QlU You-wen, FENG Zhe, FU Ming-ming, YUAN Xiao-han, LUO Chao-chao, YU Yan-bo, FENG Yan-zhong, WEl Qi, Ll Feng-lan

1 College of Life Science, Northeast Agricultural University, Harbin 150030, P.R.China

2 Heilongjiang Institute of Education, Harbin 150030, P.R.China

3 Heilongjiang Academy of Agricultural Sciences, Harbin 150086, P.R.China

Abstract Salt stress is one of the major factors affecting plant growth and yield in soybean under saline soil condition. Despite many studies on salinity tolerance of soybean during the past few decades, the detailed signaling pathways and the signaling molecules for salinity tolerance regulation have not been clari fied. In this study, a proteomic technology based on two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins responsible for salinity tolerance in soybean plant. Real-time quantitative PCR (qRT-PCR) and Western blotting (WB) were used to verify the results of 2-DE/MS. Based on the results of 2-DE and MS, we selected glucosyltransferase (GsGT4), 4-coumarate,coenzyme A ligase (Gs4CL1), mitogen-activated protein kinase 4 (GsMAPK4), dehydration responsive element binding protein (GsDREB1), and soybean cold-regulated gene (GsSRC1) in the salinity tolerant soybean variety, and GsMAPK4 for subsequent research. We transformed soybean plants with mitogen-activated-protein kinase 4 (GsMAPK4) and screened the resulting transgenics soybean plants using PCR and WB, which con firmed the expression of GsMAPK4 in transgenic soybean. GsMAPK4-overexpressed transgenic plants showed significantly increased tolerance to salt stress, suggesting that GsMAPK4 played a pivotal role in salinity tolerance. Our research will provide new insights for better understanding the salinity tolerance regulation at molecular level.

Keywords: soybean, salinity tolerance, two-dimensional gel electrophoresis, GsMAPK4

1. lntroduction

Soybean is a globally important food and oil crop. However,the productivity of soybean is severely limited by soil salinity(Manavalanet al. 2009; Weberet al. 2014). The signaling pathways and signaling molecules involved in salinity tolerance regulation in soybean have been studied for the past few decades (Yanget al. 2010, 2012). Usually, plants regulate the expression of the tolerance genesviaregulation of signal transduction pathways (Munns 2005; Ashraf 2009;Teakle and Tyerman 2010; Guanet al. 2014; Zhouet al.2014). Protein kinases play important roles in these signal transduction pathways and may include mitogen-activated protein kinase (MAPK), which regulates plant tolerance to drought, salinity and high or low temperature. Other protein kinases such as the ribosomal protein kinase (RPK)regulates specific protein synthesis, and the transcription regulation protein kinase (TRPK) regulates the expression of genes involved in plant growth, cell cycle, and the maintenance of normal chromosome structure. Calcium/calmodulin-dependent protein kinases (CDPK) are involved in the phosphorylation of transcription factors, which in turn control gene expression (Hrabaket al. 2003; Ludwiget al.2004; Colcombe and Hirt 2008; Munns and Tester 2008).However, the signaling pathways and the signaling molecules of salinity tolerance regulation in soybean remain unknown.

MAPKs are a group of serine/threonine protein kinases that are resistant to a variety of extracellular stimuli, such as cytokines, hormones, and environmental stress, and MAPK pathway is an important signaling pathway in eukaryotes.MAPK activity is controlled by the sequential activation of three protein kinases, by which a MAPK kinase kinase(MAPKKK) activates a MAPK kinase (MAPKK) that in turn activates a MAPK by phosphorylation of conserved threonine and tyrosine residues (Bergmannet al. 2004).In plants, the MAPK pathway is one of the most important signal cascade pathways that regulates plant growth,development, and tolerance during adverse conditions(Bogreet al. 2000; Agrawalet al. 2002; Ahlforset al. 2004).MAPKs are shown to regulate the environmental stresses in corn, rice, and potato (Blancoet al. 2006; Alexandrovet al. 2009; Dinget al. 2009; Vleesschauweret al. 2010).

Two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MS) is an useful method for identifying proteins in different biological samples (Stasyket al. 2005).The 2-DE/MS technique directly screens for protein pro files(proteome) in a sample. The 2-DE together with matrixassisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used to compare the protein pro files of salinity tolerant and sensitive varieties of a crop. The 2-DE/MS can be used for screening and identification of proteomic pro files in a variety of plants (Iraret al. 2014; Lauet al. 2015; Jinet al. 2016). And this technique has also been widely used to study protein pro files in soybean varieties (Chenget al. 2010; Maet al. 2014; Yuet al. 2017).

In this study, we used 2-DE to pro file the total proteins from the leaves of a salinity tolerant variety (SAT)(cv. 50109) and a salinity sensitive variety (NSAT)(cv. 85–140) of soybean (Glycine sojaSieb. and Zucc.)to identify the differentially expressed proteins in the two varieties. Our objective was to identify novel proteins involved in salinity tolerance regulation and understand the regulation mechanisms of salinity tolerance in soybean.

2. Materials and methods

2.1. Extraction of total proteins

The total proteins were extracted by the method of chloroacetic acid-acetone precipitation from leaves of a SAT(cv. 50109) and NSAT (cv. 85–140) of soybean (Glycine sojaSieb. and Zucc.) (Damervalet al. 1986). The leaves were ground in liquid nitrogen. Then, the extract buffer(acetone supplemented with 10% trichloroacetic acid and 0.07% 2-hydroxy-1-ethanethiol) was added (the volume ratio of extract buffer and the sample is 3:1) and incubated overnight at -20°C. The sample was centrifuged at 12 000 r min–1for 1 h at 4°C, and the supernatant was discarded.The cold acetone supplemented with 0.07% 2-hydroxy-1-ethanethiol was added (the volume ratio of extract buffer and the sample is 1:1), shaking blended, and then centrifuged at 12 000 r min–1for 1 h at 4°C. The supernatant was discarded and the precipitate was vacuum dried. Before the 2-DE was performed, the extracted proteins were dissolved in lysis buffer (2.7 g of urea, 0.2 g of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), and 0.5 g of DL-dithiothreitol (DTT)) with the sterilization deionized water added to the final volume of 5 mL. The concentration of total proteins for 2-DE performance was 3–10 μg L-1.

2.2. Analyses with 2-DE and MALDl-TOF/TOF MS

Proteins were precipitated with the 2D Clean-Up Kit(Amersham Biosciences/GE Healthcare) and redissolved.Samples were loaded on 18-cm strips with an immobilized pH gradient of 3 to10 and separated on an IPGPhor Unit (GE Healthcare, Uppsala, Sweden) with the following settings:200 V for 2 h, 500 V for 1.5 h, 1 000 V for 1 h, and 8 000 V for 6 h. After isoelectric focusing electrophoresis, the strips were equilibrated twice in buffer (6 mol L–1urea, 30%glycerol, 2% sodium dodecyl sulfate (SDS), 50 mmol L–1Tris-HCl buffer, pH 6.8, 0.01% bromophenol blue), first time with 2% DTT for 15 min, and then with 2.5% iodoacetamidefor 15 min. Electrophoresis was performed with a 1-mm thick,12.5% SDS-polyacrylamide gel (Huanget al. 2012). The 2-DE gels were scanned with Powerlook 2100XL (Umax,Dallas, TX) according to a previous report by Luet al. (2013).The protein spots of interest were cut from the 2-DE gel and extracted by trypsin digestion (Promega, Madison, WI)as previously described (Yanet al. 2005), and the tryptic peptide samples were sent to Shanghai Applied Protein Technology Co., Ltd., for MALDI-TOF/TOF MS peptide mass fingerprint. The method of MALDI-TOF/TOF MS was according to the previous report (Maet al. 2011; Huanget al. 2012). In brief, the proteins were digested at 37°C for 20 h with trypsin and then freeze-dried. The tandem MALDITOF/TOF MS was conducted using a 4800 MALDI-TOF/TOF MS (Bruker, United States). MS spectra were acquired using 1 200 laser shots per spot in the positive ion re flector mode over the full-scan spectrum. The six most abundant precursor ions were selected for MS/MS scans. The resulting peak lists were submitted for database sequence searches using MASCOT v2.1.03 software. Proteins with probability-based molecular weight search (MOWSE) scores(Pvalue≤0.05) were considered to be positively identi fied.The acquired MS/MS spectra were compared against the International Protein Index protein sequence database using the Turbo SEQUEST Program in the Bioworks Browser 3.3 Software Suite (Thermo, USA). Subcellular classifications were performed using gene ontology annotation according to the accession numbers of proteins in UniProt. Peptide mass fingerprint was submitted to MASCOT Sequence Query server for identification to European Bioinformatics Institute (EBI) Database.

2.3. Quantitative real-time PCR

Total RNA was extracted, and the quantitative real-time PCR (qRT-PCR) analysis was performed according to a previous report by Huang Y Let al. (2013).Actinwas used as a reference gene in this study. Primer information is shown in Table 1. The qRT-PCR reaction was performed at 95°C for 10 s, followed by 40 cycles at 95°C for 5 s,60°C for 31 s by using the two-step RT-PCR. RT-PCR analysis was performed by the 2–ΔΔCT method (Livakand Schmittgen 2001).

2.4. Vector construction

The construction of expression vector was performed using the standard techniques reported by Luoet al. (2013). The total RNA of salinity tolerant soybean was extracted, and the cDNA was synthesized by M-MLV reverse transcriptase(Promega, USA). The open reading frame (ORF) ofGsMAPK4(～1 150 bp) was ampli fied from the cDNA clone.The specific primers of theGsMAPK4were as follows:F: 5´-GCTCTAGAGAAGATGGGTAGCAAAGGCAAAC-3´(theXbaI site is underlined); and R: 5´-CCCAAGCTTC AAGAAAGAATTACTGACTGGGTGG-3´ (theHindIII site is underlined). The overexpression vector used for plant specific expression was a pCAMBIA-1302 vector(GenePharma, Shanghai, China) and the recombinant plasmids were transferred toAgrobacterium tumefaciensEHA105 strain for transformation.

2.5. Plant transformation

Soybean seeds (fromcv. 85–140 lines, as wild type, WT)were treated with 5% sodium hypochlorite solution for 10 min and then washed three times with sterilized ddH2O.The sterilized seeds were germinated and then grown in half-strength solid MS medium (pH 5.8) in a greenhouse with a photoperiod of 16-h/8-h (light/dark) at (26±2)°C.Then,GsMAPK4gene transfection was completed byAgrobacterium-mediated transformation of embryogenic callus derived from mature embryos as described by Drosteet al. (2002). First, the cloned MAPK4 vector was transformed intoEscherichiacoliby means of thermal shock, and the recombinant plasmid was namedpCAMBIAMAPK4, which was imported into agrobacterium strainEHA105. The target gene was transferred into soybean byAgrobacterium-mediated method. The pCAMBIA 1302 vector containes ahygromycin phosphotransferase(hptII) gene encoding hygromycin tolerance for selection.Selection of hygromycin resistant embryogenic tissues,embryo histodifferentiation, and conversion into plants and acclimation were carried out using a previously described method (Weberet al. 2007). Homozygous transgenic lines in the T4generation were identi fied. Soybean plants transformed with the empty vector (pCAMBIA 1302) were also used in this study. All soybean plants were grown at(26±2)°C under a photoperiod of 18-h/6-h light/dark.

2.6. Protein expression analysis

The qRT-RCR analysis was performed using the method mentioned as above and Western blotting (WB) was performed according to Huang Zet al. (2013). The specific antibodies used in the text wereMAPK4(Agrisera,SWEDEN, AS12 2107, 1:1 000) andActin(Agrisera, AS13 2640, 1:2 500).

Table 1 Primer sequences for qRT-PCR analysis

2.7. Salinity tolerance of GsMAPK4-overexpressed soybean plants

Seed germinationSoybean seeds from transgenic T4plants and WT plants were sterilized and cultured in halfstrength solid (half-Murashige and Skoog (h-MS) medium)(pH 5.8) or high saline soil (half-strength solid MS medium supplemented with 200 mmol L–1NaCl, pH 9.0, human serum albumin (HSA) medium), and the germination rate was recorded every day for one week. The experiment was divided into four groups: 1) WT seeds on h-MS medium,2) transgenic T4seeds on h-MS medium, 3) WT seeds on HSA medium, and 4) transgenic T4plants on HSA medium.Seedling stageSoybean seeds from transgenic T4and WT plants were sterilized and sown on half-strength solid MS medium for two weeks, and then the seedlings were transferred to a fresh medium (in the absence and presence of 200 mmol L–1NaCl, respectively) for vertical growth for 12 days. Then, plant growth, chlorophyll contents, and fresh biomass were recorded. The groups of this experiment were the same as above.

Mature stage analysisSeeds from transgenic T4and WT plants were sterilized and cultivated in half-strength solid MS medium for four weeks, and then the plants were divided into four groups: 1) transgenic T4seeds irrigated with 200 mmol L–1NaCl, 2) WT seeds irrigated with 200 mmol L–1NaCl, 3) transgenic T4seeds without irrigation of 200 mmol L–1NaCl, and 4) WT seeds without irrigation of 200 mmol L–1NaCl. The irrigation was made once every three days for 15 days, and plant growth, chlorophyll content, and fresh biomass were recorded and photographed.

2.8. Statistical analysis

Results were reported as mean±SE. Data statistics and individual differences among groups were analyzed usingt-test by Sigma Plot 9.0. The differences were considered significant if the value ofP＜0.05 and highly significant ifP＜0.01.

3. Results

3.1. ldentification of proteins by 2-DE and MALDl-TOF/TOFMS

Glycine sojacv. 50109 is a salinity tolerant variety with salt tolerance grade 3; Tolerance soy (cv. 85-140) is not salttolerant. The 2-DE was performed to find the differences in proteins between Tolerance soy (cv. 85-140) (control group)andGlycine sojacv. 50109 (SAT group) (Fig. 1-A).

Fig. 1 Comparison of phosphoprotein patterns between the salinity-sensitive soybean varieties (control group) and the salinitytolerant soybean varieties (salinity tolerant (SAT) group) by two-dimensional gel electrophoresis (2-DE). A, silver-stained 2-DE gels loaded with extracts of the control group and SAT group. Spots that were differentially expressed were annotated. B, zoom-in detail of the regions including the selected differentially expressed spots. 1–5, protein spots.

Through image analysis, we selected five protein spots (these protein spots exhibited ≥1.5-fold differences in abundance) of interest and identi fied them by MALDITOF/TOF MS (Fig. 1-B and Table 2). Glucosyltransferase(GsGT4), 4-coumarate: coenzyme A ligase (Gs4CL1),mitogen-activated protein kinase 4 (GsMAPK4), dehydration responsive element binding protein (GsDREB1), and soybean cold-regulated gene (GsSRC1) were identi fied by MALDI-TOF/TOF-MS.

3.2. Expression of selected proteins in the salinity tolerant soybean variety analyzed by qRT-PCR

The gene expression of proteins was analyzed by qRT-PCR.The mRNA expression of all tested five proteins was upregulated in the SAT group (Fig. 2-A). In addition,GsMAPK4expression was up-regulated in salinity tolerant soybean variety according to the WB analysis (Fig. 2-B–C,P＜0.01),and this result was consistent with the result of 2-DE.

3.3. Construction of expression vectors

The CDS area of theGsMAPK4gene was ampli fied. A～1 150 bp segment was detected, and its sequence was the same as theGsMAPK4gene (Fig. 3-A). The main structure of the pCAMBIA-MAPK4 recombinant plasmid is shown in Fig. 3-B.

3.4. Expression of GsMAPK4

The qRT-PCR was used to investigate mRNA expression ofGsMAPK4,GsGT4,Gs4CL1,GsDREB1, andGsSRC1.GsMAPK4was up-regulated in plants of theGsMAPK4transgenic group as compared with WT group (Fig. 4-A,P＜0.01). WB showed thatGsMAPK4was up-regulated inGsMAPK4transgenic group as compared with WT group (Fig. 4-B-C,P＜0.01). These results indicated that theGsMAPK4was ef ficiently transcribed and expressed successfully in soybean plants.

Table 2 Identification of the differentially expressed spots by mass spectrometry (MS)1)

Fig. 2 Expressions of five selected proteins in salinity-sensitive soybean varieties (control group) and salinity-tolerant soybean varieties (SAT group). A, comparative expression of genes as determined by RT-PCR. The ratio of the control group was regard as 1. B, Western blotting results of MAPK4. C, results of grayscale scan of MAPK4. Values are mean±SE (n=3 per group, biological replicates). ＊＊ indicates significant difference from values obtained in the control group at P＜0.01 level.

Fig. 3 The construction of MAPK4 expression vectors. A, result of cloning of MAPK4 gene. M, DNA marker. 1, MAPK4 gene.The expected size of DNA segment was about 1 150 bp. B, the main structure of the pCAMBIA-MAPK4 recombinant plasmid. LB,left border; TNOS, NOS terminator; PNOS, NOS promoter; P35S, 35S promoter; RB, right border.

Fig. 4 The expression of MAPK4, GT4, 4CL1, GmDREB1, and SRC1 in MAPK4 transgenic group (MAPK4T group). A, comparative expressions of MAPK4, GT4, 4CL1, GmDREB1, and SRC1 were determined by RT-PCR. The ratio of the control group was regard as 1. B, Western blotting results of MAPK4. C, results of gray scale scan of MAPK4. Values are mean±SE (n=3 per group,biological replicates). ＊＊ indicates significant difference from values obtained in the control group at P＜0.01 level.

3.5. Salt-alkali tolerance in GsMAPK4-transformed soybean plants

Seeds from transgenic T4and WT plants were sown in h-MS medium or HSA medium to test the effects ofGsMAPK4overexpression on responses to salinity stresses at the seed germination stage. The germination rate of the seeds of theGsMAPK4-overexpressing lines was similar to that of WT when cultivated in h-MS medium, but was higher than that of WT when cultivated in HSA medium (Fig. 5-A). The effect ofGsMAPK4overexpression on salinity stresses was assessed at the seedling stage by growing both T4and WT plants in h-MS or HSA medium. Although plant growth of both T4and WT seedlings was the same in h-MS medium,GsMAPK4overexpression lines showed better plant growth compared with WT in HSA medium (Fig. 5-B). Four-weekold plants fromGsMAPK4overexpression lines also showed improved plant growth in 200 mmol L–1NaCl (grown for 15 days) as compared with WT (Fig. 5-C).

4. Discussion

4.1. ldentification of proteins by 2-DE and MALDl-TOF/TOFMS

Fig. 5 NaCl tolerance of transgenic T4 soybean varieties at the seed germination stage. A, comparison in germination rate between the soybean seeds from transgenic T4 plants and wild-type (WT) plants cultivated in half-Murashige and Skoog(h-MS) medium or human serum albumin (HSA) medium. B,comparison in growth of seedlings between the seedlings from transgenic T4 plants and nontransgenic plants cultivated in h-MS or HSA medium. C, comparison of NaCl tolerance between transgenic plants and WT plants after treated with and without 200 mmol L–1 NaCl. h-MS medium, half-strength solid MS medium, pH 5.8; HAS medium, high saline soil, half-strength solid MS medium supplemented with 200 mmol L–1 NaCl pH 9.0.

The 2-DE method is one of the most important proteomic analysis (Rabilloudet al. 2010). At present, more than 10 000 protein spots have been detected by high-resolution 2-DE, while regular 2-DE helps identify 1 000–3 000 protein spots (Menget al. 2011; Huet al. 2013; Wanget al. 2015).The 2-DE and subsequent mass spectrometry-based identification can be applied to differentially abundant stress-responsive proteins among the analyzed samples.Moreover, 2-DE is apparently suitable for the detection of changes at the protein isoform level. At present, 2-DE/MS is widely used in the identification of plant proteomics in wheats (Iraret al. 2010; Kacemet al. 2016),Panax ginseng(Maet al. 2016), and maize (Wanget al. 2016). In this study, we used 2-DE to screen the potential regulator of salinity tolerance in soybean plants. Our results identi fied five significantly up-regulated proteins includingGsGT4,Gs4CL1,GsMAPK4,GsDREB1, andGsSRC1.

4.2. The regulation of salinity tolerance in soybean plants

Soybean plants have evolved three distinct mechanisms to combat salinity including osmotic adjustment to maintain leaf turgor, salt exclusion from leaf blades, and compartmentalization of ions at cellular or intracellular sites.However, the regulation of salinity tolerance in soybean plants is a very complicated process and involves many signaling molecules and pathways. At present, studies show that ABA-dependent signaling pathways and ABA-independent signaling pathways are the main signaling pathways involved in salinity tolerance regulation in soybean plants (Chaeet al. 2007; Dinget al. 2009; Fujitaet al. 2011).The five identi fied proteins in our study have previously been implicated in response to stress, protein synthesis, and transcriptional regulation (Chenet al. 2009; Saballoset al.2012; Rastogiet al. 2013; Liet al. 2014; Yuanet al. 2014)suggesting that these proteins are involved in the regulation of salinity tolerance in soybean plants.

4.3. GsMAPK4 is an important regulator for salinity tolerance in soybean

MAPKs are a group of serine/threonine protein kinases that play important roles in plant tolerance to adversity(Championet al. 2004; Castellset al. 2006; Lampardet al. 2009; Bartelset al. 2010), such as abiotic stresses inZea mays(Dinget al. 2009), wounding stresses in forage and turf (Dombrowskiet al. 2011) and low temperature inRheum australe(Ghawanaet al. 2010). As reported,the overexpression ofMAPKgene,ZmSIMK1, increases tolerance of maize to salt stress (Guet al. 2010).GsMAPK4is involved in theGsMAPK4pathway, an important kinasesignaling pathway in soybean plants. Previous studies have also demonstrated that kinases play a major role in salinity tolerance (Jianget al. 2010; Yanet al. 2012; Jiet al. 2013).In the present study,GsMAPK4overexpression in soybean plants enhanced their ability to tolerate salinity stresses. We tested the expression of four other proteins found in 2-DE and the result showed that the expression level of these four proteins was not significantly different inGsMAPK4transgenic soybean from that in the wild type.GsMAPK4is an important positive regulatory factor involved in salinity tolerance regulation in soybean plants.

5. Conclusion

Our current study con firmed thatGsMAPK4transgenic soybean had a good ability to tolerate salinity stresses.As a positive regulator,GsMAPK4played a pivotal role in salinity tolerance in soybean. In addition, this research will provide new insights for understanding the salinity tolerance regulation at molecular level.

Acknowledgements

This research was supported by the Science and Technology Research Project of Department of Education of Heilongjiang Province, China (12541047).