Comparative proteomic analyses reveal the proteome response to short-term drought in Italian ryegrass (Lolium multiflorum)

Drought is a major abiotic stress that impairs growth and productivity of Italian ryegrass. Comparative analysis of drought responsive proteins will provide insight into molecular mechanism in Lolium multiflorum drought tolerance. Using the iTRAQ-based approach, proteomic changes in tolerant and susceptible lines were examined in response to drought condition. A total of 950 differentially accumulated proteins was found to be involved in carbohydrate metabolism, amino acid metabolism, biosynthesis of secondary metabolites, and signal transduction pathway, such as β-D-xylosidase, β-D-glucan glucohydrolase, glycerate dehydrogenase, Cobalamin-independent methionine synthase, glutamine synthetase 1a, Farnesyl pyrophosphate synthase, diacylglycerol, and inositol 1, 4, 5-trisphosphate, which might contributed to enhance drought tolerance or adaption in Lolium multiflorum. Interestingly, the two specific metabolic pathways, arachidonic acid and inositol phosphate metabolism including differentially accumulated proteins, were observed only in the tolerant lines. Cysteine protease cathepsin B, Cysteine proteinase, lipid transfer protein and Aquaporin were observed as drought-regulated proteins participating in hydrolysis and transmembrane transport. The activities of phospholipid hydroperoxide glutathione peroxidase, peroxiredoxin, dehydroascorbate reductase, peroxisomal ascorbate peroxidase and monodehydroascorbate reductase associated with alleviating the accumulation of reactive oxygen species in stress inducing environments. Our results showed that drought-responsive proteins were closely related to metabolic processes including signal transduction, antioxidant defenses, hydrolysis, and transmembrane transport.


Introduction
Drought is the largest abiotic stress factor leading to reduce the productivity of Italian ryegrass, especially at the seedling stage. The effect of drought stress on plants is usually characterized by reduced leaf water content, decreased cell growth, and inducted oxidative stress [1,2] ability of plants to acclimate to such conditions through appropriate regulation is a key determinant of their survival [3]. Recent research has clearly demonstrated that stress responses rely on the functioning of complex gene networks. When plants regularly experience drought, extensive modification of gene accumulation occurs and results in alterations in protein synthesis (up-or down-regulation) [4]. These proteins react to stress by regulating metabolic homeostasis and detoxifying harmful elements such as reactive oxygen species (ROS) [5,6]. Hence, identification of responsive proteins involved in drought tolerance is a major interest to plant scientists. Early proteome analyses used two-dimensional gel electrophoresis for protein separation and tandem mass spectrometry for protein identification [7]. Out of a total of 455 proteins identified, 17 differentially accumulated proteins existed in L. multiflorum and F. arundinacea introgression lines [8]. With the rapid innovations in proteomics new methods have been developed for protein analysis. The iTRAQ (Isobaric tags for relative and absolute quantification) is a quantitative proteomic method for examining multiple samples in a single mass analysis, thereby enable sensitive assessment and quantification of protein levels [9,10]. Based on the iTRAQ approach, by comparing tolerant and susceptible cultivars, many proteins were discovered that had the potential to enhance resistance in plants [11,12].
Italian ryegrass (Lolium multiflorum L.) is one of most widespread cultivated cool-season forage grass in the world. Typically, it is grown in a mixture with other grass and legume species to improve pasture quality [13]. In southern China, L. multiflorum is most commonly served as an annual forage crop for feeding [14]. Although Italian ryegrass expresses some levels of drought tolerance, it still suffers a significant reduction of yield under drought conditions [15], and does not match F. arundinacea with respect to the potential of tolerance [16]. This potential, however, can be significantly improved in intergeneric L. multiflorum x F. arundinacea hybrids, and their introgression derivatives [8,17].
However, there have been few reports on the regulatory mechanisms of drought tolerance at proteome level for Italian ryegrass. By using the iTRAQ-based method, two L. multiflorum lines, drought-tolerant "Abundant 10" and drought susceptible "Adrenalin 11" were used in the study to evaluate differentially accumulated proteins under drought stress. This study provides a novel proteomic data for further dissection the regulatory mechanisms of drought tolerance in Lolium multiflorum response to short-term drought.

Plant materials and drought treatments
Two L. multiflorum lines, drought-tolerant "Abundant 10" and drought susceptible "Adrenalin 11" were used in this study [18]. Seeds were germinated on filter paper moistened with distilled water in an environment kept at 25˚C.Seedlings were then transferred into plastic pots filled with the Hoagland's nutrient solution and put into growth chambers with a 16/8 hour daynight cycle, a 25/18˚C day-night temperature, and relative humidity of 60%.
One half of the 20-day-old seedlings of the two L. Multiflorum lines were grown in aerated hydroponics containing Hoagland's nutrient solution at 25˚C to be used as the control and the remaining seedlings were treated under drought stress condition, by lying on plastic trays and naturally air-drying for 2 hours at 25˚C in the growth chamber. Ten individual plants for each L. Multiflorum line were used as biological replicate. We performed two biological replicates for each treatment in the experiment (S1 Fig). Therefore, 20 drought tolerant seedlings under control, 20 drought tolerant seedlings with drought stress treatment, 20 drought susceptible seedlings under control, and 20 drought susceptible seedlings with drought stress treatment, were immediately frozen in liquid nitrogen, and stored at -80˚C until protein extraction. quartz tip as the emitter (New Objectives, Woburn, MA, USA). Data was acquired using an ion spray with 2.5 kV voltage and, curtain gas was set at 30 psi, nebulizer gas was set at 15 psi, and the interface heater temperature was 150˚C. The MS was operated by a resolving power (RP) of 30,000FWHM for TOF/MS scans. Survey scans were obtained from 250 ms and up to 30 product ion scans (cut-off threshold was 120 counts per second (counts/s). Raw data files were transformed into MGF files using Proteome Discoverer software.

Protein identification and data analysis
The Mascot 2.3.02 search engine (Matrix Science, London, UK; version 2.3.02) was used to identify and quantify proteins. To identify proteins the following parameters were set: 1) a mass tolerance of 2 Da (ppm) was permitted for intact peptide masses, 2) the Peptides matching error was set at 0.05 Da, 3) Gln-> pyro-Glu (N-term Q), Oxidation (M), Deamidated (NQ) were set as potential variable modifications, 4) Carbamidomethyl (C), iTRAQ8plex (Nterm), iTRAQ8plex (K) were set as fixed modifications, and 5) the charge states of the peptides were set to +2 and +3. An automatic decoy database search can be performed by choosing the decoy checkbox to produce a random sequence database and to test for raw spectra.
A 95% confidence interval was used to identify peptide using the Mascot probability analysis. Each protein was identified by at least one unique peptide and each protein should contain at least two unique spectra. The protein sequence database (NCBInr (http://www.ncbi.nlm.nih. gov), SwissProt (http://www.ebi.ac.uk/swissprot), and UniProt (http://www.uniprot.org) was used for the protein identifications. The quantitative protein ratios were measured and normalized in Mascot. A 2-fold change, statistical P-values < 0.05 and false discovery rate (FDR) < = 0.01 were used as criteria for identifying differentially accumulated proteins. Functional annotations of identified proteins were performed using Blast2GO against the Non-redundant protein database (NR). The KEGG database and the clusters of orthologous groups (COG) database were applied to classify the identified proteins. The data including the number of identified peptides, peptides masses, peptides sequence, and peptides scores were provided in S1 Table. Western blotting For each protein sample 10 ug was loaded on SDS PAGE gel (1.5 mm). Migration of proteins in the PAGE gel was conducted at 150 V until the blue band from the sample buffer run out of the gel. Protein-Marker IV was also loaded to determine the molecular weight of the proteins, Proteins were then transferred onto a Polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The following antibodies were used in the western blot analysis: actin (ACT), ADP-glucose pyrophosphorylase (ADGP), β-amylase, isocitrate lyase (ICL), aquaporin, plasma membrane intrinistic protein 1-3 (PIP1), tonoplast intrinsic protein 1-1(TIP1), heat shock protein 90 (HSP90), dehydroascorbate reductase (DHAR1), and alpha-amylase (from Agrisera, Sweden). The PVDF membrane was probed with primary antibody and developed using enhanced chemilu-minescence detection (PerkinElmer, Waltham MA, USA). The blots were detected using the BeyoECL plus (P0018). The images were obtained with the ChemiDoc TM MP imaging system, and the quantifications were conducted with the software Image Lab TM V5.1.

Changes in activities of antioxidative enzymes in drought treated L. multiflorum
To investigate the effects of drought stress on the oxidative stress, the two L. multiflorum lines under control and drought treatment were tested for H 2 O 2 content, enzyme activities of PHGPx, Prx and APX. Under drought stress, H 2 O 2 content in both L. multiflorum lines showed dramatically increase compared with control seedlings (Fig 1A), especially in susceptible. A higher enzymatic activities of hospholipid PHGPx, Prx and APX were observed in the tolerant lines exposed to drought stress than under control (Fig 1B-1D). These results indicated that antioxidant enzymes contributed to alleviated oxidative stress-triggered ROS accumulation in tolerant lines during short-term drought stress treatment.

Differential accumulation analysis of L. multiflorum proteome under drought stress
Drought stress-induced changes in the proteome of the two L. multiflorum lines were captured by analyzing quantitative information generated by iTRAQ-based quantitative analysis and LC-MS/MS method. A total of 7, 089 unique peptides matching to 2, 808 proteins were identified with a Mascot probability analysis (Fig 2A). A differentially accumulated analysis revealed that 449 proteins were up-regulated and 501 were down-regulated ( Fig 2B). These proteins were categorized into biological process and molecular function based on blast2 GO program (Fig 3). The drought-regulated proteins were primarily related to metabolic process, cellular process, single-organism process and response to stimulus, which play a role in regulating the The proteome response to short-term drought in Italian ryegrass (Lolium multiflorum) catalytic activity, structural molecule activity, transporter activity, electron carrier activity and antioxidant activity ( Table 1).
Identification of proteins differentially accumulated in the two L. multiflorum Lines response to drought stress A total of 51 drought-induced proteins were obtained from both tolerant and susceptible L. multiflorum proteomes (Table 1). Of them, 27 up-regulated and 16 down-regulated proteins having the same change trends were observed in the two L. multiflorum lines, of which up-regulated proteins associated with carbohydrate metabolism and proteolysis, while down-regulated proteins mostly participated in translation and transmembrane transport. Based on COG categories, the majority of shared proteins participated in regulation of carbohydrate transport and metabolism, post translational modification, protein turnover, translation, chaperones, ribosomal structure and biogenesis, and energy production and conversion. Comparison of the differentially accumulated proteins in the two lines identified eight responsive proteins  The proteome response to short-term drought in Italian ryegrass (Lolium multiflorum) The proteome response to short-term drought in Italian ryegrass (Lolium multiflorum) having an opposite trend in the tolerant and susceptible lines, among them, three proteins as the important antioxidative enzymes, peroxiredoxin 2, peroxiredoxin 5 and dehydroascorbate reductase involved in oxidation-reduction processes were specifically identified in the tolerant lines. Other proteins, glycerate dehydrogenase (GDH), dehydroascorbate reductases (DHAR), glucose-1-phosphate (G1P) and lipid transfer protein (LTP), were also only up-regulated in the tolerant lines, but not susceptible line. The results not only revealed that drought tolerance of Italian ryegrass had a direct link to the specific proteins induced by drought stress, but also provided evidence that carbohydrate metabolism, oxidation-reduction processes, proteolysis, and transmembrane transport had a significant relationship with drought tolerance of Italian ryegrass.

Network analysis for drought responsive proteins
Proteins in plant cells and subcellular fractions play interrelated roles together in the context of networks [19]. Within the differentially accumulated proteins (susceptible vs. tolerant), a total of 76 proteins were identified using the Cytoscape software that can be mapped onto an interaction network (Table 2; Fig 4). The left region showed that responsive proteins involved in carbohydrate metabolism-related pathways, e.g. fructose and mannose metabolism, glycolysis and gluconeogenesis, citrate cycle (TCA cycle), starch and sucrose metabolism, fructose and mannose metabolism, pentose and glucuronate interconversions, galactose metabolism, pointing to a potential importance of carbohydrates in modulating the homeostasis of the drought response of Italian ryegrass. The middle part revealed that drought-related proteins enriched in nine amino acid metabolic pathways including tyrosine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, cysteine and methionine metabolism, arginine The proteome response to short-term drought in Italian ryegrass (Lolium multiflorum) The proteome response to short-term drought in Italian ryegrass (Lolium multiflorum) The proteome response to short-term drought in Italian ryegrass (Lolium multiflorum) The proteome response to short-term drought in Italian ryegrass (Lolium multiflorum) and proline metabolism, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, tryptophan metabolism, valine, leucine and isoleucine degradation. The amounts of special proteins involving both of the two pathways were observed in the right side. Interestingly, a predicted protein (CL16151.Contig2) existed in tryptophan metabolism, valine, leucine, lysine, and isoleucine degradation, and butanoate and propanoate metabolism. Furthermore, a special pathway with all the differentially accumulated protein, inositol phosphate metabolism, was only found in tolerant lines, and phosphoinositide-specific phospholipase C1 (PLC) showed obviously up-regulated in phosphatidylinositol signaling systems in tolerant lines subjected to drought condition.

Discussion
Drought-responsive proteins related to tolerance to oxidative stress The overproduction of reactive oxygen species (ROS) occurs when plants suffer from drought stress [20]. To prevent damage caused by ROS, plants can synthesize antioxidants, such as ascorbate, glutathione, and flavonoids, and enhance antioxidative enzymes [21]. Tolerance to abiotic stresses is involved with the ROS scavenging capacity in plants [22]. Phospholipid PHGPx is an antioxidant enzyme that directly reduces the phospholipid hydroperoxides in bio-membranes and protects cells from oxidative damage [23]. In our study, PHGPx was involved in the metabolism of arachidonic acid, which reduced the oxidative damage in the The proteome response to short-term drought in Italian ryegrass (Lolium multiflorum) drought tolerant lines. Prx and catalase (CAT) can reduce the accumulation of ROS in plants [24,25]. Consistent with the results of previous studies, the activity of Prx exhibited an increase trend in drought tolerant line. Dehydroascorbate reductase (DHAR) helps to enhance plant tolerance to various abiotic stresses [26]. Actually, the protective role of DHAR was found and confirmed by developing transgenic tobacco plants with cytosolic DHAR gene [27]. For Italian ryegrass, it can be speculated that DHAR has a potent protective role in defending oxidative stress. pAPX play a key role in protecting plants against oxidative stress and thus conferred abiotic stress tolerance [28]. The up-regulation of peroxisomal ascorbate peroxidase (pAPX) was observed in this study, indicating that the enzyme might reduce cell damage caused by oxidative stress in Lolium multiflorum. Similarly, monodehydroascorbate reductase (MDHAR) was a key enzyme in the ascorbate-glutathione cycle and served as an important antioxidant [29]. In our results, MDHAR was down-regulated in ascorbate and aldarate metabolism, but the exact mechanism is still not clear and needs further study.

Metabolism-related proteins contributed to enhanced drought tolerance
The levels of the readily metabolizable carbohydrates significantly increased in plants under drought stress to maintain metabolic homeostasis [30]. In this study, changes in the accumulation levels of a number of carbohydrate metabolism-associated proteins in drought tolerant lines were observed (Fig 6A). The α-glucosidase (EC 3.2.1.20) played role in catalyzing the liberation of α-D-glucose from the non-reducing end of polysaccharides [31], especially the Type I of α-glucosidase can rapidly hydrolyzes sucrose [32].  The proteome response to short-term drought in Italian ryegrass (Lolium multiflorum) The proteome response to short-term drought in Italian ryegrass (Lolium multiflorum) (EC 3.2.1.2), α-amylase (EC 3.2.1.1), α-glucosidase, and putative β-D-xylosidase (EC 3.2.1.37), showed significant up-regulation and were linked with the hydrolysis of starch and sucrose under drought condition, suggesting that the drought-tolerant lines invested more carbohydrate into immediate defense against drought stress than drought susceptible lines. A putative β-D-xylosidase gene (AtBXL1) has been reported to be involved in secondary cell wall xylan synthesis [33]. Hemicelluloses were usually grouped into xylans and β-glucans [34]. The increase of the concentrations of β-D-glucan glucohydrolase (GGH) led to decrease of cell wall β-glucan concentrations in Hordeum vulgare [35]. In our study, down-regulated GGH not only catalyzed the hydrolytic removal of β-D-glucosyl residues [36], but also associated with enhancing drought tolerance by stimulating the degradation of β-glucans in abiotic stress. Plant growth inhibition due to drought stress was accompanied by increased amino acid concentrations [37]. Our analysis revealed that drought-responsive proteins as regulators participated in amino acid metabolism in tolerant lines of Italian ryegrass (Fig 6B). Cobalaminindependent methionine synthase (MetE) (EC 2.1.1.14), glycerate dehydrogenase (GDH) (EC 1.1.1.29) and Phospho-2-dehydro-3-deoxyheptonate aldolase 1 (EC 2.5.1.54) were found to be up-regulated in tolerant lines, except for unnamed protein product (EC 3.3.1.1). Cobalaminindependent methionine synthase wass involved in methionine synthesis and connected sulfur and carbon metabolic networks [38]. The activity of MetE could modulate methionine biosynthesis in E.coli under oxidative stress conditions [39]. We observed the up-regulation of MetE specifically in tolerant lines, suggesting that this enzyme might has a positive regulatory role in methionine biosynthesis to mitigate oxidative stress. Glycerate dehydrogenase is one of the precursors of glycine betaine, which was accumulated in both plants and animals in response to drought stress [40]. Results in this study might explain that up-regulated GDH was associated with drought tolerance in Italian ryegrass. The salt-inducible cDNA had high homology to phospho-2-dehydro-3-deoxyheptonate aldolase 1 in salt-tolerant rice [41]. Our results provided evidence that phospho-2-dehydro-3-deoxyheptonate aldolase 1 as regulator contributed to drought tolerance of Italian ryegrass. It was found in previous research that proline analogues and galactose levels were higher in drought resistant varieties of potato and barley [11,42].The higher amounts of proline involved in osmoprotection and stress signaling under drought [43]. Similar to results from other studies, we also noticed that the glutamine synthetase 1a (GS1a; EC 6.3.1.2) was up-regulated indicating that it participated in proline and glutamine synthesis to enhance drought tolerance in the tolerant line, and no change was observed in the drought susceptible lines [44][45][46]. A wide range of experiments showed that plants subjected to drought stress indeed expressed highconcentrations of secondary metabolites [47]. Terpenoids constitute a large and structurally diverse group of chemicals, playing diverse functional roles in plants as hormones, electron carriers and structural component of membranes [48]. Farnesyl pyrophosphate synthase (FPS) not only played a vital role in terpenoid metabolism, but also functioned as a key regulatory enzyme to control the sterol biosynthetic pathway [49]. In our study, FPS as key regulators was significantly accumulated and involved in Terpenoid backbone biosynthesis.
Inositol phosphates, diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP3) assecond messengers play an important role in signal transduction pathway. Actually, the PI-PLC could mediate the production of the DAG and IP3 [50]. In our study, PI-PLC as up-regulator played role in both inositol phosphate metabolism and phosphatidylinositol signaling system in tolerant lines, indicating that PI-PLC contributed to drought-mediated abiotic stress tolerance in Italian ryegrass. Moreover, DAG as a structural lipid changed its abundance during drought condition in grasses from Lolium-Festuca complex was observed by Perlikowski et al. (2014) [8].

Hydrolysis proteins and transport proteins respond to drought stress
The increase of amino acid concentration was caused by proteolysis in the advanced stages of drought [51]. Our data indicated that cathepsin B was up-regulated in the vacuole of L. multiflorum under drought stress, and might relate to the degradation of proteins. Recently, cysteine protease cathepsin B (TbCatB) was described as being involved in host protein degradation in Trypanosoma brucei [52]. Cysteine proteinase (CysP) showed to respond to environmental stress, such as cold or water deficiency [52] and its accumulation was observed in the leaves of tomato plants submitted to drought-stress [53]. In agreement with all the previous researches, our results indicated that CysP as an up-regulator controled protein degradation, which allowed L. multiflorum to adapt to drought stress. Aspartic proteinase (APs) are a large family of proteolytic enzymes, which are found in almost every plant [54]. APs were participated in some biological processes, e.g., stress responses, and programmed cell death [55]. We also observed the up-regulated accumulation of APs in the drought tolerant line of Italian ryegrass subjected to drought stress. This result suggested that aspartic proteinases might be important for improving drought tolerance.
Lipid transfer proteins (LTP) are small, basic, soluble proteins and are involved in stress response processes [56]. Furthermore, LTPs may repair stress-induced damage in membranes [57], and also may be responsible for increasing wax deposition [58]. In our study, LTP accumulation had dramatically up-regulated in the tolerant line, which might relate to enhance drought tolerance. Aquaporin (AQP) is plasma membrane water-transporting protein that facilitates water movement across cell membranes against osmotic gradients [59]. AQP transports water and other small molecules through biological membranes, which is vital for plants to tolerate drought [60]. According to our results, the drought tolerant line might have a lower water evaporation and higher water transport than the drought susceptible lines in drought conditions.

Predicted (hypothetical) proteins functionally relevant to drought tolerance
The functions of hypothetical proteins are still unknown, which is a challenge not only to genome annotation but also to in depth biological interpretation [61]. Hypothetical proteins were reported previously in cereal crops under abiotic stress [12,62]. Similarly, some predicted proteins are considered as key regulators of molecular mechanisms, with an important function in stress conditions. Kaneko et al. (1997) [63] found 3,189 predicted proteins as saltresponsive proteins in the Synechocystis genome (a cyanobacterium). Yang et al (2015) [64] detected 7% predicted protein that may improve drought tolerance and maintain photosynthetic activity in Purpurea seedlings. In our study, predicted proteins (CL16151.Contig2) played a vital role in metabolic processes of amino acids, propanoate, and butanoate. Five predicted proteins (gi|326533328, gi|149392357, gi|326503406, gi|357121703, and gi|357138855) ( Table 1), were significantly accumulated in response to drought stress in two L. multiflorum lines. Based on these results, predicted proteins are valuable though with unknown functions in understanding the effects caused by complex metabolic processes.

Conclusions
The current study revealed how drought-related proteins involved in regulatory system to adapt to abiotic stress in Italian ryegrass. Some differentially accumulated proteins, such as AtBXL1, GGH, GDH, MetE, GS1a, FPS, DAG, and PI-PLC, were found to be involved in carbohydrate metabolism, amino acid metabolism, biosynthesis of secondary metabolites, and signal transduction pathway, which might contributed to enhance drought tolerance or adaptation in Lolium multiflorum. The two specific metabolic processes, arachidonic acid and inositol phosphate metabolism, were differentially accumulated only in the tolerant lines. CatB, CysP, LTP and AQP were observed as drought-regulated proteins participating in hydrolysis and transmembrane transport. The activities of PHGPx, Prx, CAT, DHAR, pAPX and MDHAR associated with alleviate the accumulation of reactive oxygen species in stress inducing environments. In addition, a significant number of predicted proteins might play a vital of role in the modulation of metabolic pathways.