Abstract

   To understand the cross-talk and specificity of the early responses of
   plants to salt and drought, we performed physiological and proteome
   analyses of Brassica napus seedlings pretreated with 245 mM NaCl or 25%
   polyethylene glycol (PEG) 6000 under identical osmotic pressure (-1.0
   MPa). Significant decreases in water content and photosynthetic rate
   and excessive accumulation of compatible osmolytes and oxidative damage
   were observed in response to both stresses. Unexpectedly, the drought
   response was more severe than the salt response. We further identified
   45 common differentially expressed proteins (DEPs), 143 salt-specific
   DEPs and 160 drought-specific DEPs by isobaric tags for relative and
   absolute quantitation (iTRAQ) analysis. The proteome quantitative data
   were then confirmed by multiple reaction monitoring (MRM). The
   differences in the proteomic profiles between drought-treated and
   salt-treated seedlings exceeded the similarities in the early stress
   responses. Signal perception and transduction, transport and membrane
   trafficking, and photosynthesis-related proteins were enriched as part
   of the molecular cross-talk and specificity mechanism in the early
   responses to the two abiotic stresses. The Ca^2+ signaling, G
   protein-related signaling, 14-3-3 signaling pathway and phosphorylation
   cascades were the common signal transduction pathways shared by both
   salt and drought stress responses; however, the proteins with executive
   functions varied. These results indicate functional specialization of
   family proteins in response to different stresses, i.e., CDPK21, TPR,
   and CTR1 specific to phosphorylation cascades under early salt stress,
   whereas STN7 and BSL were specific to phosphorylation cascades under
   early drought stress. Only the calcium-binding EF-hand family protein
   and ZKT were clearly identified as signaling proteins that acted as
   cross-talk nodes for salt and drought signaling pathways. Our study
   provides new clues and insights for developing strategies to improve
   the tolerance of crops to complex, multiple environmental stresses.

Introduction

   Brassica napus is the third most economically important oilseed crop in
   the world and is a essential source of plant oil and proteins for human
   consumption and animal feed [[37]1]. With global climate changes and
   industrialization, drought and salt have become major challenges to
   oilseed production [[38]2]. Improving the tolerance of oilseed crops to
   drought and salt is an important strategy to increase oilseed yield and
   supply. Understanding the mechanisms of both drought and salt in
   oilseed crops is critical. Compared to Arabidopsis, much less is known
   about the mechanism underlying oilseed responses to salt and drought
   stresses although some progress has been made recently [[39]2–[40]4].

   Both salt and drought severely affect crop growth and production
   [[41]5, [42]6]. Drought limits plant growth by imposing osmotic stress,
   whereas salt disrupts plant growth by causing physiological drought and
   ion toxicity [[43]7]. Elucidating the universal and specific response
   mechanisms of drought and salt will provide insights on how plants
   integrate multiple signal pathways that may cross-talk or diverge at
   various steps in the response pathway [[44]8]. Cross-talk among Ca^2+
   and ABA-signaling pathways and mitogen-activated protein kinase (MAPK)
   cascades in drought and salt stress responses have recently been
   reported [[45]8–[46]10]. As a second messenger, calcium is a major
   factor in signaling cross-talk because it can be induced by both salt
   and drought. ABA is an important phytohormone that plays a critical
   role in response to various stress signals, particularly, in regulating
   plant water balance. MAPKs connect diverse receptors/sensors to a wide
   range of abiotic stresses, including drought, salt and cold. Members of
   MAPK cascades are activated in plants in response to various stresses,
   suggesting MAPKs act as cross-talk nodes in stress signaling
   [[47]8–[48]10]. Although conventional forward and reverse genetic
   approaches have provided valuable insights into stress responses,
   technical limitations may prevent further development. To compensate
   for the defects of traditional technologies, genome-wide transcriptome
   and proteome strategies have been applied. Using these-omics
   approaches, a large number of salt and drought stress-related genes or
   proteins have been identified [[49]11, [50]12]. However, no systematic
   consensus on the specific categories of proteins that correspond to
   particular signaling events has been established so far. The complexity
   of signaling networks is far beyond our imagination. Identification of
   key factors at the nodes and branches between different stress-response
   pathways will aid in understanding the mechanism of stress responses in
   higher plants. Much effort is required to elucidate the critical points
   of convergence and divergence that enable the integration of different
   signals.

   Proteomic technologies are an effective approach to conduct
   large-scale, quantitative, and reproducible analyses of the proteomic
   profiles of a stress responses. Moreover, mass spectrometry (MS)-based
   proteomic analysis is a powerful tool to identify isoforms of specific
   proteins, thus enabling the specific and overlapping functions within a
   protein family to be distinguished. Using this strategy, most of the
   previous proteomic analyses of abiotic stress responses in higher
   plants have focused on the responses to long-term (≥ 24 h) exposure to
   stress [[51]13]. Only a few studies have focused on the early (≤ 24 h)
   transcriptome and physiological responses [[52]14–[53]17]. Very limited
   proteome data are available about the early events in the perception
   and transduction of drought and salt stress signals [[54]16, [55]18].
   Recent preliminary studies have demonstrated that the response of
   plants to stress maybe determined by the rapid perception of stress
   shock that occurs within hours [[56]14, [57]16]. However, these
   investigations did not extensively examine the systematic signaling
   network. Understanding the specificity and cross-talk between different
   stress stimuli is essential and may provide clues and insights for
   developing strategies to improve the tolerance of crops to complex
   multiple environmental stresses worldwide.

   In the present study, NaCl and PEG were used as salt and osmotic stress
   agents, respectively, to imitate environment salt and drought stresses.
   We investigated the proteomic profiles of the early response of B.
   napus to salt and drought stresses using iTRAQ-tandem mass spectrometry
   (MS/MS) technology. We observed enrichment of membrane trafficking and
   signal perception and transduction proteins, suggesting that these
   proteins were crucial for elucidating the molecular cross-talk and
   specificity mechanism in the early response to the two abiotic
   stresses. A calcium-binding EF-hand family protein and ZKT protein were
   key proteins mediating the cross-talk between salt and drought stress
   responses, whereas different potential receptors, small G proteins,
   14-3-3 proteins, kinases and phosphatase guided stress-specific late
   responses. Our results provided valuable insights to guide future
   studies of the cross-talk between the salt and drought stress
   responses.

Materials and Methods

Plant growth conditions and stress treatments

   Seeds of a winter-type B. napus line, Zhong Shuang11 (ZS11), were
   germinated in Petri dishes in the dark at 23°C for 4 days. Seedlings
   were transferred to plastic plots filled with half-strength (1/2)
   Hoagland’s nutrient medium and grown in a growth chamber in a
   controlled environment (16 h photoperiod, 350 μmol m^−2 s^−1, 22 ± 2°C,
   and 65%–70% relative humidity).

   The osmotic pressures of different concentrations of NaCl solution (200
   mM, 215 mM, 230 mM, 245 mM, 250 mM, and 260 mM) and PEG 6000 solution
   (18%, 20%, 22%, 24%, 25%, and 26%) (w/v) were detected using a
   freezing-point depression osmometer (Osmomat 030, Gonotec, Berlin,
   Germany). Isotonic solutions of 245 mM NaCl and 25% PEG 6000 (which
   showed an identical osmotic potential of -1.0 MPa) were selected for
   further stress-treatment experiments.

   For proteome analysis and physiological index determination, 15-day-old
   seedlings were randomly assigned to the control and stress treatments.
   After treatment for 4 h, true leaves were harvested for further
   experiments. For proteomic analysis, 60 seedlings in one plastic pot
   were used for each treatment; six independent experiments were
   performed for each treatment, and two biological repeats were pooled
   from the leaf samples of three experiments per treatment. For
   physiological analysis, three biological and technical repeats were
   performed for each treatment.

Physiological analysis

   The relative water content was measured according to with the
   methodology of Pang et al. (2010) [[58]11]. Total chlorophyll was
   isolated using an extraction solution (2:1 acetone:95% alcohol), and
   the chlorophyll a and b contents were measured at 646.6, 663.6, and 750
   nm using a spectrophotometer (Lambda, PerkinElmer, USA), as described
   elsewhere [[59]19]. The photosynthetic and transpiration rates of
   leaves were assessed using a LI-6400XT portable photosynthesis system
   (LI-COR Biosciences, Lincoln, NE, USA). Chlorophyll fluorescence was
   measured using an Imaging PAM 101 (Walz, Effeltrich, Germany), and
   F[v]/F[m] values were calculated according to the manufacturer’s
   instructions. Proline concentrations were estimated using the ninhydrin
   reaction method [[60]20, [61]21]. The concentrations of total soluble
   sugars and sucrose were determined as previously described [[62]21].
   Malondialdehyde (MDA) was extracted and measured as described elsewhere
   [[63]22]. For the H[2]O[2] content assay, plant leaves were homogenized
   in sodium phosphate buffer, and the supernatant and 0.1% (w/v) titanium
   sulfate reagent were mixed to precipitate the peroxide-titanium
   complex. The absorbance of the solution was quantified at a wavelength
   of 410 nm.

Protein extraction and digestion

   Approximately 1 g of fresh leaves was frozen and ground to a fine
   powder in liquid nitrogen. Total protein was extracted in lysis buffer
   [7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris-HCl (pH 8.5), 1 mM PMSF,
   and 2 mM EDTA] and then sonicated for 15 min. A 5× volume of chilled
   acetone was added to the protein mixtures, and the proteins were
   precipitated at -20°C for 2 h. The pellets obtained after
   centrifugation at 25,000×g for 20 min were resuspended in lysis buffer.
   The protein extracts were reduced with 10 mM DTT (final concentration)
   at 56°C for 1 h, followed by precipitation with a 5× volume of chilled
   acetone at -20°C for 2 h. After centrifugation at 25,000×g at 4°C, the
   pellets were dissolved in 0.5 M TEAB (Applied Biosystems, Milan, Italy)
   and sonicated on ice. After centrifugation at 25,000×g at 4°C, an
   aliquot of each supernatant was used to determine the protein
   concentration using Bradford assay reagents and a spectrophotometer
   (UV-160, Shimadzu, Kyoto, Japan). Bovine serum albumin was used as the
   protein standard. The supernatant proteins were stored at -80°C until
   proteomic analysis.

   For digestion, total protein (100 μg) was digested with Trypsin Gold
   (Promega, Madison, WI, USA) at a 30:1 protein:trypsin ratio at 37°C for
   16 h.

iTRAQ labeling and strong cation exchange (SCX) fractionation

   After trypsin digestion, peptides were dried by vacuum centrifugation.
   Peptides were reconstituted in 0.5 M TEAB and labeled using an 8PLEX
   iTRAQ^® Reagent Kit (AB Sciex Inc., Foster City, CA, USA) following the
   manufacturer’s recommendations. The untreated controls were labeled
   with iTRAQ tags 113 (biological repeat 1) and 117 (biological repeat
   2), the salt-treated samples were labeled with iTRAQ tags 114
   (biological repeat 1) and 118 (biological repeat 2), and the
   drought-treated samples were labeled with iTRAQ tags 115 (biological
   repeat 1) and 119 (biological repeat 2). The labeled peptides were
   incubated at room temperature for 2 h, then pooled and
   vacuum-centrifuged to dryness.

   SCX chromatography was performed with a LC-20AB HPLC pump system
   (Shimadzu, Kyoto, Japan). The iTRAQ-labeled peptide mixtures were
   reconstituted in 4 mL of buffer A (25 mM NaH[2]PO[4] in 25% ACN, pH
   2.7) and loaded onto a 4.6 × 250 mm Ultremex SCX column containing 5-μm
   particles (Phenomenex). The peptides were eluted at a flow rate of 1
   mL/min using a gradient of buffer A for 10 min, 5%–60% buffer B (25 mM
   NaH[2]PO[4], 1 M KCl in 25% ACN, pH 2.7) for 20 min, and 60%–100%
   buffer B for 2 min. The system was then maintained at 100% buffer B for
   1 min before equilibrating with buffer A for 10 min prior to the next
   injection. Elution was monitored by measuring the absorbance of the
   eluent at a wavelength of 214 nm, and fractions were collected at 1-min
   intervals. The eluted peptides were pooled into 20 fractions, desalted
   with a Strata X C18 column (Phenomenex) and vacuum-dried.

Liquid chromatography—electrospray ionization—tandem mass spectrometry

   Each fraction was resuspended in buffer A (5% ACN, 0.1% FA) and
   centrifuged at 20,000×g for 10 min. The average final peptide
   concentration was approximately 0.5 μg/μL. Five micrograms of peptide
   was loaded onto a LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan) using an
   autosampler and a 2-cm C18 trap column. The peptide was then eluted
   from a 10-cm analytical C18 column (inner diameter: 75 μm) packed
   in-house. The samples were loaded at 8 μL/min for 4 min, then a 35-min
   gradient was run at 300 nL/min starting from 2% to 35% B (95% ACN, 0.1%
   FA), followed by a 5-min linear gradient to 60%, followed by a 2- min
   linear gradient to 80%, maintained at 80% for 4 min, and finally
   returned to 5% in 1 min.

   Data processing was performed using a TripleTOF 5600 System (AB SCIEX,
   Concord, ON, Canada) fitted with a NanoSpray III source (AB SCIEX,
   Concord, ON, Canada) and a pulled-quartz tip as the emitter (New
   Objectives, Woburn, MA, USA). For information-dependent acquisition,
   survey scans were acquired at 250 ms, and as many as 30 product ion
   scans exceeding a threshold of 120 counts/s and with a 2+ to 5+
   charge-state were collected. The total cycle time was fixed at 3.3 s.
   The Q2 transmission window was 100 Da for 100%. Four time bins were
   summed for each scan at a pulser frequency of 11 kHz by monitoring the
   40-GHz multichannel TDC detector with four-anode channel detection. A
   sweeping collision energy setting of 35±5 eV coupled with
   iTRAQ-adjusted rolling collision energy was applied to all precursor
   ions for collision-induced dissociation. Dynamic exclusion was set for
   half the peak width (15 s), and the precursor was then removed from the
   exclusion list.

Proteomic data analysis

   The raw data files acquired from the Orbitrap were converted to MGF
   files using Proteome Discoverer 1.2 (Thermo Fisher Scientific, Bremen,
   Germany). Protein identification was performed using the Mascot search
   engine (version 2.3.02; Matrix Science, London, UK) against two B.
   napus genome databases of an unpublished database containing 83,006
   sequences and a published database containing 98,762 sequences
   ([64]http://www.genoscope.cns.fr/brassicanapus/data/). Mass tolerance
   of 0.05 Da (ppm) was permitted for intact peptide masses and 0.1 Da for
   fragmented ions, with one missed cleavage allowed in the trypsin
   digests. Gln->pyro-Glu (N-term Q), oxidation (M), and deamidated (NQ)
   were selected as the potential variable modifications, and
   carbamidomethyl (C), iTRAQ8plex (N-term), and iTRAQ8plex (K) were
   selected as fixed modifications. Specifically, an automatic decoy
   database search was performed in Mascot by choosing the decoy checkbox
   in which a random sequence from the database was generated and tested
   for the raw spectra and the true database. To reduce the probability of
   false peptide identification, only peptides with a significance score
   ≥20 at the 99% confidence interval from a Mascot probability analysis
   greater than “identity” were counted.

   The minimum requirements for differentially expressed proteins (DEPs)
   were at least two matched unique peptides and a significant change (P <
   0.05 and > or < 1.2-fold) in protein quantities between the
   stress-treated samples and control samples in at least one replicate,
   with the other replicate displaying a similar trend. Functional
   annotation of DEPs was conducted using the Blast2GO program against
   NCBInr and Swiss-Prot databases. The Kyoto Encyclopedia of Genes,
   Genomes database (KEGG; [65]http://www.genome.jp/kegg/) and the
   Clusters of Orthologous Groups of Proteins database (COG;
   [66]http://www.ncbi.nlm.nih.gov/COG/) were used to classify the
   proteins.

Protein validation by multiple reaction monitoring (MRM)

   MRM was used to validate the DEPs. MRM transitions for selected
   proteins of interest were predicted using Skyline based on a protein
   FASTA database derived from a MS/MS spectra library imported from
   ProteinPilot software (AB SCIEX, Foster City, CA, USA) [[67]23].
   Samples were digested as described elsewhere [[68]24] and spiked with
   20 fmol of α-galactosidase for data normalization. MRM analyses were
   performed on a QTRAP 5500 mass spectrometer (AB Sciex Inc., Foster
   City, CA, USA) equipped with a Waters nanoACQUITY ultra-performance
   liquid chromatography system (Waters Corp., Milford, MA). The mobile
   phase consisted of 0.1% aqueous formic acid (solvent A) and 98%
   acetonitrile with 0.1% formic acid (solvent B). Peptides were separated
   on a BEH130 C18 column (0.075 × 200 mm column, 1.7 μm; Waters) at 300
   nL/min, and eluted with a gradient of 5%−8% solvent B for 2 min, 8%−30%
   solvent B for 94 min, and 30%−80% solvent B for 3 min. For QTRAP 5500
   mass spectrometry, a spray voltage of 2100 V, nebulizer gas of 20
   p.s.i., and dwell time of 10 ms were applied. Multiple MRM transitions
   were monitored using a unit resolution in both Q1 and Q3 quadrupoles to
   maximize specificity. Data analysis was conducted using Skyline
   [[69]23]. The three most abundant transitions for each peptide were
   used for quantitation unless interference from the matrix was observed.

Statistical analysis

   Student’s t-test was used for significance evaluation when only two
   groups were compared. The statistical significance of multiple
   comparison among groups was evaluated by one-way ANOVA. All
   calculations were performed using with software SPSS 19.0 (SPSS, Inc.,
   USA).

Results

Early physiological response to drought and salt stresses

   According to previous research, under the premise of iso-osmotic
   pressure, the major difference between salt and drought stress is ion
   toxicity caused by Na^+ and Cl^- [[70]7]. To eliminate differences due
   to different osmotic stressors, we determined the osmotic potential of
   different concentrations of NaCl and PEG 6000 solutions ([71]S1 Table).
   The results indicated that 245 mM NaCl and 25% PEG 6000 exhibited
   identical osmotic potential (-1.0 MPa); therefore, 245 mM NaCl and 25%
   PEG 6000 were used in subsequent experiments. We hypothesized that the
   early responses of B. napus to both stresses would initially involve
   the promotion of primary signal perception and transduction, which
   could be more important than the later responses to stresses. We
   observed the stress responses of 15-day-old B. napus seedlings during
   the early stage of the treatments. After treatment for 4 h, we observed
   visible phenotypic changes in both stress treatments ([72]Fig 1),
   namely, hypocotyl bending and cotyledon wilting. The stress symptoms
   under drought stress were more severe than those under salt stress.
   These physiological changes represented the plant’s early response to
   these specific stresses.

Fig 1. Phenotypic changes in B. napus seedlings exposed to 245 mM NaCl or 25%
PEG 6000 for 4 h.

   [73]Fig 1
   [74]Open in a new tab

   We first evaluated the net moisture content of the leaf, stem and root
   after 4 h of stress treatment. The net moisture content decreased under
   both salt and drought stress treatments. As shown in [75]Table 1, the
   moisture content decreased by 6.9% under salt stress and by 11.3% under
   drought stress in the leaf, 4.4% under salt stress and 8.8% under
   drought stress in the root, and 2.7% under salt stress and 7.4% under
   drought stress in the stem. These results indicated that the effects of
   water loss decreased in the order leaf>root>stem and were more severe
   under drought stress than under salt stress.

Table 1. Physiological and chemical parameters of B. napus seedlings after
treatment with 245 mM NaCl or 25% PEG 6000 for 4 h.

   Index Control Salt Drought
   Relative water content (%) Leaf 87.4 ± 2.7 (a) 81.4 ± 1.6 (b) 77.5 ±
   1.4 (c)
   Stem 82.3 ± 3.2 (a) 80.1 ± 1.5 (ab) 76.2 ± 0.8 (b)
   Root 83.4 ± 1.9 (a) 79.7 ± 3.5 (b) 76. 1± 1.5 (c)
   Proline (μmol g^-1 DW) 0.764 ± 0.004 (a) 1.092 ± 0.058 (b) 1.099 ±
   0.099 (b)
   Soluble sugar (mg g^-1DW) 40.276 ± 3.331 (a) 47.694 ± 2.260 (b) 52.907
   ± 1.086 (c)
   Sucrose (mg g^-1DW) 22.646 ± 2.632 (a) 32.868 ± 3.075 (b) 36.313 ±
   2.395 (c)
   H[2]O[2] (μmol mg^-1FW) 0.186 ± 0.016 (a) 0.275 ± 0.014 (b) 0.320 ±
   0.006 (c)
   MDA (μmol g^-1 FW) 3.047 ± 0.235 (a) 4.771 ± 0.207 (b) 5.054 ± 0.334
   (c)
   [76]Open in a new tab

   DW, dry weight; FW, fresh weight.

   The values represented the mean ± SD of three biological replicates.
   The different letters (a, b, and/or c) indicate significant differences
   among non-stressed, drought-stressed, and salt-stressed B. napus plants
   based on one-way ANOVA (p < 0.05).

   Previous studies have demonstrated that proline, sucrose, and total
   soluble sugars are compatible osmolytes for the maintenance of turgor
   and for the stability of cellular structures under adverse
   environmental conditions [[77]22]. We further determined the osmolyte
   levels of B. napus leaves after 4 h of stress treatments. As shown in
   [78]Table 1, the proline levels under salt stress and drought stress
   were 42.93% and 43.85% higher than that observed under control
   conditions, respectively, both significant differences, whereas the
   differences in proline content between drought and salt stress were not
   significant. The total soluble sugar concentration in leaves was
   elevated by 18.42% under salt stress and by 31.36% under drought
   stress, and the sucrose concentration in leaves increased by 45.14%
   under salt stress and by 60.35% under drought stress. ANOVA indicated
   that the increases in total soluble sugar and sucrose concentrations
   under drought stress were significantly higher than those observed
   after salt stress treatment ([79]Table 1).

   Generally, the concentrations of MDA and H[2]O[2] are major indicators
   of stress-triggered oxidative damage and ROS accumulation [[80]25]. We
   measured the concentrations of MDA and H[2]O[2] in leaves. The
   concentrations of MDA increased by 56.58% under salt stress and by
   65.87% under drought stress, whereas H[2]O[2] levels increased by
   47.85% under salt stress and by 72.04% under drought stress. These
   results indicated that oxidative damage and ROS accumulation in leaves
   were more severe under drought stress than under salt stress,
   consistent with the phenotypic results ([81]Table 1).

   To determine whether photosynthetic systems differ in their responses
   to different abiotic stresses, we measured chlorophyll a and b
   concentrations, chlorophyll fluorescence parameters, the net
   photosynthesis rate and the transpiration rate. Under salt stress, the
   chlorophyll a and b contents in B. napus leaves decreased by 20.9% and
   18.1%, respectively, whereas under drought stress, the chlorophyll a
   and b content decreased by 30.8% and 20.9%, respectively ([82]Fig 2A).
   The photosynthetic rate was inhibited by 81.8% under salt stress and by
   94.3% under drought stress. The transpiration rate decreased by 90.3%
   under salt stress and by 90.1% under drought stress ([83]Fig 2B). salt
   and drought stress-treated leaves exhibited lower F[v]/F[m] values than
   did the control ([84]Fig 2C). The adverse effects on photosynthetic
   systems were more severe under drought stress than under salt stress.

Fig 2. Characterization of salt- and drought-treated B. napus leaves.

   [85]Fig 2
   [86]Open in a new tab

   (a) Chlorophyll (Chl) a, Chl b, and total Chl concentration and Chl a/b
   ratio of stress-treated or untreated leaves. Error bars represent SD (n
   = 3). fw, fresh weight. (b) Photosynthetic rate and transpiration rate
   of stress-treated or untreated leaves. Error bars represent SD (n = 3).
   Pn, Photosynthetic rate; Tr, Transpiration rate. (c) F[v]/F[m] values
   of stress-treated or untreated leaves. The color bar indicates
   F[v]/F[m]values from 0 to 1. Different letters (a, b, and/or c)
   indicate significant differences among non-stressed, drought-stressed,
   and salt-stressed B. napus plants based on one-way ANOVA (p < 0.05).

   In summary, these results indicated that the physiological responses
   (e.g., water loss, osmolytes accumulation, oxidative damage, and
   photosynthesis) which usually were observed during the late stages of
   stress responses reported previously also occurred in the early stage
   of stress responses in the present study, and the physiological
   response under drought stress was stronger than that under salt stress.

Proteome profile of B. napus leaves under salt and drought stresses

   To further explore the early responses to salt and drought stresses in
   B. napus, we analyzed the proteome profiles of B. napus leaves under
   salt and drought stresses by iTRAQ-LC/MS-MS. In total, 339,224 spectra
   were obtained, of which 63,645 spectra were matched to known peptides
   and 37,623 spectra were categorized as unique peptides. A total of
   5,583 unique proteins were identified ([87]S2 and [88]S3 Tables). Among
   these proteins, 48.5% (2708/5583) had at least two unique peptides, 75%
   of the peptides length were 8–15 amino acids long, 60% of the proteins
   had molecular weights of 20–70 kD, and 71% of the proteins exhibited
   greater than 5% sequence coverage ([89]S1 Fig). These results indicated
   that the data qualified for further analysis.

   Further functional classification [[90]26] revealed that the proteins
   were primarily involved in posttranslational modification, protein
   turnover, chaperon functions, energy production and conversion, amino
   acid and carbohydrate transport and metabolism, translation,
   posttranslational modification/protein turnover, and signal
   transduction ([91]Fig 3). Our results indicated that the proteins
   expressed in the early responses were involved in nearly every aspect
   of plant growth and metabolism.

Fig 3. Clusters of Orthologous Groups of proteins (COG) classification of
total proteins in B. napus leaves.

   [92]Fig 3
   [93]Open in a new tab

   A: RNA processing and modification; B: chromatin structure and
   dynamics; C: energy production and conversion; D: cell cycle control,
   cell division, chromosome partitioning; E: amino acid transport and
   metabolism; F: nucleotide transport and metabolism; G: carbohydrate
   transport and metabolism; H: coenzyme transport and metabolism; I:
   lipid transport and metabolism; J: translation, ribosomal structure and
   biogenesis; K: transcription; L: replication, recombination and repair;
   M: cell wall/membrane/envelope biogenesis; N: cell motility; O:
   posttranslational modification, protein turnover, chaperones; P:
   inorganic ion transport and metabolism; Q: secondary metabolites
   biosynthesis, transport and catabolism; R: general function prediction
   only; S: function unknown; T: signal transduction mechanisms; U:
   intracellular trafficking, secretion, and vesicular transport; V:
   defense mechanisms; Y: nuclear structure; Z: cytoskeleton.

   To explore the similarities and differences in the proteome profiles of
   the salt and drought stress responses in B. napus leaves, we compared
   the proteome profiles of B. napus leaves from salt- and drought-treated
   seedlings with those of the non-treated control. As shown in [94]Fig 4,
   of the 5,583 unique proteins detected in B. napus leaves, 188 were
   differentially expressed between the control and salt-treated samples,
   and 205 were differentially expressed between the control and
   drought-treated samples. More DEPs were identified in drought-stressed
   leaves than in salt-stressed leaves.

Fig 4. Venn diagram of the distribution of differentially expressed proteins
responsive to salt and drought stresses.

   [95]Fig 4
   [96]Open in a new tab

   The circles are proportional to the number of proteins identified in
   each treatment. The overlapping regions indicate the number of common
   proteins. The number above or below the horizontal line in each portion
   indicates the number of up-regulated or down-regulated proteins. In
   total, 188 and 205 proteins were identified by iTRAQ as salt-responsive
   and drought-responsive, respectively. Forty-five proteins were
   identified under both salt and drought treatments. Among the 143
   salt-specific DEPs, 95 were up-regulated and 48 were down-regulated.
   Among 160 drought-specific DEPs, 99 were up-regulated and 61 were
   down-regulated. Among the 45 common DEPs, 26 were up-regulated and 19
   were down-regulated.

Validation of iTRAQ data for selected candidate proteins by MRM

   To study the early responses of B. napus to salt and drought stresses,
   the treatment duration should be very short; however, the range of
   change in the protein profile may be not very obvious within such a
   short period. To retain as much valuable information as possible, we
   set comparatively less stringent criteria (see [97]Materials and
   Methods) to define DEPs in the proteome analysis. Therefore, evaluating
   the reliability of the data was of great concern.

   MRM is a fast and highly precise method for quantifying targeted
   proteins through measurements of representative peptides via mass
   spectrometry [[98]27], particularly when antibodies directed against
   the proteins of interest are unavailable. To verify the quantitative
   data derived from iTRAQ, seven DEPs were randomly selected for
   validation by MRM. The temporal expression levels of the proteins
   detected by MRM corroborated well with the iTRAQ data ([99]Fig 5).
   These results demonstrated that the iTRAQ data were highly reliable for
   further analysis and that our definition of DEPs was likely adequate to
   identify real differences between the stress treatments and control.

Fig 5. Relative expression levels of selected proteins measured by iTRAQ and
MRM in B. napus leaves under the control, 245 mM NaCl and 25% PEG 6000
treatments for 4 h.

   [100]Fig 5
   [101]Open in a new tab

   (a) FAD-binding and BBE domain-containing protein, (b) chlorophyll a-b
   binding protein CP29.3, (c) TIC110, (d) ATPase 2, (e) thylakoid lumenal
   16.5 kDa protein, (f) GPI-anchored protein, (g): carbonic anhydrase.
   Error bars represent SD (n = 3). Asterisks indicate a significant
   difference compared with the control (p < 0.05).

Overlapping changes in protein profiles in response to drought and salt
stresses

   Our previous physiological data indicated that common osmotic stress
   and the associated oxidative stress may lead to similar physiological
   and molecular responses to salt and drought stresses. To further
   determine whether similar trends exist in the proteomic profiles of the
   early-stage responses to salt and drought stresses, we analyzed the
   DEPs shared by the two stress treatments. In total, 24% (45/188) and
   22% (45/205) of the DEPs were shared by plants under both salt and
   drought stress treatments, respectively. Excluding two proteins that
   exhibited opposite trends, 43 common proteins displayed extremely
   similar change trends in response to the stress treatments ([102]Table
   2 and [103]S4 Table).

Table 2. Differentially expressed proteins common to the salt and drought
stresses responses.

   Accession[104] ^a Protein name Species Uniquepeptide Fold change of
   NaCl/CK Fold change of PEG/CK
   sp|[105]P14279 Chlorophyll a-b binding protein 5 Solanum lycopersicum 3
   1.5 1.36
   gi|62321409 Carbonic anhydrase Arabidopsis thaliana 2 1.19 1.82
   gi|62319619 Xylose isomerase Arabidopsis thaliana 10 1.29 1.2
   sp|[106]Q9SVG4 Reticuline oxidase-like protein Arabidopsis thaliana 7
   1.2 1.28
   gi|9279589 Dihydrolipoamide S-acetyltransferase Arabidopsis thaliana 3
   0.83 0.86
   sp|[107]Q2V2S7 NAD(P)H-quinone oxidoreductase subunit M Arabidopsis
   thaliana 3 0.84 0.8
   gi|119389583 Allene oxide cyclase 2 Arabidopsis thaliana 2 1.62 2.38
   sp|[108]Q93XW5 Nitrile-specifier protein 5 Arabidopsis thaliana 5 1.6
   1.5
   sp|[109]Q9SJD4 Long chain acyl-CoA synthetase 8 Arabidopsis thaliana 5
   1.24 1.35
   gi|62319394 Dihydroorotate dehydrogenase like-protein Arabidopsis
   thaliana 6 1.26 1.29
   sp|[110]Q9SXP7 Octanoyltransferase Arabidopsis thaliana 4 1.24 1.21
   gi|15229458 Cyanate hydratase Arabidopsis thaliana 3 1.21 0.85
   gi|15228596 Cysteine synthase A Arabidopsis thaliana 4 0.81 0.84
   gi|15241295 Aspartokinase 2 Arabidopsis thaliana 4 0.78 0.81
   gi|42572701 Biotin/lipoyl attachment domain-containing protein
   Arabidopsis thaliana 3 0.71 0.66
   gi|6048743 Chitinase Brassica juncea 2 1.49 1.61
   gi|347309137 Ascorbate peroxidase Brassica oleracea 6 1.17 1.22
   gi|334185637 Peroxiredoxin Q Arabidopsis thaliana 6 0.78 0.77
   gi|15229775 40S ribosomal protein S14-2 Arabidopsis thaliana 2 1.37
   1.25
   sp|[111]Q6WZ83 Histone H4 Eucalyptus globulus 7 1.41 1.22
   sp|[112]Q9SJA6 Serine/arginine-rich splicing factor RSZ22A Arabidopsis
   thaliana 4 0.78 1.22
   sp|[113]Q9SMB8 Tyramine N-feruloyltransferase 4/11 Nicotiana tabacum 3
   0.9 0.88
   sp|[114]P49313 30 kDa ribonucleoprotein Nicotiana plumbaginifolia 3
   0.83 0.75
   sp|[115]Q6ICX4 Polypyrimidine tract-binding protein homolog 3
   Arabidopsis thaliana 2 0.76 0.76
   gi|20140684 Translationally-controlled tumor protein homolog Brassica
   oleracea 2 0.88 0.82
   gi|118197456 Elongation factor 1-beta Brassica rapa 2 0.88 0.82
   gi|119720832 Ribosomal protein S27 Brassica rapa 4 0.83 0.86
   gi|15239409 60S ribosomal protein L12-3 Arabidopsis thaliana 3 0.81
   0.84
   gi|42564228 MAR-binding filament-like protein 1 Arabidopsis thaliana 2
   0.73 0.72
   gi|62321351 Leucine aminopeptidase Arabidopsis thaliana 3 1.31 1.25
   gi|15242045 Chaperonin 20 Arabidopsis thaliana 4 0.83 0.8
   sp|[116]O22870 Peptidyl-prolyl cis-trans isomerase FKBP16-3 Arabidopsis
   thaliana 4 0.78 0.81
   gi|297848776 Calcium-binding EF-hand family protein Arabidopsis lyrata
   10 1.33 1.36
   gi|18405391 ZKT protein containing PDZ, K-box and a TPR region
   Arabidopsis thaliana 11 0.83 0.82
   gi|110739803 Syntaxin related proteint Vam3p Arabidopsis thaliana 2
   1.74 1.56
   gi|62320206 Ca^2+-dependent membrane-binding protein annexin
   Arabidopsis thaliana 5 1.62 1.25
   gi|297843340 Synaptotagmin Arabidopsis lyrata 4 1.43 1.39
   sp|[117]Q0WNJ6 Clathrin heavy chain 1 Arabidopsis thaliana 2 1.2 1.24
   gi|121489751 ABC transporter precursor Phillyrea latifolia 6 1.17 1.23
   sp|[118]Q8LC95 Early nodulin-like protein 3 Arabidopsis thaliana 3 1.52
   1.64
   sp|[119]Q9LQU4 PLANT CADMIUM RESISTANCE 2 Arabidopsis thaliana 3 1.31
   1.53
   gi|159138535 Leucine zipper protein Helianthus annuus 2 1.19 1.26
   gi|147816472 Hypothetical protein Vitis vinifera 7 1.26 1.3
   gi|297800834 Hypothetical protein Arabidopsis lyrata 2 1.12 1.24
   gi|312281509 Unnamed protein Thellungiella halophila 3 0.69 0.64
   [120]Open in a new tab

   ^a Accession numbers according to NCBInr (gi) or Swiss-Prot (sp).

   The functions of those proteins were categorized according to
   biological functions that included photosynthesis, carbohydrate and
   energy metabolism, metabolism, stress and defense, transcription- and
   translation-related, protein folding and degradation, signaling,
   membrane and transport ([121]S4 Table). Approximately 24% (11/45) of
   the proteins were transcription- and translation-related, indicating
   that both stresses clearly affected gene transcription and protein
   translation. Approximately 73% of those proteins (8/11) were
   down-regulated in both stress treatments, i.e., 60S ribosomal protein
   L12-3, ribosomal protein S27, 30 kDa ribonucleoprotein, and elongation
   factor 1-beta. Furthermore, all proteins in the category of membrane
   and transport were up-regulated. These proteins included vam3p,
   annexin, synaptotagmin, clathrin heavy chain 1, and ABC transporter
   precursor, showing that up-regulation of these proteins may
   re-establish the cellular osmotic balance and in vivo
   compartmentalization under both stress treatments, consistent with a
   previous report [[122]26].

Profiles of drought- and salt-specific differentially expressed proteins

   Based on pairwise comparisons, 143 salt stress-specific DEPs, including
   95 up-regulated and 48 down-regulated proteins, and 160 drought
   stress-specific DEPs, including 99 up-regulated and 61 down-regulated
   proteins, were identified ([123]Fig 4). Further characterization of the
   functional features of the DEPs revealed that they were involved in a
   wide range of biological processes, including photosynthesis,
   carbohydrate and energy metabolism, metabolism, stress and defense,
   transcription- and translation-related, protein folding and
   degradation, signaling, membrane and transport, and “miscellaneous”
   ([124]Fig 6, [125]S5 and [126]S6 Tables).

Fig 6. Expression patterns of salt stress-specific and drought
stress-specific differentially expressed proteins in each functional
category.

   [127]Fig 6
   [128]Open in a new tab

   [129]Fig 6 presents the overall response pattern of B. napus leaves to
   specific salt or specific drought stress based on their proteomic
   profiles. Two common features were observed by pairwise comparison of
   both stress responses: 1) overall, more proteins were up-regulated than
   down-regulated, regardless of the actual stressor; 2) two key
   categories associated with growth, “metabolism” and “transcription and
   translation”, notably changed under both salt and drought stresses.
   Several differences in stress responses were also observed: 1) there
   were more drought-specific responsive DEPs than salt-specific
   responsive DEPs, and more proteins were down-regulated proteins in
   response to drought than to salt stress; 2) more photosynthesis- and
   metabolism-related proteins were down-regulated in response to drought,
   whereas more protein folding- and degradation-associated proteins and
   signaling proteins were up-regulated in response to salt. These
   proteomic features, including greater activation of transcription and
   translation, more severe attenuation of metabolism, and greater
   inhibition of photosynthesis-related proteins in response to drought,
   were consistent with the more severe physiological responses to drought
   stress in B. napus. Meanwhile, the proteomic changes revealed greater
   number of active protein folding- and degradation-associated proteins
   and signaling proteins in response to salt, consistent with the
   physiological phenomenon of enhanced salt tolerance in B. napus (Figs
   [130]1 and [131]2, [132]Table 1).

   To further understand the changes in the proteome profiles between the
   salt and drought stress responses, pathway enrichment analysis was
   performed. Compared to the control, proteins involved in signaling,
   membrane and transport and photosynthesis were largely enriched in both
   salt and drought stress responses. Therefore, these proteins were
   selected for further analysis.

DEPs related to signal perception and transduction

   Signaling components play essential roles in plant adaptions to abiotic
   stresses. In total, 25 signaling-related proteins were identified,
   including two overlapping DEPs in both treatments, 13 salt-specific
   DEPs and 10 drought-specific DEPs. The majority of these proteins were
   up-regulated ([133]Table 3). Based on the roles of these proteins in
   signal perception and transduction pathways, we classified these
   proteins into several groups, including probable receptors, small
   GTP-binding proteins, and Ca^2+ sensors ([134]Table 3).

Table 3. Signaling-related proteins differentially expressed under salt and
drought stresses.

   NaCl/CK PEG/CK
   Accession[135] ^a Protein name Fold change Species Accession[136] ^a
   Protein name Fold change Species
   (1) Probable receptor gi|110736873 GPI-anchored protein 1.13
   Arabidopsis thaliana sp|[137]Q9SYM9 Receptor-like
   serine/threonine-protein kinase 1.17 Arabidopsis thaliana
   sp|[138]Q8L710 Cysteine-rich receptor-like protein kinase 17 1.17
   Arabidopsis thaliana sp|[139]Q8GYA4 Cysteine-rich receptor-like protein
   kinase 10 1.28 Arabidopsis thaliana
   sp|[140]Q8VZG8 Probable LRR receptor-like serine/threonine-protein
   kinase 1.65 Arabidopsis thaliana sp|[141]Q9M8T0 Probable inactive
   receptor kinase 1.21 Arabidopsis thaliana
   sp|[142]O48788 Probable inactive receptor kinase 1.45 Arabidopsis
   thaliana
   sp|[143]Q9C9Y8 Probable inactive receptor kinase 1.19 Arabidopsis
   thaliana
   (2) Samll G protein related sp|[144]P31582 Ras-related protein RABF2a
   1.23 Arabidopsis thaliana gi|357470519 Ran-binding protein 0.83
   Medicago truncatula
   sp|[145]P92963 Ras-related protein RABB1c 1.21 Arabidopsis thaliana
   (3) Phospholipase sp|[146]Q39033 Phosphoinositide specific
   phospholipase C 1.22 Arabidopsis thaliana
   (4) Ca^2+ sensor gi|297848776 Calcium-binding EF-hand family protein
   1.33 Arabidopsis lyrata gi|297848776 Calcium-binding EF-hand family
   protein 1.36 Arabidopsis lyrata
   (5) 14-3-3 sp|[147]P48348 14-3-3-like protein GRF8 1.19 Arabidopsis
   thaliana sp|[148]Q01525 14-3-3-like protein GRF2 1.19 Arabidopsis
   thaliana
   (6) Kinases/phosphatases sp|[149]Q9ZSA2 Calcium-dependent protein
   kinase 21 1.56 Arabidopsis thaliana gi|56693619 Protein kinase C
   conserved region 2 1.23 Brassica napus
   sp|[150]Q9FHD7 Probable serine/threonine-protein kinase 1.39
   Arabidopsis thaliana sp|[151]Q9S713 Serine/threonine-protein kinase
   STN7 1.17 Arabidopsis thaliana
   sp|[152]Q9FR53 Serine/threonine-protein kinase TOR 1.28 Arabidopsis
   thaliana sp|[153]Q8L7U5 Serine/threonine-protein phosphatase BSL1 0.86
   Arabidopsis thaliana
   sp|[154]Q05609 Serine/threonine-protein kinase CTR1 1.25 Arabidopsis
   thaliana
   sp|[155]Q9SL76 Putative protein phosphatase 2C 1.32 Arabidopsis
   thaliana
   sp|[156]Q94AT1 Probable protein phosphatase 2C 0.85 Arabidopsis
   thaliana
   (7) Other gi|18405391 ZKT 0.83 Arabidopsis thaliana gi|18405391 ZKT
   0.82 Arabidopsis thaliana
   [157]Open in a new tab

   ^a Accession numbers according to NCBInr (gi) or Swiss-Prot (sp).

   Two common signaling-related DEPs that responded to both salt and
   drought stresses were identified: a calcium-binding EF-hand family
   protein and a novel probable ZKT protein ([158]Table 3). Three
   potential salt-specific response receptors and five potential
   drought-specific response receptors were also identified. Receptor-like
   kinases (RLKs) belong to a kinase superfamily that regulates plant
   growth and behavior [[159]28]. In the salt stress response, two RLKs
   consisting of a cysteine-rich receptor-like protein kinase 17 (CRK17)
   and a probable LRR receptor-like serine/threonine-protein kinase
   (LRR-RLK) were identified. Two RLKs, a cysteine-rich receptor-like
   protein kinase 10 (CRK10) and receptor-like serine/threonine-protein
   kinase were identified in the drought stress response. Interestingly,
   we identified a probable receptor protein in the salt stress response,
   glycosylphosphatidylinositol (GPI)-anchored protein, which has not been
   reported to be involved in salt or drought stress responses. These
   receptors were all up-regulated. Moreover, during downstream signal
   transduction, we identified three small G proteins. RABF2a and RABB1c
   were up-regulated by 23% and 21% under salt stress, whereas a
   Ran-binding protein exhibited a 17% reduction under drought stress.
   Furthermore, we found that the 14-3-3 protein GF14 kappa (GRF8) was
   up-regulated under salt stress, whereas the 14-3-3-like protein GF14
   omega (GRF2) was up-regulated under drought stress. Three
   serine/threonine-protein kinases, one calcium-dependent protein kinase
   21 (CDPK 21), and one putative protein phosphatase 2C displayed
   significant accumulation in response to salt stress. Another probable
   protein phosphatase 2C exhibited decreased expression during salt
   stress ([160]Table 3). By contrast, two kinases were up-regulated, and
   one serine/threonine-protein phosphatase, BSL1, was down-regulated
   under drought stress ([161]Table 3). In addition, we identified an
   up-regulated phosphoinositide-specific phospholipase C under salt
   stress but not under drought stress in the present study. These results
   indicated that early signal perception and transduction may be enhanced
   under salt and drought stresses.

DEPs related to ion transport and vacuolar trafficking

   ABC transporters function in the transport of hormones, lipids and
   secondary metabolites, which are widely reported to be associated with
   abiotic stress responses [[162]26, [163]29]. The present proteome
   analysis indicated that the ABC transporter precursor was up-regulated
   in both salt and drought stress treatments ([164]Table 4). The
   subfamilies ABC transporter B and ABC transporter C were specifically
   detected under salt stress, whereas the subfamily ABC transporter G was
   specifically identified under drought stress. These differences in
   expression indicated that different gene family members executed
   different functions to cope with various stresses. All ABC transporters
   were up-regulated under both stress treatments ([165]Table 4).
   Different TIC family proteins that play roles in chloroplast protein
   import trafficking were identified ([166]Table 4). In addition, two
   plasma-membrane-localized H^+-ATPases were up-regulated in B. napus
   leaves under salt stress; however, a vacuole-localized H^+-ATPase was
   down-regulated under drought stress. A Ca^2+-dependent membrane-binding
   protein, annexin D1 (ANN1), was up-regulated under both salt and
   drought stresses in B. napus leaves.

Table 4. Membrane and transport proteins differentially expressed under salt
and drought stresses.

   NaCl/CK PEG/CK
   Accession[167] ^a Protein name Fold change Species Accession[168] ^a
   Protein name Fold change Species
   (1) ABC transporter gi|121489751 ABC transporter precursor 1.17
   Phillyrea latifolia gi|121489751 ABC transporter precursor 1.23
   Phillyrea latifolia
   sp|[169]Q8LPQ6 ABC transporter B family member 28 1.15 Arabidopsis
   thaliana sp|[170]Q93YS4 ABC transporter G family member 22 1.23
   Arabidopsis thaliana
   sp|[171]Q9LZJ5 ABC transporter C family member 14 1.25 Arabidopsis
   thaliana sp|[172]Q9XIE2 ABC transporter G family member 36 1.15
   Arabidopsis thaliana
   (2) Other transporter sp|[173]Q9ZPY7 Exportin-2 1.35 Arabidopsis
   thaliana gi|13877587 2-Oxoglutarate/malate translocator
   proteinprecursor-like protein 1.22 Arabidopsis thaliana
   sp|[174]Q8LPR9 TIC110 1.22 Arabidopsis thaliana gi|28540971 TPT 1.34
   Brassica rapa
   sp|[175]Q9FMD5 TIC 40 0.89 Arabidopsis thaliana gi|30694885 TPT 1.52
   Arabidopsis thaliana
   sp|[176]Q9FJX3 Mitochondrial outer membrane protein porin 2 1.20
   Arabidopsis thaliana
   sp|[177]Q93XM7 Mitochondrial carnitine/acylcarnitine carrier-like
   protein 0.83 Arabidopsis thaliana
   (3) ATPase sp|[178]P20431 H+-ATPase 3, plasma membrane-type 1.38
   Arabidopsis thaliana sp|[179]Q8W4E2 V-type proton ATPase subunit B3
   0.83 Arabidopsis thaliana
   sp|[180]P19456 ATPase 2, plasma membrane-type 1.14 Arabidopsis thaliana
   sp|[181]Q9XES1 Calcium-transporting ATPase 4 1.32 Arabidopsis thaliana
   (4) Ion channel sp|[182]Q9SYT0 Ca^2+-dependent membrane-binding protein
   annexin D1 1.62 Arabidopsis thaliana sp|[183]Q9SYT0 Ca^2+-dependent
   membrane-binding protein annexin D1 1.25 Arabidopsis thaliana
   (5) Vesicle trafficking gi|110739803 Syntaxin related protein AtVam3p
   1.74 Arabidopsis thaliana gi|110739803 Syntaxin related protein AtVam3p
   1.56 Arabidopsis thaliana
   sp|[184]Q0WNJ6 Clathrin heavy chain 1 1.20 Arabidopsis thaliana
   sp|[185]Q0WNJ6 Clathrin heavy chain 1 1.24 Arabidopsis thaliana
   gi|297843340 Synaptotagmin 1.43 Arabidopsis lyrata gi|297843340
   Synaptotagmin 1.39 Arabidopsis lyrata
   sp|[186]Q5U247 Exocyst complex component 8 1.33 Xenopus laevis
   sp|[187]Q8L828 Coatomer subunit beta'-3 1.23 Arabidopsis thaliana
   sp|[188]P31582 Ras-related protein RABF2a 1.23 Arabidopsis thaliana
   sp|[189]Q9C827 Coatomer subunit beta'-2 1.53 Arabidopsis thaliana
   sp|[190]P92963 Ras-related protein RABB1c 1.21 Arabidopsis thaliana
   sp|[191]Q8LPK4 AP-2 complex subunit alpha-2 0.86 Arabidopsis thaliana
   gi|357470519 Ran-binding protein 0.83 Medicago truncatula
   [192]Open in a new tab

   ^a Accession numbers according to NCBInr (gi) or Swiss-Prot (sp).

   Furthermore, we identified 11 vesicle trafficking-related proteins that
   exhibited differential expression patterns under salt and drought
   stresses ([193]Table 4). Among these proteins, four proteins overlapped
   in both the salt and drought stress responses, including clathrin heavy
   chain 1, syntaxin-related protein AtVam3p, synaptotagmin and
   Ca^2+-dependent membrane-binding protein annexin D1. Exocyst complex
   component 84B was a salt-response-specific DEP. Coatomer subunit
   beta'-3, coatomer subunit beta'-2 and AP-2 complex subunit alpha-2 were
   identified as drought response-specific DEPs. In addition, three
   proteins belonging to small G protein family proteins (RABF2a, RABB1c
   and a Ran-binding protein) that function not only in single
   transduction but also in membrane trafficking were identified. Thus,
   the exchange of products between different endomembrane systems was
   adjusted under salt and drought stresses, and the adjustment modes
   differed between salt and drought stresses.

DEPs related to photosynthesis

   Previous study have showed that salt or drought stress can affect the
   photosynthetic rate and enzyme activities associated with CO[2]
   assimilation [[194]30]. In the present proteomic analysis, we also
   identified a set of DEPs associated with the photosynthetic system in
   B. napus leaves ([195]S4–[196]S6 Tables). These DEPs exhibited diverse
   responses to the stress treatments. i) Two proteins, chlorophyll a/b
   binding protein 5 and carbonic anhydrase, which functions in light
   harvesting and the Calvin cycle, were up-regulated in both stress
   treatments. ii) Four DEPs were salt response specific: two
   down-regulated chlorophyll a-b binding proteins, CP26 and Lhca2, the
   up-regulated chlorophyll a-b binding protein CP29.3, and an
   up-regulated thylakoid lumenal 16.5 kDa protein. iii) Seven DEPs were
   drought response specific, including two up-regulated and five
   down-regulated proteins. Notably, THYLAKOID FORMATION 1 (THF1), which
   is required for dynamic of PSII-LHCII supramolecular organization in
   higher plants by interacting with Lhcb, was up-regulated [[197]31].
   Three of the five down-regulated proteins were light reaction
   process-related proteins: oxygen-evolving enhancer protein 2,
   photosystem I reaction center subunit III, and PsbP domain-containing
   protein 4. The other two down-regulated proteins were ferredoxin or
   ferredoxin-like proteins. Ferredoxin, an electron carrier in the
   photosynthetic electron transport chain, accepts electrons from
   photosystem I and delivers them to essential oxido-reductive pathways.
   Taken together, most of these DEPs were up-regulated under salt stress
   treatment and down-regulated under drought stress. These results
   suggest that B. napus implements divergent mechanisms to modulate the
   photosynthetic machinery in response to different stresses.

Discussion

Systematic differences and similarities of the responses to salt and drought
stresses

   The physiological data indicated that most of the stress responses were
   similar between the salt and drought treatments, such as a decreased
   water content, accumulation of osmoprotectants such as proline and
   sugar, increased oxidative damage, and attenuation of the
   photosynthetic rate. However, the drought response was more severe than
   the salt response, revealing the major differences between salt and
   drought stresses. Excluding the potential effect of PEG, the only
   reason to explain this phenomenon is that B. napus is a relatively
   salt-tolerant species [[198]2]. This result also indicated that the
   salt stress response in B. napus is not a simple superposition of water
   stress and ion toxicity.

   Comparison of proteome profile changes between the salt and the drought
   responses revealed that DEPs associated with the photosynthetic system
   were significantly enriched. A greater number of proteins involved in
   the light reaction were down-regulated in the drought stress treatment
   compared to that of salt stress treatment, consistent with the lower
   photosynthesis rate observed under drought stress. Huang et al.
   reported that chlorophyll content decreases more slowly in the thf1
   mutant of Arabidopsis. In the present study, THF1 was specifically
   up-regulated under drought stress. Therefore, we speculated that, the
   decrease in chlorophyll a and b concentrations was greater under
   drought stress than under salt stress, which may be a result of
   up-regulation of THF1 under drought stress. Moreover, the
   photosynthetic rate fell by 94.3% under drought stress, significantly
   higher than the 81.8% decrease observed under salt stress ([199]Fig 2).
   Thus, the accumulation of organic compounds and energy supply in B.
   napus was lower for 4 h of drought stress treatment compared to 4 h of
   salt treatment, consistent with the greater number of down-regulated
   proteins in the “metabolism” category in the drought stress response in
   our proteome data.

   The proteome data did not provide direct evidence to support
   physiological data such as the higher accumulation of sugar and sucrose
   or more serious of oxidative damage in the drought response. However,
   it is well known that these types of down-stream responses usually lag
   early signal perception and transduction. By analyzing of the details
   of the potential functions of DEPs in signaling, we summarized the
   putative signal regulation networks in salt and drought stress
   responses (see [200]Fig 7 for the pathways). We speculated that the
   cross-talk and divergence of signaling pathways just underlie the
   similarities and differences in the proteome profiles and physiological
   responses under different stress treatments.

Fig 7. Cross-talk and specificity of signal perception and transduction in
response to salt and drought stress in B. napus.

   [201]Fig 7
   [202]Open in a new tab

   The most differentially expressed signaling-related proteins were
   integrated and marked in red (up-regulated) or green (down-regulated).
   The blue region in the middle represented the cross-talk pathway shared
   by both the salt and drought responses. The calcium-bind EF-hand family
   protein, which acted as a Ca^2+ sensor sensing Ca^2+ ion concentration
   in the cytoplasm, and ZKT protein, which may act as a molecular adaptor
   that was regulated by phosphorylation and modulated the ROS pathway,
   were overlapping signaling proteins in the salt and drought responses.
   Differential signal receptors, small G protein, 14-3-3 pathway members,
   kinases and phosphatases were identified in the salt and drought
   responses, indicating differences in signal perception and transduction
   occurred in the early stage of the salt and drought responses.
   Abbreviations: GPI, glycosylphosphatidylinositol (GPI)-anchored
   protein; PLC2, phospholipase C; RABF2a, Ras-related protein RABF2a;
   RABB1c, Ras-related protein RABB1c; GRF8, 14-3-3-like protein GF14
   kappa; GRF2, 14-3-3-like protein GF14 omega; CDPK21, calcium-dependent
   protein kinase 21; TOR, serine/threonine-protein kinase TOR; CTR,
   serine/threonine-protein kinase CTR, STN7, serine/threonine-protein
   kinase STN7; BSL1, serine/threonine-protein phosphatase BSL1; ZKT,
   protein containing PDZ, K-box and a TPR region.

   Signal perception is the first step for plant adaptation to abiotic
   stresses. Adverse stimuli usually are perceived by diverse receptors
   anchored on the plasma membrane. However, direct salt sensors or
   drought sensors have not been identified. Our proteome analysis
   identified an interesting protein, GPI-anchored protein, which has
   diverse functions and is important for signal transduction, cell-cell
   interaction, cell adhesion, and host defense [[203]32–[204]34]. In
   mammalian and yeast cells, some GPI-anchored proteins function as
   receptors for viruses and toxins [[205]34]. Association with
   specialized lipids, called a lipid microdomain or lipid raft, is one of
   the most striking features of GPI-anchored proteins. This lipid
   microdomain may facilitate the perception of an extracellular or
   intercellular signal and is required for the selective transport of
   proteins [[206]35–[207]37]. Downstream signal transduction is mediated
   by the specific cleavage of GPI-anchored proteins to release GPI and
   form multiple signal materials as second messengers involving the
   mobilization of Ca^2+, activation of DNA synthesis and rebuilding of
   cell structure. The specific cleaved enzymes include
   glycosylphosphatidyl-inositol-specific phospholipidase D (GPI-PLD),
   glycosylphosphatidyl-inositol-specific phospholipase C (GPI-PLC) and
   phosphatidyl-inositol-specific phospholipase C (PI-PLC) [[208]33].
   Interestingly, we indeed identified an up-regulated
   phosphoinositide-specific phospholipase C in the salt treatment but not
   the drought treatment in the present study ([209]Table 3, [210]Fig 7).
   This finding may indicate a potential novel signal perception and
   signal transduction pathway that is specific to the salt stress
   response in plant cells, i.e., salt stress signal →GPI-anchored
   proteins → PI-PLC → Ca^2+→ protein phosphorylation cascade ([211]Fig
   7). In downstream signal transduction, we identified three small G
   proteins, RABF2a, RABB1c, and Ran-binding protein. Rab and Ran belong
   to the small G protein superfamily, which function in cell signaling
   through the receptor-Ras-14-3-3-MAPK pathway and protein trafficking.
   In this study, the 14-3-3-like protein GF14 kappa (GRF8)-specific
   response to salt and the 14-3-3-like protein GF14 omega (GRF2)-specific
   response to drought were identified. 14-3-3 family proteins are master
   regulators of signal transduction that bind to phosphorylated proteins
   and transduce the phosphorylation signals to targets. Their regulating
   target proteins include kinases (e.g., CDPK and MAPK), transcription
   factors, structural proteins, ion channels, and other functional
   proteins [[212]26, [213]38]. Reversible phosphorylation reactions
   catalyzed by protein kinases and phosphatases are an important part of
   signal transduction, which involve various physiological and
   pathological processes. Four kinases, including CDPK21, TOR, CTR and a
   Probable serine/threonine-protein kinase and two PP2C phosphatases
   specifically responded to salt, whereas one protein kinase C, STN7 and
   one BSL1 phosphatase specifically responded to drought. No common
   kinases or phosphatases were identified in both stress responses,
   suggesting that different phosphorylation pathways may utilize
   different kinases/phosphatases to cope with different stresses
   ([214]Fig 7). These findings further support the above viewpoint
   regarding the functional specialization of family proteins when
   encountering different stresses. In addition, our study indicated that
   two common signaling-related DEPs, calcium-binding EF-hand family
   protein and ZKT protein, could be key nodes of cross-talk between salt
   and drought stresses. Calcium-binding EF hand family protein is a type
   of Ca^2+ sensor [[215]39] that can recognize the changes in
   [Ca^2+][cyt]and regulate the activity of specific protein targets in
   the cell [[216]40]. The observed increase in the expression level of
   the calcium sensor indicated that calcium, as a second messenger, may
   mediate both salt and drought stress signaling pathways in B. napus
   leaves ([217]Fig 7). Furthermore, ZKT protein, which contains PDZ,
   K-box and TPR domains, was down-regulated under both salt and drought
   stresses. In animals, there is no ZKT homologue, but only the PDZ
   domain containing proteins involved in regulating functions of G
   protein-coupled receptors, mediating signal transduction pathways, and
   assembling a variety of membrane-associated proteins [[218]41].
   Previous studies in animals have shown that the proteins rich in the
   PDZ domain usually act as key nodes in complex protein—protein
   interaction networks [[219]42, [220]43]. In plants, the ZKT protein
   acts as a molecular adaptor that is regulated by phosphorylation in the
   wound response [[221]44]. Zhu et al. (2007) discovered that the protein
   ENH1, which contains the PDZ and rubredoxin domains, plays a role in
   the detoxification of ROS under salt stress and may involve SOS2 in
   this detoxification pathway [[222]45]. In the present study, the PDZ
   domain-containing protein ZKT in B. napus exhibited a remarkably
   consistent change trend under both salt stress and drought stress,
   suggesting that the PDZ domain may participate in regulating cell
   signaling (From the phosphorylation cascade → ZKT → ROS) and shedding
   new light on the functions of proteins containing the PDZ domain in the
   cross-talk between salt and drought stress signal transduction in
   plants ([223]Fig 7).

Characteristics of the early salt and drought stress responses

   In the present study, 26 signaling-related and 27 transport- and
   vesicle trafficking-related proteins were identified as DEPs.
   Signaling-related proteins generally exhibit rapid turnover and low
   abundance [[224]46]. Surprisingly, many signaling-related DEPs were
   identified in leaves in the current study using the traditional iTRAQ
   approach, but not using a phosphoproteomic method or in specific
   subcellular organelles (e.g., the plasma membrane). We speculated that
   the 4-h treatment duration was appropriate for the proteomic analysis
   to inspect signaling pathways of early stress responses in the present
   experiment. It is helpful to explain why so many transcription- and
   translation-related proteins were differentially expressed, whereas the
   stress-, defense-, and metabolism-related proteins exhibited smaller
   changes in the levels of expression. These results indicated that
   stress signal perception and transduction was a key component of the
   early salt and drought stress responses in B. napus leaves and that
   transport and vesicle trafficking may play important roles in this
   process. These observation were consistent with a previous report
   regarding the early drought stress response of Arabidopsis [[225]16].
   cutive function were differentling At the early stress response stage,
   we observed that the differences in the proteome profiles of B. napus
   leaves were greater than the similarities between the salt and drought
   stress treatments. Only 24% and 22% of the DEPs in the salt- and
   drought-stress treatments, respectively, were shared, lower than the
   proportions (50%-70%) reported in previous studies of the late stress
   response [[226]10, [227]47]. By comparing the proteome profiles of
   cross-talk and the specificity of the salt and drought stress signal
   perception and transduction pathways, we identified that only two
   common proteins that act as convergence nodes in the salt and drought
   signaling pathways, a Ca^2+ sensor and ZKT protein ([228]Fig 7).
   Although both G protein-related signaling and 14-3-3 signaling pathways
   were identified in both stress responses, the detail proteins of
   executive functions were different. Thus, the differences in the
   proteomic profiles between drought-treated and salt-treated seedlings
   exceeded the similarities in the early stress response, suggesting that
   the different mechanisms underlying the salt and drought stress
   responses differ at the early stage but not at the late stage in
   plants.

Conclusion

   We used iTRAQ-MS/MS to compare the early protein profiles of responses
   of B. napus under salt and drought stresses. We identified important
   cross-talk and divergence of the early response mechanisms between
   these two stresses. First, the differences in the proteomic profiles
   between the drought- and salt-treated seedlings exceeded the
   similarities in the early stress responses. Second, signaling,
   transport and membrane trafficking, and photosynthesis pathways were
   important response events in the mechanism underlying the cross-talk
   and divergence of the early salt and drought response mechanisms of B.
   napus. Third, Ca^2+, G protein-related, 14-3-3 signaling pathways and
   phosphorylation cascades were shared signal transduction pathways in
   the salt and drought stress responses, but the specific proteins of
   executive function differed. Our results revealed functional
   specialization of protein families when plant cells encountering
   different stresses. Fourth, only calcium-binding EF-hand family
   protein, which mediates the Ca^2+ signature in the cytoplasm, and ZKT,
   which regulates the ROS pathway, were definitively identified as
   cross-talk nodes in the salt and drought signaling pathways.

Supporting Information

   S1 Fig

   (A) Peptide length distribution, (B) peptide number distribution, (C)
   protein mass distribution, and (D) distribution of protein’s sequence
   coverage.

   (TIF)
   [229]Click here for additional data file.^ (1.1MB, tif)
   S1 Table. Osmotic potential of NaCl solutions and PEG 6000 solutions of
   different concentrations.

   (XLS)
   [230]Click here for additional data file.^ (10.5KB, xls)
   S2 Table. Overview of protein identification results of B. napus leaf
   proteins by the iTRAQ-LC/MSMS method.

   (XLS)
   [231]Click here for additional data file.^ (9.5KB, xls)
   S3 Table. Raw iTRAQ data for protein identification and quantitation.

   (XLS)
   [232]Click here for additional data file.^ (10MB, xls)
   S4 Table. Common differentially expressed proteins (DEPs) in the leaves
   of B. napus treated with 245 mM NaCl or 25% PEG 6000 for 4 h.

   (XLS)
   [233]Click here for additional data file.^ (3.7MB, xls)
   S5 Table. Salt-specific DEPs in the leaves of B. napus treated with 245
   mM NaCl for 4 h.

   (XLS)
   [234]Click here for additional data file.^ (530KB, xls)
   S6 Table. Drought-specific DEPs in the leaves of B. napus treated with
   25% PEG 6000 for 4 h.

   (XLS)
   [235]Click here for additional data file.^ (592KB, xls)

Data Availability

   All relevant data are within the paper and its Supporting Information
   files.

Funding Statement

   This work was supported by the National Natural Science Foundation of
   China (grant numbers 31201152) to JLL and the National Natural Science
   Foundation of China (grant numbers 31160019) to XJP. The funders had no
   role in study design, data collection and analysis, decision to
   publish, or preparation of the manuscript.

References