Comparative proteomics of root plasma membrane proteins reveals the involvement of calcium signalling in NaCl-facilitated nitrate uptake in Salicornia europaea

Highlight

Unlike in glycophytes, NaCl facilitates the nitrate uptake in the euhalophyte S. europaea, while calcium signalling plays important roles in this process.

Abstract

Improving crop nitrogen (N) use efficiency under salinity is essential for the development of sustainable agriculture in marginal lands. Salicornia europaea is a succulent euhalophyte that can survive under high salinity and N-deficient habitat conditions, implying that a special N assimilation mechanism may exist in this plant. In this study, phenotypic and physiological changes of S. europaea were investigated under different nitrate and NaCl levels. The results showed that NaCl had a synergetic effect with nitrate on the growth of S. europaea. In addition, the shoot nitrate concentration and nitrate uptake rate of S. europaea were increased by NaCl treatment under both low N and high N conditions, suggesting that nitrate uptake in S. europaea was NaCl facilitated. Comparative proteomic analysis of root plasma membrane (PM) proteins revealed 81 proteins, whose abundance changed significantly in response to NaCl and nitrate. These proteins are involved in metabolism, cell signalling, transport, protein folding, membrane trafficking, and cell structure. Among them, eight proteins were calcium signalling components, and the accumulation of seven of the above-mentioned proteins was significantly elevated by NaCl treatment. Furthermore, cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) was significantly elevated in S. europaea under NaCl treatment. The application of the Ca²⁺ channel blocker LaCl₃ not only caused a decrease in nitrate uptake rate, but also attenuated the promoting effects of NaCl on nitrate uptake rates. Based on these results, a possible regulatory network of NaCl-facilitated nitrate uptake in S. europaea focusing on the involvement of Ca²⁺ signalling was proposed.

Introduction

Salinity is among the most severe abiotic stresses in agriculture that affects ~6% of the total land on earth (Munns and Tester, 2008). In saline soil, the salt concentration is high, whereas the nitrogen (N) content is usually deficient (Hamed et al., 2013); most crops could not survive on it. However, many halophytes can thrive in this type of infertile soil, suggesting that they may have specific N uptake and utilization mechanisms. Salicornia europaea is one of the most salt-tolerant plant species in the world (Ungar, 1987). A previous study demonstrated that S. europaea has a strong ability to utilize N; this plant can thrive under both N-limited and high N conditions (Nie et al., 2012). However, the effect of NaCl on N utilization has not been investigated in S. europaea. Elucidating the regulatory networks of efficient N uptake of S. europaea under salinity would be helpful for creating salt-tolerant crops with high N use efficiency, which is essential for the development of sustainable agriculture in marginal lands.

N is an essential macronutrient for plant growth. Among various N forms (nitrate, ammonium, amino acids, and peptides), nitrate (NO₃ ^–) is the predominant form available in aerobic soils (Marschner and Rimmington, 1988). Salinity can severely influence nitrate assimilation in plants. Generally, a high Cl^– concentration acts as an antagonist and represses nitrate uptake; thus, NaCl negatively affects nitrate uptake, assimilation, and protein synthesis which may be responsible, at least in part, for the depressed plant growth under saline conditions (Bar et al., 1997). In addition, the state of the membrane and/or membrane proteins also affects uptake of NO₃ ^– by altering plasma membrane (PM) integrity under salinity (Frechilla et al., 2001). However, in halophytic Distichlis spicata (Pessarakli et al., 2012), NaCl has no negative effect on N uptake. Another report on the euhalophyte Suaeda physophora showed that NaCl application significantly increased leaf NO₃ ⁻ concentration under N-sufficient conditions (Yuan et al., 2010). In fact, sodium-dependent NO₃ ⁻ uptake has been reported in the marine diatom Phaeodactylum tricornutum (Rees et al. 1980), cyanobacteria (Lara et al. 1993), and the marine halophyte Zostera marina (Rubio et al., 2005). These results indicated that NaCl may have a promoting effect on nitrate uptake in some halophytes, which is different from glycophytes, whereas the underlying molecular networks are still unclear.

Nitrate uptake occurs in the outer cell layers of roots and relies on nitrate transporters, which are mainly localized at the PM together with the regulatory proteins. To date, four families of nitrate transporters have been identified: nitrate transporter 1/peptide transporter family (NRT1/PTR), nitrate transporter 2 family (NRT2), chloride channel family (CLC), and slow anion channel-associated homologues (SLAC1/SLAH) (Krapp et al., 2014). More than 60 nitrate transporters have been estimated in Arabidopsis, the regulation of which involves complex networks that are not fully understood. Investigations on root PM protein accumulation under different NaCl and nitrate levels would significantly enhance our knowledge of nitrate signalling under salinity. Proteomics is a powerful tool to study PM proteins depending on two main approaches, namely gel-free and two-dimensional electrophoresis (2-DE) (Nouri and Komatsu, 2010). The gel-free method has better coverage and higher solubility for hydrophobic membrane proteins than 2-DE. However, proteins containing a single transmembrane domain or peripheral membrane-associated proteins can be more readily resolved in 2-DE. In addition, 2-DE can give information on post-translational modifications (Tang, 2012) and it is also a valuable tool in deciphering N-terminal processing of proteins (Taylor et al., 2011). Nouri and Komastu (2010) used both gel-free and 2-DE methods to study soybean PM proteins, and concluded that the two techniques are complementary for comparative analysis. Meanwhile, Tang et al. (2008) used two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) to study Arabidopsis PM proteins, and three homologous brasinosteroid-signalling kinases were successfully identified; thus, 2D-DIGE is demonstrated to be a powerful approach for studying signalling proteins localized in the PM.

Proteomic research on glycophytes has focused on PM proteins under salt stress (Cheng et al., 2009). Meanwhile, in a few studies, whole-plant proteomics have been applied in Arabidopsis (Wang et al., 2012), Zea mays (Prinsi et al., 2009), and barley (Moller et al., 2011) to detect their responses to different N conditions. However, no research has been performed to analyse the PM proteome of halophytes under different nitrate and NaCl levels. The aim of this study was to investigate the characteristics of nitrate uptake in S. europaea under NaCl treatment and to identify the underlying regulatory components. Through physiological analysis, it was demonstrated that in contrast to the situation in glycophytes, NaCl facilitates nitrate uptake in S. europaea. Comparative proteomics and cell biology studies further revealed that calcium signalling plays important roles in this process. These results open up exciting perspectives for further investigations on the specific regulatory pathways involved in efficient N uptake under NaCl conditions.

Materials and methods

Plant materials and growth conditions

Salicornia europaea seeds were collected from the coastal area of Dafeng City, Jiangsu Province, China. The plants were grown in a greenhouse with a day/night temperature regime of 25 °C/20 °C, photoperiod of 16h, and relative humidity of ~60%. At 20 d after sowing on perlite granules, seedlings were irrigated with four solutions containing different concentrations of NO₃ ^– and NaCl in modified 1/2 Hoagland solution for 30 d. Then the plants were harvested and washed with distilled water to remove the surface ions and used for further analysis. The modified 1/2 Hoagland solution contained 0.5mM KH₂PO₄, 1mM MgSO₄, 2.5mM CaCl₂, 0.05mM Fe-EDTA, 2.5mM KCl, and micronutrients, with the pH adjusted to 6.5±0.1. The four treatments were 0.1mM NO₃ ^– (LN), 0.1mM NO₃ ^–+200mM NaCl (LN+S), 10mM NO₃ ^– (HN), and 10mM NO₃ ^–+200mM NaCl (HN+S).

Measurement of fresh weight (FW), dry weight (DW), root length, and root volume

FW was measured immediately after harvesting. DW was measured after drying for 48h in an oven at 80 °C. Roots were scanned with a digital scanner (Epson, Nagano, Japan) and then analysed with WinRHIZO software (Regent Instruments Inc., Quebec, Canada).

Quantification of total Na, total N contents, and NO₃ ^– concentrations

Plants were dried for 72h in an oven at 80 °C and then weighted and subsequently ground into powder in a mixer mill (Retsch MM400, Hann, Germany). The powder was digested with a mixture of nitric acid and hydrogen peroxide using a microwave system (MARS; CEM Corporation, Matthews, NC, USA), which was used to determine total Na contents by atomic emission spectrometry (ICP-AES, Thermo, Waltham, MA, USA). N contents were determined by a Vario EL III CHNOS Elemental Analyzer (Elementar Analysensysteme GmbH, Germany). The NO₃ ^– concentration was measured as previously described (Jampeetong and Brix, 2009). Dried material (5–10mg) was extracted with 10ml of Milli Q water at 80 °C in a water bath for 20min. Then, NO₃ ^– concentrations were analysed by an AA3 continuous flow-analyser (Seal Analytical, Germany).

Analysis of nitrate reductase (NR) activities

NR activities were measured as previously described (Yaneva et al., 2002) with some modifications. The seedlings were homogenized in extraction buffer [50mM HEPES-KOH, pH 7.5, 1mM EDTA, 10mM glutathione (GSH), 0.1% (w/v) polyvinylpyrrolidone (PVP), 1mM dithiothreitol (DTT), and 1mM phenylmethylsulphonyl fluoride (PMSF)] and centrifuged for 20min at 12 000 g. The supernatant was incubated with reaction buffer (50mM HEPES-KOH, pH 7.5, 5mM KNO₃, 0.2mM NADH) in the dark at 25 °C for 20min; the reaction was terminated by addition of 1% sulphanylamide in 1.5M HCl and 0.01% N-(1-naphthyl)-ethylenediammonium dichloride. Nitrite production was determined by reading the absorbance at 540nm.

Analysis of nitrate uptake rates

Nitrate uptake rates were measured as previously described (Muños et al., 2004). Salicornia europaea plants of ~30 d old irrigated with 1/2 Hoagland solution plus 200mM NaCl were transferred to 0.1mM CaSO₄ for 1min and then to four solutions containing different NaCl and K¹⁵NO₃ (99% atom excess ¹⁵N) concentrations, as follows: 0.1mM K¹⁵NO₃, 0.1mM K¹⁵NO₃+200mM NaCl, 10mM K¹⁵NO₃, and 10mM K¹⁵NO₃+200mM NaCl for 15min, and finally, to 0.1mM CaSO₄ for 1min. Six roots for each treatment and three independent biological replications were dried at 70 °C for 48h and ground. The powder (2–3mg) was used for ¹⁵N determination by the Delta Plus-MS system (Thermo).

Thirty-day-old S. europaea plants irrigated with 1/2 Hoagland solution plus 200mM NaCl were pre-treated with 1mM of the non-specific Ca²⁺ channel blocker LaCl₃ for 24h. The LaCl₃-pre-treated and control plants were used for ¹⁵NO₃ ^– uptake assay following the above-mentioned method.

Plasma membrane (PM) isolation

PMs were isolated as previously reported (Tang, 2012). Roots were homogenized in grinding buffer (pH 7.5) (25mM HEPES, 0.33M sucrose, 10% glycerol, 0.6% PVP, 5mM ascorbic acid, 5mM EDTA, 5mM DTT, and 1mM PMSF). The homogenate was filtered through Miracloth and centrifuged at 10 000 g for 15min. Total microsomal (TM) fractions were pelleted by centrifugation at 80 000 g for 1h and resuspended in suspension buffer [5mM KH₂PO₄/K₂HPO₄ buffer (pH 7.8), 0.33mM sucrose, 3mM KCl, 1mM DTT, and 1mM protease cocktail]. Two-phase partitioning was performed by using a solution containing 6.2% polyethylene glycol (PEG) 3350, 6.2% dextran T-500, 0.33M sucrose, 3mM KCl, and 5mM KH₂PO₄/K₂HPO₄ buffer (pH 7.8). The mixture was centrifuged at 1500 g for 10min. After partitioning, the upper phase fraction was diluted with 10 vols of dilution buffer (0.33M sucrose, 25mM HEPES, and 1mM DTT) and spun at 120 000 g for 1h to collect the PMs. Then, the PM vesicles were incubated with 0.02% Brij-58 detergent on ice for 10min to invert the vesicles and release the cytosolic contaminants. Samples were then diluted 20 times with double-distilled H₂O and centrifuged at 120 000 g for 60min (Elmore et al., 2012). The pellets were resuspended in 100 μl of suspension buffer (5mM KH₂PO₄/K₂HPO₄ buffer, pH 7.8, 3mM KCl, 1mM DTT, 0.1mM EDTA, and 1 μM protease cocktail). For each sample, five independent biological replicates were prepared.

Immunodetection assay and H⁺-ATPase hydrolytic activity assay

Immunoblot analysis was performed according to standard methods (Zhang et al., 2011). Equal amounts of 5 μg of proteins from TM and PM fractions were separated by SDS–PAGE (Supplementary Fig. S1 availabe at JXB online) and transferred to nitrocellulose membranes (GE Amersham Biosciences, NY, USA) using a wet transblot system (Bio-Rad, Hercules, USA). The TM and PM proteins were probed with specific primary antibodies, and visualized using the enhanced chemiluminescence method. The primary antibodies used were anti-H⁺-ATPase (PM marker, Agrisera, Vännäs, Sweden), anti-Sar1 [secretion-associated and Ras-related protein 1, an endoplasmic reticulum (ER) marker, Agrisera], and anti-V-ATPase (tonoplast marker, Agrisera).

Hydrolytic activity of the PM H⁺-ATPase was measured according to the method of Nouri and Komastu (2010). The PM fraction was added to the reaction solution containing 30mM MES-TRIS (pH 6.5), 50mM KCl, 3mM MgSO₄, and 3mM ATP in the presence or absence of the specific H⁺-ATPase inhibitor 0.1mM Na₃VO₄ and incubated at 30 °C for 15min. The reaction was stopped by the addition of 0.5% ammonium molybdate, 1% SDS, and 0.4M H₂SO₄. Ascorbate was then added at a final concentration of 0.3%, the mixture was placed at room temperature for 30min, and the absorbance was measured at 750nm.

Labelling of proteins with Cy dye

PM proteins were precipitated with the methanol/chloroform method as previously reported (Wessel and Flügge, 1984). The protein pellets were recovered in modified 2D-DIGE buffer (7M urea, 2M thiourea, 4% CHAPS, 25mM TRIS-HCl, and 1% n-dodecyl β-d-maltoside). The pH of protein samples was adjusted to 8.8 with HCl and NaOH, and the protein concentration was determined using the Bradford method. The internal standard was prepared by mixing equal amounts of all analysed samples. Protein samples were labelled using minimal fluorescent dyes (Cy3, Cy5, and Cy2) (GE Healthcare, Pittsburgh, PA, USA) following the manufacturer’s instruction.

2D-DIGE and image scanning

The labelled protein samples were mixed with the internal standard (Supplementary Table S1 at JXB online), adjusted to a total volume of 450 μl with rehydration buffer [7M urea, 2M thiourea, 4% CHAPS, 1% DTT, and 1% immobilized pH gradient (IPG) buffer, pH 4–7], and used for isoelectric focusing (IEF). IEF was performed on an IPG strip holder with 24cm, linear gradient IPG strips with pH 4–7 (GE Healthcare) and then on an Ettan DALT System (GE Healthcare) according to the manufacturer’s instructions. The images were analysed using DeCyder 6.5 (GE Healthcare). Spots reproducible in 24 of 30 images were used to identify protein abundance change by two-way analysis of variance (ANOVA) (P<0.05). The gels used for 2D-DIGE analyses were stained with Coomassie Brilliant Blue (CBB) to observe highly abundant spots. Then, additional gels with 1mg of internal standard proteins were stained with CBB for spots that could not be determined from 2D-DIGE gels.

Protein identification and database searching

The 117 most abundant spots were digested in-gel with bovine trypsin (Roche Molecular Biochemicals, Indianapolis, IN, USA) as described previously (Wang et al., 2007). The ultrafleXtreme matrix-assisted laser desorption ionization-time of flight/time of flight-mass spectrometer (MALDI-TOF/TOF-MS; Bruker Daltonics, Billerica, MA, USA) was used to reveal the MS/MS spectra. The peptides were suspended with 10 μl of 70% acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA), and 1 μl was taken and spotted onto the AnchorChip™ MALDI target plate (Bruker Daltonics). Then 1 μl of matrix solution (1mg ml^–1 α-cyano-4-hydroxycinnamic acid in 70% ACN containing 0.1% TFA) was spotted after the sample solution dried. All spectra were obtained in positive ion reflection mode under the control of FlexControl 3.3. The matrix suppression was set to deflection, 500Da. The spectra detection mass range was set at 700–4000 m/z. External calibration involved the use of the Bruker standard peptide calibration kit. For each spot, the top 30 strongest intensity peaks in the MS spectra and single-to-noise threshold >6 were automatically selected as precursor ions for MS/MS analysis. MS spectra were acquired with 400 laser shots per spectrum, whereas MS/MS spectra were obtained using 1500 laser shots per fragmentation spectrum. Then MS and MS/MS data were transferred to BioTools 3.2 (Bruker Daltonics) and searched against three databases with the Mascot engine 2.2.03 (http://www.matrixscience.com). The three databases were the NCBInr protein database (http://www.ncbi.nlm.nih.gov/; green plants, 1 669 695 sequences in NCBI 20131226) and two S. europaea transcriptome-translated protein databases, as follows: database 1 (162 969 sequences; Ma et al., 2013) and database 2 (35 219 sequences; Fan et al., 2013). Monoisotopic and [M+H]⁺ were selected for mass values. Peptide mass tolerance was set at 50 ppm, fragment mass tolerance was set as 0.5Da, and one missing cleavage was permitted. Carbamidomethyl (C) was set as fixed modification and Oxidation (M) was set as variable modification. All proteins were matched by Mascot with scores that exceeded their 95% confidence threshold and contained at least one peptide with a score at a significance threshold (P<0.05). If several database entries of homologous proteins matched these criteria, only the entry with the highest score is reported. For proteins having only one matching peptide with a score at a significant threshold (P<0.05), the MS/MS spectra were also presented (Supplementary Fig. S3; Supplementary Table S3 at JXB online).

Protein classification and bioinformatics analysis

The identified proteins were searched against the NCBI protein database (http://www.ncbi.nlm.nih.gov/) and Uniprot (http://www.uniprot.org/) and were divided into different groups based on their molecular and biological functions. The subcellular location of each protein was determined by SUBAIII (http://suba3.plantenergy.uwa.edu.au) and published information on the homologous proteins in Arabidopsis. Principal component analysis (PCA) was performed using the extended data analysis module within the DeCyder software (GE Healthcare). Heat map was performed using MultipleExperiment Viewer 4.8.1 software based on the Log₂-transformed fold change.

RNA isolation and quantitative real-time PCR (qRT-PCR) analysis

Total RNA was extracted by Trizol reagent and treated with RNase-free DNase I. A 0.5 μg aliquot of RNA was used for the first-strand cDNA synthesis with the SuperScript III first-strand synthesis system (Invitrogen, Carlsbad, CA, USA). qRT-PCR was performed with an Mx3000P Real-Time PCR System (Agilent, USA) using SYBR qPCR Mix (Toyobo, Japan). The relative gene expression levels were calculated by the 2^–ΔΔCT method, and the α-tubulin gene was used as internal control (Lv et al., 2012). qRT-PCR of each sample was repeated three times. The oligonucleotide primers used are listed in Supplementary Table S5 at JXB online.

Detection of [Ca²⁺]_cyt

Thirty-day-old S. europaea plants irrigated with 1/2 Hoagland solution plus 200mM NaCl were used. In the 2h NaCl exposure experiment, the intact roots were incubated in loading solution containing 20 μΜ Fluo-4/AM, 50mM sorbitol, 0.2mM CaCl₂, and 0.05% Pluronic F127 (pH 4.2) at 4 °C for 4h in the dark, followed by a 2h incubation with 0.2mM CaCl₂ with or without 200mM NaCl at 20 °C in the dark. In the 24h, 3 d, and 30 d NaCl exposure experiments, the plants were first pre-treated with 200mM NaCl for 18h, 66h, and 30 d. Then, the intact roots of treated and control plants were incubated in loading solution with or without 200mM NaCl at 4 °C for 4h in the dark, after which the roots were incubated for 2h with 0.2mM CaCl₂ with or without 200mM NaCl at 20 °C in the dark. After washing with buffer, [Ca²⁺]_cyt was determined under a laser scanning confocal microscope (Leica TCS SP5, Wetzlar, Germany) at excitation and emission wavelengths of 488nm and 505–530nm, respectively. Observations were focused on the primary root tips of plants. The same area of root tips of each sample was analysed using Image J software (National Institutes of Health, Bethesda, MD, USA) to determine the fluorescence intensity (mean pixel intensity).

Statistical analysis

Statistical analysis was carried out using SPSS 18.0 software. Data were evaluated by two-way ANOVA. To determine the type of interaction between saline and nitrogen treatment, taking the FW for example, the effect of saline–high nitrogen treatment (FW_HN+S/FW_LN) was compared with the effect of each factor when applied individually (i.e. FW_HN/FW_LN×FW_LN+S/FW_LN). Interaction was synergistic if FW_HN+S/FW_LN was substantially more than FW_HN/FW_LN×FW_LN+S/FW_LN; and it was additive if FW_HN+S/FW_LN was approximately equal to FW_HN/FW_LN×FW_LN+S/FW_LN, whereas it was antagonistic if FW_HN+S/FW_LN was substantially less than FW_HN/FW_LN×FW_LN+S/FW_LN (Striker et al., 2015). For each experiment, at least three replications were used for analysis. The methods of significance testing are described in the figure legends.

Results

NaCl had a synergetic effect with nitrate on the growth of S. europaea

In this study, the effect of NaCl and nitrate on the growth of S. europaea was first investigated. Seedlings were treated with four different solutions, LN, LN+S, HN, and HN+S. Generally, NaCl has a positive influence on the growth of euhalophytes, for which zero NaCl is an abnormal condition whereas 200mM NaCl is in the range of optimal condition (Flowers and Colmer, 2008). Consistently, in this study, it was found that S. europaea grows better in the presence of 200mM NaCl under both LN and HN conditions. In addition, high N also promotes the growth of S. europaea (Fig. 1A, B). The FW, DW, root length, and root volume were measured, and two-way ANOVA (P<0.05) was used to reveal the effects of S, N, and S×N on these parameters. All the four traits showed significant S and N effects. The FW and DW increased under NaCl and NO₃ ^– treatments, and seedlings treated with HN+S exhibited the highest values, whereas LN showed the lowest (Fig. 1C, D). Moreover, a significant S×N interaction was observed for FW and DW, and the type of interaction was synergistic (Fig. 1C, D), suggesting that NaCl and nitrate had synergistic effects on the growth of S. europaea. NaCl facilitated the root growth of S. europaea, whereas high nitrate inhibited it. Both the root length and volume were largest under LN+S treatment, the values of which were 110.8% and 103.1% higher than those treated with HN, respectively (Fig. 1E, F).

Fig. 1.

Phenotypic and physiological changes of S. europaea grown under different NaCl and NO₃ ^– concentrations. S. europaea seedlings were treated with different nitrate and NaCl concentrations for 30 d, and the phenotypes (A and B), fresh weight (C), dry weight (D), root length (E), and root volume (F) were determined. Scale bars=2cm. Values were means ±SE (n=12). Different letters above the bars indicated significant differences (P<0.05). For C, D, E, and F, two-way ANOVA was performed to indicate significant S, N, and S×N effects. Double asterisks indicate significant differences at P<0.01. Combined effects are symbolized by a slash (/). Synergistic indicates the type of interaction.

The effects of NaCl on nitrate uptake and assimilation

In order to investigate the effects of NaCl on the nitrate uptake and assimilation, the total Na and total N contents per plant, the N and NO₃ ^– concentrations in shoot of S. europaea, the nitrate uptake rates, and nitrate reductase activities were measured (Fig. 2). All of the traits except NR activities showed significant S, N, and S×N effects. As expected, total Na contents were significantly increased under NaCl treatments, which were also promoted by N treatment (Fig. 2A). Under low N conditions, no significant effect of NaCl on total N content and shoot N concentrations was observed (Fig. 2B, C), but the shoot NO₃ ^– concentrations and the nitrate uptake rates increased significantly by NaCl treatment. The values increased by 83.4% and 32.8% (Fig. 2D, E), respectively. Compared with the LN condition, NR activity under LN+S increased by 23.5%, but the values were very low and the difference was not statistically significant (Fig. 2F). Under high N conditions, the shoot N concentrations were significantly decreased by NaCl treatment (Fig. 2C), but the total N contents, shoot NO₃ ^– concentration, the nitrate uptake rates, and the NR activities were all significantly increased by NaCl treatment. The values increased by 27.8, 14.6, 57.6, and 41.7%, respectively (Fig. 2B–F). Notably, NaCl and NO₃ ^– also had a synergistic interaction effect on nitrate uptake rate. These results showed that NaCl could promote nitrate uptake and assimilation in S. europaea.

Fig. 2.

Total Na and N contents, shoot N and NO₃ ^– concentrations, nitrate uptake rates, and NR activities of S. europaea grown under different NaCl and NO₃ ^– concentrations. S. europaea seedlings were treated with different nitrate and NaCl concentrations for 30 d, total Na (A) and total N (B) content of the whole plant, shoot N (C) and NO₃ ^– (D) concentration, and NR activity (F) were measured. (E) S. europaea seedlings were treated with four different NaCl and ¹⁵N labelled K¹⁵NO₃ concentrations for 15min, and the content of ¹⁵N in roots was measured. Values were means ±SE (n=4 for A, B, C, D, and F; and n=3 for E). Asterisks indicate significant differences (P<0.05) between salt-treated and untreated samples. In addition, two-way ANOVA was performed to indicate significant S, N, and S×N effects. Double asterisks indicate significant differences at P<0.01, and combined effects are symbolized by a slash (/). Synergistic, additive, and antagonistic represent different types of interaction.

Identification of NaCl- and nitrate-regulated PM proteins by 2D-DIGE

To determine the proteins regulated by NaCl and nitrate in S. europaea, a comparative proteomic analysis of root PM proteins under the four treatments was conducted. Root PM fractions were purified by the two-phase partitioning method (Tang, 2012), and their purity was evaluated by western blot analysis. Compared with the TM fraction, H⁺-ATPase was strongly enriched in the PM fraction. Conversely, V-ATPase, a tonoplast membrane protein, was more strongly enriched in the TM fraction than in the PM fraction. Sar1, an integral ER membrane protein, was only detected in the TM fraction and was absent in the PM fraction (Fig. 3A). Furthermore, the hydrolytic activity of PM H⁺-ATPase in the presence or absence of 0.1mM of the specific PM H⁺-ATPase inhibitor Na₃VO₄ was determined, and the purity of PM vesicles was 84.76±2.80%_. These results demonstrated that PM vesicles of relatively high purity were obtained.

Fig. 3.

Purity assessment and a representative 2D-DIGE image. (A) Western blot analysis of extracted PM proteins with antibodies against proteins from different cell compartments. (B) PM proteins were separated by 2D-DIGE and detected with a Typhoon 9400 Scanner. The differentially accumulated protein spots under different NaCl and nitrate concentrations were determined using two-way ANOVA, and those positively identified by MALDI-TOF-TOF are marked by arrows.

2D-DIGE with pH 4–7 strips was used to separate root PM proteins, and ~2400 protein spots were found in each image (Fig. 3B; Supplementary Fig. S2 at JXB online). The 20 protein samples representing five biological repeats of each of the four treatments were assigned to 10 DIGE gels, which were labelled with Cy2, Cy3, and Cy5. A total of 30 images were acquired (Supplementary Table S1; Supplementary Fig. S2). After matching the 30 spot maps, 717 protein spots were found in 80% of them (Supplementary Table S2). PCA based on these 717 protein spots was performed to determine the distribution and similarities of different experimental groups. The five spot maps of each treatment were exactly clustered in the score plot (Fig. 4A), indicating high reproducibility among the replicate gels. Principal component 1 (PC1) explained 47.7% of the variance and separated the experimental groups according to NaCl treatment, whereas the axis PC2 accounted for 13.7% of the variance and separated the experimental groups according to nitrate treatment.

Fig. 4.

PCA, spot significant analysis, and functional classification of the identified proteins. (A) PCA of the different experimental groups. Samples plotted on the first two principal components (PCs) are shown. (B) Venn diagram showing the distribution of significant spots following two-way ANOVA. The numbers outside the parentheses are the total numbers of significant spots and at least one absolute variation was obtained above 1.5-fold by performing comparisons among the different treatments. The numbers in parentheses are the number of protein spots identified in the MALDI-TOF/TOF-MS. (C) Functional classification of 81 identified proteins. The percentage of proteins from each category is shown. The two most abundant categories, metabolism and cell signalling, were subsequently divided into subgroups.

Two-way ANOVA (P<0.05) with false discovery rate (FDR) correction for multiple testing revealed that a total of 406 spots displayed significant S, N, and S×N effects and at least one absolute variation >1.5-fold by comparison within the different treatments. Overall, among the differentially accumulated proteins, 166, 18, and 14 spots displayed pure S, N, and S×N effects, respectively, showing that NaCl treatment had a greater impact on protein accumulation than nitrate. In addition, 69 spots displayed pure combined S and N effects, which indicated that both NaCl and nitrate affected the accumulation of these proteins, but the two factors were independent. Finally, 64 spots displayed significant S×N effect in combination with S and N effects, indicating that NaCl and nitrate had interactive effects on the accumulation of these proteins (Fig. 4B).

Functional classification of NaCl- and nitrate-regulated proteins

After removing the dim spots and noise, the 117 most abundant spots displaying significant S, N, and/or S×N effects were manually excised from the gels, and further analysed by MS/MS spectra. Since there is a lack of information on the genome and comprehensive protein sequence of S. europaea, these spectra were searched against the NCBInr green plant database and two S. europaea transcriptome-translated protein databases (Fan et al., 2013; Ma et al., 2013). Most proteins blasted by the three databases were homologous proteins from different species, and the ones with the highest scores were finally selected for annotation (Supplementary Fig. S3; Supplementary Table S3 at JXB online). In total, 81 out of the 117 spots were annotated, among which 65 proteins have been previously shown or predicted to be associated with the PM. The localization information determined by the SUBAIII database and the literature is listed in Supplementary Table S3.

Furthermore, the identified proteins were grouped into six categories based on their molecular and biological functions, namely metabolism (46%), cell signalling (27%), transport (10%), protein folding (7%), membrane trafficking (5%), and cell structure (5%). The two major categories were further classified into subcategories. Specifically, proteins related to metabolism were divided into six subcategories, as follows: N metabolism, energetic metabolism, secondary metabolism, acid phosphates, cell wall-associated proteins, and proteolysis. Proteins involved in cell signalling were assigned to three subcategories, as follows: calcium signalling components, stress response, and detoxication (Fig. 4C; Supplementary Table S3 at JXB online).

The accumulation pattern of the identified proteins

The 81 annotated, differentially accumulated proteins were further grouped into eight distinct clusters (Clusters I–VIII, Fig. 5; Supplementary Table S4 at JXB online) according to their change in abundance pattern. These clusters were illustrated by reaction norm diagrams consisting of a graphical representation highlighting the contribution of NaCl treatment (0mM and 200mM) to the observed protein abundance for different levels of nitrate treatments (0.1mM and 10mM). Cluster I contained 16 proteins whose accumulation increased significantly by NaCl treatment under both LN and HN conditions. Seven of these proteins were also significantly changed under nitrate treatments. Cluster II comprised nine proteins that were significantly repressed by the NaCl treatment under both LN and HN conditions. Four of these nine proteins changed significantly under nitrate treatments. Clusters III and IV comprised proteins whose abundance was increased or decreased significantly by NaCl treatment, but only under HN conditions. Cluster III included 10 proteins, three of which were significantly changed under nitrate treatments. Cluster IV contained six proteins, all of which were significantly changed under nitrate treatments. The largest cluster (Cluster V) comprised 19 proteins, which showed increased accumulation by NaCl treatment under LN conditions. Sixteen of these 19 proteins were also significantly changed under nitrate treatments. Only one protein in Cluster VI was repressed by NaCl treatment under LN conditions, and this protein was also down-regulated under nitrate supplement without NaCl. Cluster VII comprised seven proteins showing opposite responses to NaCl under LN and HN conditions. All of these proteins displayed a clear S×N interaction effect. Cluster VIII comprised 13 proteins whose abundance was not changed significantly by NaCl treatment whether under LN or HN conditions, but the abundance of such proteins changed significantly in response to nitrate.

Fig. 5.

Clustering analysis of differentially accumulated proteins. The 81 differentially accumulated proteins were grouped into eight clusters according to their change patterns, which are illustrated by reaction norm diagrams consisting of a graphical representation highlighting the contribution of NaCl treatment to the observed protein abundance under nitrate treatment. Each row in the heat map indicates a single protein, whose expression values were generated using Log₂ ($\overline{RV}$ _treatments/$\overline{RV}$ _LN). For each protein, the spot number and the protein name with its ANOVA effect (S, N, and/or S×N) are shown. Reaction norms were generated using the means of Log₂ ($\overline{RV}$ _treatments/$\overline{RV}$ _LN) of spots for each defined cluster. $\overline{RV}$, means of normal volume/normal standard volume.

The involvement of Ca²⁺ signalling in NaCl-facilitated nitrate uptake in S. europaea

Eight calcium signalling components were identified by 2D-DIGE, and seven of these components were up-regulated, whereas the other one was down-regulated by NaCl treatment under HN and/or LN conditions (Fig. 6). qRT-PCR was performed to examine whether the eight components were also regulated at the transcription level. As shown in Fig. 6, the mRNA transcripts of the eight proteins were all up-regulated by NaCl treatment under HN and/or LN conditions. Two of them, annexin (ANN)-like protein RJ4-like and PBI41 14-3-3, displayed consistency between mRNA and protein variation trends, whereas the other six proteins showed some discrepancies (Fig. 6).

Fig. 6.

Expression profiles of calcium signalling components at protein and mRNA levels in response to NaCl and nitrate. The images on the left are magnified views of 2D-DIGE gel sections containing the spot of the calcium signalling components. The corresponding expression files at protein and mRNA levels are listed on the right. Expression levels of mRNAs were evaluated using qRT-PCR, n=3. The protein expression values were generated using ($\overline{RV}$ _treatments/$\overline{RV}$ _LN). $\overline{RV}$, means of normal volume/normal standard volume, n=5. Values are means ±SD.

Previous studies have shown that [Ca²⁺]_cyt fluctuations and the downstream calcium signalling proteins play pivotal roles in stress responses (Plieth, 2005). Thus, the dynamic changes of [Ca²⁺]_cyt under 200mM NaCl treatment were determined in S. europaea roots using the Ca²⁺-sensitive fluorescent probe, Fluo-4/AM. In contrast to weak fluorescence detected in the control root tips, strong fluorescence was observed in roots treated with NaCl for 2h, 24h, and 3 d (Fig. 7A, B). Although the fluorescence from 30 d NaCl-treated root tips was weak, it was still significantly higher than that of the control. Statistical analysis of the mean intensity of fluorescence showed that [Ca²⁺]_cyt was significantly elevated under NaCl treatments (Fig. 7B).

Fig. 7.

The involvement of Ca²⁺ signalling in NaCl-facilitated nitrate uptake in S. europaea. (A) The effects of NaCl on relative [Ca²⁺]_cyt in S. europaea root tips. S. europaea seedlings treated with 200mM NaCl for 2h, 24h, 3 d, and 30 d were incubated with Fluo-4/AM, and root tips were observed by confocal microscopy (b, d, f, and h). The root tips without NaCl treatments served as controls (a, c, e, and g), a0 to h0 are light field images of the corresponding root tip. Scale bar=100 μm. (B) Quantification of average fluorescence intensities of root tips. Values are means ±SE (n=10). Asterisks indicate significant differences (P<0.05). (C) The effect of LaCl₃ on nitrate uptake rates of S. europaea roots. LaCl₃-pre-treated S. europaea seedlings were immersed in modified 1/2 Hoagland solutions with different NaCl and ¹⁵N-labelled KNO₃ concentrations for 15min, and the content of ¹⁵N in roots was measured. Values are means ±SE (n=3). Different letters above the bars indicate significant differences at P<0.05, which were calculated separately under LN and HN conditions.

To investigate the effects of [Ca²⁺]_cyt on nitrate uptake under NaCl treatments in S. europaea, LaCl₃ (a Ca²⁺ channel blocker) was applied to S. europaea roots, and the nitrate influx rates were detected. Pre-treatment with LaCl₃ resulted in decreased [Ca²⁺]_cyt (Supplementary Fig. S4 at JXB online), and the nitrate uptake rates of roots were significantly reduced under the three treatments besides the HN conditions compared with the control. Moreover, the reduction was much more pronounced under NaCl treatments than without NaCl treatments. After LaCl₃ pre-treatment, no significant difference in the nitrate uptake rate was observed between LN and LN+S, or between HN and HN+S (Fig. 7C). This result indicated that the decrease in [Ca²⁺]_cyt not only caused a decrease in nitrate uptake rate, but also attenuated the promotion effects of NaCl on this parameter.

Discussion

NaCl facilitates nitrate uptake in S. europaea

Generally, NaCl has inhibitory effects on nitrate assimilation in glycophytes. For example, in Arabidopsis, NO₃ ^– concentrations and NR activity in both leaves and roots were markedly decreased by NaCl during the second week of treatment (Debouba et al., 2013). However, in this study, it was found that NaCl can promote nitrate uptake in the euhalophyte S. europaea. The shoot NO₃ ^– concentration and nitrate uptake rates of S. europaea were significantly promoted by NaCl under both low N and high N conditions (Fig. 2D, E). These results are consistent with the findings for the euhalophyte S. physophora which showed that NaCl application significantly increased the leaf NO₃ ^– concentration under N-sufficient conditions (Yuan et al., 2010). The current results and previous studies together show that the effect of NaCl on nitrate uptake and assimilation may be significantly different between glycophytes and halophytes. NaCl generally has a positive influence on the growth of euhalophytes, for which zero NaCl is an abnormal condition whereas 200mM NaCl is in the range of the optimal condition (Flowers and Colmer, 2008). Previous studies showed that the growth of S. europaea is stimulated by 200–400mM NaCl (Wang et al., 2009; Lv et al., 2012). Consistently, in this study, it was found that supply of 200mM NaCl improved the growth of S. europaea under both LN and HN conditions (Fig. 1A, B). Better growth of S. europaea under salt conditions increased the demand for N, which could at least partly explain the stimulation of nitrate uptake and increased plant N content by salt treatment (Fig. 2B, E). Furthermore, there might be an NaCl-facilitated nitrate uptake mechanism in S. europaea, which is directly supported by the findings that nitrate uptake rates and shoot NO₃ ^– concentrations of S. europaea were significantly promoted by NaCl treatment under both high N and low N conditions (Fig. 2D, E). In addition, the NR activities were significantly increased by salt under the high N condition (Fig. 2F). Consistent with this study, a sodium-dependent nitrate transport at the PM of leaf and root cells was also reported in Zostera marina, which is a seagrass that grows submerged in seawater where the NaCl concentration is ~500mM (Rubio et al., 2005).

The possible roles of Ca²⁺ signalling proteins identified by comparative proteomics in S. europaea

Ca²⁺ plays vital roles in plant development and in response to environmental stimuli (DeFalco et al., 2010). Developmental and environmental cues are perceived at the cell surface, thereby eliciting [Ca²⁺]_cyt changes through concerted action of channels, pumps, and carriers (Kudla et al., 2010). Two protein families, ANN and calreticulin (CRT), involved in this process were identified in this proteomic study. ANN encodes unconventional calcium-permeable channels, which is a key component of root cell adaptation to salinity (Davies, 2014). Four ANN proteins (spots 1358, 1401, 1402, and 1480) were significantly increased in response to NaCl (Fig. 6), indicating that they may act as NaCl-regulated Ca²⁺-permeable channels in S. europaea. Interestingly, spots 1401 and 1402 represented the same protein, which have the same molecular mass but different pI values (Fig. 3B). It is speculated that the isoforms may be generated by phosphorylation of ANN. CRT is a highly conserved Ca²⁺-binding protein functioning in intracellular Ca²⁺ homeostasis and signalling and is predominantly located in the ER; CRT is reportedly associated with the extracellular membrane surface (Jia et al., 2009). CRT is an NaCl-responsive protein in potato (Aghaei et al., 2008), as well as in Arabidopsis (Jiang et al., 2007). In S. europaea, the abundance of CRT (spot 830) was up-regulated by NaCl only under HN conditions, and it was also induced by HN under NaCl application (Fig. 6), indicating that CRT may act as an NaCl- and HN-responsive protein in S. europaea regulating calcium homeostasis and signalling transduction.

The spatio-temporal Ca²⁺ signals are then decoded and transmitted by Ca²⁺-binding proteins (DeFalco et al., 2010). Calmodulin (CaM)-like (CML) and 14-3-3 proteins involved in this process were identified. CMLs are involved in Ca²⁺ signal transduction by associating with numerous downstream targets, such as protein kinase, phosphatases, transcription factors, transporters, and channels (Popescu et al., 2007). CMLs have been implicated in plant development processes and abiotic stress responses (Magnan et al., 2008). In this study, one CML13-like protein was up-regulated by NaCl under HN conditions (Fig. 6), and this protein may function in Ca²⁺ signalling as a part of the response of S. europaea to NaCl under N-sufficient conditions. In contrast, the accumulation of CML was significantly down-regulated after 48h salt treatment in roots of barely, which was accompanied by significant reduction of root Ca²⁺ content (Wu et al., 2014). These different trends may reflect diverse mechanisms of CML regulation in glycophytes and halophytes. 14-3-3 proteins are also involved in Ca²⁺ signalling, which can interact with Ser/Thr-phosphorylated proteins (Pauly et al., 2007). They have been reported to regulate plant N assimilation by tuning the activities of NRT2, NR, and glutamine synthase (GS). Two phosphorylation sites of NRT2.1 were identified as being regulated by NaCl treatment (Vialaret et al., 2014), and sequence analysis of NRT2s in tobacco and Arabidopsis has identified some possible 14-3-3-binding sites (Guo et al., 2011). The activities of cytosolic NR and GS, the key enzymes in nitrate assimilation, are regulated through phosphorylation by 14-3-3s, calcium-independent protein kinases (CIPKs), and calcium-dependent protein kinases (CDPKs) (Masclaux-Daubresse et al., 2010). NR is the target of CPK17 and 14-3-3, which is regulated by salt stress (Lambeck et al., 2010). Notably, the two 14-3-3 proteins identified in this study changed oppositely in response to NaCl treatment (Fig. 6). Similarly, in salt-tolerant rice strains, the abundance of two 14-3-3 proteins increased, whereas one 14-3-3 protein decreased under salt stress (Liu et al., 2014). These findings indicate that NaCl may regulate 14-3-3 proteins through multiple mechanisms.

Changes in [Ca²⁺]_cyt and downstream Ca²⁺ signalling are essential for NaCl-facilitated nitrate uptake in S. europaea

Salt stress is first perceived at the cell membrane level, and then an intracellular signalling cascade is triggered via secondary messengers (Kader and Lindberg, 2010). The present results suggested that Ca²⁺ acted as an essential second messenger in NaCl-facilitated nitrate uptake in S. europaea and functioned through downstream signalling components. First, [Ca²⁺]_cyt was significantly elevated in S. europaea under both short- and long-term NaCl treatment (Fig. 7A, B). Many studies have revealed an increase in [Ca²⁺]_cyt after salt stress (Kader et al., 2007; D’Onofrio and Lindberg, 2009). NaCl induces a higher increase in [Ca²⁺]_cyt in salt-tolerant rice cultivars than in salt-sensitive cultivars (Kader et al., 2007). However, in Arabidopsis root cells or corn root protoplast, NaCl induced a decrease in [Ca²⁺]_cyt within minutes (Lynch and Läuchli, 1988; Halperin et al., 2003). Thus, the regulation of [Ca²⁺]_cyt by NaCl varies with the tested species, cells, and tissues. It is likely that the amplitude, frequency, and duration of changes in [Ca²⁺]_cyt are important for decoding the specific downstream responses for salt stress response in plants. Secondly, a decline in [Ca²⁺]_cyt not only caused a decrease in nitrate uptake rate, but also eliminated the promotion effects of NaCl on this parameter (Fig. 7C), suggesting that the NaCl-induced [Ca²⁺]_cyt elevation was essential for NaCl-facilitated nitrate uptake in S. europaea. Evidence on the direct relationship between [Ca²⁺]_cyt and nitrate uptake is still lacking, but some research has shown links between them. Application of Ca²⁺ could increase nitrate uptake under salt stress, and this effect may be ascribed to its involvement in preserving the structural and functional integrity of cell membranes or in increasing the activities of nitrate transporters (Mansour, 2000). Meanwhile, plants treated with EGTA (a specific Ca²⁺ chelator) or La³⁺ (a Ca²⁺ channel blocker) showed decreased induction of the key genes involved in the nitrate assimilatory pathway, such as NR and nitrite reductase (NiR) (Vidal et al., 2010). Thirdly, eight calcium signalling proteins were identified in this proteomic study, seven of which were significantly up-regulated under NaCl treatments (Fig. 6); it is speculated that these may play important roles in NaCl-facilitated nitrate uptake in S. europaea. The important roles of Ca²⁺ signalling proteins in salt adaption have been reported in other plant species. In Arabidopsis, the phosphorylation levels of CPKs changed significantly under salt stress (Vialaret et al., 2014). Mehlmer et al. (2010) also reported that CPK3-mediated Ca²⁺ signalling is required for salt stress acclimation, and 28 potential CPK3 targets were identified by their studies. Among these, remorin, CRT, VDAC (voltage-dependent anion channel), 14-3-3 proteins, and RAB GTPase, all of which play roles in cellular ion homeostasis and signal transduction under salt stress (Mehlmer et al., 2010), were also identified in the present study. In addition, Latz et al. (2013) reported that the vacuolar two-pore K⁺ channel 1 (TPK1) was a target of CPK3 and 14-3-3 protein, and played an essential role in the regulation of the cytosolic K⁺/Na⁺ ratio under salt stress adaptations.

Based on the present results and previous studies, a possible regulatory network of NaCl-facilitated nitrate uptake in S. europaea focusing on the involvement of Ca²⁺ signalling is proposed (Fig. 8). In S. europaea roots, the NaCl stimuli induced Ca²⁺ entry across the PM by activating ANN protein and some unknown Ca²⁺ channels (Davies, 2014), thereby causing [Ca²⁺]_cyt elevation. CRT is involved in cytosolic Ca²⁺ homeostasis (Jia et al., 2009). The [Ca²⁺]_cyt elevation is sensed by calcium signalling proteins including CaM/CML, CBL/CIPKs, as well as CDPKs/CPKs (DeFalco et al., 2010). 14-3-3 protein can be activated by CPK3 or some unknown Ca²⁺ signalling components (Mehlmer et al., 2010). The calcium signalling proteins can transmit the signal into phosphorylation cascades capable of modulating gene expression, and target protein activity, which may function in ion transport/homeostasis, membrane trafficking, redox homeostasis, and so on (DeFalco et al., 2010). Thus, the elevated nitrate uptake rates and NR activity under NaCl treatment in S. europaea may be ascribed to calcium-dependent phosphorylation of proteins such as those described in the following sentences. CBL1/9 and CIPK8/23 are involved in the regulation of NRT1.1 by phosphorylation (Ho et al., 2009; Hu et al., 2009), while 14-3-3 is involved in the regulation of NRT2s (Guo et al., 2011). NRs in the cytosol are targets of CPK17 and 14-3-3, which are also regulated by salt stress (Lambeck et al., 2010). The subsequent steps of N assimilation in roots take place in plastids. GS participates in this processs and can be regulated by CIPK and 14-3-3 through phosphorylation (Masclaux-Daubresse et al., 2010).

Fig. 8.

A putative regulatory network of NaCl-facilitated nitrate uptake in S. europaea. In S. europaea roots, the NaCl stimuli induced Ca²⁺ entry across the PM by activating ANN protein and some unknown Ca²⁺ channels, thereby causing [Ca²⁺]_cyt elevation. CRT is involved in cytosolic Ca²⁺ homeostasis. The [Ca²⁺]_cyt elevation is sensed by calcium signalling proteins including CaM/CML, CBL/CIPKs, and CDPKs/CPKs. 14-3-3 protein can be activated by CPK3 or some unknown Ca²⁺ signalling components. The calcium signalling proteins can transmit the signal into phosphorylation cascades capable of modulating gene expression and target protein activity, which may function in ion transport/homeostasis, membrane trafficking, redox homeostasis, and so on. CBL1/9 and CIPK8/23 are involved in the regulation of NRT1.1 by phosphorylation while 14-3-3 is involved in the regulation of NRT2s. NRs in the cytosol are targets of CPK17 and 14-3-3, which are also regulated by salt stress. The subsequent steps of N assimilation take place in plastids. GS participates in this process and can be regulated by CIPK and 14-3-3 through phosphorylation. The components in red indicate those identified in the present study.

Conclusions

In this study, it was first found that, unlike in glycophytes, NaCl facilitates nitrate uptake in S. europaea, which may be a unique feature in halophytes. Further comparative proteomics of root PM proteins revealed 81 differentially accumulated proteins in response to salt and nitrate. Among them, there are eight calcium signalling components, and the accumulations of seven was increased in response to salinity. [Ca²⁺]_cyt was significantly elevated in S. europaea under both short- and long-term NaCl treatment. In addition, application of the Ca²⁺ channel blocker LaCl₃ not only caused a decrease in nitrate uptake rate but also reduced the promotion effects of NaCl on the nitrate uptake rates. It is proposed that NaCl induced [Ca²⁺]_cyt elevation and the downstream calcium signalling are essential for NaCl-facilitated nitrate uptake in S. europaea. The calcium signalling proteins may function through regulating the expression or activity of nitrate transporters and some key enzymes in N assimilation, which can consequently affect nitrate assimilation.

Supplementary data

Supplementary data are available at JXB online.

Figure S1. Coomassie blue-stained gel image of TM and PM samples.

Figure S2. Ten images of 2D-DIGE.

Figure S3. Scores and matched peptides of the identified proteins based on ultrafleXtreme MALDI-TOF/TOF-MS.

Figure S4. The effect of LaCl₃ on the relative [Ca²⁺]_cyt in S. europaea root tips.

Table S1. Protein samples assigned for Cy dye label and DIGE.

Table S2. Expression profile data of the 717 spots present in at least 24 of the 30 images.

Table S3. The differentially accumulated S. europaea PM proteins identified by MALDI-TOF/TOF.

Table S4. Protein distribution in the eight clusters.

Table S5. Specific primers used for real-time PCR of genes encoding calcium signalling components of S. europaea.

Glossary

Abbreviations:

ANN

annexin

ANOVA

analysis of variance

[Ca²⁺]_cyt

cytosolic Ca²⁺ concentration

CaM

calmodulin

CBL

calcineurin B-like

CDPK

calcium-dependent protein kinase

CIPK

calcium-independent protein kinase

CML

calmodulin-like

CRT

calreticulin

2D-DIGE

two-dimensional fluorescence difference gel electrophoresis

DW

dry weight

FW

fresh weight

GDH

glutamate dehydrogenase

GS

glutamine synthase

NiR

nitrite reductase

NR

nitrate reductase

NRT

nitrate transporter

PM

plasma membrane

TM

total microsomal

VDAC

voltage-dependent anion-selective channel.