A proteomic approach to understand the impact of nodulation on salinity stress response in alfalfa (Medicago sativa L.)


 Background Symbiotic nitrogen fixation in legumes is an important source of nitrogen supply in sustainable agriculture. Salinity is a key abiotic stress that negatively affects host plant growth, rhizobium-legume symbiosis and nitrogen fixation.Results To explore how the symbiotic relation impacts plant response to salinity, we assayed the proteome profile of alfalfa plants with active nodules (NA), inactive nodules (NI) or without nodules (NN) when plants were subjected to salinity stress. Our data suggested that NA plants respond to salinity stress through some unique signaling regulations. NA plants showed an upregulation of proteins related to cell wall remodeling and reactive oxygen species (ROS) scavenging and a down-regulation of proteins involved in protein synthesis and degradation. The data also showed that NA plants, together with NI plants, upregulated proteins in photosynthesis, carbon fixation and respiration, anion transport, and plant defense to pathogens.Conclusions The data suggest that the symbiotic relations conferred the host plant a better capacity to adjust the key processes, probably to more efficiently use energy and resources, deal with oxidative stress, and maintain ion homeostasis and healthy status during salinity stress.


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In the rhizobium-legume symbiotic process, host plants provide carbon compounds derived from photosynthesis to support rhizobia activity and the development of microbial populations. In return, rhizobial symbiosis improves plants' nutritional status by providing fixed nitrogen, promotes plant development, and enhances their defense against pathogens and pests and their adaptation to various environmental stresses [4]. Rhizobia produce chemical molecules that can influence plant development, including phytohormones, lipo-chito-oligosaccharide nod factors, lumichrome, riboflavin, and H 2 evolved by nitrogenase [5]. When present in soil, nod factors can stimulate seed germination, promote plant growth, and increase grain yields, as well as increase photosynthetic rates. Very low concentrations of lumichrome and H 2 released by rhizobia also promote plant growth and increase biomass. Rhizobia are known to suppress the population of soil pathogens, and the legume itself releases phenolics that can suppress pathogens and promote growth of mutualistic microbes. Rhizobial inoculation of plants has also been suggested to alleviate the effect of water stress in symbiotic legumes [6,7]. It is possible that rhizobial products such as cytokinin, which results in ABA accumulation, elicits stomata closure and thus decreases water loss via transpiration in the leaves [5,8].
It has been reported that a response observed during one type of stress may have a role in the amelioration of another stress [9]. The symbiosis thus may enhance the fitness of their hosts and improve their tolerance to abiotic stresses. Salinity, one of the most important abiotic stresses, results in low agricultural productivity, low economic returns, and soil erosions [10,11]. Legumes growing in saline environments exhibit reduced yield [12]. It was reported that rhizobium-legume symbiosis and nitrogen fixation in root nodules are more sensitive to salinity than plant growth [13]. 4 The higher sensitivity of rhizobium-legume symbiosis and nitrogen fixation may be due to two main possibilities: rhizobium is more sensitive to salinity compared to host plants, or plants sever the symbiotic relation to conserve energy in stress response. The latter leads to other frequently asked questions: i.e. how the symbiotic relation impacts plant tolerance to salinity. It has been reported that N-fixing legumes were more sensitive to salt stress than those depending on mineral nitrogen [14]. Compared with N-fertilized plants, the ones with SNF showed a larger degree of decreased yield [15], less nitrogen accumulation [16] and greater uptake of Na + and Cl − under NaCl stress, inducing ionic imbalance in host plants [17]. Interestingly, Cordovilla et al. [18] reported that peas (Pisum sativum L.) dependent on nitrogen fixation were more tolerant to salt stress than those N-fertilized. In another study, chickpea (Cicer arietinum) genotypes with a greater capacity for nodulation performed better than other genotypes under salt stress in terms of both N fixation and grain yield [19]. Opposite results were obtained with faba bean (Vicia faba L.), where growth inhibition of shoot and root was observed in nitrogen-fixing plants [18]. Apparently, more studies are needed to address how the symbiotic relationship impacts salt tolerance in legume plants and to understand the physiological and molecular processes of stress response that are affected by SNF.
Salt tolerance is a complex phenomenon that involves morphological and developmental changes as well as physiological and biochemical processes. Many salt tolerant plants adapt to salinity by minimizing direct effects of Na or Cl ions through mechanisms such as salt exclusion, salt sequestration or salt excretion [11]. However, these mechanisms are usually not sufficient in limiting the amount of salt entering the plants. Thus, many plants need to deal with the direct effects of salt toxicity. One adverse effect of salinity is production of reactive oxygen species (ROS) [20]. ROS not only damage host plants but 5 also negatively affect nitrogen fixation [21]. Prevention of ROS production in the nodule and reduction of oxidative stress could provide a protective effect on nodule structure and function in salinity stress [22]. Plants employ antioxidants (e.g., ascorbate and glutathione) and detoxifying enzymes, such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), to combat oxidative stress induced by salinity [23,24]. Dehydration often occurs concurrently with salinity stress, and accumulation of solutes, like proline and soluble sugars, helps plant systems adapt to a saline environment by reducing water loss [25]. Plants accumulate proline, soluble sugar and stress responsive proteins that participate actively in the osmotic adjustment when plants are under salt stress [26,27].
A number of approaches have been used to identify genes and proteins that are involved in plant salt tolerance, such as screening cDNA libraries to isolate salt-responsive cDNA clones and analyzing gene expression at the transcriptional and post-transcriptional levels using transcript profiling [28]. A number of genes involved in salt tolerance and symbiotic nitrogen fixation have been isolated and characterized [29]. Proteomics is a powerful tool for describing the complete proteome and for comparing how the proteome is affected by different internal and environmental factors. Proteomic responses of rice [30], grasspea [31], sorghum [32], common wheat [33], soybean [34,35], and many other plants to salt stress have been reported. Theses proteomics experiments detected many proteins involved in plant response to salt stress. For example, an increased accumulation of enzymes involved in glycolysis and carbohydrate metabolism is frequently observed, indicating a greater need for energy during stress response. Other major groups of increased proteins are ROS scavenging enzymes, suggesting dealing with oxidative stress induced by salinity is one of the important processes in stress response. Other proteins 6 with an increased abundance under salinity are enzymes involved in biosynthesis of compatible solutes [36]. However, how these stress responsive proteomes are affected by SNF has not been studied.
Alfalfa (Medicago sativa L.) is the most extensively cultivated leguminous forage in the world. Although alfalfa is considered a moderately salt tolerant species when compared to other legumes, salinity is one of the limiting factors in the production and nitrogen-fixing activity of alfalfa [37,38]. While salinity inhibits both plant production and rhizobiummediated nitrogen-fixing activity in alfalfa, it is unclear how the symbiotic interaction affects salinity response in plants. We recently demonstrated that symbiosis in alfalfa improves plant survival to salinity treatment [39]. We hypothesize that symbiotic relations result in molecular and physiological changes that contribute to greater salt tolerance in plants. To address this hypothesis, we studied proteomic changes in alfalfa plants under salinity stress with or without symbiotic relation with rhizobium. Specifically, the isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic approaches were used to quantify protein changes in alfalfa plants with active nodules, inactive nodules, or no nodules when plants were subjected to salinity stress. We also examined relative water content, antioxidant enzymatic activities, and osmotic solute content as a few key physiological changes in these plants.

Plant materials and growth conditions
Alfalfa seeds (Medicago sativa L. cv. Ladak + ) and rhizobia (Rhizobium meliloti strain Dormal) used in this study were purchased from the Clover Group of China. Seeds were surface sterilized with 70% ethanol for 30 s and 5% sodium hypochlorite for 5 min, rinsed 4 to 5 times with sterile water, and germinated on wet filter paper in Petri dishes in a growth chamber at 25/15 ℃, 55/70% relative humidity (day/night). Five-day-old seedlings were transplanted to plastic cones (9 by 30 cm) filled with sterilized quartz sand in the greenhouse with the average temperature of 25±3 ℃ and 20±2 ℃, and the relative humidity of 55±5% and 70±5% during day and night, respectively.

Rhizobia inoculation and salt treatments
After transplanting, all the seedlings were watered daily with a nutrient solution (Supplement Table S1) prepared according to Peel [49] with little modification. Sixty days after transplanting, seedlings with similar size were selected and randomly divided into three groups. The first group was inoculated with Rhizobium meliloti and watered with a Nfree nutrient solution (Supplement Table S1), which resulted in development of active nodules (NA). The second group was also inoculated with the rhizobia but was watered with a complete nutrient solution, which led to the development of inactive nodules (NI) due to inhibition of nitrogen fixation by sufficient nitrogen in the nutrient solution. The third group was not inoculated and watered with a complete nutrient solution. These plants did not develop nodules (NN). Additional details of the treatments were described in other studies [39].
Plants were subjected to salt stress 30 days after inoculation. NI and NN plants were

Determination of physiological changes in response to salt stress
Relative water contents (RWC) of alfalfa leaves under salt stress were determined according to Chen et al. [50]. Dry matter was obtained after drying the shoot tissues in an oven at 80 ℃ for 48 h. The contents of proline and soluble sugars were determined spectrophotometrically by following the method of Bates et al. [51] and Dreywood [52] respectively. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) were measured by the nitroblue tetrazolium (NBT) method [53] and the method described by Raza et al. [54], respectively. Three plants from each treatment were used for measurement in each experiment. The experiment was repeated five times. Data were analyzed for mean difference by analysis of variance (ANOVA) in SPSS 20.0 with the least significant difference (LSD) test (α = 0.05).

Protein extraction and digestion
The shoots of control (CK, treated with nutrient solutions without NaCl) and salt-stressed Iodoacetamide (IAM) and a 2-h incubation at -20 ℃ after mixing with five-volume of chilled acetone. The protein pellet was collected by centrifugation and stored at -80 ℃ for further use. For digestion, the protein pellet was resuspended in 0.5 M triethylammonium bicarbonate (TEAB) solution, and the protein concentration was determined using bicinchonic acid assay (Sigma, USA). Equal aliquots (100 µg) from each lysate were then digested with trypsin overnight at 37 ℃ and lyophilized.

iTRAQ labeling and strong cation exchange (SCX) chromatography
The iTRAQ labeling of peptide samples derived from the shoots of alfalfa plants were performed using iTRAQ reagent 8-plex kit (Applied Biosystems, USA) according to the manufacturer's protocol with two biological replicates for each experimental group. All tagged samples were incubated at room temperature for 2 h and then were pooled together and lyophilized to powder form.
For SCX chromatography, the dried iTRAQ labeled and pooled samples were reconstituted with 4 mL buffer A (25 mM NaH 2 PO 4 in 25% acetonitrile (ACN), pH 2.7) and loaded onto a 4.6×250 mm Ultremex SCX column (Phenomenex, USA) containing 5 μm particles. The peptides were eluted at a flow rate of 1 mL/min with a gradient of buffer A for 10 min, 5-35% buffer B (25 mM NaH 2 PO 4 , 1 M KCl in 25% ACN, pH 2.7) for 11 min, 35-80% buffer B for 1 min. The system was then maintained in 80% buffer B for 3 min before equilibrating with buffer A for 10 min prior to the next injection. Elution was monitored by measuring absorbance at 214 nm, and the fractions were collected every minute. The eluted peptides were pooled as 20 fractions, desalted by Strata XC18 column (Phenomenex, USA) and vacuum-dried.

LC-ESI-MS/MS identification
The mass spectroscopy analysis was performed using a TripleTOF™ 5600 mass spectrometer (AB SCIEX, CA), fitted with a Nanospray III source and a pulled quartz tip as the emitter as described before [55]. Each fraction was resuspended in buffer C (2% ACN, 0.1% formic acid ) and centrifuged at 20,000 × g for 10 min; 10 µL supernatant was and P value for each protein was presented in the table (Supplementary Table S2 and   Table S3) after the mean value of protein expression ratios of each compared groups.

Physiological responses to salt stress
We examined leaf relative water content (RWC), superoxide dismutase (SOD) and catalase (CAT) activities, proline (PRO) and soluble sugar (SS) contents in alfalfa plants with active nodules (NA), inactive nodules (NI) and without nodules (NN) under different extents of salt stress (Fig. 1). RWC of alfalfa leaves decreased with the time of salt stress and was higher in inoculated plants (NA and NI) compared to NN 11 d after salt stress treatment (Fig. 1A). The activities of SOD and CAT were found to be the highest in NA shoots under salt stress, and the difference was more noticeable on day 10 among NA, NI, and NN plants ( Fig. 1B and 1C). PRO and SS contents in NA and NI shoots were higher than those in NN after salt stress ( Fig. 1D and 1E). A greater PRO content in NA shoots was observed on day 10 after stress compared to NI shoots. NA and NI plants also displayed higher SS contents on day 10 after salt stress compared to NN plants. Overall, NA plants show different physiological responses to salt stress treatment on day 10 compared to NI and 12 NN plants, and the samples harvested on day 10 after salt stress were studied for difference in protein profiles among NA, NI and NN plants.

Protein identification
An iTRAQ-based quantitative proteomic approach was used in this study to investigate the molecular links between root nodules and alfalfa salt tolerance. With two biological replications, a total of 396,885 spectra were obtained, among which 33,896 spectra were matched to the reference genomes M. truncatula protein database (ftp://ftp.jcvi.org/pub/data/m_truncatula/Mt4.0/Annotation/ Mt4.0v1/), representing 29,981 unique spectra. A total of 9,240 peptides were identified, representing 8,717 unique peptides which can be assembled into 3,274 proteins ( Fig. 2A). Majority of the peptides (85%) are 7-17 amino acid residues long (Fig. S1). Over 50% of the 3,274 proteins identified contain at least two peptides (Fig. 2B).
The protein mass distribution was presented in Figure 2C. Proteins with 20-30 kDa were the most abundant, followed by proteins with 30-40 and kDa 10-20 kDa. Our results showed that iTRAQ was able to identify proteins with a wide range of molecular mass.
Forty one percent of the identified proteins had more than 10% of the peptide coverage, and 19% have more than 20% of the peptide coverage (Fig. 2D).

Repeatability analysis
In this iTRAQ-based proteomics, the distribution of the coefficient of variation (CV) showed that our data had a relatively high repeatability with two biological replicates. The mean  (Fig. 3).

Overall differentially expressed proteins in alfalfa tissues
A total of 3,274 non-redundant proteins in alfalfa shoots were identified (Supplementary Table S2). These proteins were used to determine the significant changes in abundance during salinity treatments. With a 1.2-fold change cutoff and p-value less than 0.05, 607 proteins are differentially expressed in the shoots when comparing the salt stress condition with normal growth conditions in each treatment (Supplementary Table S3).

Functional classification of differentially expressed proteins
Clusters of orthologous groups of proteins (COG) analysis was performed to predict the functions and phylogenetic annotations of differentially expressed proteins in NA, NI and NN tissues under salt stress (Fig. 5). NN, NI and NA all showed a higher up-regulation of proteins involved in "energy production and conversion (C)"; "amino acid transport and metabolism (E)", "carbohydrate transport and metabolism (G)", "translation, ribosomal structures and biogenesis (J)"; "posttranslational modification, protein turnover, chaperones (O)" and "general function production only (R)". NA and NI generally showed similar but higher levels of changes when compared to NN in C and E functional categories. The most highly down-regulated functional categories are the same for NN, NI and NA, i.e. "translation, ribosomal structures and biogenesis (J)"; "posttranslational modification, protein turnover, chaperones (O)" and "general function production only (R)". Salt stress appeared to have little effect on "cell motility (N)". COG analysis for NA, NI and NN plants showed that the three most down-regulated functional categories are among the six most up-regulation functional categories, and two of them, "translation, ribosomal structure, and biogenesis" and "posttranslational modification, protein turnover, chaperons", are involved in protein synthesis, modification or degradation, suggesting plants actively regulated protein quantity and activity during salt stress response, resulting in a large increase of proteins in "energy production and conversion", "amino acid transport and metabolism", and "carbohydrate transport and metabolism".

Pathway analysis for differentially expressed proteins with MapMan
To better understand how NA, NI and NN plants respond to salt stress differently, MapMan was used to compare the proteins involved in specific processes. As indicated in Figure 6, seven processes were upregulated in NA, NI and NN plants, and among them four processes are highly over-represented, and they are all related to photosynthesis. Three upregulated processes common to NA, NI and NN plants are under-represented and belong to "not assigned" categories (Fig. 6). Interestingly, the only down-regulated process common to NA, NI, and NN plant also belongs to "not assigned unknown" category.
MapMan analysis revealed 5 processes were upregulated and 12 were downregulated only in NA under salinity based on the over-represented proteins (Fig. 6). Four of the upregulated processes are related to cell wall modification. They include "cell wall degradation", "miscellaneous", "miscellaneous gluco-, galaco-and mannosidases" and

Common pathway changes under salt stress
As indicated in Figure 6, seven processes are upregulated in NA, NI and NN plants, and among them four processes are highly over-represented, and they are all related to photosynthesis. A large set of proteins involved in both light reaction and carbon fixation 16 are up-regulated due to salt treatment. An increase in photosynthesis-related proteins under salt stress were also observed (Supplementary Table S4). Thus, the results suggested an enhancement of photosynthesis may be a common strategy to respond to salt stress, potentially to boost an energy source that is required for dealing with salinity stress. Notably, five chlorophyll binding a/b binding proteins (medtr2g008610.1, medtr2g081090.2, medtr4g015570.1, medtr5g097280.1, and medtr6g060175.1) were all up-regulated more than 2-fold only in NA plants, suggesting that the enhanced light energy harvesting may be needed to meet the additional energy needs for active nitrogen fixation. In the processes of light reaction, the light-harvesting chlorophyll a/b-binding proteins (LHC-CAB) absorb light through chlorophyll excitation and then transfer the absorbed energy to photo-chemical reaction centers [40]. These five proteins are members of chlorophyll binding complex in PSII, protect chlorophyll in cells, and play important roles in photosynthesis [41]. Three upregulated processes common to NA, NI and NN plants are underrepresented and belong to "not assigned" categories (Fig. 6).
Interestingly, the only downregulated process common to NA, NI, and NN plant also belongs to "not assigned unknown" category.

Unique pathway changes in NA plants
We are more interested in the proteins that are uniquely impacted by active nitrogen fixation since we observed that active nitrogen fixation improves salt tolerance in alfalfa [39]. MapMan analysis revealed 5 processes were up-regulated and 12 were downregulated only in NA under salinity based on the over-represented proteins (Fig. 6).
Looking closely, several cell wall degradation enzymes are pectin lyases. Together with those galactosidases in the miscellaneous group, the data suggest that NA plants may actively decrease or modify pectin component in the wall. At the same time, some of the upregulated peroxidases may be involved in lignification or cross-linking of cell wall components that will strengthen cell walls [42]. It is well-known that salinity stress can severely disrupts synthesis and weakens cell wall structure [43]. These changes in NA plants may help maintain healthy status under salinity. Since peroxidases are also involved in removing ROS in plants, it is possible that some of the upregulated peroxidases are involved in reducing ROS molecules in NA plants. The physiological data ( Fig. 1B and 1C) suggested that NA plants can indeed better deal with oxidative stress. We did not, however, identify upregulation of CAT and SOD proteins in our proteomic analysis, suggesting that the higher CAT and SOD activities in NA plants under salinity are due to posttranslational regulation. A cysteine-rich receptor-like kinase (Medtr2g088980.1) was only upregulated in NA plants, suggesting that nitrogen fixing plants may activate a unique signaling pathway that can enhance their tolerance to salinity stress (Supplementary Table S7).
Among the 12 down-regulated processes, 8 of them are related to protein metabolism, including two for protein degradation, four for protein synthesis and two for protein targeting/secreting ( Fig. 6 and Supplementary Table S8). The data indicate that NA plants suppress protein synthesis and at the same time reduce protein degradation and secretion, implying NA plants may reduce the protein turnover rate to save energy from costly protein synthesis and efficiently use all the resource by reducing secretion.

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NA plants also showed down-regulation of ethylene responsive proteins (Supplementary   Table S9). Ethylene is often considered a stress hormone, involved in suppression of growth [44].Thus a better stress tolerance and growth of NA plants under salt stress may be partially due to the reduced ethylene response. Several calcium-binding EF hand proteins (medtr3g115640.1, medtr5g011850.1, medtr5g088320.1, medtr5g099170.1,   medtr8g107110.1) were down-regulated in NA, NI and/or NN plants (Supplementary Table   S10). However, NA plants showed overall the strongest reduction in these proteins, and Medicago [46]. The reason why multiple EF-hand proteins were downregulated in alfalfa and NA plants is unknown.

Common pathway changes in NA and NI plants
Our previous studies demonstrated that NI plants also showed some improvement of salt tolerance compared to NN plants [39], prompting us to look at the pathways shared between NA and NI that may contribute to their salt tolerance. The MapMan analysis clearly showed many similar responses, mostly in up-regulated proteins, between NA and NI plants under salinity stress (Fig. 6). The high similarity was demonstrated in the proteins listed in Supplementary Tables S4-S13, in contrast with the changes in NN plants.
Both NA and NI showed significant up-regulation of proteins involved in photosynthesis and respiration, including proteins in light reaction of PSI, in the Calvin cycle, such as fructose-bisphosphate adolase, in photorespiration, and in TCA cycle (Supplementary   Table S4 and Table S11). The data suggest that both NA and NI plants are actively 19 managing energy production and consumption during stress response. Alternatively, these changes in carbohydrate metabolism may have something to do with a greater accumulation of osmoticans in NA and NA plants shown in Fig. 1 (1D and 1E) Table S13).
Salinity stress induced by NaCl results in both Na + and Cltoxicity. In fact, excessive Climposes stronger toxic effect on plants [48]. Thus, these anion channer may help maintain the anion homeostasis in alfalfa under salinity.

Conclusions
In conclusion, we found that symbiotic interaction or nodulation of alfalfa altered the pattern of protein changes involved in multiple physiological processes during salinity stress response compared to the plants without nodulation. Our data suggested that NA plants may respond to salinity stress through regulating some unique signaling pathways, leading to a better tolerance to stress. The greater stress tolerance in NA plants may be associated with active cell wall remodeling and strengthening, enhanced activity of removing ROS molecules as well as suppressing protein synthesis and degradation. The data also suggest that NA plants, together with NI plants, enhance photosynthesis, carbon 20 fixation and respiration, anion transport, and plant defense to pathogens. All these changes may allow NA plants to efficiently utilize all the energy and resources to maintain the healthy status, leading to better growth under salinity compared to NI and NN plants.
Our study provides some strong supporting evidence for an improved salinity tolerance in NA and NI plants compared to NN plants [39]. While these observations are exciting, how NA plants regulate those specific processes is intriguing. Identification of signaling and regulatory mechanisms in symbiotic nitrogen-fixing alfalfa that enhance salinity tolerance in plants will be the focus of future research and may have a profound impact on agricultural production in saline soil.

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Availability of data and materials
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