African Journal of Biotechnology Vol. 10(17), pp.

3260-3268, 25 April, 2011

Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1556
ISSN 16845315 2011 Academic Journals

Full Length Research Paper

Enhanced accumulation of catharanthine and vindoline

in Catharanthus roseus hairy roots by overexpression
of transcriptional factor ORCA2
Dong-Hui Liu1,3, Wei-Wei Ren1, Li-Jie Cui1, Li-Da Zhang1, Xiao-Fen Sun2 and Ke-Xuan Tang1*
1

Plant Biotechnology Research Center, Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, FudanSJTU-Nottingham Plant Biotechnology R & D Center, School of Agriculture and Biology, Shanghai Jiao Tong University,
Shanghai 200240, China.
2
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R
& D Center, Morgan-Tan-International Center for Life Sciences, Fudan University, Shanghai 200433, China.
3
Department of Biochemistry and Molecular Biology, Shenyang Medical College, Shenyang 110034, China.
Accepted 17 December, 2010

The AP2/ERF-domain transcription factor ORCA2 from Catharanthus roseus was demonstrated earlier
to regulate the expressions of Str gene, an important gene involved in the terpenoid indole alkaloids
biosynthetic pathway in C. roseus cells. Therefore, the factor was postulated to play an important role
in the production of secondary metabolites in plants. To investigate the effect of over expression of
ORCA2 on the TIAs biosynthesis in C. roseus hair roots, transformation of ORCA2 gene was conducted
+
with the disarmed Agrobacterium rhizogenes C58C1 harboring pCAMBIA1304 , a plasmid that contains
the Orca2 gene, a Gus gene and an Hpt gene all under the control of the cauliflower mosaic virus 35S
promoter (35S-CaMV). Transgenic hairy root cultures expressing Orca2 gene were obtained and
demonstrated by genomic- polymerase chain reaction (PCR) analysis for the integration of the Orca2
gene in the C. roseus genome, by real-time quantitative PCR (RT-QPCR) and -glucuronidase (GUS)
staining for the expression of the foreign genes. Metabolite analysis using high performance liquid
chromatography (HPLC) analysis established that the average content of catharanthine and vindoline in
the transgenic hairy root extracts was increased up to 2.03 and 3.67-fold in comparison to the control
lines, respectively. However, vinblastine could not been detected in the transgenic and control hairy
root cultures by HPLC.
Key words: Catharanthus roseus, ORCA2, hairy root, overexpression, terpenoid indole alkaloids (TIAs),
AP2/ERF-domain transcription factor.
INTRODUCTION
The Catharanthus roseus plant synthesizes more than
130 different terpenoid indole alkaloids (TIAs). These
TIAs include the dimeric alkaloids vinblastine (VLB) and
vincristine (VCR), which are valuable antitumor agents,
and the monomeric alkaloid ajmalicine, which is used to

*Corresponding author. E-mail: kxtang1@yahoo.com or

kxtang@sjtu.edu.cn. Tel: +86-(0)21 34206916. Fax: +86 (0)21
34205916.

reduce hypertension (van der Heijden et al., 2004). Most

of these TIAs are produced at low levels in the natural
plants and are difficult to be chemically synthesized due
to their complex structures. The need for chemotherapy
treatment of cancers has prompted extensive efforts to
develop inexpensive and efficient approaches for production of these TIAs.
TIAs biosynthetic pathway in C. roseus is complex with
multiple steps and is under strict molecular regulation (Liu
et al., 2007). A variety of TIAs are derived from stricto-

Liu et al.

sidine which is condensed from tryptamine and secologanin and catalyzed by strictosidine synthase (STR). The
tryptamine is converted from tryptophan by tryptophan
decarboxylase (TDC) through the Shikimate pathway
(indole pathway), while the secologanin is derived from
geraniol via the terpenoid pathway. Strictosidine can be
modified by strictosidine glucosidase (SGD) to form
cathenamine, a precursor to various biologically active
alkaloids (Collu et al., 2001). It was approved that there is
an equilibrium between cathenamine and 4,21-dehydrogeissoschizine (Heinstein et al., 1979). Facchini and StPierre (2005) pointed out that many important monoterpenoid indole alkaloids, such as tabersonine and
catharanthine, are produced via 4,21-dehydrogeissoschizine, but their enzymology has not been established.
The biosynthetic route from 4,21-dehydrogeissoschizine
to tabosonine is not completely confirmed. However, it
has been established that tabersonine is transformed to
vindoline through a sequence of six enzymatic steps. In
the final dimerization step of the TIAs biosynthetic pathway, a class III basic peroxidase (CrPRX1) was approved
to catalyze the coupling of the monomeric precursors
vindoline and catharanthine into
-3,4-anhydrovinblastine (AVLB), the common precursor of all dimeric
alkaloids. AVLB is then transferred into VLB and VCR
(Sottomayor et al., 1998).
Promoter analysis of the genes that encode STR and
TDC reveals that both contain sequences involved in the
regulation by stress signals such as UV-irradiation and
fungal elicitors (Ouwerkerk et al., 1999; Pasquali et al.,
1999). Two transcription factors were isolated by yeast
one-hybrid screening with Str promoter sequence (Menke
et al, 1999). The two proteins were called ORCA1 and
ORCA2, for octadecanoid-responsive Catharanthus AP2/
ERF-domain protein (ORCA). Co-transformation experiments showed that transient overexpression of ORCA2
activated the Str promoter, whereas overexpression of
ORCA1 had little effect on Str promoter activity. Transient
expression assays also indicated that ORCA2 transactivated the Str promoter via direct binding and its
expression was rapidly inducible with jasmonate (JA) and
elicitor, whereas ORCA1 was expressed constitutively.
Considering that STR is a very important enzyme in TIAs
biosynthesis and that STR activity is controlled by
expression of ORCA2, therefore, it is necessary to verify
the relationship the between function of ORCA2 and TIAs
biosynthesis in C. roseus cells.
In this study, transcription factor Orca2 gene was transformed into C. roseus hairy root cultures to investigate
the transgenic effect of overexpression of ORCA2 on the
TIAs biosynthesis in C. roseus hairs roots. The results
showed that the transgenic hairy root extracts accumulated more catharanthine and vindoline in comparison
with the control hair root lines, but VLB could not be
detected in the transgenic and non-transgenic hairy root
cultures by HPLC analysis. The reasons for the results
are discussed.

3261

MATERIALS AND METHODS

Construction of plant expression vector
The -glucuronidase (GUS) expression cassette was excised from
the plasmid pBI121 by EcoRI and HindIII (New England Biolabs,
USA) double digestion, and then integrated into plasmid pCAMBIA
1304 (CAMBIA, Canberra, Australia) to form pCAMBIA1304+
(p1304+). The plasmid p1304+ contains two GUS expression cassettes and a hygromycin-resistant gene (hpt) expression cassette,
which are all driven by CaMV35S promoter.
Total RNA was isolated from the one month old seedlings of C.
roseus by CTAB method (Chang et al., 1993). Using oligo (dT)18 as
template primer and the total RNA as template, the first strand of
cDNA was synthesized by AMV reverse transcriptase (Takara
Company, China), and then the second strand of the cDNA was
replicated by Escherichia coli DNA polymerase I after cutting mRNA
into oligonucleotides with RNaseH (Takara Company, China).
Based on the CDS sequence of Orca2 gene (Genbank Accession
No. AJ238740), forward primer FO2 5- GAAGATCTATGTATCAA
TCAAATGCCCATAATTCC-3(Bgl II) and reverse primer RO2 5GGGTCACCTTATTGAGGACGAAGATGACACG-3(BstE II) were
designed and synthesized for the amplification of Orca2 gene. The
primer sequences contained BglII or BstEII restriction endonuclease
site separately. C. roseus cDNAs were used as template and the
PCR was performed with PrimeSTAR HS DNA polymerase kit
(Takara Company, China) by denaturing at 94C for 1 min, followed
by 30 cycles of amplification (98C for 10 s, 55C for 15 s and 72C
for 60 s) and then a 10 min final extension at 72C. The PCR
fragment was then cloned into plasmid pMD18-T (Takara Company,
China) to form plasmid pMD18-T-Orca2. After confirming the
sequence, the Orca2 gene was excised from pMD18-T-Orca2 by
BglII / BstEII double digestion.
Lastly, plant expression vector p1304+-Orca2 was constructed by
replacing a gus gene in the plasmid p1304+ with the Orca2 gene by
BglII / BstEII double digestion. The vector p1304+-Orca2 was transformed into the disarmed Agrobacterium rhizogenes C58C1 strain
carrying the plasmid pRiA4 of A. rhizogenes, and the resulting A.
rhizogenes C58C1 strains was used for the transformation study.
Cultures conditions and genetic transformation
Seeds of C. roseus, purchased from PanAmerican Seed Company
(Cherry Red, USA), were surface sterilized and placed on MS solid
medium (1962) in the greenhouse at 25C for germination. Young
leaves from one month old germinated seedlings of C. roseus were
broken with sterile surgical knife and pre-incubated on half-strength
MS solid medium for 2 to 4 h, and then cultivated with the A.
rhizogenes C58C1 strain (OD600 = 0.7) containing vector p1304+Orca2 for co-cultivation. After 48 h co-cultivation, the leaves were
maintained on the regulator-free half-strength MS solid medium
containing 500 mg/l cefotaxime to eliminate bacterial contamination.
Two weeks after the C58C1 strain infection, hairy roots were
induced from the wounded edges and surface of the leaf explants.
Single transformed roots were excised when they grew over 2 cm in
length and were maintained separately as independent clone. The
hairy root lines were grown at 25C in the dark and were routinely
subcultured to fresh regulator-free half-strength MS solid medium
every two weeks. After two months of subculture on solid medium,
hairy root cultures were obtained and transferred individually into
the regulator-free half-strength B5 liquid medium for continuous
subculture. All hairy root cultures were kept at 25C on a rotary
shaker at 100 rpm in the dark. After 30 days of subculture in liquid
medium, the hairy root cultures were filtered, washed with 10 ml
sterile distilled water and lyophilized immediately in liquid nitrogen
for molecular analysis and TIAs extraction. The control hairy root
lines were generated by transforming the leaf explants with C58C1

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Afr. J. Biotechnol.

RB

LB
poly

Hpt

35S

NO S

Gus

35S

35S

Orca 2

NO S

Figure 1. Schematic map of T-DNA region in plant binary expression vector p1304+-Orca2. LB, Left border; RB, right
border; 35S, CaMV 35S promoter from cauliflower mosaic virus; NOS, the polyadenosyl signal of the nopaline synthase
gene; poly, CaMV 35S poly-A terminator; Hpt, hygromycin-resistant gene; Gus, -glucuronidase (GUS) gene; Orca2
and Orca2, gene from C. roseus.

strain lacking vector p1304+-Orca2 and were grown for subculture

as earlier described.
Polymerase chain reaction (PCR) analysis
The bacterium-free hairy root lines were collected, dried on sterile
filter paper and quickly frozen in liquid nitrogen. Total genomic DNA
was isolated from putative transgenic and control hairy root lines by
using the CTAB DNA isolation method (Woodhead et al., 1998).
The DNA samples were then used in PCR analysis for detecting the
presence of Agrobacterium rol (rolB, rolC) genes and hpt gene in
transgenic hairy root cultures. Oligonucleotide primers for the PCR
detection of rolB, rolC and hpt gene were designed based on the
DNA sequences of these genes described by Fumer et al. (1986).
For the amplification of rol genes (rolB, rolC), primers FrolB (5GCTCTTGCAGTGCTAGATTT-3), RrolB (5-GAAGGTGCAAGCTA
CCTCTC-3), FrolC (5-CTCCTGACATCAAACTCGTC-3) and
RrolC ( 5-TGCTTCGAGTTATGGGTACA-3 ) were used. For the
detection of the hpt gene, Fhpt (5-CGATTTGTGTACGCCCGACA
GTC-3) and Rhpt (5-CGA TGTAGGAGGGCGTGGATATG-3)
were used. PCR for the detection of all the above genes was
carried out by denaturing the template at 94C for 3 min followed by
35 cycles of amplification (94C for 40 s, 55C for 30 s and 72C for
1 min) and then a 10 min final extension at 72C. The PCR
products were separated by electrophoresis in 0.8% (w /v) agarose
gel. Plasmid DNA from A. rhizoenes strain containing plasmid
p1304+-Orca2 was used as positive control and genomic DNA from
untransformed C. roseus root was used as a negative control in
PCR analysis.

Real-time quantitative analysis (RT-QPCR)

After 30 days of subculture in liquid medium, total RNA was
extracted separately from the putative transgenic and control hairy
root cultures with plant RNA mini kit (Watson Company, Shanghai,
China), and treated with RNase-free DNase (Takara Company,
China) to eliminate the potential contaminating residual DNA. The
quality and concentration of RNA samples were tested by agarose
gel electrophoresis and spectrophotometer analysis. Total RNA was
reversely transcribed by using AMV reserve transcriptase (Takara
Company, China) to generate cDNA, which was then subjected to
RT-QPCR analysis for the expression of Orca2 gene. RT-QPCR
was performed on a RoterGene 3000 instrument (Corbett
Research, Sydney, Australia). The gene-specific primers FOrca2
(5-GATCAGGATAATTACGAAGACGAAGT-3) and ROrca2 (5AGTTCCCAACCATATCCTCGATCCTT-3) were designed according to the conserved sequence of C. roseus Orca2 gene and were
used to amplify the Orca2 gene. Ubiqitin gene (house-keeping
gene) was used as an internal calibrator. FUbi (5-GTGACAA
TGGAACTGGAATGG-3) and RUbi (5-AGACGGAGGATAGCGTG
AGG-3) were used as primers to amplify the Ubiqitin gene. The RTQPCR was carried out with SYBR PrimeScript RT-PCR kit

(Perfect Real Time) according to manufacturers instructions

(Takara Company, China) as follows: 1 min predenaturation at
95C, 1 cycle; 10 s denaturation at 95C, 30 s annealing at 56C
and 15 s collection fluorescence at 72C and 42 cycles. The
products of RT-QPCR were run on 1.0% (w/v) agarose gel
electrophoresis and it showed an equal-sized band as predicted.
Quantification of the gene expression was done with comparative
computed tomography (CT) method. The quantitative analysis was
repeated three times for each sample.
GUS histochemical assay
GUS assays of the hairy roots were performed by histochemical
staining as described by Jefferson et al. (1987) with slight
modifications. Both the putative transgenic and the negative control
hairy root segments (about 10 mm in length) were incubated at
37C in the dark in an X-Gluc solution. The X-Gluc solution
contained 50 mM Na3PO4 (pH 7.0), 10 mM Na2EDTA, 0.1% (v/v)
Triton X-100, 0.1 M K3[Fe(CN)6], 0.1 M K4[Fe(CN)6], 0.25 mM L-1 5bromo-4-chloro-3-indolyl-b-D-glucuronide (X-Gluc) and 20%
methanol. The roots were subsequently washed in an ethanol
gradient at room temperature (30 min in 70% ethanol, 30 min in
40% ethanol and then 30 min in 20% ethanol). After rehydration,
the roots were kept in water and then mounted on a slide for
observation and photography.
Alkaloids
extraction
and
high
chromatography (HPLC) analysis

performance

liquid

The 30 day old transgenic and control hairy root samples cultured
in half-strength B5 liquid medium were harvested and lyophilized
overnight, respectively. The resulting dried roots were weighted,
ground to very fine powder using a mortar and pestle, and extracted
three times at room temperature with 10 ml of MeOH for 1 h in a
sonicating bath. The mixture was centrifuged at 13000 g for 15 min
at 15C (Singh et al., 2000). The supernatant was removed and the
biomass was re-extracted again prior to HPLC analysis.
The alkaloid analysis of C. roseus hairy root samples was performed on a Waters Alliance HPLC system (Alliance model 2690;
Waters Corporation, Milford, MA, USA) and separated using a C18
column with binary gradient mobile phase profile (55% 5 mmol/l
pH6.0 sodium phosphate buffer, 45% acetonitrile) (Singh et al.,
2000; Tikhomiroff and Jolicoeur, 2002). Extracts were analyzed by
HPLC with a photodiode array detector (Model 996, Waters) to
verify the identity and purity of peaks of interest. HPLC with UV
detection at a single wavelength only was employed for quantifycation of TIAs. An aliquot of 10 l injection volume provided
adequate signal at 220 nm. Authentic standards of catharanthine,
vindoline and vinblastine (Sigma, USA) were prepared separately in
methanol at a final concentration of 5 g/l and used for the
preparation of the calibration graphs. Quantification was repeated
three times for each sample.

Liu et al.

3263

Figure 2. The induction and subculture of

transgenic hairy root cultures; (A), Hairy roots
were induced from the leaf explants C. roseus
and were cultured on MS solid medium; (B),
hairy roots growing in half strength B5 liquid
medium in a 250 ml Erlenmeyer flask for ten
days; (C), hairy roots were cultivated in the
liquid medium for one month.

RESULTS
Establishment and subculture of C. roseus hairy root
cultures
+

The plant expression vector p1304 -Orca2, harboring the

coding region of the wild-type C. roseus Orca2 gene and
two selectable marker genes (hpt and Gus) on the same
T-DNA fragment, was constructed (Figure 1) and transformed into disarmed Agrobacterium tumefaciens C58C1
strain carrying the plasmid pRiA4 of A. rhizogenes.
+
Integration of Orca2 gene into the vector p1304 and

transformation of vector p1304 -Orca2 into C58C1 strain

was confirmed separately by PCR analysis and sequencing. Two weeks after infecting the leaf explants, hairy
roots were induced from the wounded edges and surface
of the leaves. Ten days later, the percentage of leaf
explants to form hairy root lines was about 70%.
50 putative transgenic hairy root lines and 30 control
hairy root lines were excised separately when they grew
over 2 cm in length and transferred to the fresh regulatorfree half-strength MS solid medium that contained 500
mg/l cefotaxime to eliminate bacterial contamination.
These hairy root lines were cultured in the dark at 25C
and were then routinely subcultured to the same MS solid
medium every two weeks (Figure 2A). After two months
of subculture, 38 independent putative transgenic hairy
root cultures and 16 control hairy root cultures were obtained and transferred individually into the regulator-free
half-strength B5 liquid medium for subculture. In
comparison with growth on the solid medium, hairy roots
cultured in the liquid medium grew more rapidly and had
higher lateral branching (Figures 2B and C). However,
the hairy root cultures changed gradually from white to
red-brown during the subculture, and it could be observed that a few of red-brown substance was secreted
from hairy roots into the culture medium after 3 to 4
weeks. As a control, adventitious roots excised from C.
roseus sterile seedlings were cultured on the growth
regulator-free half-strength MS solid medium, but these
roots grew very slowly, did not branch and perished after
2 or 3-week subculture period.
Of the 38 putative transgenic hairy root cultures, only
12 cultures could be maintained in liquid media after one
month of subculture. At the same time, 8 control hairy
root cultures were obtained. These cefotaxime-resistant
hairy root lines (12 putative transgenic and 5 control hairy
root cultures) were evaluated for growth, integration and
expression of Ri plasmid T-DNA genes, and alkaloid
contents in dry hairy root samples.
Molecular analysis of the transgenic hairy root
cultures
By using the genomic DNA from the putative transgenic
and the control hairy root cultures as template, integration
of the rol genes (rolB, rolC) and hpt gene into the genome of C. roseus hairy root cultures was confirmed by
PCR analysis (Figure 3). As expected, it was demonstrated that three fragments, with lengths of 423, 622 and
812 bp corresponding to rolB, rolC and hpt gene,
respectively, were amplified only from the putative
transgenic hairy root cultures but not from the control
hairy root samples. These results indicated that the rolB
and rolC genes from the Ri plasmid of A. rhizogenes
C58C1 and the hpt gene from the plant expression vector
+
p1304 -Orca2 were all integrated into the genome of
transgenic C. roseus hairy root cultures.
Real time-PCR is the most sensitive method for quanti-

3264

Afr. J. Biotechnol.

Relative mRNA Expression

Figure 3. PCR analysis for the presence of rolB, rolC and hpt gene in
independently transgenic hairy root cultures. M, DL2000 marker; lane +,
plasmid p1304+-Orca2 was used as positive control; lane -, untransformed C.
roseus root DNA was used as a negative control; lanes 1 to 12, 12 individual
hairy root cultures transformed with Orca2 gene.

Figure 4. Relative mRNA expression levels of Orca2 gene in transgenic hairy root cultures of
C. roseus checked by real-time PCR method. The mRNA expression value in untransformed
control sample was 1.0; O2-1 to O2-12, 12 individual hairy root cultures transformed with
Orca2 gene; Data shown are means standard deviation of three replicate measurements.

tation of gene expression levels. SYBR green I-based

quantitative real-time PCR method was used to characterize the Orca2 gene relative expression status in
transgenic C. roseus hairy root cultures. The results
indicated that the expression levels of Orca2 gene in 12
putative transgenic hairy root samples were different.
Among them, the samples No.4, No.7 and No.12 expres-

sed the maximum level of Orca2 gene (3.646-, 3.96- and

3.19-fold) in comparison with the control samples, respectively, while the sample No.10 expressed the lowest
level of Orca2 gene in all hairy root samples (the value is
about half as much expression as in the control samples)
(Figure 4). The fact indicated that expression of Orca2
gene was inhibited in the transgenic hairy root sample

Liu et al.

Figure 5. GUS staining results of C. roseus hairy root

cultures. C58C1, non-transformed (negative control) hairy root
cultures induced from C58C1; No.1, No.7, Number 1 and
Number 7 of hairy root cultures transformed with Orca2 gene,
respectively.

No.10. After detecting the gene relative expression of

Orca2 gene, the seven hairy root cultures with high
expression level were used for HPLC analysis.
In conclusion, molecular analysis experiments for the
hairy root cultures proved that the T-DNA from the disarmed A. rhizogenes C58C1 strain containing vector
+
p1304 -Orca2 had been integrated into all the putative
transgenic hairy root cultures.
GUS histochemical assay
To monitor transgenic expression in the hairy root cultures, histochemical GUS activity assays were performed
with seven putative transgenic and five negative control
hairy root cultures from cefotaxime-resistant hairy root
lines. All the putative transgenic hairy root cultures were
stained blue and different samples showed diverse GUS
activity with different blue spots. The control hairy root
samples showed no significant GUS activity (Figure 5).
These results indicate that the Gus gene in the T-DNA
had already been integrated into the genome of the
transgenic C. roseus hairy root cultures.
HPLC analysis of TIAs accumulation in hairy root
cultures
TIAs profiles (catharanthine, vindoline and VLB) of the
putative seven hairy root cultures (samples No.1, 2, 4, 6,
7, 8, 12) with relative high level expression of Orca2
gene, and negative control hairy root cultures were determined by HPLC analysis in this study. The results showed that the putative transgenic hairy root extracts accumulated more catharanthine and vindoline in comparison

3265

with the control hair root cultures. However, VLB content

could not be detected in the transgenic and control hairy
root cultures by HPLC method. The average catharanthine content in the transgenic hairy root extracts was
4.7869 0.59 mg/g DW; over 2.03-fold higher than that in
the control cultures (2.357 0.415 mg/g DW). The
sample No.7 accumulated the maximum level of catharanthine content (5.986 0.672 mg/g DW) among the
seven samples, while the sample No.4 accumulated the
lowest level of catharanthine content (3.815 0.376 mg/g
DW) (Figure 6, upper panel). The average vindoline
content in the putative transgenic hairy root cultures were
0.144 0.0157 mg/g DW, over 3.67-fold higher than that
in the control cultures (0.0392 0.0054 mg/g DW). It is
noteworthy that the transgenic sample No.2, but not the
sample No.7, accumulated the highest level of vindoline
content (0.1888 0.0024 mg/g DW) (Figure 6, lower
panel).
DISCUSSION
Transcription factors are regulatory proteins that modulate the expression of specific groups of genes through
sequence-specific DNA binding and protein-protein
interactions. They can act as activators or repressors of
gene expression, mediating either an increase or a
decrease in the accumulation of messenger RNA (Broun,
2004). More than ten transcription factor genes have
been cloned and characterized in C. roseus till now.
Among them, Orca1, Orca2 and Orca3 were characterized to control closely the expression of some genes
involved in TIAs biosynthesis. ORCA1 had been
approved to be expressed constitutively and had little
effect on Str promoter activity. ORCA2 activated the Str
promoter and its expression was rapidly inducible with
jasmonate (JA) and elicitor (Menke et al., 1999). Orca3
was isolated via a T-DNA activation tagging approach
applied to a C. roseus cell culture (van der Fits and
Memelink, 2000; van der Fits et al., 2001) and activates
Str gene expression by binding to the special sequence
of Str gene promoter (van der Fits and Memelink, 2001).
Overexpression of ORCA3 in C. roseus cultured cells
increased the expression of the TIA biosynthesis genes
Tdc, Str, Sgd, Cpr, D4h, As and Dxs. However, ORCA3
was not found to regulate G10h and Dat gene. The fact
indicates that ORCA3 is a central regulator of TIA biosynthesis, acting on several steps of the TIA pathway and
also regulating the biosynthesis of TIA precursors
(Memelink and Gantet, 2007). The function of ORCA3
had been approved in C. roseus cultured cells, however,
the function of ORCA2 during the TIAs biosynthesis has
not been reported till now. Therefore, Orca2 gene was
chosen to transform C. roseus leaves to investigate the
transgenic effect of overexpressing ORCA2 on the TIAs
biosynthesis in C. roseus hairs roots. The results indicated
that the accumulation of catharanthine and vindoline

Afr. J. Biotechnol.

Vindoline content (mg/g DW)

Catharanthline content
(mg/g DW)

3266

Figure 6. Catharanthline (upper panel) and vindoline contents (lower panel) in the transgenic
hairy root cultures of C. roseus detected by HPLC. Con, untransformed (negative control) hairy
root culture; O2-1,02-2,02-4,02-6,02-7,02-8,02-12, seven individual hairy root cultures
transformed with Orca2 gene; Data shown are means standard deviation of three replicate
measurements.

are enhanced by overexpressing ORCA2 in C. roseus

hairs roots.
It had been approved that catharanthine is distributed
equally throughout the aboveground and underground
tissues of C. roseus. However, vindoline, tabersonine as
well as the dimeric alkaloids are restricted to leaves and
stems because of the late steps of vindoline biosynthesis
which require specialized cell types, idioblast and laticifer
cells, which are located in stems and leaves
(Westekemper et al., 1980; Deus-Neumann et al., 1987).
Therefore, many cell and hairy root cultures produce
catharanthine and tabersonine, but do not produce
vindoline because there is a limitation in the conversion
from tabersonine to vindoline (Shanks et al., 1998). This

fact had been confirmed by some experiments (Bhadra

and Shanks, 1997; Brillanceau et al., 1989; Toivonen et
al., 1989).
In this study, however, the results demonstrated that
the transgenic hairy root extracts accumulated 2.03 and
3.67-fold of catharanthine and vindoline more than the
control hairy root cultures, respectively. Previously, Parr
et al. (1988) reported a similar result: an experimental
detection by immunoassay demonstrated that ajmalicine,
serpentine, vindolinine and catharanthine were prominent
components in C. roseus hairy root cultures during all
stages of the growth cycle. Vinblastine could also be
detected by a combination of HPLC and radioimmunoassay, though at a low level (0.05 g/g DW). They

Liu et al.

speculated that the differentiated characteristics of the

hairy roots have offered the potential for the production of
various monomeric indole alkaloids. It can be proposed
that the main reason for these results is the activation of
the enzymes in vindoline biosynthesis pathway in hairy
root cultures by weak light during their subculture in liquid
medium, since it was found that the hairy root samples
had been lightened by some weak scattered light around
in the culture room. It was noticed that some early
experiments had already proven that light plays a critical
role in TIAs biosynthesis in C. roseus plants during their
growth and development. Further studies showed that
phytochrome is involved in the activation of the last two
enzymes in vindoline biosynthesis; D4H (Vazquez-Flota
and De Luca, 1998) and DAT (Aerts and De Luca, 1992),
in Catharanthus seedlings. In addition, Ramani and
Jayabaskaran (2008) reported that catharanthine and
vindoline increased 3 and 12-fold, respectively, on
treatment with a 5-min UV-B irradiation in the suspension
cultures of C. roseus. The other possible reason for the
results is that the overexpression of ORCA2 in the hairy
root might have triggered the flow of TIAs pools towards
AVLB biosynthesis in which catharanthine and vindoline
are needed as precursors. Therefore, our next research
would focus on investigating the exact mechanism of light
on the vindoline biosynthesis in C. roseus hairy root cultures and on the metabolic flow during TIAs biosynthesis
in C. roseus.
ACKNOWLEDGEMENTS
This work was supported by the China 973 Program
(grant number 2007CB108805), China 863 Program
(grant number 2010AA100503), China Transgenic Research Program (grant number 2008ZX08002-001) and
the Shanghai Leading Academic Discipline Project
(project number B209).
Abbreviations
TIAs, Terpenoid indole alkaloids; PCR, polymerase chain
reaction; MS, Murashige and Skoog; ORCA,
octadecanoid-responsive Catharanthus AP2/ERF-domain
protein; CTAB, cetyltrimethyl ammonium bromide; HPLC,
high performance liquid chromatography; RT-QPCR,
real-time quantitative PCR; GUS, -glucuronidase; STR,
strictosidine synthase; TDC, tryptophan decarboxylase.
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