ORIGINAL PAPER

Stress and developmental responses of terpenoid biosyntheticgenes in Cistus creticus subsp. creticus

Irene Pateraki • Angelos K. Kanellis

Received: 28 January 2010 / Revised: 5 March 2010 / Accepted: 19 March 2010 / Published online: 3 April 2010

� Springer-Verlag 2010

Abstract Plants, and specially species adapted in non-

friendly environments, produce secondary metabolites that

help them to cope with biotic or abiotic stresses. These

metabolites could be of great pharmaceutical interest

because several of those show cytotoxic, antibacterial or

antioxidant activities. Leaves’ trichomes of Cistus creticus

ssp. creticus, a Mediterranean xerophytic shrub, excrete a

resin rich in several labdane-type diterpenes with verified

in vitro and in vivo cytotoxic and cytostatic activity against

human cancer cell lines. Bearing in mind the properties and

possible future exploitation of these natural products, it

seemed interesting to study their biosynthesis and its reg-

ulation, initially at the molecular level. For this purpose,

genes encoding enzymes participating in the early steps of

the terpenoids biosynthetic pathways were isolated and

their gene expression patterns were investigated in differ-

ent organs and in response to various stresses and defence

signals. The genes studied were the CcHMGR from the

mevalonate pathway, CcDXS and CcDXR from the meth-

ylerythritol 4-phosphate pathway and the two geranylger-

anyl diphosphate synthases (CcGGDPS1 and 2) previously

characterized from this species. The present work indicates

that the leaf trichomes are very active biosynthetically as

far as it concerns terpenoids biosynthesis, and the terpenoid

production from this tissue seems to be transcriptionally

regulated. Moreover, the CcHMGR and CcDXS genes (the

rate-limiting steps of the isoprenoids’ pathways) showed an

increase during mechanical wounding and application of

defence signals (like meJA and SA), which is possible to

reflect an increased need of the plant tissues for the cor-

responding metabolites.

Keywords Cistus creticus subsp. creticus �Secondary metabolism � Terpenoid biosynthesis �Hydroxymethylglutaryl coenzyme A reductase (HMGR) �1-deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS) �DXP reductoisomerase (DXR) � Geranylgeranyl

diphosphate synthase (GGDPS) � Gene expression

Introduction

Cistus creticus subsp. creticus is a perennial, dimorphic,

Mediterranean plant, found mainly in arid and warm areas

like maquis and garigues ecosystems. Its drought resistance

properties and the adaptation to non-friendly environments

partly are due to the dimorphic characteristics of this species

and the glandular and non-glandular trichomes that cover the

adaxial and abaxial surfaces of its leaves (Aronne and De

Micco 2001; Gulz et al. 1996). The leaf glandular trichomes

secret a resin called ‘‘ladano’’, which displays ‘‘therapeuti-

cal’’ properties. Responsible compounds for these properties

have found to be mainly several labdane-type diterpenes

isolated from this resin. More specifically, the labdane

diterpenes (13E)-labda-13-en-8a,15-diol and (13E)-labda-

7,13-dienol exhibited cytotoxic activity against human leu-

kaemic cell lines (Matsingou et al. 2006). Sclareol, another

metabolite from this class [(13R)-labda-14-en-8,13-diol]

Communicated by J. R. Liu.

I. Pateraki � A. K. Kanellis (&)

Group of Biotechnology of Pharmaceutical Plants,

Laboratory of Pharmacognosy, Department of Pharmaceutical

Sciences, Aristotle University of Thessaloniki,

541 24 Thessaloniki, Greece

e-mail: kanellis@pharm.auth.gr

Present Address:I. Pateraki

Departament de Bioquımica i Biologia Molecular,

Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain

123

Plant Cell Rep (2010) 29:629–641

DOI 10.1007/s00299-010-0849-1

induced apoptosis in leukaemia T cell lines and in human

breast cancer cells (Dimas et al. 2006). Enhanced antitumor

activities of (13E)-labda-13-en-8a,15-diol and sclareol were

recently reported by the use of liposomal technology, as a

drug carrier system of these metabolites in vivo (Hatzian-

toniou et al. 2006).

Moreover, it has been shown that metabolites with

thermotolerant or antioxidant activities, belonging to the

class of isoprenoids produced from plant species adapted in

hostile environments (like C. creticus) namely Salvia

species, Rosmarinus officinalis or Quercus ilex, are able

to help the plants to overcome environmental stresses

(Munne-Bosch and Alegre 2003). Taking in mind the

properties of the above-mentioned terpenes produced from

C. creticus but also the plant properties and the fact that

this plant species is possible to produce a number of

metabolites for its own defence, unidentified till today,

with interesting pharmaceutical properties, it seemed

interesting to study the biosynthetic pathways of these

metabolites in the specific plant. Expression sequence tags

(EST) analysis from a cDNA library from C. creticus

trichomes revealed that these tissues are biosynthetically

active in secondary metabolites production and more spe-

cifically in terpenoids and flavonoids (Falara et al. 2008).

cDNA sequences resulting from the above analysis, toge-

ther with cDNAs isolated for the present work, were used

to study the expression of specific genes participating in the

terpenoid biosynthetic pathways.

Isoprenoids (or terpenoids) are produced in plant cells

via two distinctly localized routes, one in cytoplasm and

one in plastids (Fig. 1). These pathways are named mev-

alonate pathway (MVA) and methylerythritol 4-phosphate

pathway (MEP), respectively, after the first committed

precursors these pathways use for the biosynthesis of ter-

penoids (Rodriguez-Concepcion and Boronat 2002). The

MEP pathway provides precursors mainly for the synthesis

of mono- and diterpenes, isoprene, carotenoids, the phy-

tohormones gibberellins and abscisic acid (ABA), phytol,

the side chain of chlorophylls, tocopherols, phylloquinon-

es, and plastoquinones while in the other hand, MVA

pathway mainly provides isopentenyl diphosphate for the

synthesis of sesquiterpenes, sterols, brassinosteroids, pol-

yprenols, and the moieties used for prenylated proteins

(Rodriguez-Concepcion and Boronat 2002).

In this study, the expression profiles of genes coding for

enzymes from both pathways were examined in various

C. creticus tissues, in response to abiotic stresses or to

defence signals. From the MVA, it was chosen the gene

coding for the 3-hydroxy-3-methyl-glutaryl-CoA reductase

or HMGR (EC 1.1.1.88), the enzyme catalyzing the rate-

limiting step of the pathway (Chappell et al. 1995). From

the MEP pathway, there were selected the genes encoding

the 1-deoxy-D-xylulose-5-phosphate synthase (DXS, EC

2.2.1.7) and 1-deoxy-D-xylulose-5-phosphate reducto-

isomerase (DXR, EC 1.1.1.267). DXS is the first enzyme of

the MEP pathway and is considered to be the key-regula-

tory step (Lois et al. 2000). The product of DXS is

deoxyxylulose 5-phosphate (DXP). DXR is the first com-

mitted step of the pathway for the synthesis of isoprenoids

(Lange and Croteau 1999). In addition, the two genes

responsible for the geranylgeranyl diphosphate (GGDP)

synthesis, the building block of all diterpenoids, carote-

noids, gibberellins and ABA were also examined (Pateraki

and Kanellis 2008).

From the results presented here, it can be concluded that

the expression of the assessed terpenoid biosynthetic genes

is regulated in a tissue-wise manner or according to the

developmental stage of the tissue, with the highest expres-

sion presented in the leaves’ trichomes. Furthermore, the

specific genes’ expression was affected by abiotic stresses

like heat or drought, mechanical wounding and elicitors

(namely methyl jasmonate or salicylic acid). The genes with

the strongest alterations in the transcript levels were mainly

the CcHMGR and CcDXS, which correspond to the regu-

latory steps of the MVA and MEP pathway, respectively.

Materials and methods

Plant material and treatments

Cistus creticus subsp. creticus plants were grown outdoors

in the area of Thermi, Thessaloniki, Greece, at the premises

of the National Agricultural Research Foundation or in

plant growth chambers under controlled conditions (16 h

light/8 h dark, 25�C day/18�C night temperature cycle).

Four-month-old C. creticus plantlets growing in plant

growth chambers were watered and sprayed at the same

time with (1) 100 lL meJA (Sigma-Aldrich Chemie

GmbH, Germany, Cat No W34,100-2) or (2) with 5 mM SA

(Aldrich Chemie GmbH, Germany, Cat No: S-6271).

200 lM Silwet L-77 was added in the spraying preparation.

For the heat stress, the temperature of the chamber was set

at 42�C and samples were collected after 0.5, 1, 2, 3, and 6-h

incubation. For the drought stress experiment, plantlets

growing in the growth chamber remained without water for

1, 3 and 5 days. To estimate the stress magnitude induced

by the water deficit, the relative water content (RWC) was

calculated in each sample using the following formula:

RWC = FW - DW/TW - DW, where FW is leaf fresh

weight (just after sampling), TW is leaf turgid weight (leaf

weight after 24-h incubation in distilled water) and DW is

dry weight (leaf weight after 24 h at 94�C). RWC was 72%

for the control, 65% for 1 day drought-stressed sample,

59% for 3 days drought-stressed sample and 47% for 5 days

drought-stressed sample. For mechanical wounding, leaves

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were cut in uniform stripes with scissors in planta, and

samples were collected after 0, 15, 30 min, 1 3, 6, 12 and

24 h. Plant tissue was collected, immediately frozen in

liquid nitrogen, and stored at -80�C. All the experiments

were performed in triplicates regarding biological replica-

tions. Each individual sample was a pool of approximately

ten plants.

Trichomes were physically removed from leaves with

the isolation procedure described by (Yerger et al. 1992).

Leaves were collected from outdoors cultivated plants. The

same stands for the plant material used to test the tissue

specific gene expression.

Total RNA purification

Total RNAs were extracted according to Pateraki

and Kanellis (2004)

Cloning and sequence analysis of C. creticus DXR full-

length cDNA An initial 670-bp cDNA fragment, corre-

sponding to the 30 end of the C. creticus DXR, was

amplified by PCR using the degenerate primer DXR.SEN3

[50 CC(AGCT) CC(AT) CC(AGCT) GC(AGCT) TGG

CC(AGCT) GG 30] and the oligodT primer [50 ACT AGT

CTC GAG TTT TTT TTT TTT TTT TTTT 30]. As tem-

plate for these reactions was used single-stranded cDNA

synthesized with the oligodT primer and reverse trans-

criptase SuperScript II (Invitrogen, Life Technologies,

Carlsbad, CA, USA) from DNaseI-treated (Promega,

GmbH, Mannheim, Germany) total RNA isolated from

young leaves. For the 50 end cloning of the CcDXR cDNA,

50 RACE techniques were employed using the ‘‘50 RACE

System for Rapid Amplification of cDNA Ends’’ (Invitro-

gen, Life Technologies). A gene specific primer

[CcDXR5.REV1: 50 GCC GCA TGT CAG GCC AAC

CCA TTT GTGC 30] based on the obtained CcDXR

sequence (670-bp fragment) was used together with the 50

RACE anchor primer [50 GCG TCG ACT AGT ACG GGI I

GG GII GGG IIG 30] provided by the kit. The resulted PCR

product had the expected size (approximately 1,200-bp

long) and corresponded to the CcDXR cDNA clone. As

template for the 50 and 30 RACE reactions single strand

cDNA was used, synthesized as described above. PCR

products were cloned into pDRIVE (Qiagen, Hilden, Ger-

many) vector and sequenced using the LI-COR Long Read

4200 automated sequencer and the ‘‘Sequitherm EXCEL-

II’’ kit (Epicentre, Madison, WI, USA).

Cloning and sequence analysis of C. creticus HMGR and

DXS2 cDNAs Partial cDNAs encoding the CcDXS2 and

CcHMGR were obtained from the EST analysis of a

C. creticus leaf trichome cDNA library (Falara et al. 2008).

The specific clones were sequenced from both strands, as

described above. Phylogenetic analysis of the CcDXS2 was

performed using the UPGMA method and the MEGA 4.0.1

program. Bootstrap values were calculated by distance

analysis for 1,000 replicates.

RT-PCR reactions Gene expression of the CcHMGR,

CcDXS2, CcDXR, CcGGDP1 and CcGGDP2 genes was

monitored by RT-PCR analysis techniques. First strand

cDNA synthesis was performed using an oligodT primer

and M-MLV reverse transcriptase (Invitrogen, Life Tech-

nologies) from DNaseI-treated (Promega, GmbH) total

RNAs isolated from C. creticus tissues. The gene-specific

primers used for the PCR reactions are shown in Table 1.

The constitutively expressed C. creticus eukaryotic trans-

lation elongation factor-1a cDNA (CcEF1a, Accession

num. EF062868) was used as internal control. The PCR

conditions were optimized for each pair of primers so that

sharp bands corresponding to the expected fragments were

amplified without any signs of non-specific products. The

Table 1 Primers used for

CcHMGR, CcDXS2, CcDXR,CcGGDPS1 and CcGGDPS2expression studies

Gene Primers’ Name Primers’ sequence

CcHMGR CcHMGR.For1 50 GTT CTA ACT GCA TTA CAA TGA TGG 30

CcHMGR.Rev1 50 CTA GCT GTC CGG CTG CAA GGG C 30

CcDXS2 CcDXS.For1 50 AAC AAT GTC TTG AAG CGG CAC G 30

CcDXS.Rev1 50 TGT TGC TGA GAG ATG CCT TGA CG 30

CcDXR CcDXR.For1 50 GGC TTG CCT GAT GGT GCA CTT CGA CGC 30

CcDXR.Rev1 50 GCC GCA TGT CAG GCC AAC CCA TTT GTG C 30

CcGGDPS1 CcGGDPS1.For1 50 AGT TGG CGT ATC CCC CGC CCG 30

CcGGDPS1.Rev1 50 TAC ATC TGT CTC TAA GCA GTC GC 30

CcGGDPS2 CcGGDPS2.For1 50 GAT GTG ACG AAA TCT TCC GTG 30

CcGGDPS2.Rev1 50 CTT TTA TGC TTC TTT CAT TCA TAG 30

CcEF1a CcEF1a.For1 50 GGT CCT ACT GGT TTA ACC ACT G 30

CcEF1a.Rev1 50 CTC GGA GAA GGT CTC CAC AAC C 30

Plant Cell Rep (2010) 29:629–641 631

123

PCR amplified products for the CcHMGR was 264 bp,

for the CcDXS2 317 bp, for CcDXR 319 bp, for the

CcGGDPS1 612 bp and for the CcGGDPS2 261 bp and

lastly for the CcEF1a was 411 bp. The fragments from

each amplified transcript were cloned into pGEM-T Easy

vector (Promega GmbH) and sequenced using a LI-COR

Long Read 4200 automated sequencer and ‘‘Sequitherm

EXCELII’’ kit (Epicentre) to verify the amplicon identity.

Relative transcript levels were visualized by setting the

cycle numbers so that the rate of PCR product amplifica-

tion was in the early exponential stage of the reaction. PCR

products were analyzed in 1.5% agarose gel electrophore-

sis, photographed and quantified with ImageJ program

(Abramoff et al. 2004). Four technical replications were

performed for each biological replication mentioned.

Statistical analysis was performed using the independent

groups t test for means, with confidence level 95%.

Results and discussion

Isolation of a cDNA encoding C. creticus HMGR

enzyme

Hydroxyl-methylglutaryl coenzyme A (HMG-CoA)

reductase (HMGR, EC 1.1.1.34) synthesizes mevalonic

acid through the reduction of 3-hydroxy-3-methylglutaryl-

CoA and it is the first enzyme of the MVA or cytoplasmic

committed exclusively to the biosynthesis of terpenoids in

cytoplasm, like sesquiterpenes, sterols and triterpenes. In

plants, HMGR is encoded by multigene families, the

members of which exhibit differential expression (Korth

et al. 1997). It is localized at the endoplasmic reticulum

(Campos and Boronat 1995) and it is considered the key-

regulatory enzyme of the MVA pathway (Chappell et al.

1995).

The partial cDNA HMGR clone reported here

(CcHMGR, Accession Number EF062866) was among the

2,022 ESTs sequenced from a C. creticus leaves’ trichomes

cDNA library (Falara et al. 2008). It was 1,475 bp in size

and encoded a peptide of 388 amino acids (aa) with a

30 UTR (untranslated region) of 306 bp with no obvious

polyadenylation signal. The average size of plant HMGRs

ranges from 560 to 600 aa. Phylogenetic analysis of the

deduced CcHMGR protein did not help to designate the

specific gene according to other characterized HMGR

genes (e.g. from potato or tomato) it showed though high

similarities with known plant HMGR proteins. The highest

similarities (86 and 84%) were observed with two different

HMGR peptides from Gossypium hirsutum (Accession

Numbers: AAC05088 and AAC05089, respectively).

C. creticus HMGR showed high homologies even with

mammalian or yeast HMGR enzymes and this can be

explained by the fact that the deduced peptide corresponds

to the catalytic region of the enzyme, which is highly

conserved among species, while the most divergent

hydrophobic regions (transmembrane domains), as well as

the region (linker) that connects the hydrophobic part with

the catalytic domain are missing. Two glycosylation sites,

at Asn125 and Asn371, conserved in all the enzymes

known today (Campos and Boronat 1995), are present in

the C. creticus peptide as well.

Isolation of a cDNA encoding C. creticus DXS2

enzyme

1-deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS,

EC 2.2.1.7) catalyzes the first step of the non-mevalonate

(MEP or plastidial) pathway for the terpenoids biosynthe-

sis. It synthesizes 1-deoxy-D-xylulose-5-phosphate from

the condensation of pyruvate and D-glyceraldehydes-3-

phoshate (Lois et al. 2000). It is the only enzyme of the

MEP pathway that is encoded by multigene families in

plants (Krushkal et al. 2003).

The CcDXS2 clone reported in this work was isolated as

part of the EST analysis mentioned above (Falara et al.

2008). The isolated partial cDNA was 666-bp long and

encoded a peptide of 141 aa (Accession Number

EF062865) while the average size of plant DXS enzymes is

710–720 aa. The 30 UTR was 239 bp and no putative

polyadenylation signal was observed. The isolated frag-

ment corresponded to a region containing the ‘‘transketol-

ase C-terminal domain’’, which is common to all the

enzymes belonging to the trasketolase superfamily. Pair-

wise comparisons revealed, from one hand, high homology

with known plant DXSs and from the other obvious

divergence from other close related transketolases.

C. creticus DXS2 showed the highest similarity (78%) with

the Medicago truncatula DXS homologue (Accession

Number CAD22531), and with Chrysanthemum x morifo-

lium DXS (77%, Accession Number BAE79547). In con-

trast to the CcHMGR, CcDXS2 exhibited very low

similarities with non-plant homologues (Fig. 1).

Plant DXS enzymes are grouped in two distinct classes

according to their primary structure and gene expression

pattern. Class I encloses the house-keeping DXS enzymes,

the ones related with the primary metabolism and photo-

synthesis, while the class II contains the inducible DXS

enzymes, the ones related with the secondary metabolism

(Krushkal et al. 2003). Based on a phylogenetic analysis,

the CcDXS2 cDNA appears to be a member of Class II,

related to the secondary metabolism (Fig. 2). This obser-

vation was further confirmed by the gene expression

analysis showing that induction of CcDXS2 was triggered

by different abiotic stresses, as well as by various defence

signals (see below).

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Isolation of a full-length C. creticus DXR cDNA

Deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase

(DXR, EC 1.1.1.267) is the second enzyme of the MEP

pathway; however, it is the first enzyme of the pathway

dedicated entirely to the terpenoids biosynthesis. DXR

catalyzes the synthesis of 2-C-methyl-D-erythritol 4-phos-

phate (MEP) using as substrate 1-deoxy-D-xylulose-5-

phosphate (Rodriguez-Concepcion and Boronat 2002). In

plants, DXR is encoded by a single gene, in all species

studied till today (Carretero-Paulet et al. 2002). A full-

length cDNA encoding this enzyme was cloned from

C. creticus (CcDXR) using 50 and 30 RACE PCR tech-

niques (Accession Number AY297794). The length of the

cDNA clone was 1,846 bp, while the open reading frame

was 486 aa. The 50 UTR was 180 bp and the 30 UTR was

205 bp with no obvious polyadenylation signal. Pairwise

comparisons with other DXR peptides showed high

similarity with other plant DXR enzymes (ranging from

82 to 73%) and lower similarity with the bacterial homo-

logues. In silico analysis, using the TargetP 1.1 (Emanu-

elsson et al. 2000), WolfPSort (The WoLF PSORTII web

server 2006 [http://wolfpsort.seq.cbrc.jp/]) and Predotar

V1.03 (Prediction of organelle targeting sequences, [http://

genoplante-info.infobiogen.fr/predotar/]) software revealed

that CcDXR cDNA bears in its 50 end a putative transit

peptide for plastid localization with high probability,

ranging from 66 to 50%, as it was expected since DXR is a

plastidial enzyme (Carretero-Paulet et al. 2002). From the

above analysis as well as from the alignment of the CcDXR

aa sequence with other known plant DXR peptides and

according to Carretero-Paulet et al. (2002), it seems likely

that the first 50 aa of the CcDXR comprise the transit

peptide (Fig. 3). The well conserved, for DXR enzymes,

NADPH (helps in the stability of the enzyme) binding site

at the amino-terminal region is observed also in the present

peptide (Fig. 3).

Gene expression studies of CcHMGR, CcDXS2,

CcDXR, CcGGDPS1 and CcGGDPS2

Plant secondary metabolites’ accumulation varies accord-

ing to species, tissues, growth conditions and plant/tissue

developmental stage (Ament et al. 2006; Arimura et al.

2004; Martin et al. 2003; Oudin et al. 2007; Steele et al.

1998). The mechanisms regulating the rate of their pro-

duction and accumulation are usually closely linked with

the control of the corresponding biosynthetic pathways,

which takes place in different levels namely gene tran-

scription, post-transcriptional processing, protein transla-

tion and post-translational modification. In the case of the

MEP pathway, which is responsible for the synthesis of

monoterpenes and diterpenes in the plastids, little is known

regarding its regulation and especially in the medicinal

plant C. creticus. To get an initial insight into the regula-

tion of the MEP pathway in this plant at the transcriptional

level, the expression of CcDXS2, CcDXR, CcGGDPS1 and

CcGGDPS2 was monitored at different conditions. In

addition, the gene expression of the CcHMGR, a gene of

the MVA route was also studied under the same conditions.

Tissue distribution of terpenoid biosynthetic genes

To study the expression of the above genes in different

C. creticus tissues, RT-PCR was applied to detect

CcHMGR, CcDXS2, CcDXR, CcGGDP1 and CcGGDP2

mRNA levels (Fig. 4). The tissue specificity of CcGGDP1

and CcGGDP2 was previously studied by Pateraki and

Kanellis (2008), however, for comparison reasons it is also

presented here. Total RNAs extracted from different

C. creticus tissues, such as trichomes (TR), leaves 1–2 cm

Fig. 1 A scheme displaying the enzymes participating in the

cytoplasmic and plastidial isoprenoid biosynthetic pathways. The

enzymes studied are highlighted. MEP path: DXS 1-deoxy-D-xylulose

5-phosphate synthase, DXR 1-deoxy-D-xylulose 5-phosphate reduc-

toisomerase, MCT 2-C-methyl-d-erythritol 4-phosphate cytidylyl-

transferase, CMK 4-(cytidine 50-diphospho)-2-C-methyl-d-erythritol

kinase, MDS 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase,

HDS (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase and

HDR (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase. MVA

path: AACT acetoacetyl CoA thiolase, HMGS Hydroxymethylglu-

taryl-CoA synthase, HMGR hydroxymethylglutaryl-CoA reductase,

MVK mevalonate kinase, PMK 5-phosphomevalonate kinase, PMD5-diphosphomevalonate decarboxylase, IDI isopentenyl diphosphate

isomerise, FPS farnesyl diphosphate synthase, GGPS geranylgeranyl

diphosphate synthase

Plant Cell Rep (2010) 29:629–641 633

123

long (LV), roots (RT), stems (ST), flower buds (FB), fruits

(FR) and seeds (SD) were studied. It is evident that each

gene followed a different expression profile; a common,

however, characteristic is their significant higher accumu-

lation in trichomes, except CcDXR (Fig. 4). Trichomes are

considered part of the first line of the plant defence system,

at least at the surface of the tissues, and has been demon-

strated that they are very active structures in terpenoids

biosynthesis in many plant species (Gershenzon et al.

1992) including C. creticus trichomes as it is obvious from

the data presented in this communication together with the

ones reported by Falara et al. (2008) and Ljaljevic, Kanellis

et al., (unpublished data).

A second common feature is the low or nil mRNA

transcript accumulation in roots (Fig. 4). Generally, roots

has been reported to produce low amounts of volatile

mono-, and sesqui-terpenes compared with leaves (Schnee

et al. 2002)

Transcript steady state levels of CcHMGR exhibited a

similar pattern of expression in leaves, stems, flower buds,

fruit and seeds, suggesting that this specific gene is not

tissue-regulated. C. creticus DXS2 and GGDP2 showed a

similar expression profile with maximum accumulation in

flower buds and lower in leaves and fruit. No messages

were detected in stems, seeds and roots. On the other hand,

CcDXR transcripts accumulated mostly in stems and then

in leaves, flower buds, seeds and lastly in roots. C. creticus

GGDPS1 mRNAs exhibited high abundance in leaves and

in flower buds, while very low in seeds, roots and fruit

(Fig. 4). CcDXR was detected in all tissues tested in fairly

high levels. This observation together with the similar

expression levels in leaves and trichomes could implicate

an almost constitutive pattern of the expression of this gene

(Fig. 4).

The high levels of the CcDXS2, CcDXR and CcGGDP2

transcripts in the flower buds could favour increased needs

for isoprenoids that might be directed for the protection of

this sensitive tissue as well as for the formation of the

flower pigments and volatiles (Pichersky and Gang 2000).

In Arabidopsis, 2 out of 12 putative AtGGDPS genes were

mainly expressed in flowers (Okada et al. 2000) despite the

fact that this plant is not cross-pollinated, its flowers are

white and do not produce high amount of volatiles (Aha-

roni et al. 2003; Chen et al. 2003). Moreover, the fact that

several terpene synthases’ genes in Arabidopsis exhibit

flower-specific (or mainly flower-specific) expression

(Chen et al. 2003) underlines the importance that these

metabolites have for the flower welfare. In agreement with

these findings, a Hevea bresilensis GGDPS gene showed

also the highest transcript accumulation in flowers (Takaya

et al. 2003).

It is interesting to note that, although, seeds do not

produce terpenoids during dormancy, they exhibited,

however, relatively high mRNA abundance of CcDXR and

Fig. 2 Phylogenetic

relationships of plant DXS

proteins. The phylogenetic tree

was constructed with the

UPGMA method. The numbersindicated are the bootstrap

values. The species from which

the enzymes were obtained are

indicated together with their

accession numbers. Bacterial

DXS proteins were used as

outgroups. The peptides used in

this analysis were trimmed at 50

ends, in order to be proportional

to the length of the CcDXS2

634 Plant Cell Rep (2010) 29:629–641

123

CcHMGR. The mRNA stored at dormant seeds is termed

long-lived stored mRNAs (Rajjou et al. 2004) and their role

is to participate in the immediate proteins’ synthesis during

seed germination, just prior to transcription initiation.

These proteins, and apparently among them CcDXR and

CcHMGR, appear to be necessary during seed germination

and probably contribute to the biosynthesis of gibberellins

and sterols.

Terpenoid biosynthetic genes are developmentally

regulated in leaves and trichomes

Chemical analysis of trichomes isolated from C. creticus

leaves of different age showed that the production of ter-

penoids was developmentally regulated (Falara et al.

2008). Trichomes isolated from very young (0.5–1.0 cm)

and young leaves (1–2 cm) contained the highest amount

of labdane-type diterpenes (the predominant metabolites

extracted from these tissues) compared with older leaves,

while trichomes originating from medium size leaves

showed higher sesquiterpenes content than smaller or older

leaves (Falara et al. 2008). Therefore, in order to study

whether this phenomenon is under transcriptional

Fig. 3 Alignment of CcDXR

with other plant proteins. The

hypothetical transit peptide is

underlined with a bold dashedline while the -binding region is

underlined with a boldcontinues line. Conserved

amino acid residues in all the

sequences used in this

alignment are black boxed,

while similar amino acids are in

grey boxes. Alignments were

performed with the ClustalW

program (http://www.ebi.ac.uk/

clustalw/) while the shading was

done with the BoxShade 3.21

program (http://www.ch.

embnet.org/software/

BOX_form.html/)

Fig. 4 Tissue-specific expression of CcHMGR, CcDXS2, CcDXR,CcGGDPS1, and CcGGDPS2 genes. Samples were collected from

adult plants cultivated outdoors. TR trichomes isolated from 1 to 2 cm

long leaves, LV Leaves (1–2 cm long), RT roots, ST stems, FB flower

buds, FR fruits, SD seeds. Transcript levels were analyzed with RT-

PCR techniques. CcEF1a gene was used as internal control. Errorbars indicate standard deviations (n = 12)

Plant Cell Rep (2010) 29:629–641 635

123

regulation, the expression of CcHMGR, CcDXS2, CcDXR,

CcGGDPS1 and CcGGDPS2 was monitored in different

size leaves ranging from very young (0.5–1.0 cm) to fully

expanded (3–4 cm) and their trichomes thereof (Fig. 5).

It is evident that the expression of the majority of the

genes studied was developmentally regulated. Transcripts’

accumulation in leaves, of all studied genes, except

CcHMGR, was inversely related to their size that is the

smallest (or youngest) leaves showed the highest abun-

dance of the corresponding transcripts (Fig. 5a). The

CcHMGR expression displayed an increase in medium size

leaves (Fig. 5a). It is worth mentioning that this expression

pattern parallels the sesquiterpenes’ accumulation in

C. creticus trichomes’ extracts coming from leaves of the

same developmental stages (Falara et al. 2008). These

metabolites are biosynthesized via MVA. On the other

hand, the above genes followed a different expression

pattern in whole leaves from that observed in isolated

trichomes (Fig. 5b). In this tissue, the transcript accumu-

lation of these genes was not significantly altered, with the

exception of CcDXS2, and CcDXR that were accumulated

with the highest rates in trichomes from small and small/

medium leaves, respectively, and lesser again in trichomes

from fully expended leaves.

It is known that the production of secondary metabolites

in leaves and trichomes is developmentally controlled and

this regulation is mainly depended on transcriptional con-

trol of the corresponding genes (McConkey et al. 2000).

Our results, in combination with the results presented in

Falara et al. (2008) suggest that a similar type of devel-

opmental regulation at the transcriptional level exists for

genes participating in terpenoid biosynthesis in C. creticus

leaves and trichomes. However, another factor, which can

contribute to higher terpenoid genes’ expression and higher

terpenes accumulation in young leaves could be the greater

number of trichomes bearded in young compared to older

leaves (Falara et al. 2008), thus explaining in part the

difference in gene expression between trichomes and

whole leaves. It is obvious that the leaves’ requirements in

isoprenoids content are different, depending on the age.

Younger leaves may exhibit increased needs for protective

phytochemicals like isoprenoids because there are more

sensitive and vulnerable to plant pathogens, herbivores and

adverse environmental conditions.

Abiotic stress responses of terpenoid biosynthetic genes

in C. creticus

As a part of the plant defence machinery, isoprenoids are

employed, among others, to protect plant cells from

drought or heat stress (Munne-Bosch and Alegre 2003).

Moreover, it is known that isoprenoids are induced after

mechanical wounding due to increased transcription or

enzymatic activities of genes and enzymes participating in

the biosynthetic pathways of these metabolites (McKay

et al. 2006).

Drought stress caused a similar pattern of expression of

CcHMGR, CcDXS2; their transcript accumulation was

slightly induced during the first day of the stress and then

gradually reduced to barely detectable levels after 5 days

of water deficit. C. creticus DXR mRNA messages were

continuously decreased, whereas CcGGDPS1 remained

unaffected (Fig. 6a). The expression of CcGGDPS2 was

highly induced 1 day after water stress started and then

reduced progressively till the end of the experiment. Water

deficit results, among others, in oxidative stress, which is

confronted by the production of enzymatic or non-enzy-

matic antioxidants like ascorbic acid, tocopherols or

carotenoids by plant cells (Loreto and Velikova 2001;

Munne-Bosch and Alegre 2003). Furthermore, there are

several plant species like Salvia officinalis, S. fruticosa and

R. officinalis that are able to produce carnosic acid, a

Fig. 5 Variations of CcHMGR, CcDXS2, CcDXR, CcGGDPS1, and

CcGGDPS2 transcript relative abundance in leaves of different

developmental stages (a) and in trichomes isolated from leaves of the

same stages (b). xs leaves smaller than 1 cm or trichomes isolated

from leaves of this size, s leaves of 1–2 cm long or trichomes isolated

from leaves of this size, m leaves of 2–3 cm long or trichomes

isolated from leaves of this size, lg leaves bigger than 3 cm long or

trichomes isolated from leaves of this size. Transcript levels were

analyzed with RT-PCR techniques. CcEF1a gene was used as internal

control. Error bars indicate standard deviations (n = 12). The

asterisks denote the statistical significant differences between the xs

leaves/trichomes and the rest developmental stages at P \ 0.05

636 Plant Cell Rep (2010) 29:629–641

123

diterpene that act like antioxidant and its production is

induced during water stress conditions (Munne-Bosch and

Alegre 2003). ABA, a phytohormone participating in the

signal transduction pathways for the adaptation of plants to

several abiotic stresses like drought and high salt, is syn-

thesized from the oxidative cleavage of a 9-cis-epoxyca-

rotenoid that is produced through the MEP pathway

(Schwartz et al. 2003). During water deficit conditions

ABA and tocopherols (one of the building blocks of which

is GGDP) levels were increased in C. creticus leaves

(Munne-Bosch et al. 2008) in a manner similar to the

expression pattern of the CcHMGR, CcDXS2 and

CcGGDPS2 genes (Fig. 6a), therefore, it is possible this

induction of the gene transcripts to be related with

increased needs of the plant cells in these metabolites and

phytohormones. The decrease of the mRNA levels the last

days of the stress may reflect the inability of the plants to

withstand (or overcome) extended water losses.

High temperatures provoked a slight, but significant,

increase in CcHMGR and CcDXS2 transcripts half an hour

and 1 h after exposure, followed by a continuous decrease

thereafter (Fig. 6b). However, CcDXR, CcGGDPS1 and

CcGGDPS2 were not responded to these conditions

(Fig. 6b). In addition to their antioxidant activities, iso-

prenoids have been shown to exhibit thermotolerance-

related activities. Today, it is known that isoprene, a C-5

terpenoid, and specific monoterpenes like a-pinene help

plants to overcome high temperature stresses (Penuelas

et al. 2005). Thus one may suggest that the transient

induction of CcHMGR and CcDXS2 in response to heat

could be linked with processes participating in the adap-

tation to these conditions and the production of isoprenoids

with antioxidant and thermoprotection activities that would

allow the plant to cope and overcome specific abiotic

stresses, like high temperatures and drought.

Mechanical wounding induced instantaneously the

expression of CcHMGR (in 15 min) and CcDXS2 (in 1 h).

The peak of the expression of both genes was observed 3 h

after wounding, followed by a reduction. The expression of

the rest of the genes was not affected significantly by this

treatment (Fig. 6c). In many plant species, it has been

observed increased terpenoid production after mechanical

wounding and this increase was due to transcriptional or

post-transcriptional regulation of the terpenoid biosynthetic

genes (Steele et al. 1998). Since HMGR possess a crucial

control point in the MVA, its immediate response to

mechanical injury, could be interpreted as urgent need for

phytosterols, major components of plant cell membranes

that could assist the cell membranes’ reparation after

wounding or insect attack. In addition, induction of

CcHMGR could also lead to the production of sesquiter-

pene phytoalexins, substances participating in the protec-

tion of wounded tissues from insects and from secondary

infections by plant pathogens. CcHMGR mRNA levels

immediate increase after wounding has been observed in

other plant species such as Solanum tuberosum (Yang et al.

1991). The observed increase in CcDXS2 gene transcripts

Fig. 6 Response of CcHMGR, CcDXS2, CcDXR, CcGGDPS1, and

CcGGDPS2 genes to several abiotic stresses such as a drought stress

in 4-month-old plantlets cultivated in growth chamber after 1 day

(1d), 3 days (3d) and 5 days (5d) of water withholding, b high

temperature stress in 4-month-old plantlets cultivated in growth

chamber after 0.5 h (0.5 h), 1 h (1 h), 2 h (2 h), 3 h (3 h) and 6 h

(6 h) exposure at 42�C and c mechanical wounding in planta in

4-month-old plantlets cultivated in growth chamber, where Con non-

wounded leaves, 150 leaves wounded for 15 min, 300 leaves wounded

for 30 min, 1 h leaves wounded for 1 h, 3 h leaves wounded for 3 h,

6 h leaves wounded for 6 h, 12 h leaves wounded for 12 h, 24 hleaves wounded for 24 h. Transcript levels were analyzed with

RT-PCR techniques. CcEF1a gene was used as internal control. Errorbars indicate standard deviations (n = 12). The asterisks denote the

statistical significant differences between the control and the treated

samples at P \ 0.05

Plant Cell Rep (2010) 29:629–641 637

123

could reflect a need for mono- or di-terpenes production to

heal and/or insulate the wounded area from possible

pathogen invasion as well as the emission of phytochemi-

cals that could inhibit again secondary infections (Steele

et al. 1998).

Defence signal-induced responses of terpenoid biosynthetic

genes in C. creticus

Methyl jasmonate (MeJA) and salicylic acid (SA) have

been identified as key signalling plant hormones regulating

a network of interconnecting signal transduction pathways

responsible for induced plant defence against biotic and

abiotic stresses (Smith et al. 2009; Zavala and Baldwin

2006; Zhao et al. 2004). These responses lead, among

others, to the induction of production of secondary

metabolites related to plant defence processes, like iso-

prenoids (Ament et al. 2006; Martin et al. 2002). This

induction is usually achieved through transcriptional acti-

vation of the terpenoid biosynthetic genes (Ament et al.

2006). Figure 7 reveals that both, meJA and SA affect at

least some of the genes studied here. CcDXS2 mRNA

messages were highly accumulated after methyl-JA treat-

ment and maintained at these levels during the entire

duration of the experiment (Fig. 7a). The same gene was

stimulated only after 48 h upon SA application (Fig. 7b).

In parallel, treatment with SA caused an increase in the

accumulation of CcHMGR transcripts that was evident

only after 6 h and then the transcript abundance reached

the initial levels (Fig. 7b). The rest of the genes were

unaffected by either meJA or SA. In spite their antagonistic

effect and the their different roles in plant defence pro-

cesses (Pieterse et al. 2007; Smith et al. 2009), it is obvious

that both of them are capable of inducing genes partici-

pating in both terpenoid biosynthetic pathways.

It is interesting to note that although mechanical

wounding it is known to activate the JA defensive signal

cascade (Li et al. 2001; Ryan and Moura 2002), in the

present study CcDXS2 and CcHMGR mRNA steady state

levels were induced by both mechanical wounding and SA

whereas meJA stimulated only CcDXS2 expression. These

differences could be due to the time intervals of the

applications, the concentration of the defence signals used

or other environmental factors (Pieterse et al. 2007).

These results taken together with the previously

described heat, drought and mechanical wounding

experiments show that in C. creticus similarly with other

species, at least some of the terpenoid biosynthetic genes

were induced by abiotic stresses and by defence signals,

implying that these metabolites, coming from the plas-

tidial and the cytoplasmic pathway, could possibly play a

role in defence mechanisms of this species. Therefore,

terpenoids originated from the two distinct biosynthetic

routes could have different roles in the plant defence

mechanism but apparently coordination and interaction of

these processes are necessary for the achievement of the

final goal.

It is worth mentioning that CcHMGR and CcDXS2, the

two key-regulatory genes of the MVA and MEP pathway,

respectively, were the ones mainly affected by the treat-

ments mentioned above. It is known that increase in the

expression of the HMGR or DXS genes in transgenic plants

was sufficient for the accumulation of the final pathway

products (Enfissi et al. 2005; Harker et al. 2003; Munoz-

Bertomeu et al. 2006). Thus, it is possible that the increase

in the expression of these genes, observed in this com-

munication under the experimental conditions mentioned

above, could lead to the increase of isoprenoids production,

of plastidial or cytosolic origin. Further, this is well col-

laborated with the observed elevated levels of diterpenes

Fig. 7 Response of CcHMGR, CcDXS2, CcDXR, CcGGDPS1 and

CcGGDPS2 genes to the exogenous application of methyl jasmonate

(meJA) and salicylic acid (SA). a 4-month-old plantlets cultivated in

growth chamber, sprayed with 0 lM (Con) or 100 lM meJA. Leaf

samples were collected after 6 h (6 h), 24 h (24 h) or 48 h (48 h).

b 4-month-old plantlets cultivated in growth chamber, sprayed with

0 mM (Con) or 5 mM SA (S). Leaf samples were collected after 6 h

(6 h), 24 h (24 h) or 48 h (48 h). Transcript levels were analyzed with

RT-PCR techniques. CcEF1a gene was used as internal control. Errorbars indicate standard deviations (n = 12). The asterisks denote the

statistical significant differences between the control and the treated

samples at P \ 0.05

638 Plant Cell Rep (2010) 29:629–641

123

and sesquiterpenes during development of C. creticus

leaves (Falara et al. 2008).

One, however, cannot overlook some distinct expression

profiles in response to various stresses and factors, sug-

gesting a tight regulation of these pathways according

probably to the extent of the stress and the kind of the

defence signals applied, or according to the time-course

studied (Heidel and Baldwin 2004). It is known that the

signal transduction pathways activated in each different

abiotic or biotic stress are discrete but they are also sharing

specific elements (Spoel et al. 2003; Zhao et al. 2005).

Conclusions

From the present work, it becomes evident that the terpe-

noid biosynthetic genes assessed here are developmentally

regulated in leaf trichomes and in leaves. In combination

with the results reported in Falara et al. (2008), it seems that

trichomes are the main site of terpenoid production in this

species and the final product biosynthesis and accumulation

from these tissues is regulated, at least in part, at the tran-

scriptional level. Furthermore, it is shown that the CcDXS2

and CcHMGR, the regulatory points of the MEP and MVA

pathway, were significantly induced by abiotic stresses and

more specifically from mechanical wounding but also after

application of salicylic acid while methyl jasmonate treat-

ment resulted only in the induction of the CcDXS2.

This increase in the expression of the above genes could

be attributed to an increased need of isoprenoids of both

pathways by the plant tissues under these experimental

conditions. Further identification and chemical character-

ization of the metabolites produced and accumulated in C.

creticus during these conditions could be of great interest

as these phytochemicals could have important pharma-

ceutical properties. It is likely that the adaptation of xero-

phytic species in arid or hostile ecosystems is managed,

partly, by the synthesis of a number of metabolites as

described above. Thus, these plants could serve as an

important source for uncharacterized chemicals and con-

sequently could be considered as valuable genomic

resources.

Acknowledgments This research was partially supported from a

grant (PENED 99ED 637) implemented within the framework of the

‘‘Reinforcement Programme of Human Research Manpower’’ and co-

financed by National and Community Funds (25% from the Greek

Ministry of Development-General Secretariat of Research and

Technology and 75% from E.U.-European Social Fund).

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