Plant Cell Rep (2010) 29:403411

DOI 10.1007/s00299-010-0831-y

ORIGINAL PAPER

Upregulation of phytosterol and triterpene biosynthesis

in Centella asiatica hairy roots overexpressed ginseng
farnesyl diphosphate synthase
Ok Tae Kim Sun Hee Kim Kiyoshi Ohyama
Toshiya Muranaka Yong Eui Choi
Hyeon Yong Lee Min Young Kim Baik Hwang

Received: 18 November 2009 / Revised: 28 January 2010 / Accepted: 4 February 2010 / Published online: 27 February 2010
Springer-Verlag 2010

Abstract Farnesyl diphosphate synthase (FPS) plays an

essential role in organ development in plants. However,
FPS has not previously been identified as a key regulatory
enzyme in triterpene biosynthesis. To elucidate the functions of FPS in triterpene biosynthesis, C. asiatica was
transformed with a construct harboring Panax ginseng FPS
(PgFPS)-encoding cDNA coupled to the cauliflower
Communicated by J. R. Liu.
O. T. Kim
Department of Herbal Crop Research,
National Institute of Horticultural and Herbal Science,
RDA, Eumseong 369-873, South Korea
S. H. Kim
B. Hwang (&)
Department of Biology, Chonnam National University,
Gwangju 500-757, South Korea
e-mail: bhwang@chonnam.ac.kr
K. Ohyama
Department of Chemistry and Materials Science,
Tokyo Institute of Technology, Meguro-ku,
Tokyo 152-8551, Japan
T. Muranaka
Kihara Institute for Biological Research,
Yokohama City University, Yokohama,
Kanagawa 244-0813, Japan
Y. E. Choi
Division of Forest Resources, College of Forest Sciences,
Kangwon National University, Chunchon 200-701, South Korea
H. Y. Lee
College of Bioscience and Biotechnology,
Kangwon National University, Chunchon 200-701, South Korea
M. Y. Kim
Jeonnam Agricultural Research and Extension Service,
Naju 520-715, South Korea

mosaic virus 35S promoter. Higher levels of CaDDS

(C. asiatica dammarenediol synthase) and CaCYS (C. asiatica cycloartenol synthase) mRNA were detected in all
hairy root lines overexpressing when compared with the
controls. However, no differences were detected in any
expression of the CaSQS (C. asiatica squalene synthase)
gene. In particular, the upregulation of CaDDS transcripts
suggests that FPS may result in alterations in triterpene
biosynthesis capacity. Squalene contents in the T17, T24,
and T27 lines were increased to 1.1-, 1.3- and 1.5-fold
those in the controls, respectively. The total sterol contents
in the T24 line were approximately three times higher than
those of the controls. Therefore, these results indicated that
FPS performs a regulatory function in phytosterol biosynthesis. To evaluate the contribution of FPS to triterpene
biosynthesis, we applied methyl jasmonate as an elicitor
of hairy roots expressing PgFPS. The results of HPLC
analysis revealed that the content of madecassoside and
asiaticoside in the T24 line was transiently increased by
1.15-fold after 14 days of MJ treatment. This result may
indicate that FPS performs a role not only in phytosterol
regulation, but also in triterpene biosynthesis.
Keywords Centella asiatica

Farnesyl diphosphate synthase
Methyl jasmonate

Phytosterols
Triterpene saponins

Introduction
Triterpene saponins not only evidence pharmacological
activities, including immunostimulant, hypocholesterolemic, and anticarcinogenic properties (Francis et al. 2002)
but also play physiological roles in plants, including as
a protectant against pathogen attack (Osbourn 1996).

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Centella asiatica (L.) Urban contains a variety of saponins,

including asiaticoside, asiaticoside A, centellasaponin A,
centellasaponin B, madecassoside, and sceffoleoside
(Matsuda et al. 2001). Asiaticoside, in particular, has been
suggested to exert a therapeutic effect in Alzheimers disease (Mook-Jung et al. 1999), and these saponins have
been used for the treatment of wounds, ulceration, eczema,
etc. (Brinkhaus et al. 2000). These saponins and sterols are
synthesized from mevalonic acid via the isoprenoid pathway (Fig. 1). Oxidosqualene cyclase (OSC), a common
intermediate enzyme of both triterpene and phytosterol
biosynthesis, generates a number of triterpenoid carbon
skeletons.
Farnesyl diphosphate (FPP), which is catalyzed by farnesyl diphosphate synthase (FPS), functions as a substrate
in the synthesis of essential or secondary metabolites,
including sesquiterpenoids, phytoalexins, triterpenoids, and
phytosterols. In plants, several papers have detailed the
physiological functions or contributions of FPS in secondary metabolite synthesis. By the overexpression of a
chimeric FPS gene in transgenic plants, Chen et al. (2000)
demonstrated that FPS plays a regulatory role in sesquiterpene biosynthesis. In the tomato, the FPS enzyme has
been theorized to play an essential role in the early
development of plant organs, during which time both cell
division and growth occur (Gaffe et al. 2000). FPS1Soverexpression of Arabidopsis thaliana reduces the level of
endogenous zeatin-type cytokinins in the leaves, ultimately
resulting in their phenotypic alteration (Masferrer et al.
2002). However, there is also some evidence to suggest
that FPS supplies the carbon flow into phytosterol and
carotenoid biosyntheses in tobacco overexpressing yeast

Fig. 1 Pathways of triterpene

and phytosterol biosyntheses in
C. asiatica. IPP isopentenyl
diphosphate, DMAPP
dimethylallyl diphosphate, SQS
squalene synthase, SE squalene
epoxidase, MFS multifunctional
triterpene synthase,
bAS b-amyrin synthase,
CYS cycloartenol synthase

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FPS (Daudonnet et al. 1997). Suzuki and Muranaka (2007)

have suggested that plant metabolites may be affected by
disruptions in the balance between 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and FPS expression by
overexpression, as the physiological function of FPS is
similar to that of HMGR.
To overproduce triterpene saponins in plants, both
squalene synthase (SQS), the first enzymatic step from the
isoprenoid pathway toward phytosterol and triterpenoid
biosynthesis, and OSC, which catalyzes the cyclization of
2,3-oxidosqualene, are key regulatory enzymes. When SQS
was overexpressed in ginseng and Eleutheroccus senticosus, enhanced phytosterol and triterpenoid levels were
noted in the transgenic plants (Lee et al. 2004; Seo et al.
2005). In addition, the overexpression of AsOXA1 (Aster
sedifolius b-amyrin synthase) in M. truncatula results in a
greater accumulation of triterpene saponins in the leaf and
root, and also results in the enhancement of root nodulation
(Confalonieri et al. 2009). However, the manipulation of
other genes that regulate triterpene biosynthesis has yet to
be clearly described. In a previous work, an FPS gene from
Panax ginseng was isolated and characterized (Kim et al.
2010). It was also reported that the exposure of ginseng
hairy root cultures to methyl jasmonate (MJ) results in the
upregulation of PgFPS transcripts, thereby suggesting that
PgFPS participates in triterpene biosynthesis as an intermediate enzyme. Herein, we evaluate the consequences of
PgFPS overexpression in the hairy roots of C. asiatica.
PgFPS overexpression in the transgenic roots results
in increased phytosterol content. In addition, we have
discussed the effects of FPS cDNA overexpression on
triterpene biosynthesis.

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405

For whole plant cultures of C. asiatica, four node segments

per petri dish (90 9 20 mm) were cultured on MS basal
medium (Murashige and Skoog 1962) supplemented with
3% sucrose and 0.8% agar at 23 2C with a 16-h photoperiod. After 2 weeks of cultivation, the leaves were used
as explants for the transformations.

respectively, and those used for the amplification of the

PgFPS gene (GenBank Accession No. DQ087959) were
50 -ATGAGCGATCTGAAGACGAGA-30 and 50 -TTACTT
TTGCCGCTTATATA-30 , respectively. The PCR volume
was 50 ll, containing 100 ng of each primer, 4 ll of
0.25 mM dNTPs, 3 ll of genomic DNA, and 0.5 U of
Ex-Taq DNA polymerase (Takara, Japan). The PCR reaction was conducted for 30 cycles under the following
conditions: 94C for 1 min, 58C for 1 min and 72C for
1 min.

Vector construction

Southern and northern blot analysis

The PCR product harboring a PgFPS cDNA from Panax

ginseng was digested with BamHI and SacI to yield a
1,026 bp fragment containing the complete open reading
frame. This fragment was then positioned between a CaMV
35S promoter and a NOS terminator in a pCAMBIA 1300
vector that had been digested with the same enzymes, thus
forming the binary vector, pPgFPS, which was subsequently utilized for the transformation of C. asiatica. The
correct orientation within the vector and open reading
frame integrity of the inserted cDNA was confirmed by
DNA sequence analysis. The construct was transformed
into Agrobacterium rhizogenes R1000. C. asiatica hairy
roots transformed by Agrobacterium harboring the empty
vector were used as controls in our experiments.

The genomic DNA of the hairy roots was isolated using a

DNeasy Plant Mini kit (Qiagen); 13 lg of genomic DNA
was digested with BamHI. The digest was then fractionated
via electrophoresis on 0.8% agarose gel and blotted onto a
positively charged nylon membrane. For hybridization, the
PCR-amplified PgFPS gene products were labeled with
digoxigenin (DIG)-dUTP (PCR DIG Probe Synthesis kit,
Roche, Basel, Switzerland) as a probe. DNA cross-linked
positively to nylon membranes were incubated for 12 h at
48C with the probe for hybridization, and then washed
twice at 25C for 5 min each wash in 29 SSC, 0.1% SDS,
followed by two washes for 15 min each in 0.59 SSC, 0.1%
SDS at 68C. For RNA blot analysis, total RNA was
extracted with TRIzol (Invitrogen, Carlsbad, CA, USA)
from hairy roots (500 mg) and separated on 1.5% denaturing
agarose gel, after which 15 lg of total RNA was transferred
to positively charged nylon membranes. Probing, hybridization, and washing were conducted as described above.

Materials and methods

Plant materials

Plant transformation
Formation of Centella hairy roots was carried out as
described by Kim et al. (2007). The A. rhizogenes strain
R1000 harboring the recombinant pPgFPS vector was cultivated overnight in YEP medium containing antibiotics
(50 mg/l kanamycin) at 28C. Segments of the leaves were
immersed in the bacterial suspension for 45 min. After
3 days of co-cultivation at 18C in darkness, the explants
were washed in sterile distilled water and transferred to halfstrength MS medium supplemented with 3% sucrose, 0.8%
agar, and 300 mg/l of cefotaxime (Bioworld, Dublin, OH,
USA). Induced roots were excised from the parental tissues
and transferred to a selectable medium with 300 mg/l of
cefotaxime and 20 mg/l of hygromycin (Duchefa, Haarlem,
The Netherlands) to screen for putative transformed roots.
PCR analysis of HPT and PgFPS genes
DNA from leaves of the wild-type plant and hygromycinresistant hairy roots was isolated using a DNeasy Plant
Mini kit (Qiagen, Hilden, Germany). The forward and
reverse sequences employed for the PCR amplification of
the HPT gene were as follows: 50 -GCGTGACCTATTGCATCTCC-30 and 50 -TTCTACACAGCCATCGGTCC-30 ,

RT-PCR analysis
cDNAs were synthesized from total RNA from the hairy
roots including a empty vector and three hairy root lines
including the PgFPS gene; 2 lg of RNA was reversetranscribed into cDNA using 30 U of AMV enzyme (Promega, Madison, WI, USA). For reverse transcription, the
reaction mixture and samples were incubated for 1 h at
42C. To quantify the transcripts, first-strand cDNA was
PCR amplified using gene-specific primers: 50 -ATGGGAA
GTTTAGGGGCGATTC-30 , 50 -TCATCGGTTATTCGAT
AGATTG-30 for C. asiatica CaSQS (GenBank Accession
No. AY787628), 50 -TGCACAGCATCAATAATAGCAG
CT-30 , 50 -TCAATTGGAGAGCCACAAGCGTTT-30 for
C. asiatica CaDDS (GenBank Accession No. AY520818),
50 -GCAGGGGAAAAGGCAGATGTTGAGCGA-30 , 50 -TC
ATGGCGCCAGAAGTACAC-30 for C. asiatica CaCYS
(GenBank Accession No. AY520819). For normalization,
an actin fragment amplified by the primer designed from
P. ginseng was employed as an internal standard. The
forward and reverse sequences utilized for the PCR

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amplification of actin were 50 -GATGACATGGAAAAGA

TTTGGCATC-30 and 50 -AAGGATGGCATGAGGGAGG
GCGTAA-30 , respectively. The PCR reaction was conducted as described above.
Assay of FPS activity in hairy roots
Hairy roots (1 g) were ground in liquid nitrogen and resuspended in 1 ml of Tris buffer (50 mM, pH 8.0), 5 mM
DTT, 1% polyvinylpolypirrolidone (PVP-40), and 40 mM
ascorbic acid, as described by Daudonnet et al. (1997). The
mixture was then filtered through two layers of Miracloth
(CalBiochem, La Jolla, CA, USA) and centrifuged at
17,7009g. Protein concentrations were determined via the
Bradford (1976) method. To assess FPS activity in the
hairy roots, the reaction (in a total volume of 50 ll) was
conducted using 5080 lg of protein per sample, in
accordance with the method described by Brodelius et al.
(2002). Aliquots of tissue extract were assayed in 50 mM
TrisHCl (pH 8.0), containing 15 mM MgCl2, 10 mM
b-mercaptoethanol, 20% glycerol, 55 lM geranyl diphosphate and 50 lM [1-14C]IPP (1.8 lCi/mmol). The reaction
mixture was incubated for 10 min at 30C and 5 ll of 6 M
HCl was added to stop the reaction, incubated continuously
for 30 min to hydrolyze the formed FPP to extractable
farnesol. For the neutralization of the mixtures, 7.5 ll of
6 M NaOH was added and the mixture was extracted with
0.4 ml of hexane and extracted with water (0.3 ml). An
aliquot (0.2 ml) of the hexane phase was counted by liquid
scintillation.
Quantitative analysis of squalene and phytosterol
Quantitative analysis of squalene and phytosterols (cholesterol, campesterol, b-sitosterol, and stigmasterol) was
conducted in accordance with the procedure described by
Hartmann and Benveniste (1987). Freeze-dried hairy roots
(500 mg) were extracted twice with 20 ml of ethyl acetate
at 100 rpm on a gyrator for 6 h at room temperature. The
extracts were chromatographed on a silica gel cartridge
column (SepPak, Waters, USA). The eluent was extracted
three times with 10 ml of aqueous 5% KOH and then
extracted twice more with 10 ml of aqueous 5% HCl. The
organic fraction was washed twice with 10 ml of water and
water was eliminated with anhydrous sodium sulfate. The
extract was evaporated and the residue was dissolved with
2 ml of hexane. Suspended particles were removed by
10 min of centrifugation at 6,000 rpm. An aliquot of the
solution was analyzed via GCMS. GCMS analysis was
conducted on a mass spectrometer (Hewlett Packard, USA)
connected to a gas chromatograph with an HP-1 capillary
column (25 m 9 0.25 mm, 0.33 lm methylpolysiloxane
cross-linked capillary column, Hewlett Packard). The

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analytical conditions were as follows: carrier gas He (4 ml/

min), and column temperature 100250C (20C/min). The
squalene and phytosterol contents were calculated from the
ratio of the peak area of the respective compound to that of
the standard. Authentic standards of cholesterol, campesterol, b-sitosterol, and stigmasterol were purchased from
Sigma-Aldrich (St. Louis, MO, USA).
HPLC analysis of madecassoside and asiaticoside
Hairy root extraction was conducted via the method of
Bonfill et al. (2006). Quantitative determinations of madecassoside and asiaticoside contents were conducted by
HPLC (Agilent 1100 series equipped with an auto sampler, a
diode array detector, and a quaternary pump) using a Inertsil
ODS-3 (3.0 9 150 mm, 5 lm) column (GL Sciences,
Tokyo, Japan). A linear gradient of acetonitrile in water was
used as a mobile phase, in accordance with the method
described by Randriamampionona et al. (2007). The other
HPLC conditions for the isolation of the two triterpenoids
were as follows: flow rate 1 ml/min, column temperature
40C, detector wave length 214 nm. The standards were
purchased from ChromaDex (Santa Ana, CA, USA).

Results
Induction of hairy root overexpressing PgFPS
Genetic transformation of C. asiatica was conducted
according to the method of Kim et al. (2007). Approximately 120 leaf explants were transformed with A. rhizogenes strain R1000 harboring the pPgFPS binary vector,
resulting in the production of 45 hygromycin-resistant
hairy root lines. In 16 of those 45 lines, PCR analysis
confirmed the integrations of the PgFPS and hygromycin
phosphotransferase (HPT) genes into the genome of
C. asiatica (data not shown). We ultimately selected 3 of
the 16 lines that evidenced the same level of hairy root
growth as was seen in the hairy root culture transformed by
the bacteria containing the empty vector. All of these were
analyzed to evaluate the contribution of FPS to triterpene
biosynthesis.
Characterization of PgFPS gene in C. asiatica
hairy root
Southern analysis was conducted to further verify the
introduction of the PgFPS gene into the genome of C.
asiatica (Fig. 2a). Different positions of PgFPS gene bands
were detected in the genomic DNA from three hairy roots
(in T17, T24 and T27 lanes), whereas no hybridization
band was noted to exist in the control (in the C lane).

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407
16

FPS activity
nmol/min/mg protein

a
b

12

c
8

d
4

0
C

T17

T24

T27

Fig. 3 FPS activity of C. asiatica hairy roots overexpressing FPS

(T17, T24 and T27). The control line (C) is a hairy root transformed
by Agrobacterium harboring the empty vector. The specific activity of
extracts is expressed as nmol of IPP incorporated into FPP/min/mg
protein with standard deviations. Vertical bars indicate the
mean SE of three independent samples. Different letters indicate
significant differences at P B 0.05

Fig. 2 Confirmation of PgFPS gene in C. asiatica hairy roots (T17,

T24 and T27). Southern (a) and Northern (b) blot analyses. The
control line (C) is a hairy root transformed by Agrobacterium
harboring the empty vector

Southern blot analysis of the BamHI-digested genomic

DNA from the C. asiatica hairy roots verified the presence
of the PgFPS gene. In addition, the results of northern blot
analysis revealed that PgFPS gene overexpression induced
an accumulation of its transcription in the C. asiatica hairy
roots (Fig. 2b). Consistent with this finding, these lines also
evidenced increased levels of FPS activity when compared
with the controls (Fig. 3). The FPS activity in the T17,
T24, and T27 hairy roots was 1.4, 2.4 and 1.8 times higher
than that of the control. Therefore, these results showed
that the PgFPS-overexpressing hairy root lines had been
generated successfully, and that they gave rise to high
levels of activity.

Fig. 4 RT-PCR analysis of PgFPS, CaSQS, CaDDS and CaCYS

genes in C. asiatica hairy roots overexpressing the FPS gene (T17,
T24 and T27) after 28 days of cultivation. The control line (C) is a
hairy root transformed by Agrobacterium harboring the empty vector.
The actin fragment generated by primers designed from P. ginseng
was amplified as an internal loading control

squalene synthase) gene. Our results showed that the

CaCYS gene, as the initial step leading to phytosterol
biosynthesis, was upregulated in the hairy roots.

Upregulation of CYS and DDS gene expression

Enhancement of phytosterol biosynthesis
To determine whether or not the overexpression of the
PgFPS gene contributes to the downstream gene expression associated with triterpene biosynthesis, the accumulations of CaDDS (C. asiatica dammarenediol synthase)
and CaCYS (C. asiatica cycloartenol synthase) mRNAs
were RT-PCR-analyzed in hairy roots overexpressing the
PgFPS gene. Higher levels of CaDDS and CaCYS mRNA
were detected in all overexpressing hairy root lines than in
the controls (Fig. 4). However, no differences were
detected in any expression of the CaSQS (C. asiatica

Squalene is the common precursor leading to both triterpene and phytosterol biosynthesis. Seo et al. (2005) have
reported that the overexpression of the Panax ginseng
squalene synthase (PgSS) gene in transgenic Eleutherococcus senticosus stimulated the phytosterol and triterpene
saponin production. To determine quantitatively, the
amount of squalene in C. asiatica hairy roots, GC analysis
was conducted. As shown in Fig. 5, the squalene contents
in the T17, T24, and T27 lines were increased to 1.1, 1.3,

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250

200

ab
150

100

50

T 17

T 24

T 27

Fig. 5 Squalene content in C. asiatica hairy roots overexpressing

FPS (T17, T24 and T27). The control line (C) is a hairy root
transformed by Agrobacterium harboring the empty vector. Vertical
bars indicate the mean SE of three independent samples. Different
letters indicate significant differences at P B 0.05

and 1.5 times that of the controls, respectively. This indicates that the overexpression of PgFPS in C. asiatica hairy
roots causes an increase in squalene content, which can be
considered a positive effect on triterpene and phytosterol
biosynthesis. The levels of campesterol, b-sitosterol and
stigmasterol the principal plant phytosterols (Benveniste
2004) were also analyzed. The total sterol contents in the
T24 line were approximately three times that of the controls (Fig. 6a). The cholesterol contents in the transgenic
roots were also increased by 1.3-fold (Fig. 6b). Therefore,
these results showed that FPS performs a regulatory
function in phytosterol biosynthesis, including cholesterol
biosynthesis.
Triterpene saponin contents in hairy roots overexpressing
PgFPS after MJ treatment
To determine whether or not PgFPS gene overexpression
contributes to triterpene biosynthesis, the contents of three
triterpenes (a-amyrin, b-amyrin, and lupeol) in hairy roots
overexpressing the PgFPS gene were quantified by GC
MS analysis. However, we did not detect these triterpenes
in hairy roots after 4 weeks of cultivation (data not shown).
Alternatively, we suggest that the capacity available for
triterpene saponin biosynthesis is expanded by MJ treatment in the FPS-overexpressing hairy roots, as MJ strongly
induces the expression of several genes associated with the
triterpene pathway, thus resulting in increased triterpene
saponin production (Suzuki et al. 2005; Mangas et al. 2006;
Kim et al. 2009a; Shabani et al. 2009). C. asiatica hairy
root cultures of both the control and T24 were treated with
0.1 mM MJ after pre-culture 4 weeks. The content levels
of major compounds in the PgFPS-overexpressing C. asiatica hairy roots as compared to the controls. As shown in

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Total sterol contents ( g/g, dry wt.)

Plant Cell Rep (2010) 29:403411

Phytosterols content ( g/g, dry wt.)

Squalene contents ( g/g, dry wt.)

408

A
800

T24

T27

700
600

500
400

300
200
100
0

T17

B
500

Cholesterol

450

Campesterol

400

-Sitosterol

ab

a
b

Stigmasterol
350
300
250

200

100

b b

150

c c

a a
b

50
0

T17

T24

T27

Fig. 6 GC analysis of phytosterols in C. asiatica hairy roots

overexpressing the PgFPS gene (T17, T24 and T27) after 4 weeks
of cultivation. The control line (C) is a hairy root transformed by
Agrobacterium harboring the empty vector; a total phytosterols
indicate the sum of cholesterol, b-sitosterol, stigmasterol, and
campesterol, b the level of four sterols in hairy roots. Vertical bars
indicate the mean SE of three independent samples. Different
letters indicate significant differences at P B 0.05

Table 1, content of madecassoside and asiaticoside in the

T24 line was increased by 1.15-fold after 14 days of MJ
treatment, whereas that in the controls was 1.39 times that
of the T24 line after 28 days of elicitation. This result may
indicate that PgFPS supplies carbon flow into the triterpene
and phytosterol pathways, although the two saponins were
only transiently increased.

Discussion
Three lines of C. asiatica hairy root overexpressing PgFPS
have been characterized. The introduction and expression
of an exogenous gene in the hairy roots were confirmed by
southern and northern blot analyses, respectively (Fig. 2).
Furthermore, the results of FPS enzyme assays reflected an

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409

Table 1 Quantitative determination by HPLC of madecassoside and asiaticoside contents in C. asiatica hairy root overexpressing PgFPS
Triterpenoids

After 14 days
Control

After 28 days
T24

Control

T24

Madecassoside

0.151 0.154b

0.169 0.024a

0.221 0.008a

0.140 0.007b

Asiaticoside

0.331 0.008b

0.383 0.010a

0.388 0.012a

0.330 0.008b

C. asiatica hairy root cultures of the control and T24 were treated with 0.1 mM MJ after pre-culture 4 weeks
Values presented are expressed as percentage of dry weight and means with the same letter are not significantly different at 5% by the Duncans
multiple range test

increase in activity in the cytosolic fractions prepared from

the T17, T24, and T27 lines, when compared with the FPS
activity noted in the control transgenic hairy roots (Fig. 3).
In this paper, we attempted to determine whether or not the
PgFPS gene contributes to the triterpene saponins pathway.
C. asiatica hairy roots are considered a good model for
assessing triterpene biosynthesis, as the major saponins are
not detected in the hairy roots of C. asiatica. This suggests
deficiencies in the expressions of genes associated with
triterpene saponin, such as a-/b-amyrin synthase (OSC),
cytochrome P450-independent carboxylase/hydorxylase
(P450) and glucosyltransferase (GT). Thus, C. asiatica
hairy root samples can provide us with valuable information regarding the regulation of triterpene biosynthesis by
the overexpression of a target gene on this pathway.
As shown in Fig. 4, although expressions of the CaCYS
and CaDDS genes were upregulated in the PgFPS geneexpressing transgenic lines, no differences in CaSQS gene
expression were detected. Unexpectedly, the squalene
contents in the three transgenic lines were higher than in
the control transgenic hairy roots (Fig. 5). The squalene
content in the P. ginseng adventitious roots overexpressing
squalene synthase was previously reported to be lower than
in the controls (Lee et al. 2004). In that study, it was
suggested that this might be attributable to a rapid flux of
intermediates toward phytosterol and triterpene biosynthesis. Therefore, it appears likely that squalene without the
upregulation of CaSQS gene expression in transgenic roots
is accumulated as the consequence of the expression of
another isoform of the SQS gene. It has been previously
reported that three SQS genes exist in Glycyrrhiza glabra
(Hayashi et al. 2003) and two squalene synthases, SQS1
and SQS2, were also identified (Hayashi et al. 1999). Thos
researchers suggested that SQS1 and SQS2 may participate
in triterpene and phytosterol biosyntheses to supply carbon
flow into MJ-induced soyasaponin biosynthesis, and to
maintain the flow of carbon into phytosterol biosynthesis,
whereas SQS3 is responsible only for the biosynthesis of
phytosterol. As shown in Fig. 1, squalene is converted into
2,3-oxidosqualene by squalene epoxidase (SE) enzyme.
The accumulation of squalene in C. asiatica hairy roots

overexpressing PgFPS indicates that the volume of it is

more produced than that of consumption by SE enzyme.
Consequently, the accumulation of squalene may contribute to upregulation of gene expression related to both triterpene and phytosterol biosyntheses. Recently, it has been
reported that the effect of squalene treatment on gene
expression associated with ginsenoside biosynthesis (Han
et al. 2010). In that study, squalene feeding resulted in the
accumulation of SE gene together with other genes related
to phytosterol and triterpene biosyntheses.
In higher plants, several papers have noted that the
overexpression of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) gene and the FPS gene resulted
in sterol overproduction (Schaller et al. 1995; Daudonnet
et al. 1997; Harker et al. 2003; Hey et al. 2006). As shown
in Fig. 6a, our results clearly demonstrated that cholesterol,
campesterol, b-sitosterol and stigmasterol are overproduced in PgFPS-expressing C. asiatica hairy roots. This
result is also consistent with the upregulation of the transcription level of the CaCYS gene as the first step in phytosterol biosynthesis (Fig. 4). Therefore, PgFPS performs a
critical role in the flow of carbon into phytosterol biosynthesis. The plant FPS genes comprise a multigene family.
Two genesFPS1 and FPS2encoding for three isoforms
(FPS1S, FPS1L and FPS2) in Arabidopsis thaliana have
been previously isolated (Cunillera et al. 1996, 1997). In
previous studies of the physiological functions of FPS1S
under overexpression and oxidative stress conditions,
reduced cytokinin levels and early senescence in a transgenic plant were noted (Masferrer et al. 2002; Manzano
et al. 2004). In addition, it has been demonstrated that
sterol overproduction is not observed in FPS1S-overexpressing Arabidopsis, thereby suggesting that FPS1S,
which plays a role in the synthesis of the isoprenoids
necessary for basic plant cell functions, is not a limiting
factor in sterol biosynthesis (Manzano et al. 2004).
Chen et al. (2000) previously reported that FPS is a
key regulatory enzyme in sesquiterpene biosynthesis.
However, FPS has not previously been reported to perform
a regulatory role in triterpene biosynthesis. Our HPLC
analysis results demonstrated that two triterpene saponins

123

410

(madecassoside and asiaticoside) were not detected in the

controls or in any of the PgFPS-expressing transgenic
lines. When MJ was applied to hairy root cultures of
C. asiatica overexpressing PgFPS, we anticipated that the
contribution of FPS to triterpene biosynthesis would
become clearer. As deficient genes related to OSCs, P450s,
and GTs are expressed artificially by the elicitation process,
the production of these compounds might be affected. After
14 days of elicitation, madecassoside and asiaticoside were
increased in the PgFPS-expressing C. asiatica hairy root
cultures (Table 1). It appears likely that the overexpression
of FPS would result in the enhancement of synthesis
capacity available for madecassoside and asiaticoside
biosynthesis. However, the levels of two triterpene saponins in the transgenic roots were lower than those of the
control after 28 days of elicitation. This was, presumably,
the reason that the overexpression of FPS resulted in the
feedback-induced suppression of the downstream gene. In
this paper, we present evidence suggesting that FPS participates in triterpene biosynthesis as a regulatory enzyme.
In our previous work, the function of CaDDS (C. asiatica
dammarenediol synthase) gene was characterized in a
lanosterol synthase-deficient yeast mutant (Kim et al.
2009b). The upregulation of CaDDS gene expression in
PgFPS-expressing hairy roots was detected (Fig. 4). This
result may indicate that FPS plays a role in regulation, not
only for phytosterol, but also for triterpene biosynthesis,
although the identification of dammarene-type triterpenes
in C. asiatica has yet to be achieved.

Conclusion
In this work, we have demonstrated that C. asiatica hairy
roots expressing PgFPS, a FPS from Panax ginseng, can
generate enhanced phytosterol contents. Unfortunately, our
results did not demonstrate that FPS is a key regulatory
enzyme in triterpene biosynthesis. However, taking into
consideration the upregulation of CaDDS gene expression
in PgFPS-overexpressing hairy roots and the results of MJ
elicitation on those root samples, it seems plausible that
FPS participates as a key regulatory enzyme in triterpene
biosynthesis. In future studies, it will be necessary to
identify some of the aforementioned unknown compounds,
specifically those whose levels increased in response to MJ
elicitation; it will also be necessary to elucidate the
metabolite profiles of FPS-overexpressing hairy roots. If
this can be achieved, the results may provide us with a
much better understanding of the regulatory mechanisms of
triterpene saponin biosynthesis by genetic engineering,
thus furthering the ultimate objective of large-scale production of a target compound from medicinal plants.

123

Plant Cell Rep (2010) 29:403411

Acknowledgments This work was supported by a Korea Research
Foundation Grant funded by the Korean Goverment (MOEHRD)
(KRF-2007-C00271).

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