(Panax ginseng) and Indian Ginseng (Withania ... - World Scientific

The American Journal of Chinese Medicine, Vol. 40, No. 1, 203–218 © 2012 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X12500164

Comparative Root Protein Profiles of Korean Ginseng (Panax ginseng) and Indian Ginseng (Withania somnifera) Arulkumar Nagappan,* Nithya Karunanithi,† Sundareswaran Sentrayaperumal,‡ Kwang-II Park,* Hyeon-Soo Park,* Do Hoon Lee,* Sang-Rim Kang,* Jin-A Kim,§ Kalaiselvi Senthil,† Senthil Natesan,¶ Raveendran Muthurajan¶ and Gon Sup Kim* *Research

Institute of Life Science and College of Veterinary Medicine Gyeongsang National University Jinju, Gyeongnam 660-701, Korea

†

Department of Biochemistry, Biotechnology and Bioinformatics Avinashilingam University for Women Coimbatore 641043, Tamil Nadu, India ‡

Seed Centre

¶

Department of Plant Molecular Biology and Biotechnology Centre for Plant Molecular Biology Tamil Nadu Agricultural University Coimbatore 641003, Tamil Nadu, India §Korea

National Animal Research Resource Center Gazwa, Jinju, 660-701, Republic of Korea

Abstract: Ginsenosides and withanolides are the secondary metabolites from Panax ginseng and Withania somnifera, respectively. These compounds have similar biological properties. Two-dimensional electrophoresis (2-DE) analysis was utilized to reveal the protein profile in the roots of both plants, with the aim of clarifying similarly- and differentially-expressed proteins. Total proteins of Korea ginseng (P. ginseng) and Indian ginseng (W. somnifera) roots were separated by 2-DE using a pH 4–7 immobilized pH gradient strip in the first dimension and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. The protein spots were visualized by silver staining. Twenty-one P. ginseng proteins and 35 W. somnifera proteins were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry; of these, functions were

Correspondence to: Dr. Gon Sup Kim, Research Institute of Life Science and College of Veterinary Medicine, Gyeongsang National University, 900 Gajwadong, Jinju, Gyeongnam 660-701, Korea. Tel: (þ82) 55-772-2346, Fax: (þ82) 55-772-2349, E-mail: [email protected]

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A. NAGAPPAN et al. ascribed to 14 and 22 of the P. ginseng and W. somnifera proteins, respectively. Functions mainly included general cell metabolism, defense and secondary metabolism. ATPase and alcohol dehydrogenase proteins were expressed in both plants. The results of this study, to our knowledge, are the first to provide a reference 2-DE map for the W. somnifera root proteome, and will aid in the understanding of the expression and functions of proteins in the roots of Korean ginseng and Indian ginseng. Keywords: Comparative Proteomics; Panax ginseng; Withania somnifera; Solanaceae; Two Dimensional Electrophoresis.

Introduction Korean Ginseng (Panax ginseng C.A. Meyer) is a slow-growing perennial plant with fleshy roots that belongs to the family Araliaceae (Attele et al., 1999; Huang, 1999). It has been used in Korean and Chinese traditional medicine systems for centuries (Myung et al., 2005). P. ginseng extract is comprised mainly of ginsenosides whose various medicinal benefits include decreasing cholesterol level, balancing metabolism, increasing energy and stimulating the immune system (Jung et al., 2003; Yoshikawa et al., 1998; Cho, 2010). However, biochemical and genetic investigations of ginseng proteins have been limited (Kim et al., 2001). The most abundant proteins of ginseng root (28, 26, 21 and 20 kDa) display differing isoelectric points (pIs); of these, the 28 kDa species has been identified as the ginseng major protein (GMP), although its complete sequence and function are unclear (Lum et al., 2002; Yoon et al., 2002). Withania somnifera (L.) Dunal is an important herb in the Ayurvedic and indigenous medical systems in India. It is also known as Ashwagandha, Indian ginseng, and winter cherry, and belongs to the family Solanaceae (Andallu and Radhika, 2000). The primary chemical constituents of Ashwagandha are alkaloids (isopelletierine, anaferine), steroidal lactones (withanolides, withaferins) and the compounds known as withanolides, which are responsible for the various medicinal properties including activation of the immune, muscular and neuronal systems (RajaSankar et al., 2009; Ven Murthy et al., 2010). Steroidal lactones are typically C28 ergostane-type steroids with a 22, 26-lactone that possesses a 1-oxo-group (withaferin A) (Ali et al., 1997). Withanolides are similar to ginsenosides in structure and function (Grandhi et al., 1994). Both P. ginseng and W. somnifera are touted for their longevity-enhancing and sexual-stimulation properties. Withaferin-A has been reported having anticancer properties and ginsenoside Rb 1 has been reported to release nitric oxide and decrease intracellular-free calcium in cardiac myocytes (Dasgupta et al., 2008). Recent study has demonstrated that an aqueous extract of W. somnifera root that contains numerous withanolide derivatives was neuroprotective against H2O2- and Ab-induced cytotoxicity (Kumar et al., 2010). Different ginseng species have similar pharmacological effects. In previous studies, high-performance liquid chromatography (HPLC) has been routinely used to separate different ginsenosides (Fuzzati et al., 1999; Lee and Der Marderosian, 1981; Li et al., 2000) and withanolides (Furmanova et al.,

COMPARATIVE PROTEIN PROFILES OF PANAX GINSENG

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2001; Vitali et al., 1996; Ganzera et al., 2003). Amplification of polymorphic DNA has also been reported (Tochika-Komatsu et al., 2001). However, these methods cannot distinguish the presence of different parts of ginseng in a mixture and genetic information concerning P. ginseng remains quite limited. Only about 62 W. somnifera proteins have been reported. Similarly, limited information is available concerning expressed sequence tags (ESTs) from W. somnifera leaves and roots (Kalaiselvi et al., 2010). No studies have yet been performed on the W. somnifera genome, but the genomes of the model plants Arabidopsis thaliana (Asamizu et al., 2000) and rice (Oriza sativa) (Yamamoto and Sasaki, 1997) have been completely sequenced and could be helpful. Adaptogens are harmless herbs and reported to have pharmaceutical benefits and antioxidant potentials (Chen et al., 2008). Proteomics technologies have been used to identify new bioactive components, screen the target molecules of the traditional Chinese medicine (TCM) actions and elucidate the underlying mechanisms of their effects (Cho, 2007a and 2007b; Park et al., 2011). The present study was prompted by our interest in assessing whether different plant components with similar activities could be characterized using a proteomic approach. Recent technical improvements in the proteomic techniques of two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionizationtime of flight tandem mass spectrometry (MALDI-TOF MS/MS) have made it possible to identify thousands of proteins simultaneously and investigate protein expression, proteinprotein interaction and post-translational modification of proteins (Cho, 2007a and 2007b). The present study utilized these techniques to investigate the protein profile of P. ginseng and W. somnifera roots. Materials and Methods Plant Materials and Chemicals Root samples of two different species Korean ginseng (P. ginseng) and Indian ginseng (W. somnifera) were used for this study (Fig. 1). A fresh root sample of Korean ginseng (six years old) was obtained from the Animal Bio-resources Bank (Jinju, Korea) and a fresh root sample of Indian ginseng (four years old) was obtained from the Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Deemed University for Women, Coimbatore, Tamil Nadu, India. The roots of both species were frozen in liquid nitrogen (N2), ground thoroughly with a chilled mortar and pestle in liquid N2 to obtain a fine powder and stored at −80 C until protein extraction. All the chemicals used in this study were purchased from AMRESCO (Solon, OH, USA) and Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used were of analytical grade. Sample Preparation for 2-DE One hundred milligrams of finely ground powdered roots of each species were homogenized with 0.5 ml of extraction buffer containing 7 M urea, 2 M thiourea and

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(A)

(B)

Figure 1. Diagram of the roots of P. ginseng (A) and W. somnifera (B).

4% (w/v) CHAPS. The samples were centrifuged at 22, 500 g for 1 h at 4 C and the supernatant was collected. One hundred microliters of the supernatant was used for protein precipitation by treatment with 10% (v/v) TCA for 1 h and centrifuged at 22, 500 g for 10 min at 4 C. After centrifugation, the pellets were washed twice with ice-cold acetone (100% acetone followed by 90% acetone) in 20 mM dithiothreitol (DTT). The pellets were dried in lyophilizer and pellets were dissolved in 0.5 ml of sample buffer containing 7 M urea, 2 M thiourea and 4% (w/v) CHAPS, 1% DTT and 0.5% immobilized pH gradient (IPG) buffer (pH 4–7; GE Healthcare, Milan, Italy). After complete solubilization of protein pellets, the samples were centrifuged at 22, 500 g for 30 min at 4 C; the supernatant was collected and stored at 80 C until use. The protein concentration was estimated by the Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) according to manufacturer instructions. 2-DE Separation and Protein Visualization 2D-polyacrylamide gel electrophoresis (PAGE) was performed according to the IPG principles and methods of Amersham Biosciences (Piscataway, NJ, USA) with some modifications. In the first dimension, 13-cm IPG strips (pH 4–7; Amersham Biosciences, Uppsala, Sweden) were rehydrated overnight with 250 l of IPG rehydration buffer at room temperature. One hundred and fifty micagrams of total protein were loaded onto each IPG strip and isoelectric focusing (IEF) was conducted at 20 C with a Pharmacia Multiphor II separation unit (Amersham Biosciences). The running conditions were 50 V for 1 h, followed by 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 8000 V for 1 h, 8000 V for 8 h and 50 V for 2 h. The focused strips were equilibrated twice for 15 min each time, first


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in 10 mg/ml DTT and then in 40 mg/ml iodoacetamide prepared in equilibration buffer containing 50 mM Tris–HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, and 2% (w/v) sodium dodecyl sulfate (SDS). Proteins were separated in the second dimension according to their molecular weight using 12% SDS-PAGE. The first dimension strips were attached to the second dimension gel with a 0.5% low melting point agarose solution (Amersham Biosciences). Gels were run at a constant 20 mA until the bromophenol dye front reached the end of the gel. The protein spots in analytical gels were visualized by silver staining (Amershan Biosciences). Proteins of each sample were separated in triplicate. Protein Digestion and MALDI-TOF MS Analysis Only protein spots that were abundantly present were selected for further analysis. Each selected spot was excised manually from the silver-stained gels and digested in-gel with trypsin according to the procedure of Shevchenko et al. (1996) with slight modifications. MALDI-TOF MS/MS experiments were performed using a Voyager-DE STR mass spectrometer (Applied Biosystems, Framingham, MA, USA). For identification of proteins, the peptide mass fingerprinting data were used to search for viridiplantae in MSDB databases using the Mascot program (http://www.matrixscience.com). The protein score was 10*log (P), where P is the probability that the observed match is a random event. Results Protein Extraction and 2-DE Protein Profile of in-vivo Root Tissues of P. ginseng and W. somnifera The 2-DE protein profiles of W. somnifera and P. ginseng root tissues were analyzed using pH 4–7 IPG strips and proteins were detected by silver staining. When 2-DE maps of these two ginseng species were compared, no common protein spots were found (Figs. 2A and 2B). Separated proteins were subjected to MALDI-TOF MS/MS analysis and then peptide mass fingerprinting, and the collected mass spectral data were analyzed by MSDB database searches. MALDI-TOF results of P. ginseng and W. somnifera root tissues revealed significant database matches for 14 of the 21 selected protein spots (Table 1) and 22 of 35 selected protein spots (Table 2), respectively. Some of the identified proteins were present more than once (spots p12 and p15 in P. ginseng; spots w31 and w33, and w2, w20 and w22 in W. somnifera). The duplicate results may have reflected post-translational protein modifications including or proteolytic degradation or the existence of different genes encoding the same protein (Jorge et al., 2005). Protein Identification from P. ginseng Root Tissue The 2-DE map of P. ginseng revealed abundant proteins originating between 5 and 318 kDa. Three-to-five matched peptides were detected with maximum sequence coverage of 82%. Spots p9 and p12 showed a good correlation between experimental and theoretical

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(A)

(B) Figure 2. Silver-stained 2-DE PAGE gels of Korean ginseng root proteins (A) and Indian ginseng root proteins (B). Proteins were separated with an IPG strip, pH 4–7, and 12% SDS-PAGE. Gel images were acquired with an Epson scanner.

molecular mass and pI. Spots p3, p4, p6, p13, p15, p19 and p20 showed good correlations between experimental and theoretical pI. The majority of the identified proteins were involved in general cell metabolism. Only minimum numbers of proteins were involved in defense mechanism and secondary metabolism. Of the 14 significantly identified proteins,

Mitochondrial NADH: ubiquinone oxidoreductase 13 kDa subunit Actin (Fragment) Chrysanthemum morifolium NB-ARC domain containing protein — Oryza sativa (japonica cultivar — group) Auxin-responsive protein IAA13 or indoleacetic acid-induced protein 13 Arabidopsis thaliana genomic DNA, chromosome, 5, P1 clone: MVP7 (At5g64690) — Arabidopsis thaliana (Mouse-ear cress) Ribonuclease-like storage protein or Root 28 kDa major protein (Panax ginseng (Korean ginseng)) Kinesin motor domain containing protein, expressed — Oryza sativa (japonica cultivar — group) Ribonuclease-like storage protein or Root 28 kDa major protein (Panax ginseng (Korean ginseng)) Trigger factor-like — Oryza sativa (japonica cultivar — group) Chloroplast EF-Tu (Fragment) — Pyramimonas disomata

Isoflavone synthase 1 (fragment) — Lens culinaris (Lentil)

NBS-LRR-like protein — Oryza sativa (japonica cultivar — group) Similarity to AAA-type ATPase (Hypothetical protein At3g28510) — Arabidopsis thaliana (Mouse-ear cress) Alcohol dehydrogenase (Fragment) — Hordeum spontaneum (Barley)

*p — Panax ginseng.

p20

p4 p6

Defense Mechanism

p16

Secondary Metabolism

p18 p19

p15

p13

p12

p10

p9

p1 p3 p5

General Cell Metabolism

Spot No. Identified Protein

9FPF5

Q93X69 Q9LH84

Q9M6D1

Q5Z4M6 O64424

P83618

Q2QMU6

P83618

Q9FGG4

Q10D34

Q6QIV9 Q5DW26 Q2QZR9

Accession Number (Swissprot)

AF326704.1

AY043283.1 AY099692.1

AF195804.1

AP006054.3 AB008010.1

AY496964.1

DP000011.25

AY496964.1

BT005986.1

AC096855.5

AY538677.1 AB205087.1 DP000010.2

EMBL ID

4

3 3

4

4 4

5

5

4

3

3

4 5 4

50

39 48

55

57 55

78

55

54

36

40

58 58 56

Matched Score Peptides

Table 1. List of Proteins Identified in P. ginseng by MALDI-TOF MS/MS

33.9/7

117.9/39 61.74/32

56.96/13

60.29/7 39.23/7

27.67/13

318.82/15

27.67/21.5

38.18/22.5

23.91/22.5

16.16/52 26.21/42 126.01/32.5

5.56/5.1

5.83/5.72 6.66/6.2

9.04/6.07

5.21/4.5 4.63/4.6

5.87/5.83

5/5.6

5.87/5.7

4.46/5.7

5.4/5.5

9.59/4.9 4.9/5.3 6.36/5.95

17

4 9

11

11 12

24

2

20

11

22

39 31 2

pI Sequence Molecular Mass (kDa) Theoret/Exp Coverage (%) Theoret/Exp

COMPARATIVE PROTEIN PROFILES OF PANAX GINSENG 209

6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (Zea mays) Putative retroelement (Oryza sativa)

Lysyl-tRNA synthetase (Oryza sativa) 60S ribosomal protein L9 (Gibberellin-regulated protein GA) (Pisum sativum) Ribosomal protein small subunit 4 (Dicranoweisia crispula)

Probable U3 small nucleolar RNA — associated protein 11 (Oryza sativa subsp. japonica) U6 snRNA-associated Sm-like protein (Arabidopsis thaliana) Probable U3 small nucleolar RNA — associated protein 11 Probable U3 small nucleolar RNA — associated protein 11 Maturase K (Intron maturase) (Mandragora officinarum)

Proline-rich protein-2 (Gossypium hirsutum) Calcium ATPase (Zea mays)

w6 w15

w1

Soluble inorganic pyrophosphatase 1, chloroplastic (Pyrophosphate phospho-hydrolase 1) (Arabidopsis thaliana) Major allergen Cor a 1 — Corylus avellana (European hazel) Alcohol dehydrogenase (Hordeum spontaneum)

Defense Mechanism

w11 w5

Cell Differentiation

w18 w20 w22 w25

w2

RNA Processing

w34

w7 w8

Protein Synthesis

w24 w9

General Cell Metabolism


Q39454 Q9FPF5

Q9LXC9

Q94G50 Q9LM01

Q8LCS5 Q8S1Z1 Q8S1Z1 Q70D04

Q8S1Z1

Q7YK70

Q10HI8 P30707

O24559 Q94I24


Z72440.1 AF326704.1

AY551439.1

AF277674.1 AF096871.1

AY086430.1 AP003260.5 AP003260.5 AJ585883.1

AP003260.5

AJ554014.1

DP000009.2 X65155.1

AF007582.1 AC022352.5

EMBL ID

3 3

4

3 4

3 4 5 4

5

6

5 4

5 4

32 32

34

32 32

41 41 53 37

50

56

51 42

63 51

17.7/52 33.9/35

33.6/57

35.5/38 113.5/53

9.9/21.5 27.5/20.8 27.5/20.8 60.6/17.5

27.5/57

21.7/14.5

68.1/52 22.1/50.5

43.2/20.5 131.3/42

6.10/6.55 5.56/5.4

5.71/5.8

9.68/6.4 7.00/6.4

7.82/4.9 10.03/5.1 10.03/5.4 9.64/5.5

10.03/5.9

10.36/5.6

5.9/6.7 9.21/7

6.15/6.1 8.12/5.9

31 13

16

17 3

30 24 24 10

29

44

8 32

15 3

pI Sequence Matched Score Molecular Peptides Mass (kDa) Theoret/Exp Coverage (%) Theoret/Exp

Table 2. List of Proteins Identified in Withania somnifera Roots by MALDI-TOF MS/MS

210 A. NAGAPPAN et al.

Cinnamyl-alcohol dehydrogenase (Medicago sativa) ATPase 1, plasma membrane-type (Proton pump 1) (Arabidopsis thaliana) ATPase 1, plasma membrane-type (Proton pump 1) (Arabidopsis thaliana)

12-oxophytodienoate reductase 1 (Solanum lycopersicum) F-box family protein, putative, expressed (Oryza sativa) 12-oxophytodienoate reductase 1, LeOPR1 12-oxophytodienoate reductase 1

*w — Withania somnifera.

w3 w16 w19 w21

Secondary Metabolism

w33

w28 w31


Q9XG54 Q8LNJ4 Q9XG54 Q9XG54

P20649

Q53X16 P20649


M24107.1

L46856.1 M24107.1

EMBL ID

AJ242551.1 AC079029.11 AJ242551.1 AJ242551.1

Table 2. (Continued)

5 5 4 5

3

5 3

42 52 52 44

35

49 37

42.8/57 35.09/26.2 42.8/21.2 42.8/20.8

61.5/14.5

39.5/16 104.6/12.3

5.86/6.05 9.09/6.8 5.86/4.95 5.86/5.25

6.30/4.9

7.15/6.85 6.25/5.3

13 16 13 13

4

14 4

pI Sequence Matched Score Molecular Peptides Mass (kDa) Theoret/Exp Coverage (%) Theoret/Exp

COMPARATIVE PROTEIN PROFILES OF PANAX GINSENG 211

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ten proteins had functions attributable to general cell metabolism, one protein functioned enzymatically in secondary metabolism and three other proteins had defense mechanisms. Ribonuclease-like storage protein had isoforms with different pI values on 2-DE (spots p12 and p15) (Table 1). Spots p4, p6 and p20 represented NBS-LRR-like protein, AAA-type ATPase and alcohol dehydrogenase, respectively; all were involved in defense. Spot p16 was identified as isoflavone synthase I, which is involved in secondary metabolism. Protein Identification from W. somnifera Root Tissue Analysis of the W. somnifera 2-DE map revealed that all the abundant proteins were 9.9– 113 kDa in molecular mass. Three-to-six peptides were matched, with a maximum of 63% sequence coverage. Six spots (w1, w3, w6, w15, w19, w24) showed a good correlation between experimental and theoretical pI. Only one spot (w11) correlated well in its experimental and theoretical molecular mass. W. somnifera root proteins that were functionally identified by peptide mass fingerprinting were categorized according to their biological functions, which included general cell metabolism, defense-related proteins and secondary metabolite production (Table 2). Among the 22 proteins for which a function was determined, 12 proteins were involved in general cell metabolism including probable U3 small nucleolar RNA-associated protein 11 (spots w2, w20 and w22), six proteins were involved in defense mechanism including ATPase (spots w31 and w33) and four proteins were involved in secondary metabolite production including 12-oxophytodienoate reductase. Discussion Herbal medicines are readily available in all stores cheaply. Nowadays, the use of herbal medicines among the general population is on the rise. P. ginseng and W. somnifera are widely used in traditional Indian medicines and traditional Korean medicines, respectively. P. ginseng has been used as an antioxidant, anti-inflammatory agent, anticancer remedy as well as a cardioprotective agent in traditional medicines for centuries. For thousands of years, ginseng has been used as a heart tonic in China (Dasgupta et al., 2008). W. somnifera is mainly used for a variety of musculoskeletal conditions and as a general tonic to increase energy, improve overall health and longevity, and prevent disease in athletes, the elderly, and during pregnancy (RajaSankar et al., 2009). Both P. ginseng and W. somnifera are touted for their longevity-enhancing and sexualstimulation properties. However, clinical studies are needed to find out pharmacological properties of ginseng. A large proportion of the genes in any plant genome encode enzymes of primary and specialized (secondary) metabolism. Only a small portion of the estimated hundreds of thousands of specialized metabolites has been studied in any given species. Proteome profiling studies have proven to be a powerful approach for the identification of candidate genes and enzymes, particularly those involved in secondary metabolism


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(Fridman and Pichersky, 2005). A cell is normally dependent upon a multitude of metabolic and regulatory pathways for its survival. There is no strict linear relationship between genes and the protein complement or the proteome of a cell. Proteomics is complementary to genomics because it focuses on the gene products, which are the active agents in cells (Pandey and Mann, 2000). 2-DE is an established and powerful technique for analyzing the complex mixtures of proteins. This technique was used to compare the protein profiling and reveal protein diversity of root tissues of P. ginseng and W. somnifera root tissues, two plant species that share similar pharmaceutical properties. Proteins Involved in General Metabolism General cell metabolism category includes energy production, RNA processing, protein synthesis, cell differentiation and signal transduction. Such functions are crucial for the plant survival in normal environmental conditions. In P. ginseng, spots p12 and p15 were identified as ribonuclease (RNase)-like storage protein, which provides a nitrogen source and has no RNase activity, although it has conserved active site residues; these proteins are the vegetative storage proteins of ginseng for survival in the natural environment (Kim et al., 2004). This type of isoform protein can be useful for the analysis of post-translational modifications in certain plants. Other proteins involved in general metabolism are trigger factor like protein and chloroplast EF-Tu (fragment), which are involved in protein translation. In W. somnifera, spot w24 was identified as fructose-2, 6-bisphosphatase, an enzyme that catalyzes the synthesis and degradation of fructose 2, 6-bisphosphate, which is a powerful activator of 6-phosphofructo-1-kinase, the rate-limiting enzyme of glycolysis (Atsumi et al., 2005). Many enzymes involved in protein synthesis and RNA processing were identified. Lysyl-tRNA synthetase (spot w7), ribosomal protein small subunit 4 (spot w34) and 60S ribosomal protein L9 (Gibberellin-regulated protein GA) (spot w8) are involved in protein synthesis and processing (Journet et al., 2002). Spots w2, w20 and w22 were identified as probable U3 small nucleolar RNA-associated protein 11, which is involved in RNA processing and modification (Kufel et al., 2000). U6 snRNA-associated Sm-like protein (spot w18) is involved in RNA processing (Moore et al., 1993). Maturase K (spot w25) is a splicing factor for the plant group II introns from premature RNAs (Mohr and Lambowitz, 2003). Spot w11 was identified as proline-rich protein-2, which plays an important role in the differentiation and function of particular cell types (Ye et al., 1991). Additionally, proline is also utilized for protein synthesis, and hydroxyproline, a hydroxylation derivative of proline, is enriched in structural proteins, such as collagen in animals or hydroxyprolinerich protein in plants (Hall and Cannon, 2002; Myllyharju, 2003). Ca 2þ -ATPases, including spot w5, are involved in the transduction of gravitational stimuli, which increases cytosolic-free Ca 2þ levels, which in turn triggers a signal transduction cascade resulting in a physiological response (Urbina et al., 2006).

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Proteins Involved in Defense Mechanism Field grown plants must endure different environmental stresses including pollutants, fungi, bacteria, viruses and insects, which can result in heavy loss in yield and alter the plant’s medicinal content. Plants possess defense mechanism to protect from those damages at the primary level. In P. ginseng, spot p4 was identified as a nucleotide-binding site leucine-rich repeat (NBS-LRR)-like protei, which is involved in disease resistance. The majority of disease resistance genes in plants encode NBS-LRR proteins (McHale et al., 2006). In W. somnifera, the proteins involved in defense mechanism were identified as soluble inorganic pyrophosphatases 1 (spot w1), which are important enzymes catalyzing the hydrolysis of inorganic pyrophosphate to inorganic phosphate. They participate in the assimilation of mineral nutrients (Jardin et al., 1995). George et al. (2010) suggested that inorganic phosphate metabolism might be indirectly involved in mediating drought stress responses in Nicotiana benthamiana leaves. Spot w28 was identified as 6-cinnamyl-alcohol dehydrogenase, a key enzyme in lignin biosynthesis that catalyzes the final step in the synthesis of monolignols. Lignin is a phenolic heteropolymer in secondary cell walls that plays a major role in the development of plants and their defense against pathogens (Barakat et al., 2009). ATPase and alcohol dehydrogenase were the only proteins common in both plant species. The experimental and theoretical pI and molecular weight of alcohol dehydrogenase (ADH) was similar in both species, whereas the ATPase only shared theoretical pI. ATPase (spots p6, w31 and w33) is involved in nutrient and environmental stresses (Shen et al., 2006). Alcohol dehydrogenase (spots p20 and w15) is involved in the ethanol fermentation pathway that is responsible for the reduction of acetaldehyde, which is toxic to plant tissues, in ethanol resulting a continuous regeneration of NADP in the cytoplasm (Chung and Ferl, 1999). Hence, induction of ADH can enhance survival of plants under flood conditions (Johnson et al., 1994). Proteins Involved in Secondary Metabolism There are many enzymes involved in secondary metabolism, often working in close collaboration to catalyze cascades of reactions. Besides the enzymes, transport and regulatory proteins are also involved, which makes the proteome essential in the study of metabolic pathways (Jacobs et al., 2000). Isoflavone synthase I found in P. ginseng (spot p16) catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway (Jung et al., 2000). In W. somnifera, spots assigned as w3, w19 and w21 were found to be 12-oxophytodienoate reductase 1, which is involved in the biosynthesis of jasmonic acid, a potent signal molecule in the defense system of plants, where it regulates the expression of many wound-activated and defense-related genes (Breithaupt et al., 2001). Jasmonic acid is an important phytohormone that regulates plant defense responses against herbivore attack, pathogen infection and mechanical wounding (Bu et al., 2008). Spot w16 was identified as F-box family protein, which is an important component of the


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E3 ubiquitin ligase Skp1-Cullin-F-box protein complex. It binds specific substrates for ubiquitin-mediated proteolysis. F-box proteins act as important receptors and signaling components in plant hormone signaling pathways (Yu et al., 2007). When 2-DE maps of the two different species of ginseng were compared, no common protein spots were identified. ATPase and alcohol dehydrogenase proteins were expressed in both W. somnifera and P. ginseng. Unfortunately, proteins commonly involved in withanolide and ginsenoside biosynthesis were not identified. These results suggest that different growing conditions and environments may induce the expression of different proteins. Only a few proteins were identified by MALDI-TOF/MS due to lack of a genome database of both P. ginseng and W. somnifera. Since the rate for protein identification process of an organism or species mainly depends on their available genome database, peptide mass fingerprinting analysis alone proved to be inefficient in ginseng proteome analysis. In addition, the peptide mass fingerprinting search based on the MS data from MALDI-TOF MS/MS was not sensitive enough to identify a protein because of the low coverage of protein sequences. Therefore, combined analysis using both internal sequences from MS/MS spectra and an EST sequence database will be an efficient and accurate protein identification method for ginseng proteome analysis. Such analysis could also be applied to other plants for which genomic information is not available. Acknowledgments This work was supported by National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2009-0084454) and Technology Development Program for (‘Agriculture and Forestry’), Ministry for Food, Agriculture, Forestry and Fisheries (20080439), Ministry for Health, Welfare and Family affairs, Republic of Korea (No: 0820050). References Ali, M., M. Shuaib and S.H. Ansari. Withanolides from the stem bark of Withania somnifera. Phytochemistry 44: 1163–1168, 1997. Andallu, B. and B. Radhika. Hypoglycemic, diuretic and hypocholesterolemic effect of winter cherry (Withania somnifera, Dunal) root. Indian J. Exp. Biol. 38: 607–609, 2000. Asamizu, E., Y. Nakamura, S. Sato and S. Tabata. A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries. DNA Res. 7: 175–180, 2000. Atsumi, T., T. Nishio, H. Niwa, J. Takeuchi, H. Bando, C. Shimizu, N. Yoshioka, R. Bucala and T. Koike. Expression of inducible 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase/PFKFB3 isoforms in adipocytes and their potential role in glycolytic regulation. Diabetes 54: 3349–3357, 2005. Attele, A.S., J.A. Wu and C.S. Yuan. Ginseng pharmacology: multiple constituents and multiple actions. Biochem. Pharm. 58: 1685–1693, 1999. Barakat, A., A.B. Zadworna, A. Choi, U. Plakkat, D.S. DiLoreto, P. Yellanki and J.E. Carlson. The cinnamyl alcohol dehydrogenase gene family in Populus: phylogeny, organization, and expression. BMC Plant Biol. 9: 26, 2009. Breithaupt, C., J. Strassner, U. Breitinger, R. Huber, P. Macheroux, A. Schaller and T. Clausen. X-ray structure of 12-oxophytodienoate reductase 1 provides structural insight into substrate binding and specificity within the family of OYE. Structure 9: 419–429, 2001.

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