Alteration of Panax ginseng saponin composition by overexpression and RNA interference of the protopanaxadiol 6-hydroxylase gene (CYP716A53v2).

BACKGROUND: The roots of Panax ginseng contain noble tetracyclic triterpenoid saponins derived from dammarenediol-II. Dammarene-type ginsenosides are classified into the protopanaxadiol (PPD) and protopanaxatriol (PPT) groups based on their triterpene aglycone structures. Two cytochrome P450 (CYP) genes (CYP716A47 and CYP716A53v2) are critical for the production of PPD and PPT aglycones, respectively. CYP716A53v2 is a protopanaxadiol 6-hydroxylase that catalyzes PPT production from PPD in P. ginseng.
METHODS: We constructed transgenic P. ginseng lines overexpressing or silencing (via RNA interference) the CYP716A53v2 gene and analyzed changes in their ginsenoside profiles. RESULT: Overexpression of CYP716A53v2 led to increased accumulation of CYP716A53v2 mRNA in all transgenic roots compared to nontransgenic roots. Conversely, silencing of CYP716A53v2 mRNA in RNAi transgenic roots resulted in reduced CYP716A53v2 transcription. HPLC analysis revealed that transgenic roots overexpressing CYP716A53v2 contained higher levels of PPT-group ginsenosides (Rg1, Re, and Rf) but lower levels of PPD-group ginsenosides (Rb1, Rc, Rb2, and Rd). By contrast, RNAi transgenic roots contained lower levels of PPT-group compounds and higher levels of PPD-group compounds.
CONCLUSION: The production of PPD- and PPT-group ginsenosides can be altered by changing the expression of CYP716A53v2 in transgenic P. ginseng. The biological activities of PPD-group ginsenosides are known to differ from those of the PPT group. Thus, increasing or decreasing the levels of PPT-group ginsenosides in transgenic P. ginseng may yield new medicinal uses for transgenic P. ginseng.

Introduction

Triterpenoid saponins are present in many higher plants and exhibit a wide range of biological activities depending on their structures. Certain triterpenoid saponins also have commercial value and are exploited as drugs and medicines [1], [2], [3], and saponins may have natural roles in the defense against pathogens and pests [4].

The roots of ginseng (Panax ginseng Meyer) contain pharmacologically active components. It is generally believed that ginsenoside saponins are the main compounds responsible for the pharmacological activities of P. ginseng [2], [3]. Ginsenosides are classified as protopanaxadiol (PPD) or protopanaxatriol (PPT) saponins based on their aglycone structures. PPD-group ginsenosides have glycosidic bonds at the C-3 and C-20 hydroxyl groups, and the major ginsenosides of this group include Ra1, Ra2, Rb1, Rc, Rd, and Rg3. PPT-group ginsenosides have glycosidic bonds at the C-6 and C-20 hydroxyl groups, and the major ginsenosides of this group include Re, Rf, Rg1, Rg2, and Rh1.

The first step in ginsenoside biosynthesis is the cyclization of 2,3-oxidosqualene to dammarenediol-II, which is catalyzed by dammarenediol synthase of the oxidosqualene cyclase group [5]. Dammarenediol-II is hydroxylated by two cytochrome P450 enzymes (CYP716A47 and CYP71653v2) to produce PPDs and PPTs [6], [7]. These compounds are subsequently glycosylated by glycosyltransferase (Fig. 1). The CYP716A47 enzyme in P. ginseng catalyzes the hydroxylation of dammarenediol-II at the C-12 position to yield PPD, which is further hydroxylated at its C-6 position by CYP71653v2 to yield PPT.

Fig. 1

Biosynthetic pathway for ginsenosides in Panax ginseng. Squalene epoxidase converts squalene to 2,3-oxidosqualene, which is then converted to a triterpene aglycone (dammarenediol-II) by dammarenediol synthase. Dammarenediol-II undergoes oxidation and glycosylation and is finally converted to protopanaxadiol (PPD)- and protopanaxatriol (PPT)-group ginsenosides.

PPD-group ginsenosides have biological activities that are opposite of those in the PPT group [8], [9]. For example, Rg1 (PPT group) stimulates the central nervous system, whereas Rb1 (PPD group) suppresses the activity of the central nervous system [8], [9].

Because the two CYP genes (CYP716A47 and CYP71653v2) are necessary for producing PPD and PPT aglycones, respectively, we postulated that modifying the expression of these genes in P. ginseng could alter the composition of ginsenosides. Transgenic ginseng with altered ginsenoside profiles could be used for new medicinal applications requiring precise tuning of pharmacological activity. In this study, we constructed two transgenic P. ginseng lines, with overexpression or silencing [RNA interference (RNAi)] of CYP716A53v2, and we analyzed the changes in their PPD and PPT ginsenoside levels. We found that the production of PPT-group ginsenosides was altered by both the overexpression and RNAi-based silencing of the CYP716A53v2 gene.

Materials and methods

Overexpression and RNAi silencing vector construction

The ORF (open reading frame) region of the CYP716A53v2 sequence was cloned into the pCR 8.0 vector (Invitrogen Life Technologies, Carlsbad, CA, USA) and then transferred to the destination vector pH2WG to yield the CYP716A53v2 overexpression vector. To construct the CYP716A53v2-RNAi vector, two primers including gateway adapters (Invitrogen Life Technologies) were designed to amplify the region from 1,259 bp to 1,498 bp in CYP716A53v2. The amplified polymerase chain reaction (PCR) product was cloned into the pSB1 vector and then transferred to the RNAi destination vector pB7GWIWG2(II) (which contains the BAR gene that confers Basta resistance to plant cells) in Escherichia coli DH5α, as described by the manufacturer (Invitrogen Life Technologies). The construct was sequenced and subsequently transformed into Agrobacterium tumefaciens GV3101 cells harboring plasmid pMP90 using standard molecular biology techniques.

Construction of transgenic P. ginseng

The generation of transgenic P. ginseng was conducted as described in our previous report [10]. Putative transgenic somatic embryos for both CYP716A53v2 overexpression and CYP716A53v2-RNAi were transferred to the selection medium, with additional supplementation of 20μM GA3, to induce embryo germination. The plantlets were maintained on 1/2-strength MS medium with 2% sucrose.

Selection of transgenic root lines was performed as described by Han et al [5]. As a nontransgenic control, adventitious roots were induced from the in vitro maintained nontransformed plants that were the original sources of the transgenic plants.

Reverse transcription-PCR in transgenic roots

Total RNA was isolated from nontransgenic and transgenic roots and reverse-transcribed using the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA). First-strand cDNA was used as the template for the reverse transcription (RT)-PCR analysis, which was performed as follows: 96°C for 5 min; 30 cycles of 96°C for 30 s, 60°C for 30 s, 72°C for 1 min; and a final extension at 72°C for 10 min. β-Actin cDNA (primers 5′-ATG GTC AAG GCT GGA TTT GCA-3′ and 5′-CTC GAC CAG CTA AAT CAA GAC G-3′) was used as a control for RNA integrity and loading accuracy. The RT-PCR analyses were performed twice, and representative data are shown in the figures. The primers used for amplification were 5′-ATG GAT CTC TTT ATC TCA TCT CAA-3′ and 5′-TTA AAG CGT ACA AGG TGA TAG ACG-3′ for P. ginseng CYP716A53v2, 5′-GCG TGA CCT ATT GCA TCT CC-3′ and 5′-TTC TAC ACA GCC ATC GGT CC-3′ for the hygromycin phosphotransferase gene (HPT), and 5′-AGG ACA GAG CCA CAA ACA CC-3′ and 5′-ATG CTT GTA TCC AGC TGC G-3′ for the phosphinothricin acetyl transferase gene (BAR).

Quantitative PCR in transgenic roots

Quantitative PCR (qPCR) was performed using a Qiagen Rotor Gene Q real-time PCR detector system (Qiagen, Hilden, Germany) with SYBR Green PCR Kit (Qiagen, Valencia, CA, USA). The cycling parameters for the qRT-PCR were as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 5 s and 60°C for 10 s. The qRT-PCR data shown are the average relative quantities ± standard error from at least three replicates. The relative expression level of each gene was calculated using the ΔΔCt method [11]. The P. ginseng β-actin gene was used for normalization. The primers used for the amplification were as follows: 5′-GGC CCC CTG CCA TAT CCA AAA TCC-3′ and 5′-CAA AGT GCT GCC TCG TTG TCC G -3′ for P. ginseng CYP716A53v2; 5′-ATG GTC AAG GCT GGA TTT GCA-3′ and 5′-GAG CCT CAT ATC CAA CAT ATG C-3′ for P. ginseng β-actin; 5′-ATG CGG AAA AGC AGA TCC CT-3′ and 5′-AAA TGC ATA CAG CAT CGC GG-3′ for PgSS1; 5′-AAG CTA GCG CTC TGC TCA TT-3′ and 5′-TGA TGA CGT CCG TAC TTC CG-3′ for PgSQE1; 5′-ATA TAT CAG CGG AAC GAT TGA CAC-3′ and 5′-ATT CAG CTA ATC CTT CTC CTA GCA A-3′ for PgDDS; 5′-GCA GCA GCA ATG GTG TTG TTT-3′ and 5′-TCG CCT ATC AAA GGC CAA CC-3′ for CYP716A47; 5′-TTC GCA ATC ATC GAT TCG AGA AAA A-3′ and 5′-AGA CTA CAC TGC CTG GAT TCT C-3′ for PgPNX.

Ginsenoside analysis by HPLC

Extraction and analysis of ginsenosides was performed using the method described by Han et al [6]. The HPLC separation was performed using a Cosmosil C18 column (5 μm, 4.6 × 250 mm; Agilent, Santa Rosa, CA, USA). The time course measurements and measurements of the ratios of water and acetonitrile were performed according to the protocol reported by Han et al [6]. Each ginsenoside was compared to authentic ginsenoside samples purchased from ChromaDex, Inc. (Irvine, CA, USA).

Results

Overexpression and RNA interference of CYP716A53v2 in transgenic P. ginseng

Transgenic ginseng plants overexpressing CYP716A53v2 were generated using A. tumefaciens-mediated genetic transformation (Fig. 2A). Transgenic root lines were induced and selected according to the protocol by Han et al [5]. Seven transgenic overexpression lines were finally selected for further analysis. The RT-PCR analysis revealed clear transcription of the selection marker, hygromycin phosphotransferase (HPT), in the overexpression lines (Fig. 2B). RT-PCR showed higher transcript levels of CYP716A53v2 in roots of all transgenic lines than in nontransgenic roots (Fig. 2C). Increases of > 2.5-fold were achieved in all seven transgenic roots, as measured by qPCR.

Fig. 2

Expression of introduced genes in transgenic ginseng roots overexpressing CYP716A53v2. (A) T-DNA region of the plasmid for CYP71653v2 gene overexpression under the control of the CaMV35S promoter. T35S, CaMV 35S terminator; HPT, hygromycin phosphotransferase gene; RB, right border of the T-DNA; LB, left border of the T-DNA. (B) Confirmation of transcription of the introduced genes (HPT and CYP716A53v2) by reverse transcription-polymerase chain reaction (RT-PCR). β-Actin was used as a loading control. (C) Transcription of CYP716A53v2 in nontransgenic (Nt) and transgenic lines (1–7) overexpressing CYP716A53v2, based on quantitative PCR (qPCR). Data are shown as mean values with the standard error obtained from three independent plants. Asterisks indicate significant differences between nontransgenic and transgenic plants (Student t test, p ≤ 0.05).

In transgenic ginseng plants for silencing (RNAi) CYP716A53v2 (Fig. 3A), expression of the selection marker gene, phosphinothricin acetyl transferase (BAR), was clearly shown in all RNAi lines by RT-PCR analysis (Fig. 3B). CYP716A53v2 transcript levels in all RNAi lines were clearly reduced compared to nontransgenic roots (Fig. 3C). As measured by qPCR, each of the CYP716A53v2-RNAi lines showed a decrease of at least 75% in the level of the CYP716A53v2 transcript.

Fig. 3

Expression of introduced genes in transgenic ginseng roots overexpressing CYP716A53v2-RNAi. (A) The T-DNA region of the plasmid for RNAi of the CYP71653v2 gene under the control of the CaMV35S promoter. (B) Confirmation of transcription of the introduced genes (BAR and CYP716A53v2) using genomic RT-PCR. β-Actin was used as a loading control. (C) Transcription of CYP716A53v2 in Nt and CYP716A53v2-RNAi transgenic lines 1–7, based on qPCR. Asterisks indicate significant differences between nontransgenic and transgenic plants (Student t test, p ≤ 0.05). Data are shown as mean values with the standard error obtained from three independent plants. BAR, phosphinothricin acetyl transferase; Nt, nontransgenic; qPCR, quantitative PCR; RNAi, RNA interference; RT-PCR, reverse transcription-polymerase chain reaction.

Altered ginsenoside profiles in transgenic P. ginseng

The HPLC chromatogram profiles revealed that PPT-group ginsenosides (Rg1, Re, and Rf) were clearly reduced in the transgenic RNAi line (line RNAi 3) (Fig. 4B) and were increased in the overexpressing transgenic line (line over 7; Fig. 4C) compared with those in the control (Fig. 4A). By contrast, PPD-group ginsenosides (Rb1, Rb2, Rc, and Rd) were clearly enhanced in transgenic RNAi roots (Fig. 4B) but reduced in overexpressing transgenic roots (Fig. 4C).

Fig. 4

Ginsenoside chromatogram of extracts from nontransgenic and transgenic roots. (A) Ginsenoside chromatograms of extracts from nontransgenic roots. (B) Transgenic CYP716A53v2-RNAi roots (line RNAi 3). (C) Transgenic P. ginseng roots overexpressing CYP716A53v2 (line over 7). (D) HPLC chromatogram of ginsenoside standards.

The levels of PPT-group ginsenosides (Rg1, Re, and Rf) were increased by at least 1.5-fold in all seven of the overexpression lines relative to nontransgenic control roots (Table 1, Fig. 5A). However, the levels of PPD-group ginsenosides (Rc, Rd, Rb1, and Rb2) were reduced in overexpression lines compared with the controls. The total ginsenoside levels in roots of the overexpression lines were increased compared to those in the nontransgenic controls (Fig. 5A, Table 1).

Table 1

Ginsenoside concentrations in transgenic lines overexpressing CYP716A53v2

Line	Ginsenoside concentration (mg/g dry weight)							Total
	PPT type			PPD type
	Rg1	Re	Rf	Rb1	Rc	Rb2	Rd
Nt	2.73 ± 0.28	1.03 ± 0.18	0.11 ± 0.13	1.56 ± 0.19	0.19 ± 0.17	0.51 ± 0.02	0.17 ± 0.11	6.30 ± 0.62
Over 1	6.82 ± 0.84	1.16 ± 0.12	0.22 ± 0.12	0.73 ± 0.07	0.11 ± 0.06	0.14 ± 0.01	0.12 ± 0.03	9.30 ± 0.79
Over 2	6.42 ± 0.52	1.22 ± 0.19	0.21 ± 0.22	0.64 ± 0.05	0.07 ± 0.01	0.16 ± 0.03	0.17 ± 0.02	8.89 ± 0.68
Over 3	5.55 ± 0.47	1.29 ± 0.18	0.16 ± 0.01	0.4 ± 0.05	0.1 ± 0.05	0.17 ± 0.02	0.13 ± 0.01	7.80 ± 0.5
Over 4	6.78 ± 0.61	1.35 ± 0.13	0.23 ± 0.02	0.65 ± 0.03	0.09 ± 0.01	0.16 ± 0.03	0.13 ± 0.01	9.39 ± 0.55
Over 5	4.98 ± 0.73	1.17 ± 0.15	0.17 ± 0.01	0.67 ± 0.06	0.05 ± 0.01	0.16 ± 0.02	0.18 ± 0.02	7.38 ± 0.67
Over 6	8.24 ± 0.43	1.58 ± 0.17	0.29 ± 0.03	0.56 ± 0.03	0.09 ± 0.01	0.14 ± 0.01	0.11 ± 0.01	11.01 ± 0.48
Over 7	6.17 ± 0.87	1.96 ± 0.19	0.38 ± 0.02	0.41 ± 0.03	0.03 ± 0.01	0.09 ± 0.01	0.11 ± 0.01	9.15 ± 0.71

PPD, protopanaxadiol; PPT, protopanaxatriol

Fig. 5

HPLC analysis of ginsenosides in nontransgenic and transgenic ginseng roots. (A) Ginsenoside contents in roots of transgenic lines (over 1 to 7) overexpressing CYP716A53v2. (B) Ginsenoside contents in roots of CYP716A53v2-RNAi lines (RNAi 1 to 7). Data are shown as means ± SE (n = 3 each). Significance was determined using Student t test (* p ≤ 0.05; ** p ≤ 0.01) for independent means. Nt, nontransgenic roots; SE, standard error.

In transgenic RNAi lines, the levels of PPT-group ginsenosides (Rg1, Re, and Rf) were clearly reduced compared with those in nontransgenic roots (Table 2, Fig. 5B). In line 3, the PPT-group ginsenosides were nearly absent. Interestingly, the total amount of PPD-group ginsenosides (Rc, Rd, Rb1, and Rb2) was markedly increased in all RNAi lines compared with the nontransgenic roots (Fig. 5B). The total ginsenoside content was lowered in transgenic RNAi lines compared with nontransgenic roots (Fig. 5B and Table 2).

Table 2

Ginsenoside concentration in RNAi transgenic lines silencing the CYP716A53v2 gene

Line	Ginsenoside concentration (mg/g)							Total
PPT type			PPD type
Rg1	Re	Rf	Rb1	Rc	Rb2	Rd
Nt	2.73 ± 0.28	1.03 ± 0.18	0.11 ± 0.03	1.56 ± 0.19	0.19 ± 0.17	0.51 ± 0.02	0.17 ± 0.11	6.30 ± 0.51
RNAi 1	0.45 ± 0.03	0.09 ± 0.01	—	2.62 ± 0.12	0.32 ± 0.02	0.55 ± 0.03	0.27 ± 0.03	4.30 ± 0.2
RNAi 2	0.52 ± 0.06	0.08 ± 0.01	—	2.43 ± 0.17	0.26 ± 0.01	0.46 ± 0.02	0.26 ± 0.03	4.01 ± 0.17
RNAi 3	0.07 ± 0.01	—	—	1.88 ± 0.21	0.34 ± 0.02	0.48 ± 0.04	0.28 ± 0.02	3.05 ± 0.18
RNAi 4	0.28 ± 0.02	—	—	2.26 ± 0.19	0.44 ± 0.03	1.2 ± 0.13	0.23 ± 0.02	4.41 ± 0.27
RNAi 5	0.35 ± 0.03	0.08 ± 0.01	—	2.67 ± 0.31	0.53 ± 0.04	0.86 ± 0.07	0.24 ± 0.01	4.73 ± 0.27
RNAi 6	1.29 ± 0.13	0.2 ± 0.03	—	1.83 ± 0.19	0.35 ± 0.02	0.56 ± 0.04	0.24 ± 0.03	4.47 ± 0.25
RNAi 7	1.06 ± 0.11	0.1 ± 0.01	—	2.53 ± 0.22	0.26 ± 0.01	0.39 ± 0.04	0.26 ± 0.03	4.60 ± 0.24

Nt, nonstransgenic; PPD, protopanaxadiol; PPT, protopanaxatriol; RNAi, RNA interference

Expression of genes involved in ginsenoside biosynthesis in transgenic lines

Squalene synthase is the first enzymatic step leading from the central isoprenoid pathway to ginsenoside biosynthesis [12]. It has been reported that PgSS1 in P. ginseng is a regulatory enzyme not only for phytosterol but also for triterpene biosynthesis [12]. Squalene epoxidase catalyzes the first oxygenation step in ginsenoside biosynthesis and is thought to be one of the rate-limiting enzymes in this pathway. Han et al [13] reported that PgSQE1 and PgSQE2 are regulated via different mechanisms and that PgSQE1 regulates ginsenoside biosynthesis but not phytosterol biosynthesis. The production of the ginsenoside triterpene skeleton relies on the cyclization of 2,3-oxidosqualene to dammarenediol-II catalyzed by dammarenediol synthase (PgDDS) [5]. Han et al [6] reported that CYP716A47 is responsible for the hydroxylation of dammarenediol-II at the C-12 position to yield PPD. Cycloartenol synthase (PgPNX) in ginseng is involved in the production of cycloartenol, which is the precursor for phytosterol biosynthesis.

The transcription levels of genes related to ginsenoside biosynthesis, such as PgSS1, PgSQE1, PgDDS, CYP716A47, and PgPNX, were measured with qPCR in selected lines of transgenic ginseng (overexpression and RNAi; Fig. 6, Fig. 7). Overexpression and RNA interference of CYP716A53v2 did not affect the expression of upstream genes in the pathway (PgSS1, PgSQE1, PgDDS, and CYP716A47); there was no clear change in mRNA accumulation between the nontransgenic control and the transgenic roots (Fig. 6, Fig. 7). Moreover, there was no difference in the accumulation of PgPNX mRNA between transgenic roots and nontransgenic roots (Fig. 6, Fig. 7).

Fig. 6

Quantitative polymerase chain reaction (qPCR) for genes upstream of ginsenoside biosynthesis in CYP716A53v2-overexpressing transgenic roots of Panax ginseng. Transcription of PgSS1, PgSQE1, PgDDS, CYP716A47, and PgPNX in transgenic lines.

Fig. 7

Quantitative polymerase chain reaction (qPCR) for genes upstream of ginsenoside biosynthesis in CYP716A53v2-RNAi transgenic roots of Panax ginseng. Transcription of PgSS1, PgSQE1, PgDDS, CYP716A47, and PgPNX in transgenic lines.

Discussion

Altered ginsenoside composition in transgenic P. ginseng

Transgenic ginseng plants were constructed to either overexpress or silence the CYP716A53v2 gene. RT-PCR and qPCR analyses revealed that all of the transgenic ginseng roots overexpressing CYP716A53v2 showed increased CYP716A53v2 transcription. All of the CYP716A53v2-RNAi roots showed reduced accumulation of CYP716A53v2 mRNA.

Ginsenoside analysis by HPLC revealed that the transgenic ginseng roots overexpressing CYP716A53v2 contained higher levels of PPT-group ginsenosides (Rg1, Re, and Rf), whereas the CYP716A53v2-RNAi roots contained lower levels. These results indicate that the production of PPT-group ginsenosides is affected by the engineered expression of CYP716A53v2. Overproduction of PPTs in CYP716A53v2-overexpressing roots reduced the levels of PPDs. Conversely, reduced PPT production in the CYP716A53v2-RNAi roots stimulated the production of PPDs. These results indicate that PPD and PPT production may be competing pathways that both depend on a common intermediate triterpene (dammarenediol-II).

Using qPCR, we investigated the expression of upstream genes in the CYP716A53v2 overexpression and RNAi roots. Neither overexpression nor RNAi affected the key upstream genes PgSS1, PgSQE1, PgDDS, and CYP716A47, and there was no effect on the OSC gene of the sterol pathway (PgPNX). These results strongly suggest that altering CYP716A53v2 expression by using an engineered promoter does not affect the upstream genes in the ginsenoside biosynthesis pathway. The lower levels of PPD ginsenosides in CYP716A53v2-overexpressing roots and the increase in PPD ginsenosides in CYP716A53v2-RNAi roots may result from complementary use of sapogenin (PPD and PPT) precursors in P. ginseng. However, it is not clear why the total ginsenoside content differs between the CYP716A53v2-overexpressing and CYP716A53v2-silenced transgenic roots. To produce the final PPD and PPT ginsenosides, various types of glycosyltransferases are required. Recently, genes for a few glycosyltransferases involved in ginsenoside biosynthesis have been identified [14]. It is likely that changing the expression of CYP716A53v2 affects the downstream genes (glycosyltransferases).

There are many reports of achieving increased ginsenoside accumulation by elicitor or precursor treatment or by overexpression of key ginsenoside biosynthesis enzymes in transgenic P. ginseng [15]. Furuya et al [16] reported that treatment with several intermediates and precursors involved in ginsenoside biosynthesis, e.g., mevalonate or farnesol, was effective for increasing ginsenoside production. Treatments with elicitors such as yeast extract or methyl jasmonate also enhanced the ginsenoside production [16], [17]. Lee et al [12] found that squalene synthase (PgSS1) increased the production of both phytosterols and ginsenosides. Overexpression of the PgSS1 gene in adventitious roots of transgenic P. ginseng resulted in an increase in the production of phytosterols (β-sitosterol, stigmasterol, and campesterol) and ginsenosides [12]. However, all of the above reports address the control of total ginsenoside biosynthesis. Our report is the first to describe fine-tuning the biosynthesis of specific ginsenoside groups.

The medicinal effects of these ginsenosides have been extensively studied, and they are involved in the immune system, antistress effects, antihyperglycemic activity, anti-inflammatory activity, antioxidant activity, and anticancer effects [3], [18]. In general, the PPD-group ginsenosides have pharmaceutical activities that are opposite those of the PPT group [8], [9]. Therefore, transgenic ginseng plants containing increased or reduced levels of PPT-group ginsenosides due to altered expression of CYP716A53v2 could be used as new medicinal plants with specific pharmacological activities.

The physiological role of saponins in plants is not yet fully understood. Saponins may play important roles in plant defense. The bitter taste of ginsenosides makes them antifeedants [19], [20], [21], [22], [23]. Various biological roles have been proposed for different saponins, mostly involving allelopathic activity and defense against insects and pathogens [22]. Ginsenosides have antimicrobial [24] and antifungal activities in nature [25]. Ginsenosides also have antiviral activities toward human viruses [26]. Lee et al [27] reported that transgenic tobacco producing dammarenediol-II by overexpression of the PgDDS gene confers resistance to tobacco mosaic virus by inhibiting viral replication.

The fungus Cylindrocarpon destructans causes serious root rot disease in ginseng-producing areas and is a main cause of ginseng replant failure [28]. PPT- and PPD-group ginsenosides exhibit different activities on C. destructans: PPT-group ginsenosides inhibit its growth, whereas PPD-group ginsenosides significantly enhance its growth [25]. However, simply increasing the level of total ginsenosides did not prevent the fungal pathogen from invading the ginseng [25]. Thus, specifically increasing the levels of PPT-group ginsenosides in transgenic ginseng by overexpressing CYP716A53v2 may be necessary to provide defense against C. destructans, thereby conferring resistance to root rot disease.

In conclusion, ginsenoside levels were successfully altered by changing the expression of CYP716A53v2 in transgenic ginseng. These transgenic P. ginseng plants can be used to produce new genetically engineered cultivars with specific biological traits such as changed medicinal values and defenses against ginseng pathogens and other microorganisms.

Conflicts of interest

The authors declare that they have no conflicts of interest.