J Korean Soc Appl Biol Chem (2014) 57(3), 289−295 Online ISSN 2234-344X DOI 10.1007/s13765-014-4008-1 Print ISSN 1738-2203

ARTICLE

Molecular Cloning and Characterization of (MVA) Pathway Genes and Accumulation in Panax ginseng

Yong-Kyoung Kim · Yeon Bok Kim · Jae Kwang Kim · Soo-Un Kim · Sang Un Park

Received: 7 January 2014 / Accepted: 19 March 2014 / Published Online: 30 June 2014 © The Korean Society for Applied Biological Chemistry and Springer 2014

Abstract Panax ginseng Meyer is one of the most important Keywords gene expression · ginsenosides · mevalonic acid medicinal plants in Asia, and ginseng has attracted considerable pathway · Panax ginseng · triterpene attention worldwide. Triterpene (ginsenosides) are the main bioactive compounds in P. ginseng. The isoprene units of triterpene are derived from the mevalonic acid (MVA) pathway. We cloned four genes involved in MVA pathway using rapid Introduction amplification of cDNA ends by polymerase chain reaction. Additionally, we investigated the transcript levels of 11 genes Panax ginseng C.A. Meyer is an important perennial herb plant involved in the pathway in different organs and cell that belongs to the Araliaceae family. The major bioactive suspension cultures of P. g i n s e n g . The full-length cDNA sequences compounds of P. ginseng are the ginsenosides which possess were as follows: PgHMGS (1764 bp; 1407-bp ORF), PgHMGR numerous physiological and pharmacological effects (Sticher, (1992 bp; 1722-bp ORF), PgPMK (2170 bp; 1530-bp ORF), and 1998). These include exerting central nervous stimulating and PgMVD (1759 bp; 1263-bp ORF). The highest expression level of suppressing activity as well as anticancer activity (Kubo et al., all genes was found in fine roots. The total ginsenoside contents 1992; Shinkai et al., 1996; Yun, 1996; Iishi et al., 1997; Attele et in different organs were ranked in the following descending order: al., 2002; Dey et al., 2002). To date, more than 30 ginsenosides leaf > fine root > lateral root > red berry > main root > petiole > have been discovered. stem. Campesterol and were detected in all organs Isoprenoids are natural compounds that play a vital role in plant but at different concentrations. The total phytosterol content was metabolism (Bohlmann et al., 1998; Rodriguez-Concepcion and highest in fine root (147.8 µg/100 mg dry weight (DW)), and was Boronat, 2002). They constitute one of the largest structurally lowest in the stem (86.4 µg/100 mg DW). Four enzymes in the diverse groups of natural products, with over 30,000 known MVA pathway were cloned and characterized in P. ginseng. Such compounds (McGarvey and Croteau, 1995). In higher plants, they genes play important roles in terpenoid biosynthesis and may have are synthesized by 2 distinct biosynthetic pathways: the mevalonic applications in the metabolic engineering of ginsenoside production. acid (MVA) pathway and the 1-deoxyxylulose-5-phosphate or 2- C-methyl-D-erythritol-4-phosphate pathway. Isoprenoids are Y.-K. Kim · Y. B. Kim · S.-U. Park () synthesized by the head-to-tail condensation of isopentenyl Department of Crop Science, Chungnam National University, Daejeon diphosphate (IPP) and its isomer, dimethylallyl diphosphate 305-764, Republic of Korea E-mail: [email protected] (Rohmer et al., 1995; Lichtenthaler et al., 1997a; b). Ginsenosides are synthesized via the MVA pathway. The J. K. Kim enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (CoA) synthase Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 406-772, Republic of Korea (HMGS) catalyzes the condensation of acetyl-CoA and acetoacetyl- CoA. The synthesis of MVA is catalyzed by 3-hydroxy-3- Y.-K. Kim · S. U. Kim methylglutaryl-CoA reductase (HMGR), which is a key regulatory Department of Agricultural Biotechnology and Research Institute for Agricultural Sciences, Seoul National University, Seoul 151-951, Republic enzyme, and the sequential action of MVA kinase, phospho- of Korea mevalonate kinase, and pyrophosphomevalonate decarboxylase 290 J Korean Soc Appl Biol Chem (2014) 57(3), 289−295

Fig. 1 The ginsenoside biosynthetic pathway. AACT, acetyl-coenzyme A (CoA) acetyltransferase; β-AS, beta-amyrin synthase; CAS, cycloartenol synthase; DDS, dammarenediol-II synthase; FPS, farnesyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase; HMGS, HMG-CoA synthase; IDI, isopentenyl diphosphate isomerase; MVD, mevalonate diphosphate decarboxylase; MK, mevalonate kinase; MPK, mevalonate-5-phosphate kinase; SE, epoxidase; SS, squalene synthase.

converts MVA to IPP (Rohmer et al., 1995). Mevalonate diphosphate represent an attractive strategy to produce better quality medicinal decarboxylase (MVD) is an important enzyme in the MVA compounds. pathway, catalyzing the ATP-dependent decarboxylation of Recently, ginsenoside biosynthetic pathway genes from P. mevalonate 5-diphosphate to yield IPP. Ginsenoside is then ginseng (Chen et al., 2011) and panax quinquefolius (Sun et al., synthesized by the terpenoid biosynthetic pathway, involving 2010) were discovered by next generation sequencing. Huge enzymes such as farnesyl diphosphate synthase (FPS), squalene amount of genetic information was clarified using high throughput synthase (SS), squalene epoxidase (SE), dammarenediol-II synthase method. Therefore, cloning and molecular characterization of (DDS), and beta-amyrin synthase (β-AS) (Fig. 1). genes involved in ginsenoside biosynthesis are required. In the The studies of MVA pathway genes related to phytosetrol and present study, we cloned 4 genes, HMGS, HMGR, PMK, and terpenoid biosynthesis have been demonstrated in Nicotiana MVD involved in terpenoid biosynthesis. In addition, we tabacum (Schaller et al., 1995), Candida utilis (Shimada et al., investigated the transcript levels of 11 genes involved in the 1998), and Saccharomyces cerevisiae (Cordier et al., 1999). terpenoid pathway in different organs and cell suspension cultures Secondary metabolite production has been shown to be induced of P. ginseng. Finally, the ginsenoside and phytosterol contents in by various elicitors - including methyl jasmonate (MeJA) and P. ginseng were analyzed. yeast extract - in Panax species such as P. notoginseng (Hu and Zhong, 2007; 2008), P. quinquefolius (Ali et al., 2005), and P. ginseng (Lu et al., 2001; Hu et al., 2003). Triterpene biosynthetic Materials and Methods genes were upregulated by MeJA treatment in Medicago truncatula (Suzuki et al., 2002), and Glycyrrhiza glabra (Hayashi et al., Plant materials. P. gi n s e n g samples were collected from the 2003). Furthermore, MeJA has also been used to increase experimental farm of Chungnam National University. Different ginsenoside content in cell suspension cultures (Hu et al., 2003) organs were stored in sealed clear polyethylene plastic bags at and in root culture of P. ginseng (Han et al., 2006). Overproduction −80oC until further use and freeze-dried at −80oC for 72 h. of phytosterols and by metabolic engineering may RNA isolation and cDNA synthesis. Total RNA was isolated J Korean Soc Appl Biol Chem (2014) 57(3), 289−295 291 from different organs (red berry, leaf, petiole, stem, main root, Scattering Detector (Model 300s; SofTA, USA). The separation lateral root, and fine root) by using TRI-reagent (Molecular method was carried out according to Kim et al. (2009) using a Research Center, Inc., USA) and the RNeasy Plant Mini Kit ProntoSIL column (Bischoff Chromatography, Germany; 250× (Qiagen, USA) according to the manufacturers’ protocols. Total 4.6 mm) at a flow rate of 0.8 mL/min. The conditions were RNA of each organ was reverse transcribed using SuperScript II optimized by the solvent gradient system (Kim et al., 2009). First-Strand Synthesis Kit according to the manufacturer’s Identification and quantification of ginsenosides were performed instructions (Life technologies, USA). by comparing the retention times and peak areas with those of a Cloning of cDNAs encoding MVA pathway enzymes. The 5' ginsenoside standard or by direct addition of a ginsenoside and 3' rapid amplification of cDNA ends (RACE)-PCR was standard into the sample (spike test). Standard chemicals carried out using the GeneRacer Kit (Life technologies, USA) (ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, and Rh1) with fine root RNA as the template and specific primers. The were purchased from Canfo Chemical Co., Ltd., China. All primer pairs (Supplementary Table 1) were based on conserved samples were run in triplicate. sequences from other known plant genes. All PCR products were Extraction and derivatization of triterpenes. Extraction of subcloned into the T-Blunt vector (Solgent, Korea) and sequenced triterpenes was performed according to the method of Du and Ahn (Solgent). (2002) with slight modifications. Triterpene components were Gene expression by real-time quantitative PCR. To investigate released from powdered samples (0.1 g) by the addition of 3 mL the expression pattern of MVA pathway genes from different of ethanol containing 0.1% ascorbic acid (w/v) and 0.05 mL of organs of P. ginseng, single-stranded cDNAs were synthesized 5α-colestane (10 µg/mL), mixed by vortexing for 20 s, and placed from total RNA by using the ReverTra Ace-α-Kit (Toyobo, in a water bath at 85°C for 5 min. After removal from the water Japan) and oligo (dT)20 primer according to the manufacturer’s bath, 120 µL of potassium hydroxide (80%) was added, and the protocol. The specific primers are listed in Supplementary Table samples were vortexed for 20 s and returned to the water bath for 2. PCR reactions were carried out in triplicate on a MiniOpticon 10 min. The samples were immediately placed on ice, and (Bio-Rad, USA) instrument using the QuantiTect SYBR Green deionized water (1.5 mL) was added. Each sample received 1.5 PCR system (Qiagen, USA). The PCR reaction consisted of a mL of hexane, and was then vortexed for 20 s and centrifuged denaturation step at 95oC for 15 min, followed by 40 cycles of (1,200×g, 5 min). The upper layer was pipetted into a separate amplification comprised of three steps: denaturation at 95°C for tube, and the pellet was re-extracted using hexane. The hexane 15 s, annealing at 55oC for 15 s, and extension at 72oC for 20 s. fraction was dried in a centrifugal concentrator (CVE-2000, Eyela, Fluorescent intensity data were acquired during the extension step. Japan). For derivatization, 30 µL of N-methyl-N-trimethylsilyltrifluoro- The transcript levels were evaluated by using standard curve acetamide was added with 30 µL of pyridine, and samples were conditions for all genes. incubated at 60oC for 30 min at a mixing frequency of 1,200 rpm Treatment of an elicitor. Suspension cells of P. ginseng were using a thermomixer comfort (model 5355; Eppendorf AG, grown in Gamborg’s B5 medium containing 2 mg/L of 2,4- Germany). dichlorophenoxyacetic acid (2,4-D) and sub-cultured every 2 Gas chromatography-time-of-flight mass spectrometry (GC- weeks. Subsequently, 10 mL of the cells cultured under optimal TOF-MS) analysis. Each derivatized sample (1 µL) was injected culture conditions were transferred into 100-mL Erlenmeyer into an Agilent 7890A Gas Chromatograph using an Agilent flasks containing 40 mL of medium. MeJA was first dissolved in 7683B Autosampler (Agilent, USA) with a split ratio of 5 and 100% EtOH at an optimal concentration. Subsequently, the separated on a 30 m×0.25 mm i.d. fused-silica capillary column elicitor solution was added to the culture medium to give a final coated with 0.25-µm CP-Sil 8 CB Low Bleed (Varian Inc., USA). concentration of 100 µM, and cells were incubated for 0, 6, 12, The injector temperature was 290oC and the helium gas flow rate 24, 48, 72, and 96 h. Each treatment was conducted in three through the column was 1.0 mL/min. The temperature program separate flasks, and the experiment was repeated three times. was set at 250oC, followed by a 10oC min−1 oven temperature Ginsenoside extraction and analysis. Different organs ramp to 290oC and a 10-min heating period at 290oC. The column (approximately 5 g) and suspension cells (0.5 g) were extracted effluent was later introduced into a Pegasus High Throughput with 400 and 40 mL of 70% EtOH, respectively, at 37oC for 3 TOF Mass Spectrometer (LECO, USA). Transfer line and the ion days in a shaking incubator. The extract was filtrated through filter source temperatures were 280 and 230oC, respectively. The paper (Whatman No. 42) and evaporated at 40oC using a Heidolph scanned mass range was 50–800 m/z, and the detector voltage was VV 2011 instrument (Germany). The evaporated extracts were set at 1800 V. resuspended in 5 mL of distilled water and freeze-dried. The samples were stored at room temperature and resuspended in 20 mL of distilled water for high performance liquid chromatography Results and Discussion (HPLC) analysis. Ginsenosides were analyzed using an NS-4000 HPLC system Isolation of MVA pathway genes. For cloning of conserved (Futecs Co., Korea) equipped with a SofTA Evaporative Light sequences, we performed RACE-PCR using degenerate primers 292 J Korean Soc Appl Biol Chem (2014) 57(3), 289−295

Fig. 2 Organ-specific expression of ginsenoside biosynthetic genes from P. ginseng. (A) P. ginseng different organs. (B) Tissue specific transcript levels. The bars indicate the means ± SD of 3 biological replicate. based on the conserved sequences of known HMGS, HMGR, PgHMGR, PgPMK, and PgMVD, encoding MVA pathway genes PMK, and MVD genes. The cloned PgHMGS cDNA had a 1407- from P. ginseng by using RACE-PCR. These MVA pathway bp ORF nucleotide sequence encoding 469 amino acids. PgHMGS genes displayed high homology with other plant species (Figs. showed 86% sequence identity with Cola acuminate, 82% with H. S1–4). Similarly, Sando et al. (2008) cloned and characterized brasiliensis, 80% with Solanum lycopersicum, 76% with Brassica MVA pathway genes from H. brasiliensis. juncea, and 75% with Arabidopsis thaliana (Supplementary Fig. Transcript levels in different organs of P. gins eng. To analyze 1). PgHMGR had a 1722-bp ORF sequence encoding 574 amino the expression in different organs of cloned genes [PgHMGS acids, and it shared 80% sequence identity with H. brasiliensis, (GU565098), PgHMGR (GU565097), PgPMK (KC439363), and 78% with Artemisia annua, 76% with C. acuminate, and 73% PgMVD (GU565096)] and terpene biosynthetic genes [PgFPS with A. thaliana (Supplementary Fig. 2). PgPMK had a 1530-bp (DQ087959), PgSS (AB115496), PgSE (AB122078), PgDDS ORF encoding 510 amino acids. PgPMK showed 77, 72, and 66% (AB265170), Pgβ-AS (AB009030), and PgCAS (AB009029)], we identity with H. brasiliensis, A. thaliana, and Zea mays, respectively designed gene-specific primers for each gene. (Supplementary Fig. 3). PgMVD had a 1263-bp ORF encoding All genes were constitutively expressed in all organs, but the 421 amino acids and shared sequence identities of 79% with H. transcript levels differed slightly among distinct organs. The brasiliensis, 75% with Arnebia euchroma, 73% with A. thaliana, highest expression level appeared in fine roots (Fig. 2). HMGR and 71% with Ginkgo biloba (Supplementary Fig. 4). The gene was known as rate-limiting step in MVA pathway, thus the isoelectric point (pI) and amino acid molecular weight (MW) of expression level is relatively lower than others. Most genes each enzyme were calculated by using software available at showed similar expression patterns in each organ; however, 3 website (http://www.expasy.org). The pI and MW of HMGS were oxidosqualene cyclase genes (PgDDS, PgCAS, and PgLAS) were 6.64 and 52.0 kDa, respectively, and those of HMGR were 7.07 highly expressed in the leaf. DDS, the enzyme that catalyzes the and 61.78 kDa, respectively. PMK had a pI of 5.21 and a MW of first committed step in ginsenoside synthesis, play an important 54.99 kDa, whereas those of MVD were 8.66 and 38.59 kDa, role in the pathway. The transcription of CAS related to phytosterol respectively. In the present study, we cloned 4 genes, PgHMGS, biosynthesis was higher in leaf than that of other organs and LAS J Korean Soc Appl Biol Chem (2014) 57(3), 289−295 293

Fig. 3 Transcription levels of ginsenoside biosynthetic genes after treatment of suspension cell cultures with MeJA. The bars indicate the means ± SD of 3 biological replicates. was also highly expressed in the leaf. However, was after MeJA treatment. Thus, saponins increase, whereas phytosterols not detected in the leaf. According to this result, we suggest that decrease. This result suggests that MeJA treatment might be an CAS and LAS have no correlation with cholesterol accumulation. effective approach to induce higher production of ginsenoside in The transcript level of Pgβ-AS displayed the same pattern as those cell suspension cultures of P. ginseng. of the other genes. Among the MVA and terpene biosynthetic After treatment of suspension cell culture with 100 µM MeJA, pathway genes, PgFPS showed the highest mRNA expression we also examined composition. The suspension cell level, which was 250-fold higher compared with PgHMGR. Actin culture had a lower ginsenoside content than that of the different was used as a housekeeping gene. In our previous report, organs, and only three ginsenosides (Rg1, Re, and Rb1) were terpenoid biosynthetic genes (PgFPS, PgSS, PgSE, and PgDDS) detected. After 72 h, the total saponin content gradually increased were highly expressed in the early berry stage, and phytosterol to 4-fold to that of the control (Fig. 4). synthetic genes (PgCAS and PgLAS) were highly expressed in the Many studies have reported that elicitor strongly increases green berry stage (Kim et al., 2012). Most enzymes were highly secondary metabolite production, including ginsenoside (Gundlach expressed in the fine root and it might relate to high ginsenosides et al., 1992; Hayashi et al., 2003; Suzuki et al., 2005) However, accumulation. little is known regarding the expression profiles of genes involved Transcription after MeJA treatment in suspension cell in the ginsenoside synthetic pathway. Therefore, we proposed that culture. To analyze the effect of elicitors of defense responses on it would be of interest to study the expression patterns of genes the expression of MVA pathway genes, we treated suspension involved in ginsenoside biosynthesis following treatment with an cells with MeJA and investigated the transcription of genes. After elicitor, MeJA. Accumulation of PgFPS, PgSQS, PgSE, and treatment 100 µM MeJA, the expression of 8 genes - PgHMGS, PgDDS mRNAs was enhanced after MeJA treatment (Lee et al., PgHMGR, PgPMK, PgMVD, PgFPS, PgSS, PgSE, and PgDDS - 2004; Han et al., 2006; Kim et al., 2010), suggesting that the increased gradually within 12 h in suspension culture, whereas activity of all four gene products are positively involved in expression of PgCAS and PgLAS, which are involved in phytosterol triterpene biosynthesis. The results of MeJA treatment also show synthesis, gradually decreased (Fig. 3). Generally, these enzymes that all MVA pathway genes are related to triterpene saponin share 2, 3-oxidosqualene as precursor and compete in this step biosynthesis. We demonstrated that ginsenoside biosynthetic 294 J Korean Soc Appl Biol Chem (2014) 57(3), 289−295

Amyrin was specifically observed in the red berry and root, but the amount detected was low (Table 2). In particular, the red berry showed striking features compared with the other organs. Cholesterol was also specifically detected in the root and stem. In addition, campesterol and stigmasterol were detected in all organs, but the contents differed. Stigmasterol content was highest in the fine root. Like saponin content, total phytosterol contents were highest in the fine root and lowest in the stem (Table 2). Several studies have demonstrated that ginsenoside content in the berries are higher than those in the root. Our previous report also supports that the gene expression pattern in the upper ground and underground part of the ginseng plant was also different (Kim et Fig. 4 Ginsenoside accumulation after MeJA treatment in suspension cell al., 2012). In the present study, we cloned and characterized genes culture. The bars indicate the means ± SD of 3 biological replicates. involved in terpenoid biosynthesis. Such genes play important roles in terpenoid biosynthesis and may have applications in the genes differently responded to the MeJA. metabolic engineering of ginsenoside production. Analysis of ginsenosides and phytosterols. HPLC is an efficient tool for the analysis of secondary metabolites, including ginsenosides Acknowledgments This work was supported by grant 2007-04269 from the (Kwon et al., 2001; Kim et al., 2007). The results of the present Ministry of Education, Science and Technology and Basic Research Program of the Korea Science & Engineering Foundation (No. R01-2007-000-20823-0). study showed that all ginsenosides analyzed accumulated in a similar pattern in all organs, and the total saponin content of the leaf was highest (65.05 mg/g DW), whereas the stem contained References the lowest saponin content (2.37 mg/g DW) (Table 1). The total ginsenoside contents of different organs were ranked in descending Ali MB, Yu KW, Hahn EJ, and Paek KY (2005) Differential responses of order: leaf > fine root > lateral root > main root > red berry > petiole anti-oxidants enzymes, lipoxygenase activity. Ascorbate content and the β production of saponins in tissue cultured root of mountain Panax ginseng > stem. Four phytosterols ( -sitosterol campesterol, stigmasterol, C.A. Mayer and Panax quinquefolium L. in bioreactor subjected to cholesterol) and β-amyrin were detected by GS-MS analysis. β- methyl jasmonate stress. Plant Sci 169, 83–92 .

Table 1 Ginsenoside contents (mg/g dry weight) in P. ginseng organs Ginsenosides Red berry Leaf Petiole Stem Main root Lateral root Fine root Rg1 00.22±0.01 11.05±0.80 02.81±0.20 0.55±0.02 3.30±0.50 3.00±0.10 1.17±0.07 Re 11.93±1.10 16.35±1.20 04.02±0.50 1.14±0.10 1.28±0.30 9.47±1.21 13.00±1.100 Rf 01.49±0.20 00.47±0.02 0.13±0.01 0.12±0.01 0.94±0.01 4.20±0.40 4.64±0.30 Rg2 00.36±0.03 01.22±0.10 0.35±0.07 0.12±0.02 0.21±0.01 0.86±0.06 1.40±0.90 Rh1 00.19±0.01 00.25±0.02 0.06±0.00 0.07±0.00 0.25±0.03 0.95±0.07 1.40±0.10 Rb1 00.90±0.01 00.65±0.02 0.06±0.00 0.06±0.00 1.96±0.10 10.16±0.900 17.63±0.120 Rc 00.76±0.05 04.32±0.50 0.05±0.02 0.06±0.03 0.60±0.06 4.84±0.60 8.26±0.70 Rb2 00.92±0.07 04.04±0.50 0.10±0.00 0.10±0.00 0.44±0.02 2.57±0.30 4.40±0.50 Rb3 00.22±0.02 00.48±0.03 0.05±0.00 0.07±0.00 0.17±0.01 0.41±0.05 0.60±0.07 Rd 01.18±0.20 26.24±2.00 0.23±0.02 0.09±0.01 0.20±0.02 1.36±0.14 4.97±0.50 Total 18.17±1.60 65.05±5.19 7.86±0.83 2.37±0.38 9.32±1.06 37.83±3.620 57.46±4.3600 Results are expressed as the mean ± standard error of the mean (n = 3).

Table 2 Phytosterol and â-amyrin composition (µg/100 mg dry weight) in P. ginseng organs Triterpene Red berry Leaf Petiole Stem Main root Lateral root Fine root Cholesterol nd nd nd 0.11±0.01 nd 0.12±0.01 0.19±0.01 Campesterol 2.24±0.16 1.25±0.10 1.04±0.12 0.94±0.04 1.28±0.06 1.52±0.06 5.73±0.71 Stigmasterol 7.82±1.00 10.83±0.420 6.68±0.58 7.63±0.60 6.48±0.48 8.80±0.48 23.1±1.27 β-Sitosterol 113.46±9.4700 125.21±5.2100 82.66±8.890 77.72±3.940 121.88±6.5500 115.75±3.1300 118.71±5.9000 β-Amyrin 1.38±0.10 nd nd nd 0.91±0.07 0.22±0.01 0.10±0.00 Total 124.90±10.730 137.30±5.5700 90.38±9.580 86.40±4.560 130.55±7.1400 126.42±3.6700 147.85±7.6800 Results are expressed as the mean ± standard error of the mean (n = 3); nd, not detected. J Korean Soc Appl Biol Chem (2014) 57(3), 289−295 295

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