important role in carbon dioxide absorption and carbon fixation. It does this by improving the activity of carbon-fixing related enzymes and increasing the concentration of inorganic carbon ions near carboxylase, thereby enhancing the carbon fixation rate [50]. Hence, the reduced abundance of these proteins indicated that the Calvin cycle in annual ryegrass was significantly inhibited under salt stress. However, we found that the abundances of the RuBisCO large subunit-binding protein subunit beta (spot 278), ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (spot 480), and ribulose bisphosphate carboxylase large chain (spot 535) were significantly increased abundance in perennial ryegrass under salt stress (Table 3). RuBisCO large subunit-binding protein subunit beta is able to bind and participate in the assembly process of RuBisCO and maintain carbon fixation in plants. The increased abundance of these proteins in perennial ryegrass indicated that the perennial ryegrass may promote the formation of RuBisCO to maintain its carbon fixation rate in response to salt stress ( Figure 5).
Thus, the photosynthetic capacities of annual and perennial ryegrass were both decreased under salt stress. However, perennial ryegrass was able to maintain relatively high photosynthesis capabilities compared to annual ryegrass by enhancing the rate of photosynthetic electron transport and Calvin cycle activity, which can provide more photo-assimilates, helping to cope with salt stress (Figure 1). These results showed that the adaptive strategies regarding photosynthesis were different in annual and perennial ryegrass under salt stress.

Genetic Information Processes and Protein-Destiny
Elongation factor catalyzes amino acids on the ribosome, allowing them to extend and participate in the regulation of protein synthesis. In this study, we found that the abundance of elongation factor Tu (spot 343 and 344, Table 2) in annual ryegrass, and the elongation factor G (spot 564, Table 3) and elongation factor 2 (spot 579, Table 3) in perennial ryegrass were all reduced under salt stress, indicating that salt stress inhibited the protein synthesis of both annual and perennial ryegrass.
Protein folding in annual and perennial ryegrass was also affected by salt stress. The abundance of the protein translocase subunit SecA (spot 351, 354), associated with protein synthesis, was decreased in annual ryegrass under salt stress (Table 2). While the abundances of protein disulfide isomerase (PDI, spot 547, 550) and chaperone ClpC2 (spot 572), associated with protein fate, were decreased in perennial ryegrass ( Table 3). The protein translocase subunit SecA is associated with membrane transport devices and is free in the cytosol for use in protein localization and transport [51]. The decreased abundance of this protein may affect protein synthesis in annual ryegrass. Protein disulfide isomerase can catalyze the rearrangement of disulfide bonds in proteins, prevent aggregation of denatured proteins, and participate in metabolic processes [52]. The chaperone protein ClpC2 plays an important role in protein metabolism and is mainly involved in protein modification and chemical reaction pathways. In addition, 30S ribosomal protein S1 (spot 339, Table 2) in annual ryegrass and 50S ribosomal protein (spot 491, Table 3) in perennial ryegrass were both decreased under salt stress. This is similar to previous reports that have shown that ribosomal proteins were down-regulated under osmotic stress and heat stress [53,54]. In general, the abundances of these proteins were decreased under salt stress, affecting protein folding and synthesis in annual and perennial ryegrass.
In addition, we found that 14-3-3-like protein GF14-F (spot 507), which is associated with signal transduction, was decreased in perennial ryegrass under salt stress. Previous studies have shown that 14-3-3 protein played a key role in plants responding to salt stress. It participated in the stress-activated ABA signaling pathways which activate expression of some important genes that enhance stress tolerances of plants [55]. The reduced abundance of this protein in perennial ryegrass indicated that salt stress affected the signal transduction of this species. In summary, the genetic information processes related to protein fate in perennial ryegrass was significantly inhibited ( Figure 5).

Amino Acid Metabolism and Stress Defense
Amino acid biosynthesis and metabolism is of critical importance for plant growth, development, and response to stress condition [56,57]. Our results reflected that the proteins associated with cellular processes (including amino acid metabolism and cellular structure) were differentially accumulated in annual ryegrass and perennial ryegrass under salt stress. Glutathione-S-transferase (GST) is a key enzyme in the glutathione binding reaction, which catalyzes the conjugation of reduced glutathione to a wide range of substrates. Glutathione plays an important role in response to environmental stress in plants, such as oxidative stress caused by the generation of reactive oxygen species (ROS) [58]. In this study, the abundance of glutathione-S-transferase (GST, spot 63) was reduced in annual ryegrass under salt stress ( Table 2), indicating that the ROS have scavenging ability, i.e., the oxidative stress response of annual ryegrass was inhibited by salt stress.
For perennial ryegrass, proteins associated with amino acid metabolism including GTP-binding nuclear protein (spot 61), tubulin alpha-4 chain (spot 534), glutamine synthetase (spot 162), serine/threonine protein phosphatase (spot 549), and nucleoside diphosphate kinase (spot 476) were all reduced in abundance under salt stress ( Table 3). The tubulin alpha-4 chain is a protein associated with the cytoskeleton that can be involved in the division and elongation of plant cells [59]. The abundance of this protein in perennial ryegrass decreased, indicating that salt stress could damage the cytoskeleton and potentially lead to the inhibition of plant growth. GTP-binding proteins are involved in a variety of life processes in cells, such as cell communication, ribosome binding, and protein synthesis [60]. The reduced abundance of this protein, therefore, may have inhibited the amino acid metabolism of perennial ryegrass under salt stress. However, these proteins were not differentially accumulated in annual ryegrass. Overall, the proteomic data pertaining to amino acid metabolism revealed that protein synthesis and protein processing were only inhibited in perennial ryegrass under salt stress.
Moreover, the abundances of several proteins associated with stress defense were also differentially accumulated in annual and perennial ryegrass. The abundance of 2-methylene-furan-3-one reductase (spot 310, Table 2; 130, Table 3), associated with oxidoreductase activity, was reduced in both annual and perennial ryegrass. This protein can utilize NADH and NADPH as electron donors to catalyze redox reactions ( Figure 5). We also found that abundance of 2-Cys peroxide reductase BAS1 (spot 312) in annual ryegrass was decreased ( Table 2). 2-Cys peroxide reductase BAS1 is a nuclear-encoded chloroplast protein, which plays an important role in the detoxification of peroxides [61]. Therefore, this result indicated that the ability of stress defense in annual ryegrass was weakened by salt stress. Thiamine thiazole synthase (spot 510) catalyzes the transfer of conserved cysteine residues of proteins to thiazole intermediates, which are involved in the biosynthesis of the thiamine precursor thiazole ( Table 3). The expression of this protein was decreased in perennial ryegrass under salt stress, indicating that salinity stress affected the stress defense ability in perennial ryegrass. In addition, the abundance of annexin (spot 102) was decreased in perennial ryegrass, indicating that salt stress affected the cell secretory process of this species (Table 3). However, this protein was not differentially accumulated in annual ryegrass. The abundance of germin-like proteins 8-14 (GLPs, spot 320) was also affected by salt stress as it decreased in annual ryegrass ( Table 2). A number of GLPs have been reported to be associated with abiotic stresses, as they were able to remove excess ROS and enhance cell wall structures to alleviate oxidative damage [62]. These results indicated that salt stress enhanced cellular response to oxidative damage in annual ryegrass seedlings.
For perennial ryegrass, we found that the abundance of RNA polymerase II transcriptional subunit 37e (spot 559, 560) was reduced (Table 3). This protein catalyzes ribonucleic acid transcription, and the synthesis of mRNA, hnRNA, and microRNA precursors [63,64]. In addition, we found that the abundance of auxin-binding protein ABP19a were up-regulated in annual (spot 318 and 322) and perennial ryegrass (spot 485) under salt stress (Tables 2 and 3). Auxin-binding proteins (ABPs) are a class of low abundance proteins in plants that bind active auxins with high specificity and affinity. It is possible that ABP could initiate the auxin signal pathways, leading to various cellular responses through ABP-auxin binding, which would thus accord it with a plant hormone receptor function [65]. The ABP19a is located on the plasma membrane and binds to auxin to activate the auxin signal pathways and regulating plant cell formation [66]. The up-regulated of ABP-19a in annual and perennial ryegrass under salt stress indicated that in ryegrass the auxin signal pathways are activated both in annual and perennial ryegrass to response salt stress, which could alleviate the growth inhibition caused by salt stress.

Conclusions
Although annual ryegrass and perennial ryegrass are similar in many regards, even at the genomic level, they have very different uses (annual ryegrass for foraging and perennial ryegrass for turf purposes). In brief, this study reported, for the first time, the physiological and molecular response mechanisms of these two-ryegrass species to salt stress during the seedling stage. The results clearly showed that salt stress reduced growth and photosynthesis in the seedlings of both species, but annual ryegrass was inhibited to a larger degree. In addition, proteomic analysis revealed 33 and 37 differentially accumulated proteins in annual and perennial ryegrass, respectively, under salinity stress. Under salt stress, TCA cycle, pentose phosphate pathway, genetic information process, and amino acid metabolism were all decreased both in annual and perennial ryegrass. In addition, glycolysis, ATP synthesis, and photosynthesis reflected significant differences between annual and perennial ryegrass in response to salt stress. These findings provide new insights into physiological mechanisms by which annual ryegrass and perennial ryegrass respond salt stress.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/9/12/843/s1, Supplementary Material 1: Mass Spectrometry Analysis Results; Figure S1: Map of annual ryegrass (A, B) and perennial ryegrass (C, D) protein spots. Representative 2-DE gels of protein samples from annual ryegrass and perennial ryegrass seedlings without salinity treatment (A, C) and 200 mM NaCl treatment (B, D). The statistically significant differential spots are labeled with arrows and match IDs, red and green arrows indicate increased and reduced abundance of protein spots, respectively.; Figure S2: Map of annual ryegrass (A, B) and perennial ryegrass (C, D) protein spots. Representative 2-DE gels of protein samples from annual ryegrass and perennial ryegrass seedlings without salinity treatment (A, C) and 200 mM NaCl treatment (B, D). The statistically significant differential spots are labeled with arrows and match IDs, red and green arrows indicate increased and reduced abundance of protein spots, respectively.
Author Contributions: X.P. did the experiment and wrote the manuscript. D.Y. analyzed the data. J.Y. did a part of experiment. N.Z. gave some good suggestions on the manuscript. J.L. designed this experiment and revised the manuscript. J.W. revised the manuscript and provided the funds support.