stress and developmental responses of terpenoid biosynthetic genes in cistus creticus subsp....
TRANSCRIPT
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: [email protected]
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
630 Plant Cell Rep (2010) 29:629–641
123
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).
632 Plant Cell Rep (2010) 29:629–641
123
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).
References
Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with
ImageJ. Biophotonics Int 11:36–42
Aharoni A, Giri AP, Deuerlein S, Griepink F, de Kogel W-J,
Verstappen FWA, Verhoeven HA, Jongsma MA, Schwab W,
Bouwmeester HJ (2003) Terpenoid metabolism in wild-type and
transgenic Arabidopsis plants. Plant Cell 15:2866–2884
Ament K, Van Schie CC, Bouwmeester HJ, Haring MA, Schuurink
RC (2006) Induction of a leaf specific geranylgeranyl pyrophos-
phate synthase and emission of -4, 8, 12-trimethyltrideca-1, 3, 7,
11-tetraene in tomato are dependent on both jasmonic acid and
salicylic acid signaling pathways. Planta 224:1197–1208
Arimura G-i, Ozawa R, Kugimiya S, Takabayashi J, Bohlmann J
(2004) Herbivore-induced defense response in a model legume.
Two-spotted spider mites induce emission of (E)-b-ocimene and
transcript accumulation of (E)-b-ocimene synthase in Lotusjaponicus. Plant Physiol 135:1976–1983
Aronne G, De Micco V (2001) Seasonal dimorphism in the
Mediterranean Cistus incanus L. subsp. incanus. Ann Bot
87:789–794
Campos N, Boronat A (1995) Targeting and topology in the
membrane of plant 3-hydroxy-3-methylglutaryl coenzyme A
reductase. Plant Cell 7:2163–2174
Carretero-Paulet L, Ahumada I, Cunillera N, Rodriguez-Concepcion
M, Ferrer A, Boronat A, Campos N (2002) Expression and
molecular analysis of the Arabidopsis DXR gene encoding
1-deoxy-D-xylulose 5-phosphate reductoisomerase, the first
committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate
pathway. Plant Physiol 129:1581–1591
Chappell J, Wolf F, Proulx J, Cuellar R, Saunders C (1995) Is the
reaction catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A
reductase a rate-limiting step for isoprenoid biosynthesis in
plants? Plant Physiol 109:1337–1343
Chen F, Tholl D, D’Auria JC, Farooq A, Pichersky E, Gershenzon J
(2003) Biosynthesis and emission of terpenoid volatiles from
Arabidopsis flowers. Plant Cell 15:1–14
Dimas K, Papadaki A, Tsimplouli C, Hatziantoniou S, Alevizopoulos
K, Pantazis P, Demetzos C (2006) Labd-14-ene-8, 13-diol
(sclareol) induces cell cycle arrest and apoptosis in human breast
cancer cells and enhances the activity of anticancer drugs.
Biomed Pharmacother 60:127–133
Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000)
Predicting subcellular localization of proteins based on their
N-terminal amino acid sequence. J Mol Biol 300:1005–1016
Enfissi EMA, Fraser PD, Lois L-M, Boronat A, Schuch W, Bramley
PM (2005) Metabolic engineering of the mevalonate and non-
mevalonate isopentenyl diphosphate-forming pathways for the
production of health-promoting isoprenoids in tomato. Plant
Biotechnol J 3:17–27
Falara V, Fotopoulos V, Margaritis T, Anastasaki T, Pateraki I,
Bosabalidis M, Artemios D, Kafetzopoulos D, Demetzos C,
Pichersky E, Kanellis A (2008) Transcriptome analysis
approaches for the isolation of trichome-specific genes from
the medicinal plant Cistus creticus subsp. creticus. Plant Mol
Biol 68:633–651
Gershenzon J, McCaskill D, Rajaonarivony JIM, Mihaliak C, Karp F,
Croteau R (1992) Isolation of secretory cells from plant
glandular trichomes and their use in biosynthetic studies of
monoterpenes and other gland products. Anal Biochem 200:130–
138
Gulz PG, Herrmann T, Hangst K (1996) Leaf trichomes in the genus
Cistus. Flora 191:82–104
Harker M, Holmberg N, Clayton JC, Gibbard CL, Wallace AD,
Rawlins S, Hellyer SA, Lanot A, Safford R (2003) Enhancement
of seed phytosterol levels by expression of an N-terminal
truncated Hevea brasiliensis (rubber tree) 3-hydroxy-3-methyl-
glutaryl-CoA reductase. Plant Biotechnol J 1:113–121
Hatziantoniou S, Dimas K, Georgopoulos A, Sotiriadou N, Demetzos
C (2006) Cytotoxic and antitumor activity of liposome-
Plant Cell Rep (2010) 29:629–641 639
123
incorporated sclareol against cancer cell lines and human colon
cancer xenografts. Pharmacol Res 53:80–87
Heidel AJ, Baldwin IT (2004) Microarray analysis of salicylic acid
and jasmonic acid signalling in responses of Nicotiana attenuatato attack by insects from multiple feeding guilds. Plant Cell
Environ 27:1362–1373
Korth KL, Stermer BA, Bhattacharyya MK, Dixon RA (1997) HMG-
CoA reductase gene families that differentially accumulate
transcripts in potato tubers are developmentally expressed in
floral tissues. Plant Mol Biol 33:545–551
Krushkal J, Pistilli M, Ferrell KM, Souret FF, Weathers PJ (2003)
Computational analysis of the evolution of the structure and
function of 1-deoxy–xylulose-5-phosphate synthase, a key
regulator of the mevalonate-independent pathway in plants.
Gene 313:127–138
Lange BM, Croteau R (1999) Isoprenoid biosynthesis via a meva-
lonate-independent pathway in plants: cloning and heterologous
expression of 1-deoxy–xylulose-5-phosphate reductoisomerase
from peppermint. Arch Biochem Biophys 365:170–174
Li L, Li C, Howe GA (2001) Genetic analysis of wound signaling in
tomato. Evidence for a dual role of jasmonic acid in defense and
female fertility. Plant Physiol 127:1414–1417
Lois LM, Rodriguez-Concepcion M, Gallego F, Campos N, Boronat
A (2000) Carotenoid biosynthesis during tomato fruit develop-
ment: regulatory role of 1-deoxy-D-xylulose 5-phosphate syn-
thase. Plant J 22:503–513
Loreto F, Velikova V (2001) Isoprene produced by leaves protects the
photosynthetic apparatus against ozone damage, quenches ozone
products, and reduces lipid peroxidation of cellular membranes.
Plant Physiol 127:1781–1787
Martin D, Tholl D, Gershenzon J, Bohlmann J (2002) Methyl
jasmonate induces traumatic resin ducts, terpenoid resin biosyn-
thesis, and terpenoid accumulation in developing xylem of
norway spruce stems. Plant Physiol 129:1003–1018
Martin DM, Gershenzon J, Bohlmann J (2003) Induction of volatile
terpene biosynthesis and diurnal emission by methyl jasmonate
in foliage of Norway spruce. Plant Physiol 132:1586–1599
Matsingou C, Dimas K, Demetzos C (2006) Design and development
of liposomes incorporating a bioactive labdane-type diterpene. In
vitro growth inhibiting and cytotoxic activity against human
cancer cell lines. Biomed Pharmacother 60:191–199
McConkey ME, Gershenzon J, Croteau R (2000) Developmental
regulation of monoterpene biosynthesis in the glandular tric-
homes of peppermint. Plant Physiol 122:215–224
McKay S, Godard K-A, Toudefallah M, Martin DM, Alfaro R, King
J, Bohlmann J, Plant AL (2006) Wound-induced terpene
synthase gene expression in Sitka spruce that exhibit resistance
or susceptibility to attack by the white pine weevil. Plant Physiol
140:1009–1021
Munne-Bosch S, Alegre L (2003) Drought-induced changes in the
redox state of a-tocopherol, ascorbate, and the diterpene carnosic
acid in chloroplasts of Labiatae species differing in carnosic acid
content. Plant Physiol 131:1816–1825
Munne-Bosch S, Falara V, Pateraki I, Lopez-Carbonell M, Cela J,
Kanellis AK (2008) Physiological and molecular responses of
the isoprenoid biosynthetic pathway in a drought-resistant
Mediterranean shrub, Cistus creticus exposed to water deficit.
J Plant Physiol 166:136–145
Munoz-Bertomeu J, Arrillaga I, Ros R, Segura J (2006) Up-regulation
of 1-deoxy-D-xylulose-5-phosphate synthase enhances produc-
tion of essential oils in transgenic spike lavender. Plant Physiol
142:890–900
Okada K, Saito T, Nakagawa T, Kawamukai M, Kamiya Y (2000)
Five geranylgeranyl diphosphate synthases expressed in different
organs are localized into three subcellular compartments in
Arabidopsis. Plant Physiol 122:1045–1056
Oudin A, Mahroug S, Courdavault V, Hervouet N, Zelwer C,
Rodrıguez-Concepcion M, St-Pierre B, Burlat V (2007) Spatial
distribution and hormonal regulation of gene products from
methyl erythritol phosphate and monoterpene-secoiridoid path-
ways in Catharanthus roseus. Plant Mol Biol 65:13–30
Pateraki I, Kanellis AK (2008) Isolation and functional analysis of
two Cistus creticus cDNAs encoding geranylgeranyl diphos-
phate synthase. Phytochemistry (in press)
Penuelas J, Llusia J, Asensio D, Munne-Bosch S (2005) Linking
isoprene with plant thermotolerance, antioxidants and monoter-
pene emissions. Plant Cell Environ 28:278–286
Pichersky E, Gang DR (2000) Genetics and biochemistry of
secondary metabolites in plants: an evolutionary perspective.
Trends Plant Sci 5:439–445
Pieterse CMJ, Koornneef A, Leon Reyes A, Ritsema T, Verhage A,
Joosten R, De Vos M, Van Oosten V, Dicke M (2007) Cross-talk
between signaling pathways leading to defense against patho-
gens and insects. In: Lorito M, Woo SL, Scala F (eds) Biology of
plant-microbe interactions. The American Phytopathological
Society, APS Press, TX
Rajjou L, Gallardo K, Debeaujon I, Vandekerckhove J, Job C, Job D
(2004) The Effect of a-Amanitin on the Arabidopsis seed
proteome highlights the distinct roles of stored and neosynthe-
sized mRNAs during germination. Plant Physiol 134:1598–1613
Rodriguez-Concepcion M, Boronat A (2002) Elucidation of the
methylerythritol phosphate pathway for isoprenoid biosynthesis
in bacteria and plastids. A metabolic milestone achieved through
genomics. Plant Physiol 130:1079–1089
Ryan CA, Moura DS (2002) Systemic wound signaling in plants: a
new perception. Proc Natl Acad Sci USA 99:6519–6520
Schnee C, Kollner TG, Gershenzon J, Degenhardt J (2002) The Maize
gene terpene synthase 1 encodes a sesquiterpene synthase
catalyzing the formation of (E)-beta -Farnesene, (E)-Nerolidol,
and (E, E)-Farnesol after herbivore damage. Plant Physiol
130:2049–2060
Schwartz SH, Qin X, Zeevaart JAD (2003) Elucidation of the indirect
pathway of abscisic acid biosynthesis by mutants, genes, and
enzymes. Plant Physiol 131:1591–1601
Smith JL, Moraes CMD, Mescher MC (2009) Jasmonate- and
salicylate-mediated plant defense responses to insect herbivores,
pathogens and parasitic plants. Pest Manag Sci 65:497–503
Spoel SH, Koornneef A, Claessens SMC, Korzelius JP, Van Pelt JA,
Mueller MJ, Buchala AJ, Metraux J-P, Brown R, Kazan K, Van
Loon LC, Dong X, Pieterse CMJ (2003) NPR1 modulates cross-
talk between salicylate- and jasmonate-dependent defense path-
ways through a novel function in the cytosol. Plant Cell 15:760–
770
Steele CL, Katoh S, Bohlmann J, Croteau R (1998) Regulation of
oleoresinosis in grand fir (Abies grandis): differential transcrip-
tional control of monoterpene, sesquiterpene, and diterpene
synthase genes in response to wounding. Plant Physiol
116:1497–1504
Takaya A, Zhang YW, Asawatreratanakul K, Wititsuwannakul D,
Wititsuwannakul R, Takahashi S, Koyama T (2003) Cloning,
expression and characterization of a functional cDNA clone
encoding geranylgeranyl diphosphate synthase of Hevea brasil-iensis. Biochim Biophys Acta 1625:214–220
Yang Z, Park H, Lacy GH, Cramer CL (1991) Differential activation of
potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes
by wounding and pathogen challenge. Plant Cell 3:397–405
Yerger EH, Grazzini RA, Hesk D, Cox-Foster DL, Craig R, Mumma
R (1992) A rapid method for isolating glandular trichomes. Plant
Physiol 99:1–7
Zavala JA, Baldwin IT (2006) Jasmonic acid signalling and herbivore
resistance traits constrain regrowth after herbivore attack in
Nicotiana attenuata. Plant Cell Environ 29:1751–1760
640 Plant Cell Rep (2010) 29:629–641
123
Zhao J, Zheng S-H, Fujita K, Sakai K (2004) Jasmonate and ethylene
signalling and their interaction are integral parts of the elicitor
signalling pathway leading to b-thujaplicin biosynthesis in
Cupressus lusitanica cell cultures. J Exp Bot 55:1003–1012
Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction
leading to production of plant secondary metabolites. Biotechnol
Adv 23:283–333
Plant Cell Rep (2010) 29:629–641 641
123