Proteomic analysis of canola root inoculated with bacteria under salt stress.

J O U RN A L OF P ROTE O M IC S 1 24 ( 20 1 5 ) 8 8 –11 1

Available online at www.sciencedirect.com

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Proteomic analysis of canola root inoculated with bacteria under salt stress Farzad Banaei-Asla,b , Ali Bandehaghb , Ebrahim Dorani Uliaeib , Davoud Farajzadehc , Katsumi Sakatad , Ghazala Mustafaa , Setsuko Komatsua,⁎ a

National Institute of Crop Science, National Agriculture and Food Research Organization, Tsukuba 305-8518, Japan Department of Plant Breeding and Biotechnology, University of Tabriz, Tabriz 51666-16471, Iran c Department of Cellular and Molecular Biology, Azarbaijan Shahid Madani University, Tabriz 53751-71379, Iran d Maebashi Institute of Technology, Maebashi 371-0816, Japan b

AR TIC LE I N FO

ABS TR ACT

Article history:

Plant-growth promoting bacteria can ameliorate the negative effects of salt stress on

Received 30 January 2015

canola. To better understand the role of bacteria in canola under salt stress, salt-sensitive

Accepted 4 April 2015

(Sarigol) and salt-tolerant (Hyola308) cultivars were inoculated with Pseudomonas fluorescens

Available online 17 April 2015

and protein profiles of roots were compared. Bacterial inoculation increased the dry weight and length of canola roots under salt stress. Using a gel-free proteomic technique, 55

Keywords:

commonly changed proteins were identified in Sarigol and Hyola308 roots inoculated with

Proteomics

bacteria under salt stress. In both canola cultivars, proteins related to amino acid

Canola

metabolism and tricarboxylic acid cycle were affected. Hierarchical cluster analysis divided

Root

the identified proteins into three clusters. Proteins related to Clusters II and III, which were

Plant-growth promoting bacteria

secretion-associated RAS super family 1, dynamin-like protein, and histone, were increased

Salt

in roots of both Sarigol and Hyola308 inoculated with bacteria under salt stress. Based on pathway mapping, proteins related to amino acid metabolism and the tricarboxylic acid cycle significantly changed in canola cultivars inoculated with or without bacteria under salt stress. These results suggest that bacterial inoculation of canola roots increases tolerance to salt stress by proteins related to energy metabolism and cell division. Biological significance Plant-growth promoting bacteria as an emerging aid can ameliorate the negative effect of salt stress on canola. To understand the role of bacteria in canola under salt stress, salt sensitive Sarigol and tolerant Hyola308 cultivars were used. Dry weight and length of canola root were improved by inoculation of bacteria under salt stress. Using gel-free proteomic technique, 55 commonly changed proteins identified in Sarigol and Hyola308 inoculated with bacteria under salt stress. In both canola cultivars, the number of proteins related to amino acid metabolism and tricarboxylic acid cycle was more than other categories with higher change in protein abundance. Hierarchical cluster analysis divided into 3 clusters. Cluster II including secretion-associated RAS super family 1 and dynamin-like protein and Cluster III including histones H2A were increased by bacterial inoculation in both cultivars.

Abbreviations: LC, liquid chromatography; MS, mass spectrometry; P5CS, delta 1-pyrroline-5-carboxylate synthase. ⁎ Corresponding author at: National Institute of Crop Science, National Agriculture and Food Research Organization, Kannondai 2-1-18, Tsukuba 305-8518, Japan. Tel.: + 81 29 838 8693; fax: +81 29 838 8694. E-mail address: [email protected] (S. Komatsu).

http://dx.doi.org/10.1016/j.jprot.2015.04.009 1874-3919/© 2015 Elsevier B.V. All rights reserved.

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J O U RN A L OF P ROT EO M IC S 1 2 4 ( 20 1 5 ) 8 8 –1 11

Furthermore, pathway mapping highlighted the importance of S-denosylmethionine synthetase and malate dehydrogenase that decreased in canola inoculated with bacteria under salt stress. These results suggest that bacterial inoculation helps the canola to endure salt stress by modulating the proteins related to energy metabolism and cell division. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Due to the apparent health benefits of reducing saturated fat intake in the human diet, canola is being increasingly used as a source of edible vegetable oil [1] and represents a third of all vegetable oil produced worldwide [2]. Because of the limited agricultural land, increasing plant productivity is necessary to generate sufficient plant oil for use in food and animal feed [3]. Although maximum yields of canola are obtained under normal soil and environmental conditions, the quantity and quality of seed yields are affected by environmental stress [4,5]. Improving the tolerance of canola to stressful conditions would lead to increased yields of higher quality oil. Soil salinity is a major agricultural concern as it causes substantially or partially unproductive lands [6]. High salt concentrations of soil are often associated with ion imbalances and hyperosmotic pressure, which eventually lead to oxidative stress conditions for plants [7]. Tolerance against salinity stress is a complex trait that is governed by numerous mechanisms at the cellular, tissue, organ, and plant level [8]. Canola is categorized as a moderately salt-tolerant plant, with amphidiploid species being relatively salt tolerant in comparison with diploid species [9]. In particular, the amphidiploid species Brassica napus was found to have superior resistance among canola species in early growth stages in a comparative study of salt tolerance [10]. To improve the tolerance and increase the yields of canola exposed to salt stress, it is necessary to determine the underlying salt responsive and tolerance mechanisms. Plant roots serve as a niche for the proliferation of certain species of soil bacteria. These plant–microbe interactions can be beneficial, neutral, or deleterious for plant growth [11]. For example, bacteria can improve plant growth and vigor in stressful environments by the fixation of nutrients [12], reducing the toxicity of heavy metals [13], controlling of soil-borne pathogens [14], and altering plant hormonal balance [15]. One well-characterized plant growth-promoting bacterium is the Pseudomonas fluorescens FY32, which increases the growth and yield of crops, particularly root length under salt stress [15,16]. Plant-growth promoting bacteria have the potential to aid crop production by reducing the deleterious effects of salt stress. Soil bacteria moderate the harmful effects of salinity on canola. Proteomic analysis of canola roots revealed that many proteins involved in photosynthesis, anti-oxidative processes, transportation across membranes, and pathogenesis-related responses were differentially expressed in the presence of bacteria and salt [17]. In experiments with salt-tolerant (Hyola308) and salt-sensitive (Sarigol) cultivars, Bandehagh et al. [18] demonstrated that the levels of proteins related to oxidative stress and energy production were changed in response to salt stress. Using a similar approach, plasma-membrane, stress, and protein synthesis-associated proteins were found to play an important role in resistance against drought stress [19]. To

better understand the stress-responsive mechanisms of canola, stress-tolerant and -sensitive cultivars are therefore useful materials. To increase the quantity and quality of canola seed yields, it is necessary to develop cultivars with higher stress tolerance. In this study, to better understand the salt-responsive mechanisms of canola and plant growth-promoting bacteria in moderating the harmful effects of salt stress, salt-tolerant Hyola308 and salt-sensitive Sarigol canola cultivars were used. A proteomic technique was used to identify responsive proteins in canola inoculated with the plant-growth promoting bacterium P. fluorescens FY32 [20] under salt stress. In addition, to determine the role of the key proteins involved in the canola response to salt stress and bacterial inoculation, hierarchical cluster and pathway analyses were performed.

2. Materials and methods 2.1. Plant material Canola seeds (B. napus L. cultivars Hyola308, Sarigol, RGS003, Amica, Hyola420, and Olga) were obtained from the Seed and Plant Improvement Institute (Karaj, Iran). Seeds were sterilized [21], germinated under aseptic conditions, transplanted, and cultured in a hydroponic system with sterilized Hoagland's solution [22]. The greenhouse was controlled as follows: temperature (25 ± 2 °C during the day and night), relative humidity (50% during the day and 60% at nights), light (14 h daily), and nutrient solution (pH 6.5 ± 0.5 using hydrochloric acid/potassium hydroxide).

2.2. Preparation of bacteria suspension The bacterial strain P. fluorescens FY32 was aerobically grown in 250 mL Luria-Bertani medium [23] at 30 °C for 18 h with continuous shaking. The bacterial cells were harvested by centrifugation at 8000 ×g for 10 min at 4 °C and re-suspended in 25 mL sterilized 0.03 M MgSO4 on ice [20]. To determine the population count of cultured bacteria, McFarland's method [24] was used. The absorbance of cell suspension was measured at 600 nm and the turbidity of bacterial suspension was adjusted to a concentration of approximately 1010 cfu mL−1 using sterilized 0.03 M MgSO4.

2.3. Salt treatment and bacterial inoculation One-week-old canola plants were transferred to a hydroponic system, which was sterilized and washed twice prior to transplantation. After transplantation, 10 mL of bacterial suspension in 0.03 M MgSO4 was injected into each reservoir containing 10 L of nutrient solution. One week after inoculation, plants were treated without or with 150 and 300 mM NaCl.

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Nutrient solution, bacterial suspension, and NaCl treatments were renewed every 2 weeks. For morphological experiments, the dry weight, length, sodium, potassium, and chloride contents of roots were measured 3 weeks after the initial NaCl treatment. After the measurement of root length, roots were placed into an oven at 72 °C for 2 days before the measurement of dry weight. After analysis of the morphological traits, the most tolerant and sensitive cultivars were selected for proteomic and enzymatic analyses. Three independent experiments were performed as biological replications.

2.4. Measurement of sodium, potassium, and chloride contents of root The dried samples (0.1 g) were ground and mixed with 0.2 N nitric acid. The slurry was passed through paper filter and sodium and potassium were determined with a flame photometer (Cole Parmer Instrument, IL, USA) [25]. For the determination of chloride, samples were ground and mixed with ferric nitrate solution and saturated mercury thiocyanate solution. The absorbance of samples was measured at 460 nm after 15 min [26]. Sodium, potassium, and chloride concentrations were calculated based on standard calibration curve.

2.5. Protein extraction and digestion for proteomic analysis A sample (0.1 g) of dried root was ground to powder in liquid nitrogen using a mortar and pestle. The powder was transferred to a solution of 10% trichloroacetic acid and 0.07% 2-mercaptoethanol in acetone and thoroughly mixed by vortexing. The resulting suspension was sonicated for 10 min and then incubated at − 20 °C for 60 min. After incubation, the suspension was centrifuged at 9000 ×g for 20 min at 4 °C. The supernatant was discarded and the pellet was transferred into a solution of 0.07% 2-mercaptoethanol in acetone for 60 min at − 20 °C. The suspension was centrifuged at 20,000 ×g for 5 min at 4 °C and the obtained pellet was washed twice with 0.07% 2-mercaptoethanol in acetone. The final pellet was dried using a Speed-Vac concentrator (Savant Instruments, Hicksville, NY, USA) and was then re-suspended in lysis buffer consisting of 7 M urea, 2 M thiourea, 5% CHAPS, and 2 mM tributylphosphine. The protein concentration was determined using the Bradford method [27] with bovine serum albumin as the standard. The extracted proteins (100 μg) were purified using a methanol–chloroform method according to Komatsu et al. [28]. After adjusting the volume of the protein sample to 100 μL, 400 μL methanol was added and mixed before adding 100 μL chloroform and mixing the sample again. To induce phase separation, 300 μL water was added to the sample and mixed thoroughly by vortexing. After centrifugation at 20,000 ×g for 10 min at room temperature, the upper aqueous phase was discarded and 300 μL methanol was added to the remaining organic phase. The sample was further centrifuged at 20,000 ×g for 10 min, the resulting supernatant was removed, and the pellet was dried in air for 10 min. The dried pellet was re-suspended in 50 mM NH4HCO3, and proteins in the sample were reduced by treatment with 50 mM dithiothreitol for 30 min at 56 °C and were then alkylated with 50 mM iodoacetamide for 30 min at 37 °C in the dark. Alkylated proteins were digested using trypsin and lysyl

endopeptidase (1:100 enzyme/protein concentration) at 37 °C for 16 h. The digested peptides were acidified with 10 μL of 20% formic acid, desalted using a C18-pipette tip (Nikkyo Technos, Tokyo, Japan), and used for nano-liquid chromatography (LC) mass spectrometry (MS) analysis.

2.6. Nano-liquid chromatography mass spectrometry analysis Peptides in 0.1% formic acid were loaded onto a C18 PepMap trap column (300 μm ID × 5 mm, Dionex) of an Ultimate 3000 LC system (Dionex, Germering, Germany). Peptides were eluted from the trap column and were then separated using 0.1% formic acid in acetonitrile at a flow rate of 200 nL/min on a C18 Tip column (75 μm 1D × 120 mm, Nikkyo Technos) with a spray voltage of 1.5 kV. Peptide ions in the spray were detected and the peptides were analyzed on a nanospray LTQ XL Orbitrap MS (Thermo Fisher Scientific, San Jose, CA, USA) operated in data-dependent acquisition mode with the installed Xcalibur software (version 2.1, Thermo Fisher Scientific). Elution was performed with a linear acetonitrile gradient (15%–40% over 115 min) in 0.1% formic acid. Full-scan mass spectra were acquired in the MS over 400– 1500 m/z with a resolution of 30,000. A lock mass function was used to obtain high mass accuracy [29]. As the lock mass, the ions C24H39O+4 (m/z 391.28429), C14H46NO7Si+7 (m/z 536.16536), and C16H52NO8Si+8 (m/z 610.18416) were used. The top 10 most intense ions were selected for collision-induced fragmentation in the linear ion trap at a normalized collision energy of 35%. Dynamic exclusion was employed within 90 s to prevent repetitive selection of peptides [30]. The acquired MS spectra were used for protein identification.

2.7. Protein identification Proteins were identified using the Mascot search engine (version 2.4.1, Matrix Science, London, UK) of an Arabidopsis database (TAIR10, http://www.arabidopsis.org/, 35,386 entries). The acquired raw data files were processed and converted to Mascot generic files using Proteome Discoverer software (version 1.4.0.288, Thermo Fisher Scientific). In the Mascot searches, carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionine was set as a variable modification. For peptides, trypsin was specified as the proteolytic enzyme and one missed cleavage was allowed. Peptide mass tolerance was set at 10 ppm, fragment mass tolerance was set at 0.8 Da, and peptide charges were set at + 2, + 3, and +4. An automatic decoy database search was performed as part of the search. Mascot results were filtered with the Mascot percolator to improve the accuracy and sensitivity of peptide identification [31]. False discovery rates for peptide identification of all searches were less than 1.0%. Peptides with a percolator ion score of more than 13 (p < 0.05) were used for protein identification. The Mascot search results were exported in XML format for SIEVE analysis (version 2.1.0, Thermo Fisher Scientific).

2.8. Analysis of differentially abundant proteins using acquired mass spectrometry data Using SIEVE software, the relative abundances of peptides and proteins were compared between samples. For the analysis, the


chromatographic peaks detected by MS were aligned, and the peptide peaks were detected as a frame using the following settings: frame time width (5 min); frame m/z width (10 ppm); and produce frames on all parent ions scanned by MS/MS. Chromatographic peak areas of each sample within a single frame were compared and the ratios between samples in each frame were determined. The frames detected in the MS/MS scan were matched to the imported Mascot search results. The ratio of peptides between samples was determined from the variance-weighted average of the ratios in frames that matched the peptides in the MS/MS spectrum. The ratios of peptides were further integrated to determine the ratio of the corresponding

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protein. In the differential analysis of protein abundance, total ion current was used for normalization. The least requirement for the identification of a protein was a minimum of two matched peptides. Significant changes in the abundance of proteins between samples were analyzed (p < 0.05). The identified peptides were used to calculate protein abundance.

2.9. Functional analysis of proteins To understand the functional role of the identified proteins, functional categorization was performed using MapMan bin codes [32].

Canola cultivars (Hyola308, Sarigol, RGS003, Amica, Hyola420, Olga) 0

Germination

1

Transplantation Without or with bacteria inoculation Without or with150 and 300 mM NaCl treatment

2

Morphological analysis Traits measurement • Root length • Root dry weight • Root sodium content • Root potassium content • Root chloride content

5 week

Proteomic analysis Root non-inoculation inoculation 0 150 300 0 150 300 mM NaCl R1 R2 R3 Protein extraction/purification Reduction/Alkylation with dithiothreitol/iodoacetamide Digestion with trypsin/lysyl endopeptidase Analysis using nano LC-MS/MS Identification using Mascot and TAIR database

Fig. 1 – Experimental design for the morphological and proteomic analyses of canola cultivars inoculated with bacteria under salt stress. One-week-old canola seedlings were transferred to a hydroponic system and inoculated without or with bacteria. After 1 week, the plants were treated without or with 150 and 300 mM NaCl for 3 weeks, and morphological and proteomic analyses were then performed. Three independent experiments were performed as biological replications.

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2.10. Cluster analysis of protein abundance Protein abundance ratios in the samples exposed to different treatments were used for cluster analysis by a hierarchical clustering method (a centroid linkage method based on a Euclidean distance metric).

2.11. Kyoto Encyclopedia of Genes and Genomes pathway mapping Pathway mapping of identified proteins was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) [33].

containing 100 mM sodium pyrophosphate pH 8.5, 0.017 mM acetaldehyde, 1.5 mM NAD. Aldehyde dehydrogenase activity was calculated with the formula: units/mL = (ΔA460 × total volume × dilution factor) / (6.22 × sample volume). For the measurement of S-adenosylmethionine synthetase activity Kramer et al. [35] method was used with minor modifications. For S-adenosylmethionine synthetase activity assay, the reaction buffer consisted of 100 mM Tris–HCl (pH 8.0), 20 mM MgCl2, 150 mM KCl, 5 mM dithiothreitol, and 1 mM methionine was used. The activity was spectrophotometrically measured at 340 nm and calculated with the formula: units/mL = (ΔA340 × total volume × dilution factor) / (12.44 × sample volume). The protein concentration was measured by the Bradford method [27].

2.12. Enzyme activity analysis 2.13. Statistical analysis Aldehyde dehydrogenase activity was measured according to Liu et al. [34] with minor modifications. Assay of aldehyde dehydrogenase activity was performed spectrophotometrically at 460 nm by the conversion of NAD into NADH in a buffer

A

The statistical significance of comparisons between multiple groups was evaluated with the one-way ANOVA test. The statistical significance of the results was evaluated by the

140

Root length (mm)

120 100 80 60 40 20 0

0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 mM NaCl Sarigol

B

Hyola 420

Olga

RGS003

Amica

Hyola308

0.20

Root dry weight (g)

0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 mM NaCl Sarigol

Hyola 420

Olga

RGS003

Amica

Hyola308

Fig. 2 – Effect of bacterial inoculation under salt stress on the dry weight and length of roots of six canola cultivars. One-week-old canola seedlings (Hyola308, Sarigol, RGS003, Amica, Hyola420, and Olga) were transferred to a hydroponic system and inoculated without (white columns) or with (black columns) bacteria, and were then treated without or with 150 and 300 mM NaCl. Root dry weight (A) and root length (B) were measured after 3 weeks of NaCl treatments. Data are means ± SD from 3 independent biological replications. The different letters indicate significant changes between different NaCl treatments for non-inoculated and inoculated roots according to one-way ANOVA (p < 0.05). Asterisks above the error bars indicate significant changes between inoculated and non-inoculated cultivars by the Student's t-test (*p < 0.05, **p < 0.01).

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3. Results 3.1. Morphological changes of canola inoculated with bacteria under salt stress

A Sodium content (mg g -1 FW)

Student's t-test when two groups were compared. A p value of < 0.05 was considered as statistically significant.

70 60 50

3.2. Differentially abundant proteins in canola roots inoculated with bacteria under salt stress To identify the proteins involved in mediating salt tolerance in canola, a proteomic technique was used to analyze the protein profiles of plants inoculated with bacteria under salt stress. One-week-old canola cultivars were transferred to a hydroponic system and inoculated without or with bacteria. After one week, plants were treated without or with 150 and 300 mM NaCl for three weeks, and root proteins were extracted for proteomic analysis. In Sarigol, a total of 637 and 756 salt-responsive proteins, including 390 common proteins, were significantly changed in non-inoculated and inoculated roots, respectively (Supplemental Tables 3 and 4). Among the differentially abundant proteins in Sarigol, 247 and 366 proteins were uniquely identified in non-inoculated and inoculated roots, respectively (Fig. 4). In Hyola308, a total of 456 and 225 salt-responsive proteins, including 74 common

*

30

*

20 10

*

*

0 0

150

300

0

Potassium content (mg g -1 FW)

B

300 mM

150

Sarigol

Hyola308

60 50 40

*

30 20 10 0 0

150

300

0

Sarigol

C

150

300 mM

Hyola308

80 70

Chloride content (mg g -1 FW)

To investigate the changes that occur in canola cultivars inoculated with bacteria under salt stress, morphological and physiological traits were measured. One-week-old canola cultivars were inoculated without or with bacteria in a hydroponic growth system and then after one week treated without or with 150 and 300 mM NaCl (Fig. 1). Dry weight and length of roots were measured after 3 weeks of NaCl treatment (Supplemental Tables 1 and 2). The root length of cultivars Sarigol and Hyola308 was decreased by NaCl treatments without bacterial inoculation (Fig. 2A). However, the root dry weight of Amica and Hyola308 was not significantly decreased compared to Sarigol and Hyola420 under NaCl treatment (Fig. 2B). The same morphological features were measured for roots inoculated with bacteria, revealing that the dry weight and length of canola roots were significantly improved compared to non-inoculated roots under NaCl treatment (Fig. 2). To get better understanding towards the response of Sarigol and Hyola308 to bacterial inoculation under NaCl treatments, sodium, potassium, and chloride contents in canola roots were analyzed. Sodium and chloride contents of root significantly increased in both inoculated and non-inoculated Sarigol compared to Hyola308 under 150 and 300 mM NaCl. While potassium content decreased in inoculated and non-inoculated Sarigol under 150 and 300 mM NaCl. Sodium, potassium, and chloride contents in inoculated and non-inoculated cultivars were not significantly changed in control. On the other hand, sodium and chloride contents significantly decreased in inoculated cultivars compared to non-inoculated cultivars (Fig. 3). Based on these results, the canola cultivars Sarigol and Hyola308 were assigned as salt-sensitive and salt-tolerant cultivars, respectively.

*

40

60 50 40

*

*

150

300

30 20

*

10 0 0

150 Sarigol

300

0

mM

Hyola308

Fig. 3 – Effect of bacterial inoculation on sodium, potassium, and chloride contents of canola roots under salt stress. Sarigol and Hyola308 used as sensitive and tolerant cultivars, respectively. One-week-old canola seedlings were transferred to a hydroponic system and inoculated without (white columns) or with (black columns) bacteria, and were then treated without or with 150 and 300 mM NaCl. Sodium (A), potassium (B), and chloride (C) contents of canola roots were measured after 3 weeks of NaCl treatments. Data are shown as means ± SD from three independent biological replications. Asterisks above the error bars indicate significant changes between inoculated and non-inoculated cultivars by the Student's t-test (*p < 0.05).

proteins, were significantly changed in non-inoculated and inoculated roots, respectively (Supplemental Tables 5 and 6). Among these differentially abundant proteins, 382 and 151 proteins were uniquely identified in non-inoculated and inoculated roots, respectively (Fig. 4).

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To gain a better understanding of the biological processes that are altered by inoculation of canola roots with a growth-promoting bacterium under salt stress, the differentially abundant proteins identified in roots were functionally

classified using MapMan bin codes [30]. The abundance of 637 proteins identified in non-inoculated Sarigol roots under salt stress were functionally categorized as being predominantly involved in protein synthesis/degradation (21%), amino acid

Sarigol non-inoculation

Hyola308

756 proteins

637 proteins

390

247

40

30

20

10

0

inoculation 225 proteins

456 proteins

382

366

Number of proteins 170 150 130 50

non-inoculation

inoculation

151

74

Number of proteins 0

10

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40

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60

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80

protein amino acid metabolism cell organization cell wall signalling TCA stress transport DNA RNA glycolysis mito. ETC development major CHO metabolism fermentation photosunthesis redox nucleotide metabolism lipid metabolism secondary metabolism hormone metabolism metal handling minor CHO metabolism oxidative pentose phosphate N-metabolism C1-metabolism S-assimilation others misc not assigned

Fig. 4 – Functional distribution of identified proteins in canola roots inoculated with bacteria under salt stress. Sarigol and Hyola308 used as sensitive and tolerant cultivars, respectively. One-week-old canola seedlings were transferred to a hydroponic system and inoculated without (white columns) or with (black columns) bacteria, and were then treated without or with 150 and 300 mM NaCl. Proteins were extracted from root after 3 weeks of NaCl treatments and used for gel-free proteomic analysis. The identified proteins were functionally classified using MapMan bin codes. Abbreviations: Protein, protein synthesis/degradation/post-translational modification/targeting; TCA, tricarboxylic acid cycle; DNA, chromatin structure; RNA, RNA binding/transcription; mito. ETC, mitochondrial election transport chain; CHO, carbohydrates; N-metabolism, nitrogen metabolism; C1-metabolism, carbon 1-metabolism; S-assimilation, sulfur assimilation; others, gluconeogenesis, tetrapyrrole synthesis, polyamine metabolism, co-factor and vitamin metabolism, and biodegradation of xenobiotics; and misc, miscellaneous.


metabolism (6%), and cell organization (6%). The differentially abundant 756 proteins detected in inoculated Sarigol roots were categorized in protein synthesis/degradation (23%), signaling (7%), amino acid metabolism (6%), cell organization (6%), and tricarboxylic acid cycle (6%) (Fig. 4). The differentially abundant 456 proteins identified in non-inoculated Hyola308 roots were categorized as being predominantly involved in protein synthesis/degradation (15%), amino acid metabolism (7%), cell organization (7%), cell wall (7%), signaling (6%), and tricarboxylic acid cycle (6%); whereas, the 225 significantly changed proteins detected in inoculated Hyola308 roots predominantly belonged to protein synthesis/degradation (28%), signaling (7%), stress (7%), cell organization (6%), and cell wall (6%) categories (Fig. 4). Protein synthesis/degradation and cell organization-related proteins were differentially changed in both non-inoculated and bacterial-inoculated canola roots; however, the number of proteins related to amino acid metabolism and tricarboxylic acid cycle markedly differed in response to bacterial inoculation compared to the other functional categories.

3.3. Salt stress-responsive protein profiles of canola roots inoculated with bacteria Among the 390 commonly changed proteins identified in non-inoculated and inoculated Sarigol roots, 110 proteins displayed a greater than 2-fold change in abundance in response to salt stress (300 mM NaCl). Among these differentially abundant proteins, 15 proteins were increased in abundance and were mainly classified in protein synthesis/ degradation (6 proteins) and stress (3 proteins) functional categories. The remaining 95 decreased proteins were mainly classified in protein synthesis/degradation (18 proteins), amino acid metabolism (12 proteins), cell organization (10 proteins), cell wall (8 proteins), tricarboxylic acid cycle (8 proteins), and glycolysis categories (7 proteins) (Table 1), as determined in 3 biological replications (Supplemental Tables 7 and 8). In non-inoculated and inoculated Hyola308 roots, a total of 74 proteins were commonly changed in response to salt stress and included 48 proteins that changed in abundance more than 2-fold. Of these proteins, 3 proteins related to DNA synthesis (2 proteins) and signaling (1 protein) were increased, whereas 45 proteins that were mainly classified in protein synthesis/degradation (10 proteins), glycolysis (7 proteins), amino acid metabolism (6 proteins), and tricarboxylic acid cycle categories (5 proteins) were decreased under NaCl treatment (Table 2), as determined in 3 biological replications (Supplemental Tables 7 and 8). The abundance of 55 proteins was commonly changed in Sarigol and Hyola308 roots inoculated with bacteria under salt stress (Table 3), as determined in 3 biological replications (Supplemental Tables 7 and 8). In Sarigol, out of 10 increased proteins, 3 and 2 proteins were classified in signaling and DNA functional groups, respectively, whereas the 45 decreased proteins were predominantly related to protein synthesis/ degradation (8 proteins), glycolysis (8 proteins), amino acid metabolism (6 proteins), cell organization (5 proteins), tricarboxylic acid cycle (4 proteins), and cell wall (4 proteins). In Hyola308, 7 proteins were increased and included 2 DNA-related proteins, whereas the 48 decreased proteins

95

were mainly categorized in protein synthesis/degradation (9 proteins), glycolysis (7 proteins), amino acid metabolism (5 proteins), and cell organization (5 proteins) functional groups (Table 3). Under 150 and 300 mM NaCl, the abundance of 15 proteins related to protein synthesis/degradation and stress was increased; while, 95 proteins related to protein synthesis/ degradation and amino acid metabolism were decreased in inoculated Sarigol (Table 1). On the other hand, the abundance of 41 proteins related to protein synthesis/degradation and tricarboxylic acid cycle was increased; while, 65 proteins related to protein synthesis/degradation and amino acid metabolism were decreased in non-inoculated Sarigol. Under 300 mM NaCl, 23 proteins related to protein synthesis/ degradation and stress were increased; while, 87 proteins related to protein synthesis/degradation, amino acid metabolism, and cell organization were decreased (Table 1). The abundance of 8 and 3 proteins, mostly related to DNA, was increased in 150 and 300 mM, respectively. On the other hand, 40 and 45 proteins related to protein synthesis/degradation, amino acid metabolism, glycolysis, and tricarboxylic acid cycle decreased in abundance under 150 and 300 mM NaCl, respectively (Table 2). In non-inoculated Hyola308, 26 and 25 proteins related to protein synthesis/degradation and glycolysis were increased in 150 and 300 mM NaCl, respectively. However, under 150 and 300 mM NaCl, 22 and 23 proteins related to amino acid metabolism and tricarboxylic acid cycle were decreased (Table 2).

3.4. Clustering analysis of differentially abundant proteins in inoculated canola roots under salt stress Proteins identified in canola inoculated with bacteria under salt stress were examined by hierarchical clustering analysis based on their abundance ratios. The clustering analysis grouped 55 proteins that were commonly changed in Sarigol and Hyola308, into 3 clusters (I, II, and III) (Fig. 5). Cluster I consisted of 48 proteins that were remarkably decreased in the bacterial inoculated Hyola308 roots under NaCl treatment. The main categories of this cluster were protein synthesis/degradation (19%), glycolysis (14%), amino acid metabolism (10%), cell organization (10%), and tricarboxylic acid cycle (8%). Cluster II consisted of 5 proteins, including secretion-associated RAS super family 1 and dynamin-like protein, which were slightly increased in inoculated roots of Hyola308 under NaCl treatment. Cluster III consisted of 2 histones that were increased in the bacterial inoculated roots of both Hyola308 and Sarigol under NaCl treatment (Fig. 5 and Table 3). Importantly, proteins related to Clusters II and III such as secretion-associated RAS super family 1, dynamin-like protein, and histone were increased in roots of both salt-sensitive and -tolerant canola cultivars inoculated with bacteria.

3.5. KEGG pathway analysis of proteins identified in inoculated canola roots under salt stress To understand the metabolic pathways in canola associated with the bacterial inoculation of roots under salt stress, identified proteins were mapped using the KEGG database

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Table 1 – Inoculation-responsive proteins identified in Sarigol roots exposed to varying salt treatments. Ratio c Non-inoculation No.

XW6-Ribosomal protein S5 family protein Ribosomal protein S5 family protein Ribosomal protein S5 family protein Ribosomal protein S5 family protein Ribosomal protein S13/S18 family Ribosomal protein S3Ae Vacuolar ATPase subunit F family protein Copper/zinc superoxide dismutase 1 Beta-amylase 5 MLP-like protein 329 Aldolase-type TIM barrel family protein MLP-like protein 28 MLP-like protein 31 RNA polymerase I-associated factor PAF67 Histone H2A 7 Sucrose synthase 1 Xyloglucan endotransglucosylase/hydrolase 7 Zincin-like metalloproteases family protein Isopropylmalate dehydrogenase 3 Nucleotide-rhamnose synthase/epimerase-reductase Phenylalanine ammonia-lyase 2 Succinate dehydrogenase 1-1 Arogenate dehydratase 5 PHE ammonia lyase 1 Succinyl-CoA ligase-alpha subunit Eukaryotic translation initiation factor 3 Phenylalanine ammonia-lyase 4 Regulatory particle non-ATPase 12A Heat shock protein 70B Reversibly glycosylated polypeptide 2 Glutamate dehydrogenase 3 Phosphoenolpyruvate carboxylase 3 Glycosyl hydrolases family 32 protein pfkB-like carbohydrate kinase family protein Eukaryotic translation initiation factor 3 ATP sulfurylase 1 Subtilase family protein ATP synthase subunit beta Cobalamin-independent synthase family protein FASCICLIN-like arabinogalactan 2 Peroxidase superfamily protein S-adenosyl-L-methionine-dependent methyltransferases

0

150

300

0

150

300 b

AT1G58380.1 AT1G58983.1 AT3G57490.1 AT2G41840.1 AT1G22780.1 AT4G34670.1 AT4G02620.1 AT1G08830.1 AT4G15210.1 AT2G01530.1 AT5G64250.1 AT1G70830.1 AT1G70840.1 AT5G25754.1 AT5G27670.1 AT5G20830.1 AT4G37800.1 AT5G10540.1 AT1G31180.1 AT1G63000.1 AT3G53260.1 AT5G66760.1 AT5G22630.1 AT2G37040.1 AT5G23250.1 AT5G44320.1 AT3G10340.1 AT1G64520.1 AT1G16030.1 AT5G15650.1 AT3G03910.1 AT3G14940.1 AT1G62660.1 AT2G31390.1 AT4G20980.1 AT3G22890.1 AT2G05920.1 ATCG00480.1 AT5G17920.1 AT4G12730.1 AT5G05340.1 AT4G34050.1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.43 0.43 0.43 0.44 0.37 0.33 3.05 1.67 1.03 0.85 0.84 2.57 2.93 1.73 1.39 0.27 0.54 0.48 0.61 1.17 0.82 1.25 0.73 0.81 1.23 0.94 0.78 1.12 1.00 0.94 1.60 1.13 1.82 0.47 0.67 0.76 0.72 1.14 0.75 1.00 0.54 0.54

0.10 0.10 0.10 0.12 0.20 0.50 2.80 2.35 1.55 1.25 1.23 7.83 5.35 1.72 1.59 0.10 0.32 0.65 0.39 0.68 0.56 1.23 0.48 0.54 0.47 0.67 0.52 0.86 0.21 0.63 1.01 1.19 1.90 0.23 0.62 0.49 0.32 0.92 0.27 0.62 0.48 0.34

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

5.87 5.87 5.87 5.85 4.90 2.16 2.14 3.12 1.34 1.43 1.16 1.14 1.35 1.96 1.37 0.01 0.54 0.37 0.53 0.69 0.66 0.38 0.90 0.61 0.26 0.54 0.61 0.78 0.23 0.21 0.24 0.30 0.37 0.41 0.51 0.69 0.16 0.24 0.15 0.26 0.48 0.40

6.21 6.21 6.21 6.04 5.47 2.75 2.54 2.49 2.48 2.36 2.30 2.20 2.12 2.10 2.05 0.48 0.48 0.48 0.47 0.47 0.47 0.47 0.47 0.46 0.46 0.46 0.46 0.45 0.45 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.43 0.43 0.43 0.42 0.42 0.41

Protein ID

Function d Protein Protein Protein Protein Protein Protein Transport Redox Major CHO metabolism Stress Misc Stress Stress Not assigned DNA Major CHO metabolism Cell wall Protein Amino acid metabolism Cell wall Secondary metabolism Tricarboxylic acid Amino acid metabolism Secondary metabolism Tricarboxylic acid Protein Secondary metabolism Protein Protein Cell wall N-metabolism Glycolysis Major CHO metabolism Major CHO metabolism Protein S-assimilation Protein Photosynthesis Amino acid metabolism Cell wall Misc Secondary metabolism


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Description

Inoculation

a

64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

Glutamine synthetase 1;4 Peroxidase superfamily protein Non-ATPase subunit 9 pfkB-like carbohydrate kinase family protein PDI-like 1-2 Ribosomal protein S5/Elongation factor G/III/V Late embryogenesis abundant protein-group 2 Target of Myb protein 1 Peroxidase family protein Methionine synthase 2 S-adenosylmethionine synthetase family protein 3-Phosphoglycerate dehydrogenase ACT domain-containing protein Reversibly glycosylated polypeptide 1 Gamma carbonic anhydrase like 1 Peroxidase superfamily protein Aldolase superfamily protein Sulfotransferase 16 Hydroxymethylglutaryl-CoA synthase D-mannose binding lectin protein D-mannose binding lectin protein with Apple-like carbohydrate-binding domain Aldolase superfamily protein UDP-glucose 6-dehydrogenase family protein Pseudouridine synthase/archaeosine transglycosylase Mitochondrial lipoamide dehydrogenase 1 Lipoamide dehydrogenase 2 Selenium-binding protein 2 Serine hydroxymethyltransferase 4 Methylenetetrahydrofolate reductase 2 Cysteine proteinases superfamily protein Transducin/WD40 repeat-like superfamily protein RNA 3-terminal phosphate cyclase/enolpyruvate transferase-alpha/beta RNA 3-terminal phosphate cyclase/enolpyruvate transferase-alpha/beta NmrA-like negative transcriptional regulator Eukaryotic translation initiation factor 4A1 eif4a-2 Granulin repeat cysteine protease family protein UDP-glucose dehydrogenase 1 Pyruvate kinase family protein Pyruvate kinase family protein DEA(D/H)-box RNA helicase family protein Aldolase superfamily protein Lactate/malate dehydrogenase family protein S-adenosylmethionine synthetase 2 Insulinase (Peptidase family M16) protein

AT5G16570.1 AT2G38390.1 AT1G29150.1 AT1G06030.1 AT1G77510.1 AT1G56070.1 AT2G44060.1 AT1G21380.1 AT4G30170.1 AT3G03780.1 AT3G17390.1 AT1G17745.1 AT5G04740.1 AT3G02230.1 AT5G63510.1 AT5G42180.1 AT3G52930.1 AT1G74100.1 AT4G11820.1 AT1G78850.1 AT1G78860.1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1.40 11.86 1.65 0.53 0.87 1.03 0.82 0.67 1.35 0.72 0.90 1.15 1.35 0.89 1.35 0.28 0.61 1.53 0.92 1.49 1.58

0.37 10.71 1.25 0.37 1.04 0.79 0.55 0.58 0.78 0.24 0.31 0.53 0.83 0.54 1.05 0.23 0.61 1.33 0.45 0.71 0.66

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.62 0.31 0.12 0.40 0.34 0.01 0.05 0.59 0.23 0.11 0.04 0.56 0.07 0.11 0.74 0.33 0.16 0.50 0.29 0.63 0.62

0.41 0.41 0.41 0.40 0.40 0.40 0.39 0.39 0.38 0.38 0.38 0.37 0.36 0.35 0.33 0.32 0.31 0.30 0.29 0.29 0.28

N-metabolism Misc Protein Major CHO metabolism Redox Protein Development Cell Misc Amino acid metabolism Amino acid metabolism Amino acid metabolism Amino acid metabolism Cell wall Mitochondrial electron transport Misc Glycolysis Not assigned Secondary metabolism Misc Misc

AT2G36460.1 AT3G29360.1 AT5G43780.1 AT1G48030.1 AT3G17240.1 AT4G14040.1 AT4G13930.1 AT2G44160.1 AT3G48340.1 AT1G18080.1 AT2G45300.1

1 1 1 1 1 1 1 1 1 1 1

0.44 0.62 0.86 1.35 1.33 0.52 0.78 0.66 1.17 1.36 0.72

0.78 0.46 0.63 1.03 0.99 0.23 0.02 0.48 0.66 0.97 0.33

1 1 1 1 1 1 1 1 1 1 1

0.12 0.07 0.78 0.86 0.63 0.43 0.04 0.03 0.38 0.66 0.21

0.28 0.27 0.26 0.26 0.26 0.26 0.26 0.26 0.25 0.24 0.21

Glycolysis Cell wall S-assimilation Tricarboxylic acid Tricarboxylic acid Metal handling C1-metabolism C1-metabolism Protein RNA Amino acid metabolism

AT1G48860.1

1

0.72

0.31

1

0.21

0.21

Amino acid metabolism

AT1G75280.1 AT3G13920.1 AT1G54270.1 AT5G43060.1 AT1G26570.1 AT3G52990.1 AT2G36580.1 AT1G72730.1 AT1G69740.1 AT1G04410.1 AT4G01850.1 AT3G02090.1

1 1 1 1 1 1 1 1 1 1 1 1

0.08 1.23 1.23 1.16 0.65 1.32 1.31 1.23 0.96 0.62 0.82 1.28

0.10 1.19 1.19 0.13 0.42 0.81 0.77 1.23 0.06 0.04 0.04 0.99

1 1 1 1 1 1 1 1 1 1 1 1

0.07 0.52 0.51 0.07 0.05 0.98 0.97 0.30 0.26 0.07 0.11 0.61

0.17 0.17 0.17 0.15 0.15 0.14 0.14 0.11 0.10 0.10 0.10 0.10

Secondary metabolism Protein Protein Protein Cell wall Glycolysis Glycolysis Protein Tetrapyrrole synthesis Tricarboxylic acid Amino acid metabolism Protein

97

(continued on next page)


43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

98

Table 1 (continued) Ratio c Non-inoculation 150

300

0

150

300 b

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.99 0.79 0.98 0.54 0.86 0.74 0.78 0.75 0.74 0.77 0.71 0.71 0.72 0.85 0.63 1.05 1.30 0.96 0.84 0.56 0.48 0.31 0.87

0.49 0.03 0.48 0.03 0.98 0.51 0.58 0.52 0.50 0.53 0.53 0.47 0.53 0.19 0.63 0.80 0.22 1.43 0.72 0.32 0.93 0.10 0.21

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.13 0.05 0.12 0.05 0.22 0.10 0.08 0.12 0.12 0.10 0.08 0.08 0.09 0.01 0.12 0.56 0.89 0.01 0.04 0.02 0.01 0.35 0.01

0.10 0.09 0.09 0.08 0.08 0.06 0.06 0.06 0.06 0.06 0.05 0.04 0.04 0.03 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.00 0.00

Description

Protein ID

88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110

GTP binding Elongation factor Tu family protein S-adenosylmethionine synthetase 1 GTP binding Elongation factor Tu family protein Lactate/malate dehydrogenase family protein Ubiquitin-specific protease 12 Tubulin beta chain 4 Tubulin beta chain 2 Tubulin beta 8 Tubulin beta-7 chain Tubulin beta-9 chain Beta-6 tubulin Tubulin beta-1 chain Tubulin beta-5 chain Glyceraldehyde-3-phosphate dehydrogenase C subunit 1 VHA-A-vacuolar ATP synthase subunit A Isocitrate dehydrogenase Eukaryotic initiation factor 4A-III Monodehydroascorbate reductase 1 Cytosolic NADP-dependent isocitrate dehydrogenase Methionine adenosyltransferase 3 Annexin 1 FASCICLIN-like arabinogalactan-protein 11 Glyceraldehyde-3-phosphate dehydrogenase C2

AT1G07920.1 AT1G02500.1 AT1G07930.2 AT5G43330.1 AT5G06600.1 AT5G44340.1 AT5G62690.1 AT5G23860.1 AT2G29550.1 AT4G20890.1 AT5G12250.1 AT1G75780.1 AT1G20010.1 AT3G04120.1 AT1G78900.1 AT1G54340.1 AT3G19760.1 AT3G52880.1 AT1G65930.1 AT2G36880.1 AT1G35720.1 AT5G03170.1 AT1G13440.1

b c d

Function d Protein Amino acid metabolism Protein Tricarboxylic acid Protein Cell Cell Cell Cell Cell Cell Cell Cell Glycolysis Transport Tricarboxylic acid Protein Redox Tricarboxylic acid Amino acid metabolism Cell Cell wall Glycolysis

Protein ID, according to Arabidopsis database. The numbers of 0, 150, and 300 indicate NaCl concentrations. Ratio, relative abundance from Inoculation-responsive protein in Sarigol root with varying salt treatments. Protein, protein synthesis/degradation; CHO, carbohydrates; DNA, chromatin structure; N, nitrogen; S, sulfur; C1, one carbon; RNA, RNA binding/regulation of transcription; and misc, miscellaneous.


0

No.

a

Inoculation

a


(Fig. 6). Amino acid metabolism and tricarboxylic acid cycle-related proteins were predominantly changed in response to bacterial inoculation of plants under salt stress. The proteins related to amino acid metabolism such as cysteine synthase A, alanine transaminase, aspartate aminotransferase, glutamate 5-kinase, glutamate-5-semialdehyde dehydrogenase, and arginase were highlighted in both saltsensitive and -tolerant cultivars without bacterial inoculation. Whereas glyceraldehyde 3-phosphate dehydrogenase, 2,3bisphosphoglycerate-dependent phosphoglycerate mutase, S-adenosylmethionine synthetase, and isocitrate dehydrogenase were highlighted in canola cultivars inoculated with bacteria under salt stress. The tricarboxylic acid cycle related proteins pyruvate dehydrogenase, dihydrolipoamide dehydrogenase, citrate synthase, aconitate hydratase, and ATP citrate lyase in both canola cultivar without bacterial inoculation while malate dehydrogenase in inoculated cultivars were highlighted.

3.6. Inoculation-responsive protein profiles of canola cultivars under varying salt treatments To understand the response of bacterial inoculation on canola in different salt treatments, differentially abundant proteins in inoculated and non-inoculated canola cultivars were studied in different salt treatments, individually. In Sarigol and Hyola308, the abundance of 86 and 49 proteins significantly changed in non-inoculated and inoculated plants under NaCl treatments (Supplemental Tables 9 and 10). Out of significant proteins in Sarigol and Hyola308, 11 proteins related to protein synthesis, glycolysis, and major carbohydrate metabolism commonly changed in both cultivars (Supplemental Table 11). Under control and 150 mM NaCl, glutathione S-transferase PHI 2 increased; while, sucrose synthase, pfkB-like carbohydrate kinase family protein, and GTP binding elongation factor Tu family protein decreased in the response of bacterial inoculation in Sarigol. However, in Hyola308, glutathione S-transferase PHI 2, sucrose synthase, and GTP binding Elongation factor Tu family protein increased under 150 and 300 mM NaCl in response to bacterial inoculation (Fig. 7).

3.7. Enzymatic activity analysis To understand the response of inoculated Sarigol and Hyola308 under NaCl treatments, aldehyde dehydrogenase and Sadenosylmethionine synthetase were selected for enzyme activity analysis. Aldehyde dehydrogenase activity significantly decreased in inoculated compared to non-inoculated Hyola308 under control and 150 mM NaCl treatments. Activity of this enzyme was decreased in inoculated Hyola308 compared to inoculated Sarigol under NaCl treatments; however, its activity was not changed significantly between inoculated and noninoculated Sarigol. The activity of S-adenosylmethionine synthetase was decreased in Hyola308 compared to Sarigol in inoculation and non-inoculation under NaCl treatments. Activity of S-adenosylmethionine synthetase was decreased in inoculated compared to non-inoculated Hyola308 under 150 and 300 mM NaCl (Fig. 8).

99

4. Discussion 4.1. Effect of bacterial inoculation on morphological and physiological changes of canola under salt stress Salt stress is reported to have harmful effects on the morphological, physiological, and biochemical traits of canola [36–38]. For example, salt stress reduces the total photosynthetically active leaf area, which affects overall carbon balance and consequently limits plant growth [39]. The reduction in leaf area under salt stress was related to less cell activity in the leaf elongation zone [40]. Under salt stress, the cellular concentrations of K+ and Ca2+ decrease in the roots, shoots, and leaves of canola, whereas those of Na+ and Cl− are increased [36]. Jafarzadeh and Aliasgharzad [41] reported that root length was increased in 2 deci Siemens per meter salt stress while progressively decreased with increasing the NaCl concentration up to 30 deci Siemens per meter. As both the root dry weight and length of the salt-tolerant cultivar Hyola308 were greater than those of salt-sensitive cultivars, specific changes in the roots of Hyola308 might confer tolerance to salt stress. Plant-growth promoting bacteria are associated with plant roots and augment plant productivity under salt and drought stresses [42]. The inoculation of canola roots with plant-growth promoting bacteria improved germination and plant growth under salt stress [15]. Pseudomonas is a nonpathogenic saprophyte which inhabits in soil and water and it can colonize on the surface of plant roots [43]. Based on these reports, canola root was used for morphological and proteomic analyses because P. fluorescens FY32 was directly interacted with roots. The present results clearly demonstrate that Hyola308 is a salt-tolerant cultivar with better growth compared to the salt-sensitive cultivar Sarigol. Notably, the suppression of root growth under salt treatment was partially alleviated by the inoculation of plants with P. fluorescens FY32. The present results are consistent with the findings of Jalili et al. [15], who demonstrated that the inoculation of canola roots with plant-growth promoting bacteria improved germination and seedling growth under salt stress. Ashraf and Rauf [25] reported that sodium and chloride increased while potassium decreased in plant tissues, progressively, with increasing the NaCl treatments. However, it was reported that potassium is decreased in plants under salt stress [44]. In fact, antagonism of sodium with potassium is one of the common phenomena occurring in plants subjected to saline conditions [45]. One possible mechanism of tolerant plants to prevent up-taking sodium under salt stress is assumed to be the activation of H+-ATPase in the membranes [46]. In the present study, sodium content of Hyola308 roots was less than that of Sarigol under salt stress. Sodium content decreased in Hyola308 roots in response to bacterial inoculation, suggesting that might be related to tolerant mechanism of Hyola308 under salt stress.

4.2. Salt-stress response on amino acid metabolism, glycolysis, and tricarboxylic acid cycle related proteins in bacterial inoculated canola under salt stress To determine the tolerance mechanisms underlying the beneficial effects of bacterial inoculation on canola cultivars under salt

100

Ratio c Non-inoculation No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Description Histone H2A 6 Histone H2A 7 Secretion-associated RAS super family 1 Aldolase superfamily protein UDP-glucose 6-dehydrogenase family protein S-adenosyl-L-homocysteine hydrolase Sucrose synthase 3 S-adenosyl-L-homocysteine (SAH) hydrolase 2 ATP-citrate lyase A-2 Heat shock protein 70 (Hsp 70) family protein Pyruvate kinase family protein Heat shock protein 70 Selenium-binding protein 2 Heat shock protein 70 (Hsp 70) family protein Heat shock protein 70 (Hsp 70) family protein Pyruvate kinase family protein Selenium-binding protein 1 ADP glucose pyrophosphorylase 1 Sucrose synthase 4 Glycosyl hydrolase family protein Heat shock protein 70B Isocitrate dehydrogenase GTP binding Elongation factor Tu family protein GTP binding Elongation factor Tu family protein Non-ATPase subunit 9 NADH-ubiquinone dehydrogenase-mitochondrial-putative

Inoculation

a

0

150

300

0

150

300 b

Function d

AT5G59870.1 AT5G27670.1 AT1G09180.1 AT3G52930.1 AT3G29360.1 AT4G13940.1 AT4G02280.1 AT3G23810.1 AT1G60810.1 AT5G02490.1 AT3G52990.1 AT3G12580.1 AT4G14040.1 AT3G09440.1 AT1G56410.1 AT2G36580.1 AT4G14030.1 AT5G48300.1 AT3G43190.1 AT5G10560.1 AT1G16030.1 AT1G54340.1 AT1G07920.1 AT1G07930.2 AT1G29150.1 AT5G37510.1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.31 0.09 1.53 1.09 0.57 0.59 0.15 0.60 0.29 1.07 1.18 1.11 0.83 1.02 1.10 1.23 1.25 2.39 0.20 1.35 1.04 0.72 0.38 0.38 1.17 0.93

1.97 1.87 1.55 1.02 0.35 0.72 0.29 0.72 0.44 1.16 1.18 1.03 0.21 1.13 1.23 1.21 1.25 2.34 0.34 1.30 1.14 0.81 0.06 0.04 1.10 0.84

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1.97 1.72 1.86 0.34 0.70 0.89 0.42 0.83 0.67 0.77 1.14 0.81 0.53 0.75 0.70 1.10 1.00 0.27 0.22 0.26 0.46 1.11 0.93 0.89 0.21 0.14

3.03 2.96 2.09 0.47 0.47 0.45 0.44 0.42 0.33 0.31 0.30 0.30 0.29 0.28 0.27 0.27 0.27 0.27 0.25 0.25 0.17 0.16 0.13 0.13 0.12 0.09

DNA DNA Signaling Glycolysis Cell wall Amino acid metabolism Major CHO metabolism Amino acid metabolism Tricarboxylic acid Protein Glycolysis Protein Metal handling Stress Protein Glycolysis Metal handling Major CHO metabolism Major CHO metabolism Cell wall Protein Tricarboxylic acid Protein Protein Protein Mitochondrial electron transport

Protein ID


Table 2 – Inoculation-responsive proteins identified in Hyola308 roots exposed to varying salt treatments.

a b c d

Serine hydroxymethyltransferase 4 Mitochondrial heat shock protein 70-1 NmrA-like negative transcriptional regulator family protein Cobalamin-independent synthase family protein Methionine synthase 2 S-adenosylmethionine synthetase 1 Lactate/malate dehydrogenase family protein Lactate/malate dehydrogenase family protein Sucrose synthase 1 Phosphoglyceratemutase-2-3-bisphosphoglycerate-independent Triosephosphate isomerase Heat shock protein 60 Annexin 1 Secretion-associated RAS 1B Glyceraldehyde-3-phosphate dehydrogenase C subunit 1 Secretion-associated RAS super family 2 ADP/ATP carrier 1 Ras-related small GTP-binding family protein Cytosolic NADP-dependent isocitrate dehydrogenase Methionine adenosyltransferase 3 VHA-A-vacuolar ATP synthase subunit A Glyceraldehyde-3-phosphate dehydrogenase C2

AT4G13930.1 AT4G37910.1 AT1G75280.1 AT5G17920.1 AT3G03780.1 AT1G02500.1 AT1G04410.1 AT5G43330.1 AT5G20830.1 AT1G09780.1 AT3G55440.1 AT3G23990.1 AT1G35720.1 AT1G56330.1 AT3G04120.1 AT4G02080.1 AT3G08580.1 AT3G62560.1 AT1G65930.1 AT2G36880.1 AT1G78900.1 AT1G13440.1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.67 1.08 0.50 0.64 0.64 0.61 1.06 1.20 0.18 1.25 1.05 1.30 0.68 1.51 1.30 1.47 1.23 1.52 0.72 0.62 1.07 1.29

0.69 0.94 0.50 0.58 0.57 0.36 1.09 1.16 0.32 1.05 0.01 1.20 0.76 1.53 1.17 1.52 1.16 1.55 0.80 0.49 0.37 1.18

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.23 1.25 0.05 0.38 0.53 0.29 0.07 0.05 0.07 0.05 0.01 0.03 0.01 0.01 0.07 0.01 0.05 0.01 0.02 0.01 0.09 0.01

0.08 0.07 0.07 0.05 0.05 0.05 0.04 0.04 0.03 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00

Protein ID, according to Arabidopsis database. The numbers of 0, 150, and 300 indicate NaCl concentrations. Ratio, relative abundance from Inoculation-responsive protein in Hyola308 root with varying salt treatments. DNA, chromatin structure; CHO, carbohydrates; TCA, tricarboxylic acid cycle; protein, protein synthesis/degradation; C1, one carbon; and cell, cell organization.

C1-metabolism Protein Secondary metabolism Amino acid metabolism Amino acid metabolism Amino acid metabolism Tricarboxylic acid Tricarboxylic acid Major CHO metabolism Glycolysis Glycolysis protein Cell Protein Glycolysis Signaling Transport Signaling Tricarboxylic acid Amino acid metabolism Transport Glycolysis


27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

101

102

Sarigol Ratio No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Protein ID

a

AT1G02500.1 AT1G04410.1 AT1G07920.1 AT1G07930.2 AT1G09180.1 AT1G09780.1 AT1G13440.1 AT1G15950.1 AT1G16030.1 AT1G23800.1 AT1G26570.1 AT1G29150.1 AT1G35720.1 AT1G49240.1 AT1G53310.1 AT1G54340.1 AT1G56330.1 AT1G56410.1 AT1G65930.1 AT1G75280.1 AT1G78900.1 AT2G36580.1 AT2G36880.1 AT3G03780.1 AT3G04120.1 AT3G09440.1 AT3G09840.1

Description S-adenosylmethionine synthetase 1 Lactate/malate dehydrogenase family protein GTP binding Elongation factor Tu family protein GTP binding Elongation factor Tu family protein Secretion-associated RAS super family 1 Phosphoglycerate mutase-2-3-bisphosphoglycerate-independent Glyceraldehyde-3-phosphate dehydrogenase C2 Cinnamoyl CoA reductase 1 Heat shock protein 70B Aldehyde dehydrogenase 2B7 UDP-glucose dehydrogenase 1 Non-ATPase subunit 9 Annexin 1 Actin 8 Phosphoenolpyruvate carboxylase 1 Isocitrate dehydrogenase Secretion-associated RAS 1B Heat shock protein 70 (Hsp 70) family protein Cytosolic NADP-dependent isocitrate dehydrogenase NmrA-like negative transcriptional regulator family protein VHA-A-vacuolar ATP synthase subunit A Pyruvate kinase family protein Methionine adenosyltransferase 3 Methionine synthase 2 Glyceraldehyde-3-phosphate dehydrogenase C subunit 1 Heat shock protein 70 (Hsp 70) family protein Cell division cycle 48

M.P. 10 11 10 9 3 12 12 4 5 2 6 9 10 12 24 6 6 10 15 3 25 12 18 19 10 15 23

b

Hyola308 d

Ratio

0

150

300

M.P.

0

150

300 c

Function e

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.05 0.07 0.13 0.12 1.67 0.48 0.01 0.96 0.23 0.75 0.05 0.12 0.01 0.54 0.34 0.56 1.43 0.45 0.04 0.07 0.12 0.97 0.02 0.11 0.01 0.44 0.56

0.09 0.10 0.10 0.09 1.95 0.54 0.00 1.40 0.45 1.18 0.15 0.41 0.00 0.52 0.52 0.02 1.81 0.65 0.01 0.17 0.02 0.14 0.01 0.38 0.03 0.69 0.59

10 13 10 9 3 10 14 7 5 4 5 9 11 13 26 7 6 10 16 3 25 11 18 20 12 16 23

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.29 0.07 0.93 0.89 1.86 0.05 0.01 0.24 0.46 0.71 0.69 0.21 0.01 0.90 1.32 1.11 0.01 0.70 0.02 0.05 0.09 1.10 0.01 0.53 0.07 0.75 0.95

0.05 0.04 0.13 0.13 2.09 0.01 0.00 0.85 0.17 0.59 0.55 0.12 0.01 0.70 1.32 0.16 0.01 0.27 0.01 0.07 0.00 0.27 0.00 0.05 0.01 0.28 0.58

Amino acid metabolism Tricarboxylic acid Protein Protein Signaling Glycolysis Glycolysis Secondary metabolism Protein Fermentation Cell wall Protein Cell Cell Glycolysis Tricarboxylic acid Protein Protein Tricarboxylic acid Secondary metabolism Transport Glycolysis Amino acid metabolism Amino acid metabolism Glycolysis Stress Cell


Table 3 – List of identified proteins from bacterial-inoculated Sarigol and Hyola308 roots exposed to different salt treatments.

a b c d e

AT3G12580.1 AT3G15730.1 AT3G29360.1 AT3G43190.1 AT3G47000.1 AT3G52930.1 AT3G52990.1 AT3G53230.1 AT3G55440.1 AT3G62560.1 AT4G02080.1 AT4G02280.1 AT4G13930.1 AT4G13940.1 AT4G14040.1 AT4G37910.1 AT5G02490.1 AT5G03340.1 AT5G15490.1 AT5G16570.1 AT5G17920.1 AT5G20830.1 AT5G20980.1 AT5G27670.1 AT5G39320.1 AT5G42080.1 AT5G43330.1 AT5G59870.1

Heat shock protein 70 Phospholipase D alpha 1 UDP-glucose 6-dehydrogenase family protein Sucrose synthase 4 Glycosyl hydrolase family protein Aldolase superfamily protein Pyruvate kinase family protein ATPase-AAA-type-CDC48 protein Triosephosphate isomerase Ras-related small GTP-binding family protein Secretion-associated RAS super family 2 Sucrose synthase 3 Serine hydroxymethyltransferase 4 S-adenosyl-L-homocysteine hydrolase Selenium-binding protein 2 Mitochondrial heat shock protein 70-1 Heat shock protein 70 (Hsp 70) family protein ATPase-AAA-type-CDC48 protein UDP-glucose 6-dehydrogenase family protein Glutamine synthetase 1;4 Cobalamin-independent synthase family protein Sucrose synthase 1 Methionine synthase 3 Histone H2A 7 UDP-glucose 6-dehydrogenase family protein Dynamin-like protein Lactate/malate dehydrogenase family protein Histone H2A 6

17 15 8 14 6 12 14 22 7 5 6 4 12 10 9 9 13 28 11 6 23 22 6 2 11 14 6 3

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.49 0.77 0.07 0.13 1.00 0.16 0.98 0.63 0.76 1.61 1.41 0.24 0.04 0.77 0.43 0.76 0.53 0.58 0.51 0.62 0.15 0.01 0.51 1.37 0.50 0.66 0.05 1.33

0.61 1.17 0.27 0.57 1.23 0.31 0.14 0.64 0.70 1.83 1.69 0.63 0.26 0.87 0.26 0.60 0.64 0.61 0.61 0.41 0.43 0.48 0.65 2.05 0.61 0.74 0.08 1.46

19 17 7 12 4 13 13 23 7 5 6 2 12 11 9 9 14 26 11 6 25 20 6 2 12 13 8 3

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.81 0.89 0.70 0.22 1.36 0.34 1.14 0.99 0.01 0.01 0.01 0.42 0.23 0.89 0.53 1.25 0.77 0.95 0.78 1.31 0.38 0.07 0.81 1.72 0.64 1.27 0.05 1.97

0.30 0.95 0.47 0.25 1.79 0.47 0.30 0.61 0.01 0.01 0.01 0.44 0.08 0.45 0.29 0.07 0.31 0.62 0.56 0.59 0.05 0.03 1.70 2.96 0.52 1.26 0.04 3.03

Protein ID, according to Arabidopsis database. M.P., matched peptides. The numbers of 0, 150, and 300 indicate NaCl concentrations. Ratio, relative abundance from inoculated Sarigol and Hyola308 root in different salt treatments. Protein, protein synthesis/degradation; cell, cell organization; CHO, carbohydrates; C1, one carbon; N, nitrogen; DNA, chromatin structure; and misc, miscellaneous.

Protein Lipid metabolism Cell wall Major CHO metabolism Cell wall Glycolysis Glycolysis Cell Glycolysis Signaling Signaling Major CHO metabolism C1-metabolism Amino acid metabolism Metal handling Protein Protein Cell Cell wall N-metabolism Amino acid metabolism Major CHO metabolism Amino acid metabolism DNA Cell wall Misc Tricarboxylic acid DNA


28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

103

104


Sarigol

Hyola308

0 150 300 0 150 300 mM

I

II

III

19 7 23 13 17 36 37 38 20 21 6 25 2 49 54 1 12 40 48 31 8 3 4 18 28 26 44 24 9 42 10 11 52 30 46 14 35 27 45 41 33 39 16 22 34 43 47 29 5 32 15 53 50 51 55

Fig. 5 – Cluster analysis of identified proteins in canola cultivars inoculated with bacteria under salt stress. Sarigol and Hyola308 used as sensitive and tolerant cultivars, respectively. One-week-old canola seedlings (Sarigol and Hyola308) were transferred to a hydroponic system and inoculated without or with bacteria, and were then treated without or with 150 and 300 mM NaCl. Proteomic analysis revealed that 390 proteins in Sarigol and 74 proteins in Hyola308 were commonly changed in non-inoculated and inoculated roots. A total of 55 commonly changed proteins in Sarigol and Hyola308 were examined by cluster analysis, which grouped the proteins into three clusters (I, II, and III). The numbers in the panel correspond to the proteins listed in Table 3.

stress, gel-free proteomic analysis of salt-sensitive and -tolerant canola cultivars was performed. Proteins related to amino acid metabolism were identified in the roots of the salt-sensitive cultivar Sarigol without and with bacterial inoculation. However, in salt-tolerant cultivar Hyola308, amino acid metabolism-related proteins were only identified in the roots of plants inoculated with bacteria (Fig. 3). The increased abundance of proteins related to amino acid metabolism in response to NaCl treatment was previously reported [47]. In the present analyses, the abundance of delta 1-pyrroline-5-carboxylate synthase (P5CS) in Hyola308 was less than that detected in Sarigol roots with and without bacterial inoculation (Supplemental Tables 3, 4, 5, and 6). In proline biosynthesis in plants, P5CS is the rate-limiting enzyme under stressful conditions. Elevated proline levels lead to reduced free radical accumulation in response to osmotic stress and increase plant growth under salt stress [48]. As proline is an important osmoprotectant in plants exposed to high soil salinity [49], an increased abundance of proline synthesis-related proteins in canola might play an important role in mediating tolerance to salt stress. Proteins related to amino acid metabolism and the tricarboxylic acid cycle significantly changed in canola cultivars inoculated with or without bacteria under salt stress conditions. Notably, S-adenosylmethionine synthetase and malate dehydrogenase displayed the same behavior in both salt-sensitive and -tolerant cultivars inoculated with bacteria (Fig. 5). Under stressful conditions, ethylene biosynthesis is rapidly accelerated, which adversely affects root growth and consequently, the growth and development of the entire plant [50]. Ethylene is derived from the amino acid methionine, which is converted to S-adenosylmethionine by S-adenosylmethionine synthetase [51]. Sánchez-Aguayo et al. [52] reported that S-adenosylmethionine synthetase is induced in tomato under salt stress. In the present study, the abundance of S-adenosylmethionine synthetase decreased in canola cultivars inoculated with bacteria (Table 3), suggesting that this enzyme might be involved in salt tolerance mechanisms in canola. Tricarboxylic acid cycle-related proteins were significantly changed in Hyola308 roots without bacterial inoculation. The tricarboxylic acid cycle is a key component of respiratory metabolism in both photosynthetic and heterotrophic plant organs. All of the major genes of the tomato tricarboxylic acid cycle have been recently cloned, allowing the generation of a suite of transgenic plants in which the majority of the enzymes in the pathway are progressively decreased [53]. In the present study, more tricarboxylic acid cycle-related proteins were increased in Hyola308 compared to Sarigol (Supplemental Tables 3 and 5). A reduction of tricarboxylic acid cycle-related metabolites under salt stress was reported [54,55]. The activation of energy metabolism is necessary to supply energy for the biosynthesis of stress-responsive proteins and osmolytes, as well as for active salt ion transport under salt stress conditions [56]. As an increased rate of several catabolic processes, including glycolysis and tricarboxylic acid cycle, has been reported in plants under salt stress, salt-tolerant plants biosynthesize energy-rich compounds more efficiently [57,58]. Proteins related to the biosynthesis of proline might be related to mechanisms of salt-tolerance in canola cultivars under salt stress.


105

106


Fig. 6 – Metabolic pathways of identified proteins in canola roots inoculated with bacteria under salt stress. Sarigol and Hyola308 used as sensitive and tolerant cultivars, respectively. One-week-old canola seedlings were inoculated without or with bacteria and treated without or with 150 and 300 mM NaCl. The positions of identified proteins in the pathways involved in amino acid metabolism (A) and tricarboxylic acid cycle (B) are highlighted using the KEGG database. Proteins in red and green were identified in non-inoculated and inoculated canola cultivars, respectively.

Tricarboxylic acid cycle-related enzymes are used to generate energy in all aerobic organisms. Malate dehydrogenases catalyze the NAD/NADH-dependent interconversion of

the substrates malate and oxaloacetate. This reaction plays a key part in the malate/aspartate shuttle across the mitochondrial membrane and in the tricarboxylic acid cycle within the

107


0.00

Sarigol

GST2

Hyola308

Sarigol

GTP

Hyola308

Sarigol

GTP

Hyola308

Sarigol

GTP

Hyola308

Sarigol

PFKB

SUS4

Hyola308

Sarigol Hyola308

Sarigol

SAMS3

Hyola308

Sarigol

SUS1

Hyola308

Sarigol

RGP2

Hyola308

Sarigol

FBA6

Hyola308

Sarigol

FBA8

Hyola308

Ratio of protein abundance 0.50 1.00 1 2

3

4

5

0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300

0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300 0 150 300

0 150 300 0 150 300

mM NaCl

Fig. 7 – Relative abundance of inoculation responsive proteins in canola roots under salt treatments. Sarigol and Hyola308 used as sensitive and tolerant cultivars, respectively. One-week-old canola seedlings were transferred to a hydroponic system and inoculated without or with bacteria, and were then treated without or with 150 and 300 mM NaCl. Ratio of protein abundance was calculated in inoculated compared to non-inoculated under varying salt treatments (Supplemental Table 11). Abbreviations: GST2, glutathione S-transferase PHI 2; GTP, GTP binding Elongation factor Tu family protein; PFKB, pfkB-like carbohydrate kinase family protein; SUS, sucrose synthase; SAMS3, S-adenosylmethionine synthetase family protein; RGP2, reversibly glycosylated polypeptide 2; and FBA, Aldolase superfamily protein.

108


Aldehyde dehydrogenase (nmol mg-1protein)

A

metabolites [62]. The conversion of glyceraldehyde-3phosphate to 1,3-bisphosphoglycerate by glyceraldehyde-3phosphate dehydrogenase is a central step in glycolysis that links the energy consuming and energy producing steps of the pathway [63]. The accumulation of glycolysis-related proteins in tolerant canola cultivars might play a role in acclimation to salt stress.

1.8 1.6 *

1.4

*

1.2 1 0.8 0.6 0.4 0.2 0 0

150

300

0

Sarigol

150

300

mM

Hyola308

S-adenosylmethionine synthetase (unit mg-1protein)

B 20 18 16 14 12 10 8

*

6

*

4 2 0 0

150 Sarigol

300

0

150

300

mM

Hyola308

Fig. 8 – Enzyme activities in canola roots inoculated with bacteria under salt stress. Sarigol and Hyola308 were used as sensitive and tolerant cultivars, respectively. One-week-old canola seedlings were transferred to a hydroponic system and inoculated without or with bacteria, and were then treated without or with 150 and 300 mM NaCl. The extracted proteins of non-inoculated and inoculated canola cultivars were assayed for aldehyde dehydrogenase (A) and S-adenosylmethionine (B) activities. Data are shown as means ± SD from three independent biological replications. Asterisks above the error bars indicate significant changes between inoculated and non-inoculated cultivars by the Student's t-test (*p < 0.05).

mitochondrial matrix [59]. Although the activity of malate dehydrogenases is increased in chickpea leaves under salt stress, this enzyme has reduced activity in response to salt treatment in nodules associated with plant symbiosis with plant-growth promoting bacteria [60]. In the present study, the abundance of malate dehydrogenase decreased in canola cultivars under salt stress, suggesting that this enzyme might be related to the improved tolerance of canola plants to salt stress in response to bacterial inoculation. Glycolysis-related proteins were mostly decreased in the roots of both salt-sensitive and -tolerant cultivars inoculated with bacteria (Table 3). Notably, the abundance of glyceraldehyde3-phosphate dehydrogenase was decreased in the salt-sensitive cultivar without bacterial inoculation under salt stress, but was increased in the salt-tolerant cultivar under the same conditions (Table 2). The abundance of glyceraldehyde-3-phosphate dehydrogenase was also found to be decreased in a saltsensitive barley cultivar under salt stress [61]. Glyceraldehyde3-phosphate dehydrogenase catalyzes a key reaction in the glycolysis pathway and plays an important role in the cellular production of reductants, energy, and carbohydrate

4.3. Effect of bacterial inoculation on identified cell division and energy metabolism related proteins in canola under salt stress Dynamin-like proteins are GTP-binding proteins [64] and are encoded by ADL1 genes in the Arabidopsis genome. ADL1Ap functions in vesicular trafficking and cytokinesis during several stages of plant development, including embryogenesis, seedling development, and reproduction [65], whereas ADL2a and ADL2b have been suggested to be involved in mitochondrial division [66]. The formation and rearrangement of the cytoskeleton, vesicle transportation, and secretion of cell wall compounds are important cellular processes for tip growth and hair elongation of roots [67]. The GTP-binding protein SAR1A (secretion-associated and RASrelated protein 1A) is a member of the ADP-ribosylation factor family of small GTPases, which are involved in vesicular trafficking from the endoplasmic reticulum to the Golgi apparatus [68]. Small GTPases are also reportedly involved in root hair initiation in Arabidopsis [69] and barley [70]. The increased abundance of proteins related to cell division in canola cultivars inoculated with bacteria might be an important response that leads to increased tolerance under salt stress conditions. Decreased abundance of histone H2A has been observed in soybean under flooding and drought stresses [71]. Histones, which are small, basic proteins involved in chromatin formation, can be modified by different post-translational modifications, such as acetylation/deacetylation, methylation/demethylation, and phosphorylation [72]. An evolutionarily conserved variant of histone H2A, called H2AX, is one of the key components of chromatin. H2AX and its variants are rapidly phosphorylated on chromatin surrounding double-stranded DNA breaks [73] and play a key role in the recruitment of DNA repair proteins [74]. In the present study, two DNA synthesis-related proteins were increased in canola roots inoculated with bacteria and exposed to salt stress, suggesting that H2A proteins might be related to the beneficial growth effects observed under salt stress in these plants. Taken together, these results suggest that inoculation with bacteria increases plant tolerance by increasing cell division in roots under salt stress.

4.4. Effect of bacterial inoculation on aldehyde dehydrogenase and S-adenosylmethionine synthetase in canola roots under salt stress Aldehyde dehydrogenases belong to a family of NAD(P)+dependent enzymes that catalyze the oxidation of various toxic aldehydes to carboxylic acids. Ishitani et al. [75] reported that aldehyde dehydrogenase activity and mRNA levels increased almost 8-fold and 2-fold, respectively, in leaves and roots of barley grown in salt stress. Many observations have indicated that osmotic stresses such as salt,


drought and water stress lead to the rise of endogenous ABA levels, and consequently to changes in gene expression [76]. S-adenosylmethionine is an important metabolite participating in many cellular processes. It is also an important effector in the regulation of the biosynthesis of threonine and methionine [77]. Time course study of the effect of salt stress on S-adenosylmethionine synthetase indicated the great up-regulation of its genes [78]. In the present study, aldehyde dehydrogenase and S-adenosylmethionine synthetase were decreased in Hyola308 compared to Sarigol in both inoculation and non-inoculation condition under salt stress. Furthermore, the activity of these enzymes was decreased in Hyola308 under salt stress. Decreased levels of these enzymes in Hyola308 and also in bacterial inoculation might be related to tolerance mechanism of this cultivar to overcome the harmful effect of salt stress.

5. Conclusion In this study, proteins affected by the inoculation of canola roots with bacteria under salt stress conditions were analyzed to better understand the mechanisms of stress tolerance and the cellular pathways influenced by plant-growth promoting bacteria. Based on morphological analysis, Sarigol and Hyola308 were designated as salt-sensitive and -tolerant cultivars, respectively. Sodium content of salt-tolerant cultivar was less than that of salt-sensitive under salt stress. The main findings of this study are as follows: (i) the abundance of glycolysis-related proteins decreased in inoculation with bacteria compared to non-inoculation conditions in both salt-sensitive and -tolerant cultivars; (ii) secretion-associated RAS super family 1, dynamin-like protein, and histones increased in response to inoculation with bacteria under salt stress in both salt-sensitive and -tolerant cultivars; (iii) S-adenosylmethionine synthetase and malate dehydrogenase decreased in salt-sensitive and -tolerant cultivars inoculated with bacteria compared to non-inoculation under salt stress; (iv) the proteins related to amino acid metabolism and tricarboxylic acid cycle were differentially abundant in bacterial-inoculated compared to non-inoculated saltsensitive cultivar; and (v) aldehyde dehydrogenase and S-adenosylmethionine synthetase were decreased in salttolerant compared to salt-sensitive cultivar and also decreased in inoculated salt-tolerant cultivar. Taken together, proteins related to amino acid metabolism and glycolysis were the most responsive proteins in bacterial inoculation of canola under salt stress suggesting that might be related to adaptation of inoculated canola under salt stress. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2015.04.009.

Accession code The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository [79] with the data set identifier PXD001580.

109

Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments The authors thank Dr. Y. Nanjo, Dr. K. Nishizawa, Mr. X. Yin, and Ms. M.W. Oh of the National Institute of Crop Science for their useful discussions. The authors are also thankful to Mr. P. Moti-Noparvar of University of Tabriz for his help in the sample preparation. This study was partially supported by a scholarship from University of Tabriz to F. Banaei-Asl.

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Transcriptome analysis of canola (Brassica napus) under salt stress at the germination stage.

Proteomic Analysis Reveals Proteins Involved in Seed Imbibition under Salt Stress in Rice.

Gel-free proteomic analysis of soybean root proteins affected by calcium under flooding stress.

Alterations in root proteome of salt-sensitive and tolerant barley lines under salt stress conditions.

Comparative Proteomic Analysis Reveals Differential Root Proteins in Medicago sativa and Medicago truncatula in Response to Salt Stress.

iTRAQ-Based Quantitative Proteomic Analysis of Cotton Roots and Leaves Reveals Pathways Associated with Salt Stress.

Physiological and proteomic analyses of Saccharum spp. grown under salt stress.

Leaf Physiological and Proteomic Analysis to Elucidate Silicon Induced Adaptive Response under Salt Stress in Rosa hybrida 'Rock Fire'.

The Arabidopsis root stele transporter NPF2.3 contributes to nitrate translocation to shoots under salt stress.

Non-specific phospholipase C5 and diacylglycerol promote lateral root development under mild salt stress in Arabidopsis.

Accelerated parathion degradation in soil inoculated with acclimated bacteria under field conditions.

Comparative proteomic analysis of early salt stress responsive proteins in roots and leaves of rice.

Proteomic Analysis of Soybean [Glycine max (L.) Merrill] Roots Inoculated with Bradyrhizobium japonicum Strain CPAC 15.

Study on salt tolerance with YHem1 transgenic canola (Brassica napus).

Comparative proteomic analysis of amaranth mesophyll and bundle sheath chloroplasts and their adaptation to salt stress.

Salt Stress Affects the Redox Status of Arabidopsis Root Meristems.

Genome duplication improves rice root resistance to salt stress.

Proteomic analysis of the salt-responsive leaf and root proteins in the anticancer plant Andrographis paniculata Nees.

Differential Proteomic Analysis Reveals the Effect of Calcium on Malus baccata Borkh. Leaves under Temperature Stress.

Physiological and proteomic analysis in chloroplasts of Solanum lycopersicum L. under silicon efficiency and salinity stress.

Proteomic analysis of Staphylococcus aureus biofilm cells grown under physiologically relevant fluid shear stress conditions.

Quantitative proteomic analysis of cell envelope preparations under iron starvation stress in Aeromonas hydrophila.

Proteomic analysis on roots of Oenothera glazioviana under copper-stress conditions.

Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis.