Acta Physiol Plant

DOI 10.1007/s11738-010-0546-2

ORIGINAL PAPER

Identification of cDNAs for jasmonic acid-responsive genes

in Polygonum minus roots by suppression subtractive
hybridization
Mian Chee Gor Ismanizan Ismail Wan Aida Wan Mustapha
Zamri Zainal Normah Mohd Noor Roohaida Othman
Zeti Azura Mohamed Hussein

Received: 14 December 2009 / Revised: 10 May 2010 / Accepted: 8 June 2010

Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2010

Abstract Elicitation, the plant-based biotechnology

approach that utilizes the ability of plant roots to absorb
and secrete a vast variety of bioactive compounds, was
studied on Polygonum minus using jasmonic acid (JA) as
an elicitor. To understand the overall molecular responses
of P. minus roots to JA induction, a subtracted cDNA
library was constructed using the suppression subtractive
hybridization (SSH) method. From a total of 1,344 randomly selected colonies, 190 clones were shown to be
differentially expressed using Reverse Northern hybridization. BLAST analysis revealed that clones were similar
to genes associated with the biosynthesis of aromatic
compounds through the oxylipin pathway, such as alcohol
dehydrogenase and lipoxygenase. Putative clones involved
in the shikimate pathway, including S-adenosyl-L-methionine synthetase and S-adenosyl-L-homocysteine hydrolase,
were identified with predicted roles in phenylpropanoids
biosynthesis. Genes responding to abiotic stress unique to

Communicated by S. Abe.
M. C. Gor  I. Ismail  Z. Zainal  N. M. Noor  R. Othman 
Z. A. M. Hussein
School of Biosciences and Biotechnology, Faculty of Science
and Technology, Universiti Kebangsaan Malaysia,
43600 Bangi, Selangor, Malaysia
I. Ismail (&)  Z. Zainal  N. M. Noor  R. Othman 
Z. A. M. Hussein
Institute of Systems Biology (INBIOSIS), Universiti
Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
e-mail: maniz@ukm.my
W. A. W. Mustapha
School of Chemical Sciences and Food Technology,
Faculty of Science and Technology, Universiti Kebangsaan
Malaysia, 43600 Bangi, Selangor, Malaysia

JA elicitation, such as ELI3-1, glutathione S-transferase

and peroxidase 1, were also identified. The kelch-repeat
containing F-box family protein, a possible transcription
factor in response to JA elicitation was also found. The
results of the RT-PCR showed that the eight selected
clones were strongly up-regulated, except for lipoxygenase,
which showed a slightly higher expression of the transcript
levels in response to the JA elicitation.
Keywords Differentially expressed genes  Elicitor 
Secondary metabolites  Aromatic compound 
Abiotic stress

Introduction
Plants produce a wide range of secondary metabolites upon
pathogen infection or in response to herbivore attacks
(Namdeo 2007). Some of these bioactive compounds may
have a novel chemical structure that can be used in the
fields of pharmaceuticals, food additives, cosmetics, perfumes, pesticides, dyes, and fine chemicals (Balandrin and
Klocke 1988). To date, most of these secondary metabolites have been isolated from wild or cultivated plants
because chemical synthesis is either extremely difficult or
not economically feasible. However, the lack of reproducible activity for more than 40% of these plant extracts
(Poulev et al. 2003) has led to the limited commercial
success of the plant phytochemical industry. This major
constraint could be overcome with biotechnological techniques to artificially induce the production of secondary
metabolites in in vitro cultures, such as the use of biotic or
abiotic elicitors.
Polygonum minus Huds. was chosen as the suitable
candidate for this study because it contains highly aromatic

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Acta Physiol Plant

compounds and is rich in essential oils. P. minus, commonly known as kesum, has been recognized by the
Malaysian government in the Herbal Product Blueprint as
an essential oil-producing crop (Wan Hassan 2007). Only
one previous study has investigated in this species,
reporting that the essential oil from kesum leaves was
found to contain about 76.59% aliphatic aldehydes that
consisted of two dominant compounds, decanal and dodecanal, and contained 0.18% b-caryophyllene, a sesquiterpene (Karim 1987). Although many efforts have attempted
to identify chemical compounds from the leaves of other
Polygonum species, the importance of roots should not be
neglected. Roots of many medicinal plants are often used
as crude drugs because they are capable of accumulating
high levels of secondary metabolites that have medicinal
activities (Yazaki 2001). Not surprisingly, some studies
have successfully identified valuable compounds from
Polygonum roots that possessed medicinal properties.
For example, researchers have found phytoestrogens from
P. cuspidatum and P. hydropiper roots (Matsuda et al.
2001), phytohormones, and anthraquinones from P. multiflorum roots (Yu et al. 2006) and indigo from P. tinctorium
roots (Chae et al. 2000).
P. minus is a species that recently gained the attention of
scientists and this is the first study to investigate secondary
metabolite biosynthesis in its roots at the molecular level.
Plant roots are one of the plant parts that are less studied
and are relatively unexploited compared to shoots. Thus,
the unexplored root exudates, which contain diverse
chemical substances, are a target in the search for novel
bioactive compounds for drug discovery and molecular
farming. The emerging biotechnology using root systems is
gaining interest among scientists because of their ability to
import and export molecules between root cells and the
rhizosphere (Gleba et al. 1999). In addition, roots are
physically unprotected in the soil environment, and as they
are surrounded by many microorganisms, roots may produce a vast amount of biological metabolites that possess
antimicrobial or aromatic properties to ensure plant survival (Poulev et al. 2003). Elicitation of hydroponically
grown roots is thus a strategy that could enhance the production of these metabolites or even new compounds that
are not synthesized in normal growth conditions. The
application of elicitors on plant shoots has serious limitations because the hydrophobic surfaces and impermeable
characteristics of leaves result in low uptake of chemical
elicitors. On the contrary, elicitors can be easily added into
growth media and absorbed by roots and can easily harvest
and screen for bioactive compounds. Roots also contain
low levels of pigments and other compounds found in
leaves, such as tannins, which may interfere in chemical
screening and extraction (Gleba et al. 1999; Poulev et al.
2003).

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We are interested in finding the genes involved in the

biosynthesis of aromatic compounds or other valuable
metabolites found in kesum roots using the elicitation
strategy. In this study, JA was chosen as the elicitor to
solicit a genetic response because it is a key endogenous
secondary signal that activates plant-defense-related genes
(Gundlach et al. 1992). JA and its methyl ester, known as
methyl jasmonate (MeJA), have been used extensively to
study the increase of secondary metabolite production in
plants. For example, JA was proven to increase phenylpropanoid and naphtodianthrone production in Hypericum
perforatum L. cell suspensions (Gadzovska et al. 2007) and
anthraquinone accumulation in Morinda elliptica cell cultures (Chong et al. 2005). Also, MeJA was shown to
increase shikonin and dihydroechinofuran production in
Lithospermum cell cultures (Yazaki et al. 1997), silymarin
production in Silybum marianum cell cultures (SanchezSampedro et al. 2005) and polyacetylene accumulation in
Ambrosia maritima hairy roots and cell cultures (Zid and
Orihara 2005). It is clear that JA activity could induce the
biosynthesis of a variety of compounds, including the three
main secondary metabolite classes: phenylpropanoids,
alkaloids, and terpenoids (Pauwels et al. 2009).
To date, most studies of kesum have focused on
phytochemistry. Hence, there has been a serious lack of
understanding on the mechanism of secondary metabolites
production in kesum. Information regarding secondary
metabolite biosynthetic pathways in many plant species is
still very limited and remains unclear. Attempts have been
made to identify the genes encoding the enzymes involved
in the biosynthetic pathways that produce secondary
metabolites (Park et al. 2004; Devitt et al. 2006). Genes
involved in the flavonoid biosynthesis pathway in tea,
such as chalcone synthase, flavone 3-hydroxylase, flavonoid 30 ,50 -hydroxylase, flavonol synthase, dihydroflavonol
4-reductase, and leucoanthocyanidin reductase were successfully identified by the suppression subtractive hybridization (SSH) technique using young and mature tea leaves
(Park et al. 2004). In papaya, genes associated with isoprenoid biosynthesis, such as S-linalool synthase and
hydroxymethylglutaryl-coenzyme A synthase were discovered using the expressed sequence tags (EST) approach.
In addition, cinnamate-4-hydroxylase, which is the core
enzyme in phenylpropanoid biosynthesis, and lipoxygenase
and alcohol dehydrogenase, which are involved in acyl
lipid catabolism pathways were also successfully identified
in the data set (Devitt et al. 2006). From a biological perspective, the combination of root elicitation and SSH
techniques could lead to the discovery of genes involved in
secondary metabolite biosynthesis in plants. Further
manipulation of these genes could create transgenic plants
that are capable of producing high levels of desired natural
products.

Acta Physiol Plant

In our effort to discover genes involved in secondary

metabolite biosynthetic pathways in response to exposure
to JA, the SSH technique was employed to elucidate the
molecular responses of kesum root tissues to JA elicitation.
Identification of the differentially expressed genes in the
JA-treated kesum roots will increase understanding of
the regulation of secondary metabolism and perhaps lead to
the discovery of potential candidate genes for use in future
plant genetic engineering.

Materials and methods

Plant materials and culture conditions
Polygonum minus Huds. plantlets were used for both
JA elicitation and subtracted cDNA library construction.
In vitro plantlets were from the same clone and obtained from
the Malacca Biotechnology Institute (Malacca, Malaysia).
The plantlets were micropropagated from auxiliary buds by
multiple-node stem cutting. The leaves were removed and
the stems were cut into multiple-node segments. Each segment was then cultured in jam jars on Murashige and Skoogs
(MS) (Duchefa, Netherlands) medium (Murashige and
Skoog 1962), supplemented with 30 g l-1 sucrose (Hmbg,
Germany) and then solidified with 0.3% gelrites (Duchefa,
Netherlands). The pH of the MS medium was adjusted to 5.8
with 1 M NaOH (Merck, Germany). The node segments
were grown in a growth chamber at 25 2C with a
photoperiod of 16 h of light and 8 h of darkness. The
plantlets were subcultured at standard intervals of 2 months.

The RNA integrity was monitored by 1.2% (w/v) agarose/

formaldehyde gel electrophoresis. The cDNA was synthesized using the SMART cDNA Synthesis Kit (Clontech,
USA) according to the manufacturers manual. The cDNA
prepared from JA-treated roots was used as a tester while
the cDNA derived from the non-treated roots was served as
a driver during the construction of the cDNA library.
Suppression subtractive hybridization (SSH)
SSH (Diatchenko et al. 1996) was performed using a PCRSelect cDNA subtraction kit (Clontech, USA) according to
the manufacturers protocol. The positive control provided
with the kit was analyzed in parallel with the experimental
samples to reduce false positives. The cDNA obtained was
digested with Rsa I restriction enzyme to form shorter and
blunt-ended fragments. The tester cDNA was divided into
two populations; each of the populations was ligated with
adaptors 1R or 2R accordingly. The driver cDNA was not
ligated with the adaptors. Two rounds of hybridization and
PCR amplifications were performed to reduce the background and to further enrich the differentially expressed
sequences.
Construction of subtracted cDNA library
The subtracted cDNA library was constructed using the
TOPO cloning kit (Invitrogen, USA). The differentially
expressed cDNA sequences were ligated into pCR2.1, a T/A
cloning vector, and were transformed into E. coli TOP10
competent cells. A total of 1,433 white colonies was randomly picked and stored at -80C.

JA elicitation
PCR amplification of cDNA inserts
Approximately 2-month-old in vitro plantlets were used for
JA elicitation. The elicitor was first dissolved in ethanol
(EtOH) and then sterilized by filtration. The in vitro
plantlets were then transferred into jam jars containing
50 ml of the MS liquid medium with 150 lM of JA
(Duchefa, Netherlands). The plantlets were grown hydroponically using the same conditions as described above. At
the same time, control plantlets were grown in an MS
liquid medium without any JA addition to serve as a reference. After 3 days of JA treatment, the roots were harvested, blotted dry and kept at -80C for further RNA
extraction.
Total RNA extraction and cDNA synthesis
The root samples were ground into powder in liquid
nitrogen using mortar and pestle. Total RNA was extracted
from the JA-treated and the non-treated roots following a
previously published method (Pateraki and Kanellis 2004).

Plasmids were extracted using the Montage Plasmid Miniprep96 kit (Qiagen, USA) according to the manufacturers
instructions. The cDNA inserts were amplified with M13
forward and reverse primers. A 25 ll PCR reaction was set
up by mixing 14.9 ll of distilled water, 2.5 ll (109) of PCR
buffer, 4.0 ll (25 mM) of MgCl2, 1 ll (10 lM) of each of
the M13F and M13R primers, 0.5 ll (10 mM) of dNTP,
and 0.1 ll (5 u ll-1) of Taq DNA Polymerase (Promega,
USA). The PCR reactions were pre-denatured at 95C for
5 min, followed by 30 cycles of denaturation at 94C for
1 min, annealing at 55.8C for 30 s, and an extension at
72C for 2 min. The final extension was performed at 72C
for 5 min.
Reverse Northern hybridization of cDNA clones
The cDNA probe was randomly labeled with Digoxigenin11-dUTP for both the driver and the tester cDNA

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Acta Physiol Plant

separately using the DIG nonradioactive nucleic acid

labeling and detection system (Roche, Germany) following
the manufacturers instructions. Two microliters of samples containing 100 ng of DIG-labeled single-stranded
cDNA fragments were arrayed in duplicates onto the nylon
membrane. After air-drying, the cDNA templates were
fixed using a UV-crosslinker (UVP Hybrilinker HL-200)
for 1 min. After probe hybridization and washing steps, the
target cDNAs were detected by incubating the membranes
in a color substrate NBT/BCIP solution in the dark until the
desired signals were seen. The results for the cDNA clones
were screened with both the unsubtracted driver cDNA and
the unsubtracted tester cDNA for comparison. Those with
the most marked differential expression clones were
selected for sequencing.
Sequencing and sequence analysis
The differentially expressed clones identified by Reverse
Northern hybridization were sent for sequencing (FirstBase, Malaysia) using universal M13 forward and reverse
primers. The sequences obtained were compared against
the GenBank database at NCBI (http://www.ncbi.nlm.
nib.gov) using BLAST (Altschul et al. 1997). The cDNA
sequences with an E value \10-5 were considered significant. All of these unique cDNA sequences have been
submitted to the EST division of GenBank with accession
numbers from GR505448 to GR505519 (Table 2) and can
be accessed at the NCBI EST database (http://www.ncbi.
nlm.nih.gov/dbEST).
Semi-quantitative RT-PCR
The mRNA expression pattern of GR505453 (elicitor-activated gene), GR505465 (alcohol dehydrogenase), GR505460
(kelch-repeat containing F-box family protein), GR505459
(glutathione S-transferase), GR505464 (peroxidase), GR505
472 (lipoxygenase), GR505467 (S-adenosyl-L-methionine
synthetase), and GR505471 (S-adenosyl-L-homocysteine
hydrolase) were examined by semi-quantitative reverse transcription-polymerase chain reaction analysis (RT-PCR). The
amplification of ubiquitin 11 (Ubi) cDNA from Arabidopsis
thaliana (NM_116744) was used as an internal control. Two
pairs of oligonucleotide primers were designed to amplify the
cDNA clones that had been selected for the expression analysis
using Primer-Premier 5 software (Table 1).
The unsubtracted driver cDNA and the unsubtracted
tester cDNA were used as templates for PCR amplification.
The PCR reaction was made up of 15.2 ll distilled water,
5 ll PCR buffer 59, 2.0 ll 25 mM MgCl2, 0.5 ll 0.2 mM
dNTPs, 1 ll 10 lM forward primer, 1 ll 10 lM reverse
primer, 1 ll ss cDNA, and 0.2 ll 1 u Taq Polymerase

123

(Promega, USA). For amplification, samples were predenatured at 95C for 3 min, followed by 30 cycles of
1 min at 95C, 30 s at 64C, 1 min at 72C, and a final
extension at 72C for 1 min.

Results and discussions

Identification of JA-responsive genes
In this study, the SSH technique was employed to identify
the genes that were differentially expressed due to JA
elicitation by the construction of a subtractive cDNA
library from the RNA isolated from the untreated roots and
from those treated with 150 lM JA for 3 days. We were
only interested in identifying the genes that were up-regulated when the kesum was exposed to jasmonic acid.
Thus, only the forward subtraction was performed, where
the cDNAs synthesized from the control and the JA-treated
plantlets were used as driver and tester, respectively. A
total of 1,344 white colonies were randomly picked from
the resultant subtractive cDNA library, and the cDNA
inserts were amplified by PCR using the M13 forward and
reverse universal primers. Of those, 960 showed a single
band after PCR amplification. The insert sizes ranged from
250 to 1,200 bp. These clones were subsequently analyzed
by differential screening using Reverse Northern hybridization to reduce false positives.
Of these 960 clones, 195 clones hybridized strongly, 213
clones hybridized moderately, while 552 clones hybridized
weakly with the unsubtracted tester clones (Fig. 1). Almost
all of the clones showed weak or no signal when compared
with the unsubtracted driver probe. These results indicated
that the subtracted cDNA fragments represented differentially expressed genes that were induced by JA in kesum
roots. DNA sequence analysis revealed that 174 out of the
190 clones had readable sequences, in which 130 clones
were unigenes that showed similarity to cDNAs whose
sequences were present in the NCBI database (Table 2).
A total of 18 clones showed no similarity to any known
sequences, and 26 clones had no significant results.
Analysis of JA-induced cDNA sequences
In this study, we report the first set of differentially
expressed genes involved in secondary metabolism of
kesum roots, obtained from a cDNA subtraction library of
JA-elicited roots. All of the 130 unigenes were classified
into 11 categories according to their putative molecular
functions based on our knowledge of plant biochemistry,
plant physiology, and plant molecular biology (Fig. 2). The
largest set of genes was assigned to the stress-related genes

Acta Physiol Plant

Table 1 Gene-specific primers
and primers for ubiquitin as
internal control gene

Genes

Gene-specific primers

Product size (bp)

ELI3-1

F: 50 -TGGTGAAAACAATGAAGCCCAACA-30

243

R: 50 -CGATGGACTATGTGAACACCGCAAT-30
ADH

F: 50 -TGGTTCCTGGTAAAAGCCGTTGC-30

212

R: 50 -CCACGGATTATTCCATTGACCCTG-30
F-box

F: 50 -TCCACTGTTGGGAAACTGCCTGA-30
0

R: 5 -CAATGACTCCTTCACCATAAGCCCTG-3
GST

124
0

F: 50 -TCCACCCAAAATCCAACCGAAT-30

235

R: 50 -TCTTGCCCCGAGACCCTTATCA-30
POD

F: 50 -ATGATTGCTTCGTTCGGGGTTG-30

241

R: 50 -CCTGACGGGACATCGTAGCCAA-30
LOX

F: 50 -TCTTCATAAACTCCCACCCAAAATCC-30

99

R: 50 -TAAGGAGTTGGAGGGGAAGAGGTTT-30
SAMS

F: 50 -AACCCATCAGGTCGCTTCGTCA-30

184

R: 50 -AAAGACGGACAACGGCTCAGGG-30
SHH

F: 50 -AACAGGGGCGTCATTATCTTGGC-30
R: 50 -TGGTTGGTGAAGGAGCACGACA-30

Ubi

F: 50 -TTGAGGACGGCAGAA-30

98
446

R: 50 -GAAAGAAACGGAAACATAG-30

Fig. 1 Differential screening of

clones from the subtractive
library. PCR products were
hybridized with DIG-labeled
cDNA probes derived from
a unsubtracted tester cDNA and
b unsubtracted driver cDNA

(25%). This was followed by the following groups: other

metabolism (20%), unknown genes (9%), transcription
factors (8%), amino acid metabolism (6%), signal transduction and kinase (5%), carbohydrate metabolism (2%),
transporter (2%), energy (2%), and regulation of gene
expression (2%). Clones that showed similarity to the
cDNA sequences from other plant species, but did not have
any specific function, were assigned to the other category
(19%). The functional categories showed that these cDNAs
might be involved in different biological processes. Clones
that showed no similarity to any sequences in the GenBank
were considered as species-specific novel genes and further
characterization is crucial to investigate the function of

these genes. These clones may represent unique genes that

were transcribed in response to the JA treatment that are
involved in the metabolic pathway elicited by JA. Since
kesum is an aromatic plant and little is known about the
expression of genes involved in biosynthesis of aromatic
compounds under natural conditions or under JA stress, we
focused our analysis on these putative clones.
Biosynthesis of secondary metabolites, such as the
production of aromatic compounds, is known to be initiated
by the modification of primary plant metabolism. Therefore, genes corresponding to lipid, amino acid, and carbohydrate metabolism have been considered as enzymes
that are involved in the synthesis of precursors or substrates

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Acta Physiol Plant

Table 2 Putative identities of jasmonic acid-induced cDNA sequences expressed in P. minus roots
Gene Bank
Accession

Size
(bp)

Homology

Organism

Number
of clones

E value

Transcription factor
GR505449

265

F-box containing protein

Populus tremula

3e-12

GR505460

595

Kelch-repeat containing F-box family protein

Arabidopsis thaliana

1e-22

GR505470

423

GAMYB-binding protein (gbp5)

Hordeum vulgare

2e-25

GR505481

298

ERF-like transcription factor

Coffea canephora

2e-09

GR505492

583

BURP domain containing protein

Solanum tuberosum Phaseolus vulgaris

9e-05

8e-06

GR505503 583 BURP domain containing protein

Signal transduction and kinase
GR505514

285

Protein kinase

Malus domestica

1e-55

GR505518

563

Multicopy suppressor IRA1 (MSI1)

Arabidopsis thaliana

3e-48

GR505519

712

Calmodulin-binding protein

Arabidopsis thaliana

7e-116

GR505451

666

MeJA-elicited root cell suspension culture

Medicago trunculata

2e-18

GR505452

539

MeJA-elicited hairy roots culture

Panax ginseng

3e-22

GR505453

503

ELI3-1 (elicitor-activated gene 3)

Arabidopsis thaliana

2e-29

GR505454

589

Auxin-induced protein

Nicotina tabacum

6e-13

GR505455

269

EST from mild drought-stressed leaves

Populus tremula

5e-16

GR505456

267

CDNA clone from senescing leaves

Populus tremula

3e-05

GR505457

406

Cell cultures in osmotic stress

Bouteloua gracilis

0.0

GR505458

669

Gene for heat-shock protein

Glycine max

3e-17

GR505459

656

Glutathione S-transferase (GST14)

Glycine max

2e-21

GR505461
GR505462

570
716

Putative glutathione S-transferase T1

cDNA clones from water-stressed seedlings

Lycopersicon esculentum
Zea mays

3
1

3e-10
2e-07

GR505463

747

Anionic peroxidase H

Zea mays

1e-09

GR505464

546

Peroxidase 1

Scutellaria baicalensis

3e-48

Stress-related

Carbohydrate metabolism
GR505465

684

Alcohol dehydrogenase

Prunus armeniaca

3e-68

GR505448

603

Alcohol dehydrogenase 1 (adh1)

Zea mays

1e-75

GR505466

436

b-fructofuranosidase

Arabidopsis thaliana

2e-14

Acid amino metabolism

GR505467

457

S-adenosyl-L-methionine synthetase

Beta vulgaris

1e-135

GR505468

446

S-adenosyl-L-methionine synthetase

Actinidia chinensis

2e-29

GR505469

443

S-adenosyl-L-methionine synthetase

Elaeagnus umbrellata

6e-18

GR505471

741

S-adenosyl-L-homocysteine hydrolase

Mesembrayanthemum crystallinum

0.0

Other metabolism
GR505473

313

Glyoxal oxidase-related mRNA

Arabidopsis thaliana

5e-06

GR505474

328

Dihydrolipoamide dehydrogenase 1

Arabidopsis thaliana

3e-20

GR505475
GR505476

460
742

Nodulin-35 (N-35) gene encoding a subunit of uricase II

Nodulin family protein (NLP)

Glycine max
Gossypium hirsutum

1
1

3e-09
4e-49

GR505477

520

Glycosyltransferase family protein 47

Arabidopsis thaliana

2e-05

GR505478

524

Cytochrome oxidase subunit 1 (COI) gene

Persicaria maculosa

0.0

GR505479

391

Cytochrome oxidase subunit 1 (COI)

Plumbago sp.

6e-163

GR505480

557

Cytochrome c oxidase

Gossypium barbadense

0.0

GR505482

401

NADH dehydrogenase

Beta vulgaris

0.0

GR505483

344

Urate oxidase

Vitis vinifera

1e-20

GR505484

748

Glucan-endo-1,3-beta-glucosidase

Nicotina tabacum

5e-29

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Table 2 continued
Gene Bank
Accession

Size
(bp)

Homology

Organism

Number
of clones

E value

952

Lipoxygenase (lox gene)

Capsicum annuum

7e-47

GR505485

430

Type-AAA ATPase family protein

Arabidopsis thaliana

1e-41

GR505486

393

Glyceraldehyde-3-phosphate dehydrogenase

Zea mays

3e-11

399

Root-specific metal transporter

Lycopersicon esculentum

5e-12

Zea mays

1e-11

GR505472
Energy

Transporter
GR505487

GR505488
291
Auxin efflux carrier protein
Regulation of gene expression
GR505489

377

Ribosomal protein S12

Fagopyrum esculentum

8e-162

GR505490

675

18S rRNA gene

Polygonum sp. Soltis

0.0

GR505491

497

Unnamed protein product

Vitis vinifera

1e-58

GR505493

538

Hypothetical protein

Zea mays

2e-21

GR505494

714

Hypothetical protein (ORF1)

Eristalis tenax

9e-19

GR505495

495

Hypothetical protein (ORF1)

Platynereis dumerilii

2e-68

GR505496

311

Unknown protein

Arabidopsis thaliana

2e-23

GR505497

696

Hypothetical protein (ORF1)

Culex quinque fasciatus

1e-56

GR505450

480

Hypothetical protein

Vitis vinifera

1e-15

GR505498

352

No homology

GR505499

525

No homology

GR505500
GR505501

489
350

No homology
No homology

1
1

GR505502

314

No homology

GR505504

419

No homology

GR505505

634

No homology

GR505506

662

No homology

GR505507

635

No homology

GR505508

644

No homology

GR505509

656

No homology

GR505510

636

No homology

GR505511

637

No homology

GR505512

657

No homology

GR505513

379

No homology

GR505515

541

No homology

GR505516

381

No homology

GR505517

633

No homology

Unknown genes

Novel genes

for secondary metabolite production. In the present study,

we found a unigene that encoded for lipoxygenase, the first
enzyme in the oxylipin pathway for JA biosynthesis (Devitt
et al. 2006). It was found to be involved in the production
pathways of volatile compounds as the indirect plant
defensive response to herbivory (Kessler and Baldwin
2001). Genes categorized into amino acid metabolism
included enzymes that are involved in phenylpropanoid
biosynthesis pathways, such as S-adenosyl-L-methionine
synthetase and S-adenosyl-L-homocysteine hydrolase

(Dewick 2001). The genes involved in carbohydrate

metabolism encoded alcohol dehydrogenase and b-fructofunasidase. The up-regulation of transcription of alcohol
dehydrogenase may be associated with the biosynthesis of
phenylpropenes, another important group of volatiles
(Devitt et al. 2006). This result suggests that the genes
involved in the biosynthesis of secondary metabolites can
be induced by JA elicitation in kesum roots.
Exogenous JA application has been known as a stress
treatment to plants and served as a stimulus to activate the

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Fig. 2 Clones classification
according to their putative
molecular functions

Others
19%

Transcription
factor
8%

Signal transduction
and kinase
5%

Unknown genes
9%
Stress-related
25%

Regulation of
gene expression
2%
Other metabolism
20%

Energy
2%

Amino acid
metabolism
6%

Transporter
2%
Carbohydrate
metabolism
2%

expression of genes involved in the synthesis of plant

secondary metabolites. As expected, a large number of
clones encoded for stress-related genes were identified
upon JA elicitation in this study. These clones showed
similarity to (1) a glutathione S-transferase, a non-catalytic
carrier that functions to maintain hormone homeostasis or
sequester vacuolar anthocyanin upon pathogen attack
(Moons 2003); (2) an ELI3-1 transcript, an elicitor-activated gene that is transcribed spontaneously in response to
any form of elicitation as a plant-defense mechanism (Ellard-Ivey and Douglas 1996); (3) a peroxidase, which is a
catalytic enzyme induced upon the production of reactive
oxygen species to protect cells against oxidative stress
(Thimmaraju et al. 2006); and (4) an auxin-induced protein, which may play a role in plant growth and development (Stefan et al. 2003). Some clones showed similarity to
the MeJA-induced cDNA sequences in Medicago trunculata (77% identity) and Panax ginseng (69% identity).
Others genes showed similarity to clones that encoded for
proteins involved in abiotic stress, such as heat-shock
proteins, drought stress, osmotic stress, water stress and
leaf senescence cDNA sequences were induced in response
to JA elicitation. This result implies that many different
stress factors will lead to the same gene expression (Sandermann et al. 1998). This result also suggests that JA, or
its derivative MeJA, are signal molecules that regulate
kesum defense responses in response to stressful environments, resulting in the activation of defense-related genes
in plants.
Validation of differentially expressed genes
by RT-PCR
RT-PCR analysis was performed for eight selected clones to
compare the transcript expression between the JA-treated

123

and untreated roots. To verify whether the gene expression

corresponding to the cDNA sequences generated by SSH
were differentially expressed in P. minus under JA stress,
four clones involved in the biosynthesis of aromatic compounds, three clones related to abiotic stress, and one clone
representing a transcription factor were examined. The
clones that showed similarity to genes associated with
aromatic compound biosynthesis were GR505472 (lipoxygenase, LOX), GR505465 (alcohol dehydrogenase, ADH),
GR505467 (S-adenosyl-L-methionine synthetase, SAMS)
and GR505471 (S-adenosyl-L-homocysteine hydrolase,
SHH). The clones that were similar to abiotic stress
response genes were GR505453 (ELI3-1), GR505459
(glutathione S-transferase, GST) and GR505464 (peroxidase, POD) and the clone involved in protein degradation
pathway was GR505460, which had a cDNA sequence
similar to the kelch-repeat containing F-box family protein
(F-box).
In general, the expression patterns were consistent with
the results of the Reverse Northern analysis. The expression patterns can be divided into three types: (1) strong upregulation in JA-treated roots and slight up-regulation in
normal roots, i.e., GR505453 and GR505459; (2) strong
up-regulation in JA-treated roots but very little or no
expression in normal roots, i.e., GR505465, GR505467,
GR505471, GR505464, and GR505460; and (3) slight upregulation in JA-treated roots compared to non-treated
roots, i.e., GR505472. Ubiquitin 11 was used as an internal
control and the expression level did not differ between
samples (Fig. 3).
Three of the kesum sequences from our study showed
significant similarity to the enzymes associated with the
biosynthesis of flavor volatiles. Two clones encoded for
alcohol dehydrogenase (72% identity) and one clone
encoded for lipoxygenase (81% identity), which are

Acta Physiol Plant

JA-treated
samples

non-treated
samples
GR505465 (ADH)

GR505472 (LOX)

GR505467 (SAMS)

GR505471 (SHH)

GR505453 (ELI3-1)

GR505459 (GST)

GR505464 (POD)

GR505460 (F-box)

Ubiquitin 11

Fig. 3 Semi-quantitative RT-PCR analysis of expression patterns of

genes responsive to jasmonic acid metabolism of JA-treated P. minus
roots subtracted library. Expression pattern for each selected clone
was examined in both JA-treated and non-treated roots samples.
Ubiquitin 11 was used as a control to demonstrate equal cDNA used
as templates. Clones involved in aromatic compounds biosynthesis
are GR505465 (ADH alcohol dehydrogenase); GR505472 (LOX
lipoxygenase); GR505467 (SAMS S-adenosyl-L-methionine synthetase) and GR505471 (SHH S-adenosyl-L-homocysteine hydrolase).
Clones related to abiotic stress are GR505453 (ELI3-1 ELI3-1 gene),
GR505459 (GST glutathione S-transferase) and GR505464 (POD
peroxidase). GR505460 is a clone similar to F-box protein (kelchrepeat containing F-box family protein)

enzymes that are involved in the oxylipin pathway. Acyl

lipids are degraded by these two enzymes to become short
carbon chains, such as aldehydes, alcohols, esters, and
ketones (Creelman and Mulpuri 2002). Surprisingly, our
study of kesum roots have further confirmed the results
obtained by Karim (1987), who reported that approximately 76% of the essential oil in kesum leaves consisted
of aliphatic aldehydes, such as decanal and dodecanal,
which contribute to the unique aroma of kesum (Karim
1987). Our results also strengthened the GCMS data

previously reported for P. odoratum leaves (Dung et al.

1995; Hunter et al. 1997). In natural systems, these two
genes may occur in kesum roots but are not routinely
expressed. However, our study showed that their expression in roots could be up-regulated by exposure to JA.
According to a model proposed by Yilmaz (2001) in a
tomato-ripening study, lipoxygenase (EC 1.13.11.12) will
catalyze the deoxygenation of linoleic and linolenic acid
into hydroperoxides and subsequently to aldehydes and
alcohols. Alcohol dehydrogenase (EC 1.1.1.1) will then
catalyze the oxidation of aldehydes to the respective
alcohols or vice versa (Yilmaz 2001). A previous study of
apple volatiles also showed that alcohol dehydrogenase
could catalyze alcohol oxidation and aldehyde reduction
using NAD and NADH as cofactors (Dixon and Hewett
2000). In this study, JA elicitation may have caused the
release of free fatty acids from the root cell membranes, as
JA treatment could mimic the herbivory condition. These
fatty acids may have served as substrates for the production
of volatile aromatic compounds in kesum.
Lipoxygenase has also been isolated from cucumbers
infected by spider mites, and the genes identified were
linked to the formation of the volatile compound (Z)-3hexenyl acetate (Mercke et al. 2004). This gene was also
identified in ripe papayas (Devitt et al. 2006) and apples
(Dixon and Hewett 2000). Alcohol dehydrogenase was also
proposed to be involved in the formation of phenylpropenes, another group of flavor volatiles (Devitt et al. 2006).
Seven clones showed similarity to S-adenosyl-L-methionine synthetase (87% identity) and one clone was similar to
S-adenosyl-L-homocysteine hydrolase (87% identity); these
were involved in the synthesis of aromatic shikimic acid
through the shikimate pathway. Both of these clones demonstrated the type 2 expression pattern, where their expression in kesum roots was only up-regulated upon JA
elicitation and no expression was observed under control
conditions. Shikimic acid is the precursor for phenylpropanoid biosynthesis, another important group of secondary
metabolites (Dewick 2001). Both S-adenosyl-L-methionine
synthetase (SAMS, EC 2.5.1.6) and S-adenosyl-L-homocysteine hydrolase (SHH, EC 3.3.1.1) are enzymes that play
a role in the synthesis of S-adenosyl-L-methionine (SAM),
the main donor of the methyl group for many specific methyl
transferase reactions, such as the transmethylation of alkaloids (Kutchan 1995). In plants, SAM is also associated with
the production of phenylpropanoids (Kawalleck et al. 1992).
Again, our data corroborate previous reports that identified
flavonoids in leaves of the Polygonum family (including
P. minus), which are synthesized by the phenylpropanoid
metabolic pathway (Urones et al. 1990), P. stagninum (Datta
et al. 2002) and P. hydropiper (Peng et al. 2003). The data
suggest that the genes that are normally expressed in leaves
could be triggered by JA in roots.

123

Acta Physiol Plant

On the other hand, SAM is also the intermediate molecule in ethylene and polyamine biosynthesis (Ravanel
et al. 1998). Nevertheless, the expression of these two
genes was also shown to be up-regulated in the petunia
flower and they were linked to the production of benzenoid, a compound that contributes to the flowers scent
(Schuurink et al. 2006). Hence, it was believed that the
expression of the SAMS and SHH genes found in this study
was related to the production of phenylpropanoids,
alkaloids, ethylene or polyamine after JA elicitation. The
activated-methyl cycle was predicted to be closely related
to JA signaling and must be further investigated.
Clones encoding the genes related to abiotic stress,
such as glutathione S-transferase (GST), ELI3-1 and
peroxidase (POD) were evaluated to investigate the correlation between JA stress and other stress factors. Both
GST and ELI3-1 demonstrated a type 1 expression pattern. Ten clones were similar to GST in this subtracted
library. GST (EC 2.5.1.18) is a cytosolic enzyme found in
all eukaryotes. This gene is always detected in stressed
plants, such as heavy metal and salt-treated rice seedlings
(Moons 2003), water-stressed maize seedlings (Zheng
et al. 2004), drought-stressed horse gram (Chandra Obul
Reddy et al. 2008) and fungus-elicited rice seedlings
(Xiong et al. 2001). Therefore, and not surprisingly, the
expression of GST was observed in normal kesum roots
because the in vitro culture itself was a stress condition
for kesum plantlets. It was strongly up-regulated in JAtreated roots as a defense mechanism, suggesting an
overlap in the plant responses of plants to various stress
factors. Also, GST catalyzes the conjugation between
synthetic electrophilic compounds and the glutathione
tripeptide (c-glutamyl-cysteinyl-glycine, GSH). The polar
S-glutathionylated product will be actively transported
into the vacuole by an ATP-binding cassette. Thus, GST
is part of the detoxification mechanism in plants. In fact,
it is the main ingredient for a variety of commercial
herbicides (Andrews et al. 2005). Therefore, it is crucial
to investigate kesum as a potential plant for phytoremediation purposes.
ELI3-1 is another gene related to the abiotic stress
caused by JA elicitation that will lead to phytoalexin and
pathogenesis-related protein accumulation, phenolic compound production, and cell wall reconstruction. Seventeen
of the ELI genes were identified in parsley cells cultured
and treated with the Phytophthora megasperma fungus
(Trezzini et al. 1993). In addition, MeJA elicitation has
successfully activated ELI3, TyrDC, HRGP, and BMT
genes in parsley (Ellard-Ivey and Douglas 1996). This
observation proved that the expression of ELI3-1 could
also be induced by JA elicitation.
Peroxidase (POD, EC 1.11.1.7) plays an important role
in the oxidation process, such as in peroxidative or

123

oxidative oxidation and catalytic hydrosilylation (Umaya

and Kobayashi 2003; Veitch 2004). It is an enzyme that
catalyzes the oxidation of phenylpropanoids (Thimmaraju
et al. 2006). It also functions in the plant-defense system,
and it could be triggered by an elicitor (Gomez-Vasquez
et al. 2004; Perera and Jones 2004). The clones that were
similar to POD in this study showed a type 2 expression
pattern in the RT-PCR analysis. Its expression has been
shown to induce the production of terpenoids in cucumbers
infected by spider mites via oxidative degradation (Mercke
et al. 2004). Our observation corroborates the results
reported in kesum (Karim 1987) and P. odoratum (Dung
et al. 1995; Hunter et al. 1997), where various sesquiterpene hydrocarbons and their oxygenated compounds were
identified. Therefore, it was predicted that stress stimuli,
such as JA, could regulate the induction of important
classes of plant secondary metabolites in kesum. In addition, its oxidative reaction showed that POD can be used as
a component in the reagent for clinical diagnosis and various laboratory experiments (Thimmaraju et al. 2006) and
thus increases the value of kesum.
Interestingly, the kelch-repeat containing F-box family
protein and the TIR1 protein that is contained in the F-box
found in this subtractive cDNA library are induced by JA
(Craig and Tyers 1999; Parry and Estelle 2006). The kelchrepeat containing F-box family protein is involved in the
proteinprotein interaction in the ubiquitin protein degradation process via the ubiquitin-mediated pathway. The
protein degradation process is important to regulate the cell
cycle, transcription, and signal transduction, as a mechanism for the root cells to adapt to JA elicitation stress (Sun
et al. 2007). The functions of these proteins have been
demonstrated in Arabidopsis and they may serve as transcription factors in genes expressed in response to JA
treatment. While these proteins all play the same role in
regulating JA, they also regulate species-specific secondary
metabolite pathways (Pauwels et al. 2009). These proteins
must be characterized and examined for the mechanism
that drives secondary metabolites production in response
to JA.
In conclusion, the subtractive cDNA library data set
presented here provides the first collection of a set of genes
that respond to JA that may be involved in the secondary
metabolite production in kesum roots and also those participating in plant-defense mechanisms. The identification
of genes associated with flavor volatiles and the production
of other aromatic compounds will provide a better understanding of the secondary metabolite biosynthetic pathways and their regulation in kesum. Furthermore, the
observed stress-related genes induced by JA elicitation
indicate that plants respond to abiotic stresses in parallel
with the biosynthesis of certain secondary metabolites.
Characterization of these genes, including those that encode

Acta Physiol Plant

alcohol dehydrogenase, lipoxygenase, S-adenosyl-L-methionine synthetase, S-adenosyl-L-homocysteine hydrolase,

glutathione S-transferase, peroxidase, ELI3-1, and F-box
protein will be studied in details by expression in E. coli
cells, and their functions could be explored with GC or
HPLC to confirm the synthesis of the corresponding
compounds. Studies to identify corresponding genes in
kesum leaves are ongoing and will provide a useful comparison to gain a more complete understanding of secondary metabolites regulation in kesum.
Acknowledgments This work was supported by Ministry of Higher
Education of Malaysia through FRGS grant (UKM-RB-FRGS00012006) awarded to Ismanizan Ismail. Miss Gor Mian Chee is a recipient of the National Science Foundation (NSF) fellowship granted by
Ministry of Science, Technology and Innovation of Malaysia.

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