Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Proteome Dynamics and Physiological Responses to Short-Term Salt Stress in Brassica napus Leaves

  • Huan Jia ,

    Contributed equally to this work with: Huan Jia, Mingquan Shao

    Affiliation State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing, China

  • Mingquan Shao ,

    Contributed equally to this work with: Huan Jia, Mingquan Shao

    Affiliation State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing, China

  • Yongjun He,

    Affiliation State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing, China

  • Rongzhan Guan,

    Affiliations State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing, China, Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing, Jiangsu, China

  • Pu Chu , (PC); (H. Jiang)

    Affiliation State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing, China

  • Haidong Jiang (PC); (H. Jiang)

    Affiliation State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing, China

Proteome Dynamics and Physiological Responses to Short-Term Salt Stress in Brassica napus Leaves

  • Huan Jia, 
  • Mingquan Shao, 
  • Yongjun He, 
  • Rongzhan Guan, 
  • Pu Chu, 
  • Haidong Jiang


Salt stress limits plant growth and crop productivity and is an increasing threat to agriculture worldwide. In this study, proteomic and physiological responses of Brassica napus leaves under salt stress were investigated. Seedlings under salt treatment showed growth inhibition and photosynthesis reduction. A comparative proteomic analysis of seedling leaves exposed to 200 mM NaCl for 24 h, 48 h and 72 h was conducted. Forty-four protein spots were differentially accumulated upon NaCl treatment and 42 of them were identified, including several novel salt-responsive proteins. To determine the functional roles of these proteins in salt adaptation, their dynamic changes in abundance were analyzed. The results suggested that the up-accumulated proteins, which were associated with protein metabolism, damage repair and defense response, might contribute to the alleviation of the deleterious effect of salt stress on chlorophyll biosynthesis, photosynthesis, energy synthesis and respiration in Brassica napus leaves. This study will lead to a better understanding of the molecular basis of salt stress adaptation in Brassica napus and provides a basis for genetic engineering of plants with improved salt tolerance in the future.


Salt stress is one of the most severe environmental challenges and limits crop production throughout the world [1]. Research to determine the mechanism of salinity adaptation and improving salt tolerance of plants has attracted increasing attention. NaCl is the main component of salinity [2] and exposure to high concentrations of NaCl can trigger various adverse effects, including water deficit, ionic toxicity, nutritional disorders, plant growth stunt, photosynthesis and protein synthesis depression, excess reactive oxygen species (ROS) generation and oxidative stress [36]. To cope with the deleterious effects of salt stress, plants have evolved a series of regulatory mechanisms. Ion homeostasis maintenance, compatible solute accumulation, antioxidant systems, hormonal control, Ca2+ signaling and SOS signaling pathways are important in salt-stress tolerance [6,7].

Salt stress can induce extensive proteome alteration in plants and proteomic analysis has proved to be an effective approach to study plant salt-stress tolerance [1,8]. Recently, a database containing 2171 salt-responsive proteins was constructed based on proteomics studies from 34 plant species, which has helped our understanding of the mechanisms underlying plant salt response and tolerance [9]. Two-dimensional polyacrylamide gel electrophoresis (2-DE) remains the primary approach in proteomic research [8] and has been applied commonly in studies on salt stress in plants [1012].

Brassica napus is the third most important source of edible oil in the world and has considerable economic and nutritional values as an oilseed crop [13]. However, Brassica napus seedlings are sensitive to salt stress and their growth is markedly inhibited by salinity [14]. Few proteomic studies on the salt-stress response have been reported for Brassica napus. A previous study on canola under salt stress detected 44 and 31 differentially accumulated proteins in leaf proteome of the salt-tolerant genotype Hyola 308 and salt-susceptible genotype Sarigol, respectively; 46 proteins were identified using mass spectrometry (MS) analysis [15]. Recently, a proteomic analysis of seedling roots from Hyola 308 and Sarigol detected 20 and 21 proteins that responded to salt-stress treatments, respectively; However, only 19 proteins were identified [16]. The proteome dynamics during salt treatment and the roles of the salt-responsive proteins in salt stress adaptation remain unclear and require further exploration.

To better understand the mechanism of salt-stress adaptation in Brassica napus, proteomic and physiological responses to salt stress were analyzed in seedling leaves. Salt-responsive proteins were separated by 2-DE and identified by MS analysis. The functions of these proteins and the dynamic changes in their abundance are investigated and discussed.

Materials and Methods

Plant growth and salt treatments

Seeds of Brassica napus (var. Nannongyou No.3, salt tolerant) were collected from the Nanjing Agriculture University Agronomy farm in Nanjing city, Jiangsu Province, China. Plants were grown in greenhouse pots filled with a mixture of sand and vermiculite (1:1 v/v) under a light intensity of 120 μmol m-2 s-1 with a 16 h/8 h photoperiod and temperatures of 23 ± 2°C. The plants were irrigated daily with half-strength Murashige and Skoog solution. Six-week-old seedlings were treated with half-strength Murashige and Skoog solution containing either 200 mM NaCl (salt-stressed treatment group) or no NaCl (unstressed control group) for 24 h, 48 h and 72 h. The third leaves were harvested from control and salt-treated plants, immediately frozen in liquid nitrogen and kept at −80°C until use. At least three independent biological replicates were prepared for physiological and proteomic analyses.

Biomass and water content

The fresh weight (FW) of Brassica napus seedlings and leaves was measured immediately after harvesting. To determine the determination of water content (WC), the plant samples were maintained at 105°C for 5 min and then at 80°C for 72 h in an oven; the dry weight (DW) of the samples was then measured. The WC of the sample was calculated using the following formula: WC = (FW − DW)/FW×100%.

Pigment determination

Chlorophyll (Chl) was extracted from 0.5 g samples of fresh leaves using 80% acetone. The extract was centrifuged at 12 000 rpm at 4°C for 5 min and the supernatant was collected. Absorbance of the extract was measured at 663 nm and 646 nm spectrophotometrically and the contents of total Chl (Chl a + Chl b), Chl a, and Chl b of leaves were determined as previously described [17]. Three biological replicates were prepared for analysis.

Leaf gas-exchange measurement

The third leaves of control and salt stressed plants were used for gas-exchange analysis. Net photosynthesis and stomatal conductance were determined by a Li-Cor 6400 portable photosynthesis system (Li-Cor Inc. Lincoln, NE, USA) using the built-in light source set at 1,000 μmol photons m-2 s-1 [18]. During the measurements, the leaf temperature was adjusted to 25°C.

Protein extraction and quantification

Protein extraction was performed using the trichloroacetic acid/acetone method, as described previously [19] with some modifications. Leaf samples (1 g) were ground in liquid nitrogen with a mortar and pestle. The tissue powder was transferred to 50 mL centrifuge tubes and suspended in 10% trichloroacetic acid (TCA) and 65 mM DTT in ice-cold acetone and incubated at −20°C overnight. Proteins were pelleted by centrifugation at 40,000 × g for 25 min at 4°C, and then resuspended in cold acetone containing 65mM DTT. The mixture was kept at −20°C for 1 h before centrifugation at 40,000 × g for 15 min at 4°C. Protein pellets were washed twice with cold acetone and lyophilized in a vacuum. The resulting pellet was solubilized in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, and 0.2% w/v Bio-Lyte) for 2 h at room temperature and then centrifuged at 16,000 × g for 30 min. The supernatant was collected and stored at −80°C until use. The protein concentration of each sample was measured using the Bradford method [20].

2-DE and image analysis

For isoelectric focusing (IEF) in the first dimension, traces of bromophenol blue were added to the protein samples. A total of 450 μg of protein samples was loaded into the immobilized pH gradient (IPG) strips (pH 4–7, 17 cm, Bio-Rad, Hercules, CA, USA). The strips were then covered with mineral oil and rehydrated at 50 V for 14 h at 20°C. Electrophoresis was carried out at 250 V for 1 h, followed by 500 V for 1 h, 1000V for 1h, 2000V for 1h, 10,000 for 4h with a linear ramp and 10,000 V for 80,000 Vh with a rapid ramp. Focused IPG strips were equilibrated as previously described [21] and after equilibration, the strip was placed on the top of 11.5% SDS-PAGE and sealed with 0.5% agarose. Electrophoresis was carried out at 10 mA/gel for 30 min followed by 20 mA/gel until the bromophenol blue dye reached the bottom of the gel. The gels were stained with silver nitrate [22] and analyzed using PDQuest software (version 8.0.1, Bio-Rad, Hercules, CA, USA) for spot detection and protein quantification according to the user manual. Three gel replicates were used for the 2-DE gels quantification. The proteins with at least a two-fold change between the control and treatment samples were considered differentially accumulated proteins (p < 0.05 by Student’s t-test). Spots with significant changes were selected and manually excised from the gel for further protein identification.

In-gel digestion and protein identification

The selected spots were in-gel digested with trypsin as previously described [23,24] with minor modification. Briefly, protein spots were washed twice with ultrapure water, destained twice with 25mM NH4HCO3 in 50% acetonitrile (ACN), dehydrated with 50% ACN and 100% ACN, and then lyophilized in an ALPHA 2–4 LSC Freeze Dryer (Martin Christ, GmbH, Osterode, Germany). The gels pieces were reduced with 10 mM DTT in 25 mM NH4HCO3 at 56°C for 1 h, alkylated with 50 mM iodoacetamide in 25 mM NH4HCO3 at 37°C for 30 min, dried with 50% ACN and 100% ACN and digested with trypsin (Promega, Madison, WI, USA) in 25 mM NH4HCO3 overnight at 37°C. The peptides were extracted twice with 5% trifluoroacetic acid (TFA) in 67% ACN after digestion. All extracts were pooled and lyophilized in the concentrator. The tryptic peptides were prepared by dissolving the extracts with 0.1% TFA and stored at −80°C until MS analysis.

MS analysis was conducted using a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrometer 4800 Proteomics Analyzer (AB SCIEX, Framingham, MA, USA). Data were analyzed using GPS 3.6 Explorer software (AB SCIEX, Framingham, MA, USA) and searched using MASCOT 2.1 software (Matrix Science Ltd., London, UK) against the NCBInr database, which contained accessible Arabidopsis and Brassica protein sequences. The peptide MS and MS/MS tolerances were set as 15 ppm and 0.25 Da, respectively. The search parameters were as follows: specificity of protease digestion was set to trypsin, and a maximum of one missed cleavage was allowed per protein. Carbamidomethyl on Cys was set as a fixed modification and oxidation of Met was set as a variable modification. The probability-based Mowse score was used to determine the confidence of protein identification and only significant hits with a confidence interval (CI) greater than 95% (p < 0.05) were accepted as correctly identified.

Hierarchical cluster analysis

Hierarchical clustering of the relative abundance profiles of differentially accumulated protein was performed on the log2-transformed fold change values of protein spots, using Cluster 3.0 [25]. The complete linkage algorithm was used for data aggregation [21] and TreeView version 1.6 was used to generate a heatmap [18].

Enzymatic assays

Superoxide dismutase (SOD) activity assay.

To assess SOD activity, 0.5 g of leaf sample was ground in an ice-chilled mortar with 0.1 M potassium phosphate buffer (pH 7.8) and was centrifuged at 12,000 × g for 20 min at 4°C. The supernatant was used for the enzymatic assay, according to a previously described method [26]. Briefly, 50 μl of the supernatant was added to the 100 mM phosphate buffer (pH 7.8) containing 1.3 μM riboflavin, 13 mM methionine and 65 μM nitro blue tetrazolium (NBT). After the reaction mixture was illuminated for 15 min with luminescent lamps, absorbance was measured at 560 nm. One unit of SOD was defined as the amount of enzyme that induced 50% inhibition of the NBT reduction, and SOD activity was expressed as unit g fresh weight-1.

Chitinase activity assay.

Chitinase activity in leaf samples was measured as previously described [27]. The crude protein extracts were prepared using sodium acetate buffer (pH4.5). After centrifugation at 10,000 × g for 15 min at 4°C, the supernatants were used for the colorimetric determination of N-acetylglucosamine (GlcNAc) with 1% colloidal chitin (w/v) as the substrate. Chitinase activity was calculated from a GlcNAc (Funakoshi, Tokyo, Japan) standard curve. One unit of chitinase activity was defined as the release of 1 μmol of GlcNAc per hour at 37°C.

ATPase activity assay.

ATPase activity in leaf samples was measured as previously described with modifications [28]. Briefly, 1 g leaf sample was ground in an ice-chilled mortar with 0.05 M potassium phosphate buffer (pH 7.8) containing 0.4 M sucrose and 0.01M NaCl. The supernatants were collected by centrifugation at 1500 × g for 5 min and the pellets were resuspended in 0.05 M potassium phosphate buffer (pH 7.8), 0.4 M sucrose and 0.01 M NaCl at a Chl concentration of 0.5 mg ml−1. ATPase activity was determined in a reaction mixture that contained 50 mM Tris-HCl (pH 8.0), 5 mM ATP, and 5 mM CaCl2. After incubation at 37°C for 10 min, 20% trichloroacetic acid was added into the mixture to stop the reaction and the concentration of inorganic phosphate (Pi) was determined as described previously [29].


Effect of salt stress on plant growth

Salt stress with 200 mM NaCl significantly (p < 0.01) decreased the FW, DW and WC of the Brassica napus ‘Nannongyou No.3’ seedlings (Fig 1). We further analyzed the effect of salinity on Brassica napus leaves, and the results showed that compared with the control plants, the FW and WC of the third leaves was reduced by 43% and 3%, respectively, after salt treatment for 3 d. However, the effect of salinity on the DW of the third leaves was not statistically significant (Fig 1).

Fig 1. Effects of salt-stress treatments on plant biomass in B. napus ‘Nannongyou No. 3’.

Values shown are means ± SD from three biological replicates. ** indicates a significant difference (compared with the control) at p < 0.01.

Effect of salt stress on pigments and photosynthesis

No significant decrease in chlorophyll a (Chl a), chlorophyll a (Chl b), total chlorophyll and carotenoid concentrations was observed in the leaves of salt-stressed ‘Nannongyou No.3’ seedlings after salt treatment for 3 d, compared with untreated controls (Fig 2). No significant changes were detected in the Chla to Chlb ratio, and the chlorophyll to carotenoid ratio after salt-stress treatment (Fig 2).

Fig 2. Effect of salt-stress treatments on pigments in B. napus ‘Nannongyou No. 3’.

Values shown are means ± SD from three biological replicates.

Leaf gas-exchange measurements revealed that net photosynthesis (Pn) was dramatically reduced by salinity (Table 1). Salt-stress treatment significantly decreased the stomatal conductance (Gs), intercellular CO2 concentration (Ci) and transpiration rate (Tr) by 84%, 27% and 80%, respectively.

Table 1. Effects of salt stress on leaf gas exchange in Brassica napus.

Leaf proteome dynamics in response to salt stress

The effects of salt stress on the leaf protein profiles of ‘Nannongyou No.3’ were analyzed using 2-DE. To analyze proteome dynamics in response to salt stress, we measured several time points, from 24 to 72 h, after 200 mM NaCl treatment (S1 Fig). Approximately 800 protein spots were detected on silver-stained 2-DE gels in the pH range of 4–7 by the PDQuest software (Fig 3). Differential protein abundance between the control and salt stressed plants under different NaCl treatment time points was assessed. Forty-four protein spots were observed to be reproducibly and significantly (p < 0.05) altered in abundance by more than two-fold in at least one time point after salt treatment. Among these 44 spots, 42 were identified by MS/MS analysis (Table 1, S1 Table and S1 Dataset). ATP synthase (chloroplast) was identified in seven spots (spot 13, 18, 28, 31, 32, 36 and 37), and 60-kDa chaperonin was identified in three spots (spot 4, 16, and 19), whereas chloroplast ribulose-1, 5-bisphosphate carboxylase/oxygenase activase (spot 7 and 30), heat shock protein 70 (spot 20 and 25), and chitinase (spot 26 and 29) were identified in two spots. These phenomena are commonly observed in 2-DE gel analysis and may result from the presence of different isoforms, differential post-translational modification or degradation in the protein extracts; they can also be related to allelic polymorphism [3033].

Fig 3. Two-dimensional electrophoresis (2-DE) gel images of proteins extracted from leaf 3 of B. napus ‘Nannongyou No. 3’ treated with 200 mM NaCl.

The 42 salt-stress-responsive protein spots identified by tandem mass spectrometry (MS/MS), which are numbered and indicated by arrows, correspond to the spot numbers in Table 2.

The identified salt-stress-responsive proteins were further divided into seven categories on the basis of their functions (Fig 4), including Chl biosynthesis (2.38%), photosynthesis (28.57%), energy synthesis (19.05%), respiration (4.76%), protein metabolism (23.81%), and damage repair and defense response (21.43%). At the 24-h time point, 12 protein spots exhibited increased abundance and 16 spots exhibited decreased abundance. The number of down-accumulated proteins upon salt stress decreased and was much lower than the number of salt-induced proteins. Only five spots were down-accumulated, while 15 protein spots were up-accumulated, at the 48-h time point. The changes persisted and at the 72-h time point, the abundance of 18 protein spots was increased and only four spots showed decreases in abundance.

Fig 4. Functional classification of differentially accumulated proteins identified in B. napus leaves under salt stress.

The salinity-responsive proteins displayed different dynamic patterns during the time course experiments. For example, no significant change in protein abundance was detected for chitinase (spot 29) at 24 h; however, it was up-accumulated at 48 h (Fig 5A). The abundance of the ATP synthase beta subunit (spot 37) was significantly decreased at 24 h, and then increased at 48 h (Fig 5B). However, for FtSH2 (spot 35) and transketolase 1(spot 39), no significant alteration was detected at 48 h, compared with the increased abundance of these two spots at 24 h and 72 h (Fig 5C and 5D). To further reveal the leaf proteome dynamics in response to salt stress, hierarchical cluster analysis was performed (Fig 6). The results indicated that leaves at 24 h and 48 h were grouped into a single group, and leaves at 72 h were in another cluster. The 42 differentially accumulated protein spots in response to salt stress were grouped in two main clusters. The first cluster (Cluster I) included 14 protein spots whose abundance mainly decreased upon high salinity. Most of these proteins are involved in Chl biosynthesis, photosynthesis, respiration and energy synthesis. This cluster could be separated into two subgroups according to the changing patterns of the spots. In subgroup A, the abundance of most down-accumulated proteins at time point 24 h was increased at time point 48 h and 72 h, while the highest depression effect of salinity on the accumulation of proteins in subgroup B was observed at time point 72 h. The second cluster (Cluster II) included 28 protein spots whose abundance was increased by salt treatment. Most of them belong to protein families for protein metabolism, damage repair and defense response. This cluster could be further divided into three subgroups. The first subgroup (subgroup C) included 10 protein spots showing the highest up-regulation upon salt stress treatment at time point 72 h. The accumulation levels of 11 spots in the second subgroup (subgroup D) and seven spots in the third subgroup (subgroup E) was strongly induced by salinity at time point 24 h and 48 h, respectively.

Fig 5. Dynamic patterns of randomly selected salt-responsive proteins at 24 h, 48 h, and 72 h after 200 mM NaCl treatment on B. napus ‘Nannongyou No. 3’ leaves.

Fig 6. Hierarchical cluster analysis of the dynamic profiles of the 42 identified proteins.

Fold changes of protein abundance were log 2 transformed. Columns 1, 2, and 3 represent 200 mM NaCl treatment for 24 h, 48 h, and 72 h, respectively. The rows represent individual proteins. Protein names and spot numbers are labeled to the right of the corresponding heat maps. The proteins that increased and decreased in abundance are indicated in red and green, respectively. Proteins showing no significant changes are indicated in black. The intensity of the colors increases with increasing accumulation differences, as shown to the left of the bar. I and II indicated Cluster I and II, while A-E indicated subgroup A-E.

Changes in enzyme activities in response to salt stress

The changes in protein abundance identified by 2-DE analysis were further validated by enzyme activity assay. Salt stressed leaves at different time points (24 h, 48 h and 72 h after 200mM NaCl treatment) were tested. In agreement with the data from the proteomics analysis, the activities of SOD and chitinase were significantly increased at 72 h and 48 h of salt stress treatment, respectively, while the ATPase activity was significantly reduced at all three time points (Fig 7).

Fig 7. Effects of salt-stress treatments on enzyme activities in leaves of B. napus ‘Nannongyou No. 3’ seedlings.

Values shown are means ± SD from three biological replicates. ** indicates a significant difference (compared with the control) at p < 0.01.


In this study, the proteomic and physiological responses to salt stress in Brassica napus seedling leaves were analyzed. Seedling biomass was reduced after NaCl treatment (Fig 1), implying that plant growth was repressed by salinity. NaCl treatment resulted in a reduction of the Gs and Tr (Table 1), which might be associated with the water deficiency in salt-stressed leaves. Forty-four salt-stress-responsive proteins were detected, of which 42 were identified (Table 2). The identified proteins were classified into different functional categories. Most of the up-accumulated proteins during NaCl treatment were associated with protein metabolism, damage repair and defense response, which might account for the alleviation of the adverse effect of salt stress on Chl biosynthesis, photosynthesis, energy synthesis and respiration in Brassica napus leaves.

Table 2. Identification of differentially accumulated proteins under salt stress in leaves of Brassica napus.

Chl biosynthesis and photosynthesis under salt stress

Salt stress can impair photosynthesis indirectly by reducing Chl content [6]. However, the impact of salinity on Chl biosynthesis remains unclear. A recent study on rice seedlings suggested that downregulation of Chl biosynthesis by salt stress could be attributed to decreased activities of Chl biosynthetic pathway enzymes [34]. Glutamate 1-semialdehyde aminotransferase (GSA-AT) is a key enzyme in plant Chl synthesis [35], and GSA-AT antisense transformants showed varying degrees of Chl deficiency [36,37]. In this study, the abundance of GSA-AT (spot 1) decreased after 24 h of salt-stress treatment. However, the GSA-AT protein level recovered to control levels at 48 h and 72 h of NaCl treatment (Fig 6), which might be attributed to salt tolerance mechanisms in Brassica napus seedlings. Consistent with this finding, the reduction in Chl content in salt-stressed seedlings was not significant after 72 h of treatment (Fig 2).

Photosynthesis is one of the primary processes affected by salinity [38,39]. The decrease in photosynthesis capacity in salt-stressed plants might result from damage to the photosynthetic apparatus and decreased CO2 availability caused by stomatal limitations [40,41], other than by reduction of the Chl content. In this study, leaf gas-exchange measurements revealed that net photosynthesis was significantly reduced in salt-stressed seedlings (Table 1). Proteomic analysis showed that 12 proteins related to photosynthesis were salinity responsive, including six photosynthetic proteins and six enzymes crucial for CO2 fixation (Table 2). Interestingly, most of the proteins involved in the light reaction, including photosystem I subunit VII (spot 3), Lhcb6 protein (spot 12), 33 kDa oxygen-evolving protein (spot 14), Chl a/b binding protein (spot 17), and 23kD protein of oxygen evolving system of photosystem II (spot 23), were up-accumulated in response to salt stress. However, the protein abundance of enzymes in CO2 assimilation, including Ribulose bisphosphate carboxylase (RuBisCO) large chain (spot 38), sedoheptulose-1, 7-bisphosphatase (spot 5) and phosphoribulokinase (spot 10), was significantly decreased after salt-stress treatment, especially at the 24-h time point. In support of this finding, the reduction in stomatal conductance and transpiration rate was far larger than the reduction in intercellular CO2 concentration after salt stress treatment (Table 1). These observations suggested that the salt-induced reduction in photosynthesis capacity could be mainly attributed to impaired CO2 fixation in Brassica napus seedlings. Photosynthesis limited by a reduction in the rate of CO2 assimilation by salinity has been reported in wheat [11], rice [42] and Arabidopsis [43].

Two spots (spot 7, 30) corresponding to RuBisCO activase (RCA) were significantly up-accumulated in Brassica napus seedlings after salt stress treatment, especially at the 48 h time point. RCA is a key regulatory enzyme that catalyzes the activation of RuBisCO [41]. RCA has been implicated in the maintenance of CO2 assimilation at low CO2 levels because of the reduction in stomatal conductance caused by salinity [44,45]. The induction of RCA by salinity has been reported previously [15,46] and might explain the recovery of the abundance of RuBisCO at the 48-h and 72-h time point of salt-stress treatment. Therefore, the up-accumulation of RCA might contribute to salt tolerance in Brassica napus seedlings.

Energy synthesis and respiration under salt stress

ATP synthase is a salt-responsive enzyme and plays crucial roles in plant salt tolerance [22,47]. In this study, proteomic analysis identified eight spots (spots 13, 18, 27, 28, 31, 32, 36, 37) as ATP synthase subunits, whose abundances changed under salt-stress conditions. Most of these proteins were down-accumulated, especially at the 24 h time point, and the enzyme activity analysis result was consistent with this finding (Fig 7). These results suggested that energy synthesis might be repressed in salt-stressed Brassica napus seedlings. In agreement with this observation, the downregulation of ATP synthase by salt stress has been reported in soybean leaves [48]. However, the response of ATP synthase to salinity depends on the plant species and genotype. For example, the studies in rice [49] and black locust [50] showed that ATP synthase was induced upon salinity. ATP synthase was significantly down-regulated in a salt tolerant cowpea cultivar, but up-regulated in a salt-sensitive cowpea cultivar after salt treatment [44].

Plant respiration response to salt stress has been studied [51]. However, the regulation of respiration by salinity stress appears to be complex, depending on species, tissue or cell type, and stress levels [4,32]. In this study, the abundance of the respiration enzyme pyruvate dehydrogenase (spot 6) was 2.2-fold higher after 72 h of salt stress treatment, while malate dehydrogenase (spot 34) was decreased by 0.66-fold at the 24 h time point. The pyruvate dehydrogenase in mung bean seedlings was reduced in 50 mM NaCl but increased in 100 mM and 150 mM NaCl, while the activity of malate dehydrogenase was decreased in salt-stressed seedlings [52]. The up-regulation of pyruvate dehydrogenase and malate dehydrogenase was reported in tomato leaves under salt stress treatment, especially in a salt-tolerant genotype [53].

Protein metabolism under salt stress

Salt stress can severely affect protein synthesis [54] and disrupt protein folding in the endoplasmic reticulum (ER), leading to the accumulation of unfolded proteins and ER stress in plants [55]. Proteomic analysis in the present study indicated that elongation factor Tu (spot 33), which is involved in protein biosynthesis and chaperones (cyclophilin, 60 kDa chaperonin/HSP60 and HSP70) related to protein folding, was significantly induced in salt-stressed leaves. By contrast, a protein associated with protein degradation (20S proteasome) was decreased by salinity stress treatment.

The up-accumulation of elongation factor Tu might enhance protein biosynthesis and thus repair the damage of salt stress on photosynthetic proteins in chloroplasts. Similar observations have been reported in cucumber [21] and soybean seedlings [12]. Chaperonins assist protein folding and assembly [56], and may function to protect and repair vulnerable protein targets under stress conditions [33,57,58]. In contrast to our findings, a previous proteomic analysis of Brassica napus seedling roots identified chaperonin hsp60 as a down-regulated protein in response to salinity [16]. This differential response could reflect the varying plant genotypes or different tissues used. Cyclophilins are implicated in protection against multiple abiotic stresses, and have been identified as salt-stress-responsive proteins in rapeseed [15], barley [19] and creeping bentgrass [59], consistent with our results. The results suggested that these proteins related to protein metabolism might participate in salt-stress tolerance in Brassica napus seedlings.

Damage repair and defense response under salt stress

Oxidative stress caused by the generation of excess ROS is a well-documented indirect form of damage caused by salt stress [60]. The up-accumulation of antioxidant enzymes, such as FeSOD 1 (spot 8), glutathione transferase (spot 9), and dehydroascorbate reductase (spot 22), might promote ROS scavenging and mitigate the oxidative damage under salt stress in Brassica napus leaves. The thiazole biosynthetic enzyme (THI) protein (spot 11) plays a role in thiamine biosynthesis, and its involvement in DNA damage repair and stress-tolerance mechanisms has been observed in bacteria, yeast and Arabidopsis thaliana [6163]. The up-accumulation of THI might help to restore DNA stability and alleviate oxidative stress caused by NaCl treatment in Brassica napus leaves. Cell division protein FtSH was suggested to play a role in attenuating the detrimental effects of salinity on the photosynthetic machinery [4], and the enhanced abundance of FtSH2 in salt-stressed Brassica napus leaves might have contributed to the restoring of the photosynthetic proteins at 48 h and 72 h after salt-stress treatment (Fig 5).

Three up-accumulated proteins related to the defense response were identified in Brassica napus leaves, including cinnamyl alcohol dehydrogenase (spot 24), chitinase (spot 26 and 29) and N-glyceraldehyde-2-phosphotransferase-like protein (spot 40). Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis and enhanced CAD might increase the extent of lignification, which might represent a salt-adaptation response in plant roots [6466]. Functional analysis in tea plants suggested that CAD in leaves might play a role in defense against biotic stresses and adaptation to abiotic stresses [67]. However, the function of CAD in salt tolerance in plant leaves is still poorly understood. Chitinase belongs to the pathogenesis-related (PR) protein family, which plays important roles in biotic and abiotic stress resistance [57]. The up-regulation of chitinase by salt stress has been reported in Brassica rapa [68], Nicotiana tabacum [10] and Halogeton glomeratus [69].The increased abundance of chitinase in salt-stressed leaves was further validated and supported by the enzyme assay in the present study (Fig 7). These results suggested that chitinase might help to enhance resistance against salt stress in Brassica napus. A study in Brassica carinata showed that N-glyceraldehyde-2-phosphotransferase was up-regulated in the resistant genotype upon pathogen attack [70]. The involvement of N-glyceraldehyde-2-phosphotransferase in salt stress resistance/tolerance is unclear, and to the best of our knowledge, it represents a novel salt-stress-responsive protein in Brassica napus leaves.


The present study identified 42 proteins affected by salt stress in Brassica napus, including several novel salt stress responsive proteins in plant leaves, such as cinnamyl alcohol dehydrogenase and N-glyceraldehyde-2-phosphotransferase. Proteome dynamics under different time points after NaCl treatment were analyzed. The results suggested that Chl biosynthesis, photosynthesis, respiration and energy synthesis were negatively affected by salt stress at the 24 h time point, and then recovered at the 48 h and 72 h time point. By contrast, protein biosynthesis, damage repair and defense response were significantly induced by NaCl during the stress treatment, implying that proteins in these categories might alleviate the damage caused by NaCl and promote salt adaptation in Brassica napus. In this study, analysis of the physiological responses to salt stress supported the proteomic data. This study expanded our knowledge of the mechanisms underlying salt-stress adaptation and will promote molecular breeding of salt-tolerant plants in the future.

Supporting Information

S1 Dataset. The MS/MS spectra for the proteins identified by single peptide.


S1 Fig. Two-dimensional electrophoresis (2-DE) gel images of proteins extracted from leaf 3 of B. napus ‘Nannongyou No. 3’.


S1 Table. Quantification summary of differentially accumulated proteins under salt stress in leaves of B. napus.

Fold change is expressed as the intensity ratio of treatment/control protein spot. Values are presented as means ± SD of the replicates.


Author Contributions

Conceived and designed the experiments: PC RG. Performed the experiments: H. Jia MS YH. Analyzed the data: PC RG. Contributed reagents/materials/analysis tools: RG PC H. Jiang. Wrote the paper: PC H. Jiang.


  1. 1. Tahir I, Sabir M, Iqbal M. Unravelling Salt Stress in Plants Through Proteomics. In Ahmad P. editors. Salt Stress in Plants: Signalling, Omics and Adaptations; 2013. pp 47–61.
  2. 2. Golldack D, Li C, Mohan H, Probst N. Tolerance to drought and salt stress in plants: Unraveling the signaling networks. Front Plant Sci. 2014;5:151. pmid:24795738
  3. 3. Yadav S, Irfan M, Ahmad A, Hayat S. Causes of salinity and plant manifestations to salt stress: a review. J Environ Biol. 2011;32(5):667–85. pmid:22319886
  4. 4. Barkla BJ, Castellanos-Cervantes T, de Leon JL, Matros A, Mock HP, Perez-Alfocea F, et al. Elucidation of salt stress defense and tolerance mechanisms of crop plants using proteomics-current achievements and perspectives. Proteomics. 2013;13(12–13):1885–900. pmid:23723162
  5. 5. Shavrukov Y. Salt stress or salt shock: which genes are we studying? J Exp Bot. 2013;64(1):119–27. pmid:23186621
  6. 6. Tang X, Mu X, Shao H, Wang H, Brestic M. Global plant-responding mechanisms to salt stress: physiological and molecular levels and implications in biotechnology. Crit Rev Biotechnol. 2014 April 16.
  7. 7. Ismail A, Takeda S, Nick P. Life and death under salt stress: same players, different timing? J Exp Bot. 2014;65(12):2963–79. pmid:24755280
  8. 8. Sobhanian H, Aghaei K, Komatsu S. Changes in the plant proteome resulting from salt stress: toward the creation of salt-tolerant crops? J Proteomics. 2011;74(8):1323–37. pmid:21440686
  9. 9. Zhang H, Han B, Wang T, Chen S, Li H, Zhang Y, et al. Mechanisms of plant salt response: insights from proteomics. J Proteome Res. 2012;11(1):49–67. pmid:22017755
  10. 10. Razavizadeh R, Ehsanpour AA, Ahsan N, Komatsu S. Proteome analysis of tobacco leaves under salt stress. Peptides. 2009;30(9):1651–9. pmid:19573571
  11. 11. Gao L, Yan X, Li X, Guo G, Hu Y, Ma W, et al. Proteome analysis of wheat leaf under salt stress by two-dimensional difference gel electrophoresis (2D-DIGE). Phytochemistry. 2011;72(10):1180–91. pmid:21257186
  12. 12. Ma H, Song L, Shu Y, Wang S, Niu J, Wang Z, et al. Comparative proteomic analysis of seedling leaves of different salt tolerant soybean genotypes. J Proteomics. 2012;75(5):1529–46. pmid:22155470
  13. 13. Purty RS, Kumar G, Singla-Pareek SL, Pareek A. Towards salinity tolerance in Brassica: an overview. Physiol Mol Biol Plants. 2008;14(1–2):39–49. pmid:23572872
  14. 14. Steppuhn H, Volkmar KM, Miller PR. Comparing Canola, Field Pea, Dry Bean, and Durum Wheat Crops Grown in Saline Media. Crop Sci. 2001;41(6):1827–33.
  15. 15. Bandehagh A, Salekdeh GH, Toorchi M, Mohammadi A, Komatsu S. Comparative proteomic analysis of canola leaves under salinity stress. Proteomics. 2011;11(10):1965–75. pmid:21480525
  16. 16. Ali Bandehagh EDU, Hosseini Salekdeh Ghasem. Proteomic analysis of rapeseed (Brassica napus L.) seedling roots under salt stress. Annals of Biological Research. 2013;4(7):212–21.
  17. 17. Zhang X, Wollenweber B, Jiang D, Liu F, Zhao J. Water deficits and heat shock effects on photosynthesis of a transgenic Arabidopsis thaliana constitutively expressing ABP9, a bZIP transcription factor. J Exp Bot. 2008;59(4):839–48. pmid:18272919
  18. 18. Chu P, Yan GX, Yang Q, Zhai LN, Zhang C, Zhang FQ, et al. iTRAQ-based quantitative proteomics analysis of Brassica napus leaves reveals pathways associated with chlorophyll deficiency. J Proteomics. 2015;113:244–59. pmid:25317966
  19. 19. Fatehi F, Hosseinzadeh A, Alizadeh H, Brimavandi T, Struik PC. The proteome response of salt-resistant and salt-sensitive barley genotypes to long-term salinity stress. Mol Biol Rep. 2012;39(5):6387–97. pmid:22297690
  20. 20. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. pmid:942051
  21. 21. Li B, He L, Guo S, Li J, Yang Y, Yan B, et al. Proteomics reveal cucumber Spd-responses under normal condition and salt stress. Plant Physiol Biochem. 2013;67:7–14. pmid:23524299
  22. 22. Liu CW, Chang TS, Hsu YK, Wang AZ, Yen HC, Wu YP, et al. Comparative proteomic analysis of early salt stress responsive proteins in roots and leaves of rice. Proteomics. 2014;14(15):1759–75. pmid:24841874
  23. 23. Yan S, Tang Z, Su W, Sun W. Proteomic analysis of salt stress-responsive proteins in rice root. Proteomics. 2005;5(1):235–44. pmid:15672456
  24. 24. Wang H, Yang Y, Chen W, Ding L, Li P, Zhao X, et al. Identification of differentially expressed proteins of Arthrospira (Spirulina) plantensis-YZ under salt-stress conditions by proteomics and qRT-PCR analysis. Proteome Sci. 2013;11(1):6. pmid:23363438
  25. 25. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A. 1998;95(25):14863–8. pmid:9843981
  26. 26. Zhou Y, Chu P, Chen H, Li Y, Liu J, Ding Y, et al. Overexpression of Nelumbo nucifera metallothioneins 2a and 3 enhances seed germination vigor in Arabidopsis. Planta. 2012;235(3):523–37. pmid:21971996
  27. 27. Boller T, Gehri A, Mauch F, Vogeli U. Chitinase in bean leaves: induction by ethylene, purification, properties, and possible function. Planta. 1983;157(1):22–31. pmid:24263941
  28. 28. McCarty RE. ATP synthase of chloroplast thylakoid membranes: a more in depth characterization of its ATPase activity. J Bioenerg Biomembr. 2005;37(5):289–97. pmid:16341773
  29. 29. Taussky HH, Shorr E. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem. 1953;202(2):675–85. pmid:13061491
  30. 30. Rossignol M, Peltier JB, Mock HP, Matros A, Maldonado AM, Jorrin JV. Plant proteome analysis: a 2004–2006 update. Proteomics. 2006;6(20):5529–48. pmid:16991197
  31. 31. Podda A, Checcucci G, Mouhaya W, Centeno D, Rofidal V, Del Carratore R, et al. Salt-stress induced changes in the leaf proteome of diploid and tetraploid mandarins with contrasting Na+ and Cl- accumulation behaviour. J Plant Physiol. 2013;170(12):1101–12. pmid:23608743
  32. 32. Wang L, Liu X, Liang M, Tan F, Liang W, Chen Y, et al. Proteomic analysis of salt-responsive proteins in the leaves of mangrove Kandelia candel during short-term stress. PLoS One. 2014;9(1):e83141. pmid:24416157
  33. 33. Shi S, Chen W, Sun W. Comparative proteomic analysis of the Arabidopsis cbl1 mutant in response to salt stress. Proteomics. 2011;11(24):4712–25. pmid:22002954
  34. 34. Turan S, Tripathy BC. Salt-stress induced modulation of chlorophyll biosynthesis during de-etiolation of rice seedlings. Physiol Plant. 2015; 153:477–91. pmid:25132047
  35. 35. Grimm B. Primary structure of a key enzyme in plant tetrapyrrole synthesis: glutamate 1-semialdehyde aminotransferase. Proc Natl Acad Sci U S A. 1990;87(11):4169–73. pmid:2349227
  36. 36. Hartel H, Kruse E, Grimm B. Restriction of Chlorophyll Synthesis Due to Expression of Glutamate 1-Semialdehyde Aminotransferase Antisense RNA Does Not Reduce the Light-Harvesting Antenna Size in Tobacco. Plant Physiol. 1997;113(4):1113–24. pmid:12223663
  37. 37. Tsang EW, Yang J, Chang Q, Nowak G, Kolenovsky A, McGregor DI, et al. Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene. Plant Mol Biol. 2003;51(2):191–201. pmid:12602878
  38. 38. Chaves MM, Flexas J, Pinheiro C. Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann Bot. 2009;103(4):551–60. pmid:18662937
  39. 39. Munns R, James RA, Lauchli A. Approaches to increasing the salt tolerance of wheat and other cereals. J Exp Bot. 2006;57(5):1025–43. pmid:16510517
  40. 40. Sudhir P, Murthy SDS. Effects of salt stress on basic processes of photosynthesis. Photosynthetica. 2004;42(2):481–6.
  41. 41. Ashraf M, Harris PJC. Photosynthesis under stressful environments: An overview. Photosynthetica. 2013;51(2):163–90.
  42. 42. Moradi F, Ismail AM. Responses of photosynthesis, chlorophyll fluorescence and ROS-scavenging systems to salt stress during seedling and reproductive stages in rice. Ann Bot. 2007;99(6):1161–73. pmid:17428832
  43. 43. Stepien P, Johnson GN. Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte thellungiella: role of the plastid terminal oxidase as an alternative electron sink. Plant Physiol. 2009;149(2):1154–65. pmid:19052149
  44. 44. de Abreu CE, Araujo Gdos S, Monteiro-Moreira AC, Costa JH, Leite Hde B, Moreno FB, et al. Proteomic analysis of salt stress and recovery in leaves of Vigna unguiculata cultivars differing in salt tolerance. Plant Cell Rep. 2014;33(8):1289–306. pmid:24770441
  45. 45. Parker R, Flowers TJ, Moore AL, Harpham NV. An accurate and reproducible method for proteome profiling of the effects of salt stress in the rice leaf lamina. J Exp Bot. 2006;57(5):1109–18. pmid:16513811
  46. 46. Yang L, Ma C, Wang L, Chen S, Li H. Salt stress induced proteome and transcriptome changes in sugar beet monosomic addition line M14. J Plant Physiol. 2012;169(9):839–50. pmid:22498239
  47. 47. Yi X, Sun Y, Yang Q, Guo A, Chang L, Wang D, et al. Quantitative proteomics of Sesuvium portulacastrum leaves revealed that ion transportation by V-ATPase and sugar accumulation in chloroplast played crucial roles in halophyte salt tolerance. J Proteomics. 2014;99:84–100. pmid:24487036
  48. 48. Sobhanian H, Razavizadeh R, Nanjo Y, Ehsanpour AA, Jazii FR, Motamed N, et al. Proteome analysis of soybean leaves, hypocotyls and roots under salt stress. Proteome Sci. 2010;8:19. pmid:20350314
  49. 49. Zou J, Liu C, Chen X. Proteomics of rice in response to heat stress and advances in genetic engineering for heat tolerance in rice. Plant Cell Rep. 2011;30(12):2155–65. pmid:21769604
  50. 50. Wang Z, Wang M, Liu L, Meng F. Physiological and proteomic responses of diploid and tetraploid black locust (Robinia pseudoacacia L.) subjected to salt stress. Int J Mol Sci. 2013;14(10):20299–325. pmid:24129170
  51. 51. Rai S, Agrawal C, Shrivastava AK, Singh PK, Rai LC. Comparative proteomics unveils cross species variations in Anabaena under salt stress. J Proteomics. 2014;98:254–70. pmid:24406298
  52. 52. Saha P, Kunda P, Biswas AK. Influence of sodium chloride on the regulation of Krebs cycle intermediates and enzymes of respiratory chain in mungbean (Vigna radiata L. Wilczek) seedlings. Plant Physiol Biochem. 2012;60:214–22. pmid:23000814
  53. 53. Manaa A, Mimouni H, Wasti S, Gharbi E, Aschi-Smiti S, Faurobert M, et al. Comparative proteomic analysis of tomato (Solanum lycopersicum) leaves under salinity stress. 2014; 15:21803–24.
  54. 54. El-Shintinawy F, El-Shourbagy M. Alleviation of changes in protein metabolism in NaCl-stressed wheat seedlings by thiamine. Biologia plantarum. 2001;44(4):541–5.
  55. 55. Wang M, Xu Q, Yuan M. The unfolded protein response induced by salt stress in Arabidopsis. Methods Enzymol. 2011;489:319–28. pmid:21266238
  56. 56. Beckmann RP, Mizzen LE, Welch WJ. Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly. Science. 1990;248(4957):850–4. pmid:2188360
  57. 57. Fernandez-Garcia N, Hernandez M, Casado-Vela J, Bru R, Elortza F, Hedden P, et al. Changes to the proteome and targeted metabolites of xylem sap in Brassica oleracea in response to salt stress. Plant Cell Environ. 2011;34(5):821–36. pmid:21276013
  58. 58. Qureshi MI, Qadir S, Zolla L. Proteomics-based dissection of stress-responsive pathways in plants. J Plant Physiol. 2007;164(10):1239–60. pmid:17662502
  59. 59. Xu C, Sibicky T, Huang B. Protein profile analysis of salt-responsive proteins in leaves and roots in two cultivars of creeping bentgrass differing in salinity tolerance. Plant Cell Rep. 2010;29(6):595–615. pmid:20361191
  60. 60. Lyon BR, Lee PA, Bennett JM, DiTullio GR, Janech MG. Proteomic analysis of a sea-ice diatom: salinity acclimation provides new insight into the dimethylsulfoniopropionate production pathway. Plant Physiol. 2011;157(4):1926–41. pmid:22034629
  61. 61. Machado CR, de Oliveira RL, Boiteux S, Praekelt UM, Meacock PA, Menck CF. Thi1, a thiamine biosynthetic gene in Arabidopsis thaliana, complements bacterial defects in DNA repair. Plant Mol Biol. 1996;31(3):585–93. pmid:8790291
  62. 62. Machado CR, Praekelt UM, de Oliveira RC, Barbosa AC, Byrne KL, Meacock PA, et al. Dual role for the yeast THI4 gene in thiamine biosynthesis and DNA damage tolerance. J Mol Biol. 1997;273(1):114–21. pmid:9367751
  63. 63. Ribeiro DT, Farias LP, de Almeida JD, Kashiwabara PM, Ribeiro AF, Silva-Filho MC, et al. Functional characterization of the thi1 promoter region from Arabidopsis thaliana. J Exp Bot. 2005;56(417):1797–804. pmid:15897230
  64. 64. Sanchez-Aguayo I, Rodriguez-Galan JM, Garcia R, Torreblanca J, Pardo JM. Salt stress enhances xylem development and expression of S-adenosyl-L-methionine synthase in lignifying tissues of tomato plants. Planta. 2004;220(2):278–85. pmid:15322882
  65. 65. Chen AP, Zhong NQ, Qu ZL, Wang F, Liu N, Xia GX. Root and vascular tissue-specific expression of glycine-rich protein AtGRP9 and its interaction with AtCAD5, a cinnamyl alcohol dehydrogenase, in Arabidopsis thaliana. J Plant Res. 2007;120(2):337–43. pmid:17287892
  66. 66. Zhao Q, Tobimatsu Y, Zhou R, Pattathil S, Gallego-Giraldo L, Fu C, et al. Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula. Proc Natl Acad Sci U S A. 2013;110(33):13660–5. pmid:23901113
  67. 67. Deng WW, Zhang M, Wu JQ, Jiang ZZ, Tang L, Li YY, et al. Molecular cloning, functional analysis of three cinnamyl alcohol dehydrogenase (CAD) genes in the leaves of tea plant, Camellia sinensis. J Plant Physiol. 2013;170(3):272–82. pmid:23228629
  68. 68. Ahmed NU, Park JI, Jung HJ, Kang KK, Hur Y, Lim YP, et al. Molecular characterization of stress resistance-related chitinase genes of Brassica rapa. Plant Physiol Biochem. 2012;58:106–15. pmid:22796900
  69. 69. Wang J, Meng Y, Li B, Ma X, Lai Y, Si E, et al. Physiological and proteomic analyses of salt stress response in the halophyte Halogeton glomeratus. Plant Cell Environ. 2015; 38:655–69. pmid:25124288
  70. 70. Subramanian B, Bansal VK, Kav NN. Proteome-level investigation of Brassica carinata-derived resistance to Leptosphaeria maculans. J Agric Food Chem. 2005;53(2):313–24. pmid:15656667