ORIGINAL PAPER

Identification of cDNAs for jasmonic acid-responsive genesin Polygonum minus roots by suppression subtractivehybridization

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 ran-

domly selected colonies, 190 clones were shown to be

differentially expressed using Reverse Northern hybrid-

ization. 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-methio-

nine synthetase and S-adenosyl-L-homocysteine hydrolase,

were identified with predicted roles in phenylpropanoids’

biosynthesis. Genes responding to abiotic stress unique to

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, per-

fumes, pesticides, dyes, and fine chemicals (Balandrin and

Klocke 1988). To date, most of these secondary metabo-

lites have been isolated from wild or cultivated plants

because chemical synthesis is either extremely difficult or

not economically feasible. However, the lack of repro-

ducible 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 tech-

niques 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

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

123

Acta Physiol Plant

DOI 10.1007/s11738-010-0546-2

compounds and is rich in essential oils. P. minus, com-

monly 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 dode-

canal, and contained 0.18% b-caryophyllene, a sesquiter-

pene (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. multi-

florum 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 pro-

duce a vast amount of biological metabolites that possess

antimicrobial or aromatic properties to ensure plant sur-

vival (Poulev et al. 2003). Elicitation of hydroponically

grown roots is thus a strategy that could enhance the pro-

duction of these metabolites or even new compounds that

are not synthesized in normal growth conditions. The

application of elicitors on plant shoots has serious limita-

tions 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).

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 phenyl-

propanoid and naphtodianthrone production in Hypericum

perforatum L. cell suspensions (Gadzovska et al. 2007) and

anthraquinone accumulation in Morinda elliptica cell cul-

tures (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 (Sanchez-

Sampedro 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, flavo-

noid 30,50-hydroxylase, flavonol synthase, dihydroflavonol

4-reductase, and leucoanthocyanidin reductase were suc-

cessfully identified by the suppression subtractive hybrid-

ization (SSH) technique using young and mature tea leaves

(Park et al. 2004). In papaya, genes associated with iso-

prenoid biosynthesis, such as S-linalool synthase and

hydroxymethylglutaryl-coenzyme A synthase were dis-

covered 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 per-

spective, 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

123

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 seg-

ment was then cultured in jam jars on Murashige and Skoog’s

(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 ± 2�C with a

photoperiod of 16 h of light and 8 h of darkness. The

plantlets were subcultured at standard intervals of 2 months.

JA elicitation

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 hydro-

ponically 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 ref-

erence. After 3 days of JA treatment, the roots were har-

vested, blotted dry and kept at -80�C 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).

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

formaldehyde gel electrophoresis. The cDNA was synthe-

sized using the SMART cDNA Synthesis Kit (Clontech,

USA) according to the manufacturer’s 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 PCR-

Select cDNA subtraction kit (Clontech, USA) according to

the manufacturer’s 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 back-

ground 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 ran-

domly picked and stored at -80�C.

PCR amplification of cDNA inserts

Plasmids were extracted using the Montage Plasmid Mini-

prep96 kit (Qiagen, USA) according to the manufacturer’s

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 95�C for

5 min, followed by 30 cycles of denaturation at 94�C for

1 min, annealing at 55.8�C for 30 s, and an extension at

72�C for 2 min. The final extension was performed at 72�C

for 5 min.

Reverse Northern hybridization of cDNA clones

The cDNA probe was randomly labeled with Digoxigenin-

11-dUTP for both the driver and the tester cDNA

Acta Physiol Plant

123

separately using the DIG nonradioactive nucleic acid

labeling and detection system (Roche, Germany) following

the manufacturer’s instructions. Two microliters of sam-

ples 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 (First-

Base, 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 signif-

icant. 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-acti-

vated 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 tran-

scription-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

(Promega, USA). For amplification, samples were pre-

denatured at 95�C for 3 min, followed by 30 cycles of

1 min at 95�C, 30 s at 64�C, 1 min at 72�C, and a final

extension at 72�C 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-reg-

ulated 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 hybrid-

ization 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 differen-

tially 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

123

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

metabolism (20%), unknown genes (9%), transcription

factors (8%), amino acid metabolism (6%), signal trans-

duction 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. There-

fore, genes corresponding to lipid, amino acid, and car-

bohydrate metabolism have been considered as enzymes

that are involved in the synthesis of precursors or substrates

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-30 124

R: 50-CAATGACTCCTTCACCATAAGCCCTG-30

GST 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 98

R: 50-TGGTTGGTGAAGGAGCACGACA-30

Ubi F: 50-TTGAGGACGGCAGAA-30 446

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

Acta Physiol Plant

123

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 1 3e-12

GR505460 595 Kelch-repeat containing F-box family protein Arabidopsis thaliana 1 1e-22

GR505470 423 GAMYB-binding protein (gbp5) Hordeum vulgare 1 2e-25

GR505481 298 ERF-like transcription factor Coffea canephora 1 2e-09

GR505492 583 BURP domain containing protein Solanum tuberosum Phaseolus vulgaris 2 9e-05

GR505503 583 BURP domain containing protein 4 8e-06

Signal transduction and kinase

GR505514 285 Protein kinase Malus domestica 2 1e-55

GR505518 563 Multicopy suppressor IRA1 (MSI1) Arabidopsis thaliana 1 3e-48

GR505519 712 Calmodulin-binding protein Arabidopsis thaliana 2 7e-116

Stress-related

GR505451 666 MeJA-elicited root cell suspension culture Medicago trunculata 1 2e-18

GR505452 539 MeJA-elicited hairy roots culture Panax ginseng 4 3e-22

GR505453 503 ELI3-1 (elicitor-activated gene 3) Arabidopsis thaliana 1 2e-29

GR505454 589 Auxin-induced protein Nicotina tabacum 7 6e-13

GR505455 269 EST from mild drought-stressed leaves Populus tremula 1 5e-16

GR505456 267 CDNA clone from senescing leaves Populus tremula 1 3e-05

GR505457 406 Cell cultures in osmotic stress Bouteloua gracilis 3 0.0

GR505458 669 Gene for heat-shock protein Glycine max 2 3e-17

GR505459 656 Glutathione S-transferase (GST14) Glycine max 7 2e-21

GR505461 570 Putative glutathione S-transferase T1 Lycopersicon esculentum 3 3e-10

GR505462 716 cDNA clones from water-stressed seedlings Zea mays 1 2e-07

GR505463 747 Anionic peroxidase H Zea mays 1 1e-09

GR505464 546 Peroxidase 1 Scutellaria baicalensis 1 3e-48

Carbohydrate metabolism

GR505465 684 Alcohol dehydrogenase Prunus armeniaca 1 3e-68

GR505448 603 Alcohol dehydrogenase 1 (adh1) Zea mays 1 1e-75

GR505466 436 b-fructofuranosidase Arabidopsis thaliana 1 2e-14

Acid amino metabolism

GR505467 457 S-adenosyl-L-methionine synthetase Beta vulgaris 3 1e-135

GR505468 446 S-adenosyl-L-methionine synthetase Actinidia chinensis 3 2e-29

GR505469 443 S-adenosyl-L-methionine synthetase Elaeagnus umbrellata 1 6e-18

GR505471 741 S-adenosyl-L-homocysteine hydrolase Mesembrayanthemum crystallinum 1 0.0

Other metabolism

GR505473 313 Glyoxal oxidase-related mRNA Arabidopsis thaliana 1 5e-06

GR505474 328 Dihydrolipoamide dehydrogenase 1 Arabidopsis thaliana 2 3e-20

GR505475 460 Nodulin-35 (N-35) gene encoding a subunit of uricase II Glycine max 1 3e-09

GR505476 742 Nodulin family protein (NLP) Gossypium hirsutum 1 4e-49

GR505477 520 Glycosyltransferase family protein 47 Arabidopsis thaliana 1 2e-05

GR505478 524 Cytochrome oxidase subunit 1 (COI) gene Persicaria maculosa 4 0.0

GR505479 391 Cytochrome oxidase subunit 1 (COI) Plumbago sp. 3 6e-163

GR505480 557 Cytochrome c oxidase Gossypium barbadense 3 0.0

GR505482 401 NADH dehydrogenase Beta vulgaris 7 0.0

GR505483 344 Urate oxidase Vitis vinifera 1 1e-20

GR505484 748 Glucan-endo-1,3-beta-glucosidase Nicotina tabacum 1 5e-29

Acta Physiol Plant

123

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-fruc-

tofunasidase. 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

Table 2 continued

Gene Bank

Accession

Size

(bp)

Homology Organism Number

of clones

E value

GR505472 952 Lipoxygenase (lox gene) Capsicum annuum 1 7e-47

Energy

GR505485 430 Type-AAA ATPase family protein Arabidopsis thaliana 1 1e-41

GR505486 393 Glyceraldehyde-3-phosphate dehydrogenase Zea mays 2 3e-11

Transporter

GR505487 399 Root-specific metal transporter Lycopersicon esculentum 1 5e-12

GR505488 291 Auxin efflux carrier protein Zea mays 2 1e-11

Regulation of gene expression

GR505489 377 Ribosomal protein S12 Fagopyrum esculentum 1 8e-162

GR505490 675 18S rRNA gene Polygonum sp. Soltis 2 0.0

Unknown genes

GR505491 497 Unnamed protein product Vitis vinifera 1 1e-58

GR505493 538 Hypothetical protein Zea mays 2 2e-21

GR505494 714 Hypothetical protein (ORF1) Eristalis tenax 1 9e-19

GR505495 495 Hypothetical protein (ORF1) Platynereis dumerilii 2 2e-68

GR505496 311 Unknown protein Arabidopsis thaliana 1 2e-23

GR505497 696 Hypothetical protein (ORF1) Culex quinque fasciatus 3 1e-56

GR505450 480 Hypothetical protein Vitis vinifera 1 1e-15

Novel genes

GR505498 352 No homology 1

GR505499 525 No homology 1

GR505500 489 No homology 1

GR505501 350 No homology 1

GR505502 314 No homology 1

GR505504 419 No homology 1

GR505505 634 No homology 1

GR505506 662 No homology 1

GR505507 635 No homology 1

GR505508 644 No homology 1

GR505509 656 No homology 1

GR505510 636 No homology 1

GR505511 637 No homology 1

GR505512 657 No homology 1

GR505513 379 No homology 1

GR505515 541 No homology 1

GR505516 381 No homology 1

GR505517 633 No homology 1

Acta Physiol Plant

123

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-acti-

vated gene that is transcribed spontaneously in response to

any form of elicitation as a plant-defense mechanism (El-

lard-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 pro-

tein, which may play a role in plant growth and develop-

ment (Stefan et al. 2003). Some clones showed similarity to

the MeJA-induced cDNA sequences in Medicago truncu-

lata (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 (San-

dermann 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 environ-

ments, 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

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 com-

pounds, 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 (lipoxy-

genase, 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 (peroxi-

dase, 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 expres-

sion patterns can be divided into three types: (1) strong up-

regulation 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 up-

regulation 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

Transcription factor

8%

Signal transduction and kinase 5%

Stress-related 25%

Amino acid metabolism 6%

Carbohydrate metabolism 2%

Other metabolism 20%

Transporter 2%

Energy 2%

Regulation of gene expression 2%

Unknown genes 9%

Others 19%

Fig. 2 Clones classification

according to their putative

molecular functions

Acta Physiol Plant

123

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 approxi-

mately 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 GC–MS 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 expres-

sion 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)-3-

hexenyl 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 phenylprop-

enes, another group of flavor volatiles (Devitt et al. 2006).

Seven clones showed similarity to S-adenosyl-L-methio-

nine 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 dem-

onstrated the type 2 expression pattern, where their expres-

sion in kesum roots was only up-regulated upon JA

elicitation and no expression was observed under control

conditions. Shikimic acid is the precursor for phenylpropa-

noid 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-homo-

cysteine 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 alka-

loids (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.

JA-treated non-treated samples samples

GR505453 (ELI3-1)

GR505465 (ADH)

GR505460 (F-box)

GR505459 (GST)

GR505464 (POD)

GR505472 (LOX)

GR505467 (SAMS)

GR505471 (SHH)

Ubiquitin 11

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

genes responsive to jasmonic acid metabolism of JA-treated P. minusroots 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 (LOXlipoxygenase); GR505467 (SAMS S-adenosyl-L-methionine synthe-

tase) 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 (PODperoxidase). GR505460 is a clone similar to F-box protein (kelch-

repeat containing F-box family protein)

Acta Physiol Plant

123

On the other hand, SAM is also the intermediate mol-

ecule 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 benze-

noid, a compound that contributes to the flower’s 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 cor-

relation between JA stress and other stress factors. Both

GST and ELI3-1 demonstrated a type 1 expression pat-

tern. 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 JA-

treated 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 phytoreme-

diation 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 com-

pound 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

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 sesquiter-

pene 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 addi-

tion, its oxidative reaction showed that POD can be used as

a component in the reagent for clinical diagnosis and var-

ious 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 kelch-

repeat containing F-box family protein is involved in the

protein–protein interaction in the ubiquitin protein degra-

dation process via the ubiquitin-mediated pathway. The

protein degradation process is important to regulate the cell

cycle, transcription, and signal transduction, as a mecha-

nism 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 tran-

scription 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 par-

ticipating in plant-defense mechanisms. The identification

of genes associated with flavor volatiles and the production

of other aromatic compounds will provide a better under-

standing of the secondary metabolite biosynthetic path-

ways 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

123

alcohol dehydrogenase, lipoxygenase, S-adenosyl-L-methi-

onine 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 com-

parison to gain a more complete understanding of sec-

ondary metabolites regulation in kesum.

Acknowledgments This work was supported by Ministry of Higher

Education of Malaysia through FRGS grant (UKM-RB-FRGS0001-

2006) awarded to Ismanizan Ismail. Miss Gor Mian Chee is a reci-

pient of the National Science Foundation (NSF) fellowship granted by

Ministry of Science, Technology and Innovation of Malaysia.

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