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. 2013 Oct 9;21(2):185–190. doi: 10.1016/j.sjbs.2013.09.011

Proteomic analysis of differentially expressed proteins induced by salicylic acid in suspension-cultured ginseng cells

Jiaman Sun a,b, Junfan Fu a,⁎, Rujun Zhou a
PMCID: PMC3942860  PMID: 24600313

Abstract

In this study, optimized 2-DE sample preparation methodologies were established for suspension-cultured ginseng cells. Three commonly used protein extraction methods (Trichloroacetic acid-acetone, urea/thiourea and phenol extraction method) were evaluated for proteomic analysis of suspension cultures of ginseng. A comparative analysis of suspension-cultured ginseng cells proteome induced by salicylic acid (SA) was reported. The results demonstrated that phenol extraction method was the best method based on protein extraction efficiency and the good quality of 2-DE patterns for suspension-cultured ginseng cells. Fifteen differentially expressed proteins induced by salicylic acid in suspension-cultured ginseng cells were identified by MALDI-TOF-MS. These identified proteins were involved in defense and stress response, energy metabolism, signal transduction/transcription, protein synthesis and metabolism, and photosynthesis. Chaperonin 60, related to defense responses, was more abundant in suspension-cultured ginseng cells after application of SA. Vacuolar ATPase subunit B was newly induced in SA treatment.

Keywords: Ginseng cells, Salicylic acid, Proteome

1. Introduction

Ginseng (Panax ginseng C.A. Meyer) is widely cultivated as a medicinal herb in northeast China (Wang, 2001). The ginseng dried roots have been widely used as a traditional medicine since ancient times because of its stimulative and tonic properties (Ali et al., 2006). Plant cell culture is an important plant biotechnology tool for the growth of plant material. With increasing demand for ginseng worldwide, plant cell culture is necessary for the growth of ginseng (Ali et al., 2007). As is known, salicylic acid (SA) is one of the key components activating resistance pathways (Vlot et al., 2009). Exogenous application of SA could not only induce antioxidant enzyme activities and formation of pathogenesis-related proteins, but also stimulates the expression of defense genes in many plants (Hwang et al., 1997; Lu and Chen, 2005; Wen et al., 2005; Fernandes et al., 2006; Thulke and Conrath, 1998; Murphy et al., 2000; Rajjou et al., 2006). Many studies focused on the effect of SA on enhancing plants resistance. However, little research paid attention to the proteome of suspension-cultured ginseng cells induced by SA.

In this study, we established the suspension culture system of ginseng and made a comparison among three protein extraction methods to find out the best method of protein extraction for 2-DE analysis in suspension-cultured ginseng cells. Moreover, the differentially expressed proteins induced by SA in suspension-cultured ginseng cells were analyzed and identified by MALDI-TOF-MS.

2. Materials and methods

2.1. Chemicals

IPG gel strips, IPG buffer, urea, thiourea, CHAPS, iodoacetamide, DTT, acrylamide, and TBP were obtained from Bio-Rad (USA). Salicylic acid and other chemicals were obtained from Sigma (St. Louis, MO, USA).

2.2. Preparation of suspension-cultured ginseng cells and SA treatment

Two-year-old fresh ginseng roots were washed clean and then sterilized in 75% ethanol for 2 min followed by 7 min in 0.1% HgCl2. The surface-sterilized root segments were placed on solid Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing a full complement of salts and vitamins (Rahman and Punja, 2005). Kinetin (KT) (0.2 mg/L) and 2,4-dichlorophenoxyacetic acid (2,4-D) (3 mg/L) were used as the growth regulators. Cultures were maintained at 25 ± 2 °C for 4 weeks for callus induction (Ali et al., 2006). Suspension cultures were initiated as described by Punja et al. (2004). The actively proliferating suspension cultures were treated with 1 mM SA and harvested for 48 h after treatment. Suspension-cultured ginseng cells treated with deionized water were used as the control. These treated samples were frozen in liquid nitrogen and stored at −80 °C until protein extraction.

2.3. Protein extraction

To find out an ideal method of protein extraction for 2-DE analysis in suspension-cultured ginseng cells, three protein extraction methods (trichloroacetic acid (TCA)-acetone, urea/thiourea, and phenol extraction method) were investigated.

2.3.1. TCA-acetone precipitation

TCA-acetone precipitation was performed according to the protocol described by Kim et al. (2003). The suspension-cultured ginseng cells were ground to a fine powder with a pestle in liquid nitrogen. The powder was incubated in a sample buffer I (0.3% SDS, 50 mM Tris–HCl pH 8.0, 200 mM DTT) at 100 °C for 10 min, and then transferred onto ice and incubated with 0.1 volume of sample buffer II (RNase A 0.25 mg/mL, DNase I 1 mg/mL, 50 mM Tris–HCl pH 8.0, 50 mM MgCl2) for 10 min. After centrifugation at 13,000g for 30 min, the supernatant was precipitated with acetone and incubated overnight at −20 °C. After centrifugation (13,000g, 30 min), the pellet was washed three times with ice-cold 80% (v/v) acetone and lyophilized.

2.3.2. Urea/thiourea method

Total protein extraction was performed according to the protocol described by Rabilloud (1998) with minor modification. Briefly, the suspension-cultured cells were ground to a fine powder and homogenized with extraction buffer (8 M urea, 2 M thiourea, 2 mM disodium EDTA salt, 4% (w/v) CHAPS, 65 mM DDT, 2% (v/v) ampholyte (pH 3–10), and 1% TBP) by vortexing for 3 h at 30 °C, followed by centrifugation (13,000g) for 30 min. The supernatant was precipitated with ice-cold acetone and incubated overnight at −20 °C. The precipitated proteins were washed with cold 80% (v/v) acetone three times and lyophilized.

2.3.3. Phenol extraction method

The method was manipulated from the protocol described by Carpentier et al. (2005) with some modifications. The suspension-cultured ginseng cells were ground to a fine powder and extracted with extraction buffer (0.7 M sucrose, 0.5 M Tris–HCl, pH 8.0, 10 mM disodium EDTA salt, 4 mM ascorbic acid, 2% (v/v) 2-ME, and 1% isopropanol) on ice, incubated at 4 °C for 30 min. The homogenate was vortexed with ice-cold Tris-buffered phenol (pH 8.0), and the upper phenol phase was mixed with ice-cold 0.1 M ammonium acetate in methanol, vortexed briefly and incubated at −20 °C overnight before centrifuging at 13,000g for 30 min. The pellet was washed with cold 80% (v/v) acetone, lyophilized and stored at −80 °C until use.

2.3.4. 2-Dimensional electrophoresis analysis

The protein concentration was determined according to Bradford’s method (1976) after resuspended in a solubilization buffer (9 M urea, 2 M thiourea, 2% (w/v) CHAPS, 50 mM DTT, 1% tributylphosphine (TBP) and 2% (v/v) Bio-Lyte 3/10 Bio-Rad ampholytes). IEF was carried out using 17 cm pH 5–8 linear IPG strips in a PROTEAN IEF Cell (Bio-Rad). The loading sample volume used was 350 μl of protein extract, corresponding to a protein amount of 0.8 mg per strip. Focusing was carried out in 4 steps (250 V for 3 h, 500 V for 3 h, 1000 V for 1 h, and 10,000 V for 9 h). SDS–PAGE was performed using 12% polyacrylamide gels and runs at 5 mA/gel for 30 min and then at 60 mA/gel for 6 h in a PROTEIN II electrophoresis kit (Bio-Rad). The experiment was performed three times.

Coomassie brilliant blue (CBB) R-350 (Sigma, USA) was used to stain the 2-DE gels after electrophoresis. Images of the 2-DE gels were scanned using an Image Scanner (PowerLook 2100XL, UMAX). The image analysis of the gels was performed using PDQuest™ 2-D Analysis Software 8.0 (Bio-Rad). Only spots with an abundance ratio of at least two were considered as the differentially expressed proteins. The selected protein spots were subjected to identification by MALDI-TOF-MS.

2.4. In-gel digestion and protein identification

Protein spots were excised from the CBB-stained gels, washed with 100 mM NH4HCO3/30% CAN solution, and then dried in a vacuum concentrator. The proteins were digested in-gel overnight at 37 °C with a digestion buffer of 5 ng μl−1 Trypsin (Promega, USA) in 100 mM NH4HCO3 per sample. Gel pieces were extracted by 0.1% (v/v) TFA in 60% (v/v) ACN three times. The collected solutions were concentrated by a centrifugal vacuum concentrator. A 4800 Plus MALDI TOF/TOF™ Analyzer (Applied Biosystems, USA) was used for MALDI-TOF-MS analysis. The accelerated voltage in the ion source was operated at 20 kV and minimum signal to noise ratio of 50 was picked out for MS/MS. The peptide masses were queried against protein database in NCBInr using MASCOT program (Matrix Science, UK).

3. Results

3.1. Measurement of the results of three protein extraction methods

Three commonly used protocols were evaluated for protein extraction. Comparison was done based on protein yield and protein purity. As shown in Fig. 1, the yield of protein was: 8.53 ± 0.51 (TCA-acetone), 11.83 ± 2.01 (urea/thiourea), and 16.68 ± 1.63 (phenol extraction) mg of proteins g−1 suspension cultures fresh weight. The protein from phenol extraction method had higher purity (50.03 ± 4.30%) with relatively fewer impurities compared to other two extraction methods.

Figure 1.

Figure 1

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Effects of different protein extraction methods on protein yield and protein purity.

3.2. Evaluation of three protein extraction methods according to 2-DE

The quantity and quality of the protein extracted by the three methods were investigated using 2-DE analysis. The representative 2-DE gels from each method are shown in Fig. 2. The gel of sample extracted by phenol extraction method (Fig. 3C) had higher-resolved spots and less horizontal streaking than those from TCA-acetone precipitation or urea/thiourea method (Fig. 3A and B). In addition, the average number of protein spots (Fig. 2) observed in 2-D gel using phenol extraction method (690 ± 14) was higher than that of urea/thiourea method (461 ± 27) and TCA-acetone precipitation (106 ± 9). The 2-D patterns generated by TCA-acetone precipitation displayed few protein spots and much streaking, which was in accordance with its low yield and purity. These results indicated that phenol extraction method produced better spots resolution and minimal streaking and is more suitable for obtaining the quantity and quality of suspension-cultured ginseng cell proteins needed for proteomic analysis.

Figure 2.

Figure 2

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Effects of different protein extraction methods on the number of protein spots.

Figure 3.

Figure 3

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2-DE gels of protein extracts obtained using TCA-acetone (A), urea/thiourea methods (B) and phenol extraction (C) from suspension-cultured ginseng cells. Equal amounts (0.8 mg) of proteins were separated on the 17 cm, pH 5–8 linear gradient IPG strips. SDS–PAGE was performed with 12% gels.

3.3. Proteins were induced by SA in suspension-cultured ginseng cells

A comparative analysis of the proteome of 1 mM SA treated with suspension-cultured ginseng cells was performed to elucidate the proteomic response of SA treatment on suspension cultures of ginseng. Proteins were extracted from suspension cultures by phenol extraction method, separated by 2-DE, stained with CBB, and analyzed by using PDQuest™ 2-D Analysis Software. The representative 2-D patterns are shown in Fig. 4 and the position of differentially expressed protein spots was marked. Image analysis revealed about 700 protein spots in the gels across pI and molecular mass range of 5–8 and 14.4–94 kDa (Fig. 4). Spots appeared in identical positions on different gels were considered to be the same protein. The abundances of 23 protein spots in suspension-cultured ginseng cells were differentially expressed after SA treatment. Fifteen of these protein spots were identified by MALDI-TOF-MS analysis followed by MASCOT database searches of the NCBI protein database (Table 1). Among these identified protein spots, spot 0502 was newly induced, 12 proteins were up-regulated (spots 5207, 4802, 5701, 1608, 8608, 1409, 9901, 1302, 6604, 8709, 0307 and 3403) and 2 proteins were down-regulated (spots 3213 and 3407) in suspension-cultured ginseng cells directly treated with SA, in comparison to the control. Function annotation of all the identified proteins was grouped under defense and stress response (1 protein), energy metabolism (4 proteins), signal transduction/transcription (3 proteins), protein synthesis and metabolism (4 proteins), photosynthesis (1 protein) and others (2 proteins).

Figure 4.

Figure 4

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Protein profiles in 2-DE gels obtained for the suspension-cultured ginseng cells. (A) Control, (B) suspension cultures treated with 1 mM SA solution. Samples were analyzed by employing immobilized pH 5–8 linear gradient IPG strips, and 12% polyacrylamide SDS gels electrophoresis. The numbered protein spots identified by mass spectrometry are shown in Table 1.

Table 1.

Identification of differentially expressed proteins from suspension-cultured ginseng cells induced by salicylic acid.

Spot No.a Protein name Accession No. Mr. (kDa)
 pI
 Protein Scoreb
Theoretic Experimental Theoretic Experimental
Defense and stress response
↑1608 Chaperonin cpn60 gi|357442729 61.5 49.5 6.27 5.51 154



Energy metabolism
↑4802 Methionine synthase gi|8439545 84.9 79.9 5.93 6.33 361
↑5701 Methionine synthase gi|8439545 84.89 65.4 5.93 6.57 540
↑5207 Malate dehydrogenase gi|225461618 37.1 22.4 8.76 6.73 447
↑8608 Pyrophosphate-dependent phosphofructokinase alpha subunit gi|3790102 68.13 55 6.71 7.85 260



Signal transduction/transcription
↑1409 Predicted: eukaryotic initiation factor 4A-11 gi|225429488 47.1 35.9 5.38 5.44 578
↑9901 TUDOR-SN protein 1 gi|343172567 23.32 23.1 9.23 7.81 234
NI 0502 Vacuolar ATPase subunit B gi|118429132 54.2 43.7 4.93 4.56 145



Protein synthesis and metabolism
↑6604 Branched-chain-amino-acid aminotransferase-like protein 2-like gi|356533600 70.9 69.7 6.96 6.93 134
↑1302 Predicted: adenosine kinase 2 gi|225449018 38.2 25.5 5.31 5.36 267
↓3407 Predicted: elongation factor Tu, chloroplastic-like gi|356513781 52.6 33.4 6.33 6.17 398
↓3213 Esterase D, putative gi|255565327 28.7 27.8 5.33 6.13 93



Photosynthesis
↑3403 Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gi|33323298 45.4 34.2 8.18 6.04 127



Others
↑8709 Predicted protein gi|224071575 65.8 57.5 6.17 7.84 97
↑0307 No results
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↑, spot up-regulated in SA-treated samples; ↓, spot down-regulated in SA-treated samples, NI, newly induced.

a

Spot numbers are given in Fig. 4.

b

MASCOT protein score from the MALDI-TOF-MS analysis.

4. Discussion

In the phenol extraction method, nucleic acids, cell debris and carbohydrates could be dissolved preferentially by the aqueous phase. The method is an effective procedure to remove interfering compounds. Carpentier et al. (2005) revealed that the phenol extraction method was useful for eliminating interfering compounds from banana and another latex-containing plant. In our study, phenol extraction method produced better spots resolution and minimal streaking, and it is more suitable for obtaining the quality and quantity of suspension-cultured ginseng cell proteins. Thus, phenol extraction method is an ideal method for protein extraction of suspension-cultured ginseng cells.

The abundance of defense and stress related protein chaperonin cpn60 (spot 1608) was increased by SA in suspension-cultured ginseng cells. Chaperonin 60 (Cpn60), also known as heat-shock protein 60 (Hsp60), is involved in protein folding and cell survival in stress conditions (Timperio et al., 2008; Jackson-Constan and Keegstra, 2001). It plays important roles in the resistance response. Chaperonin 60 alpha chain precursors were also detected in increased amount on SA-treated Vigna mungo plants, which were susceptible to Mungbean Yellow Mosaic India Virus (Kundu et al., 2011). Some researches indicate that mitochondrial chaperonin cpn60 was detected in the main root and culture hairy root of ginseng (Kim et al., 2003, 2004). In this study, increased abundance of Chaperonin 60 in SA treated suspension-cultured ginseng cells may have significant implications on triggering defense mechanism against stress.

Four identified protein spots corresponded to energy and metabolisms pathway. The SA treatment increased the abundance of methionine synthase (spots 4802 and 5701), malate dehydrogenase (spot 5207), and pyrophosphate-dependent phosphofructokinase alpha subunit (spot 8608) in suspension-cultured ginseng cells. These proteins are directly involved in glycolysis and tricarborylic acid cycle. Protein spots 5701 and 4802 were identified as the same protein, methionine synthase, which catalyzes the final reaction of the methionine biosynthetic pathway.

Proteins involved in signal transduction/transcription, such as eukaryotic initiation factor 4A-11 (spot 1409) and TUDOR-SN protein 1 (spot 9901), were also up-regulated in suspension-cultured ginseng cells after SA treatment. Vacuolar ATPase subunit B (spot 0502) was newly induced in SA treatment. Adenosine kinase 2 (spot 1302) and branched-chain amino acid aminotransferase-like protein 2-like (spot 6604) were more abundant among the protein synthesis and metabolism related proteins. Elongation factor Tu plays a specific role in protein synthesis. We identified a protein spot (spot 3407) corresponding to a decrease in EF-Tu in response to SA treatment. This was not consistent when the expression of EF-Tu was increased by various stimuli (Cho et al., 2007). So, further study is needed to explain the precise role of EF-Tu in response to SA. Spot 0307 could not be identified by MALDI-TOF-MS, probably due to the unavailability of the specific protein sequences in public databases.

In summary, phenol extraction method was proved to be the best method for protein extraction of suspension-cultured ginseng cells. Fifteen differentially expressed proteins induced by salicylic acid in suspension-cultured ginseng cells were identified. They were involved in defense and stress response, energy metabolism, signal transduction/transcription, protein synthesis and metabolism, and photosynthesis.

Acknowledgments

We thank Professor Hai-Yan Fan (Shenyang Agricultural University, China) for her skillful technical advice and Guang-Chao Yu for help with the 2-DE analysis.

Footnotes

Peer review under responsibility of King Saud University.

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