ORIGINAL PAPER

EST analysis and annotation of transcripts derivedfrom a trichome-specific cDNA library from Salvia fruticosa

Fani M. Chatzopoulou • Antonios M. Makris •

Anagnostis Argiriou • Jorg Degenhardt •

Angelos K. Kanellis

Received: 26 January 2010 / Revised: 24 February 2010 / Accepted: 2 March 2010 / Published online: 24 March 2010

� Springer-Verlag 2010

Abstract Greek sage (Salvia fruticosa Mill., Syn. Salvia

triloba L.) is appreciated for its essential oil which is used

as an aromatic spice and active against a wide range of

microorganisms and viruses. The essential oil is dominated

by terpenoids and flavonoids which are produced and

stored in glandular trichomes on the plant surface. The

present study aims to give insights into the metabolic

activities of S. fruticosa trichomes on a transcriptome level.

A total of 2,304 clones were sequenced from a cDNA

library from leaves’ trichomes of S. fruticosa. Exclusion of

sequences shorter than 100 bp resulted in 1,615 high-

quality ESTs with a mean length of 592 bp. Cluster

analysis indicated the presence of 197 contigs (908 clones)

and 707 singletons, generating a total of 904 unique

sequences. Of the 904 unique ESTs, 628 (69.5%) had

significant hits in the non-redundant protein database and

were annotated. A total of 517 (82.3%) sequences were

functionally classified using the gene ontologies (GO) and

established pathway associations to 220 (24.3%) sequences

in Kyoto encyclopedia of genes and genomes (KEGG). In

addition, 52 (5.8%) of the unique ESTs revealed a GO

biological term with relation to terpenoid (78 ESTs), phe-

nylpropanoid (43 ESTs), flavonoid (18 ESTs) or alkaloid

(10 ESTs) biosynthesis or to P450s (26 ESTs). Expression

analysis of a selected set of genes known to be involved in

the pathways of secondary metabolite synthesis showed

higher expression levels in trichomes, validating the tissue

specificity of the analyzed glandular trichome library.

Keywords Salvia fruticosa � Greek sage � EST analysis �Plant trichomes � Secondary metabolism � Gene expression

Introduction

Greek sage (Salvia fruticosa Mill.), also known as S. triloba

L., belongs to the Lamiaceae family. This family includes

important herbs, such as mint, basil, oregano, rosemary,

savory and lavender, which are known for their aromatic

and therapeutic properties since ancient times. S. fruticosa

is endemic in the Eastern Mediterranean and is used as

herbal tea as well as in the fragrance and pharmaceutical

industry (Rivera et al. 1994). Plant extracts such as its

essential oil constituents have been used in a wide range of

studies and showing hypoglycemic activity (Perfumi et al.

1991; Yaniv et al. 1987), cholinergic activity (Savelev et al.

2004), antimycotic activity (Abou-Jawdah et al. 2002),

Communicated by J. R. Liu.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00299-010-0841-9) contains supplementarymaterial, which is available to authorized users.

F. M. Chatzopoulou � 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

A. M. Makris

Department of Natural Products,

Mediterranean Agronomic Institute of Chania,

PO Box 85, 731 00 Chania, Crete, Greece

J. Degenhardt

Institute for Pharmacy, Martin Luther University

Halle-Wittenberg, Hoher Weg 8, 06120 Halle, Germany

A. M. Makris � A. Argiriou

Institute of Agrobiotechnology,

Center for Research and Technology, Hellas,

6th Km Charilaou Thermi Rd, 570 01 Thermi, Greece

123

Plant Cell Rep (2010) 29:523–534

DOI 10.1007/s00299-010-0841-9

antifungal activity against several human or plant pathogens

(Adam et al. 1998; Ali-Shtayeh and Abu Ghdeib 1999;

Daferera et al. 2000; Pitarokili et al. 2003; Sokovic et al.

2002), as well as anti-inflammatory, antimicrobial, antiviral

and cytotoxic activities (Kaileh et al. 2007; Longaray

Delamare et al. 2007; Sivropoulou et al. 1997). Furthermore,

the occurrence of phenolic compounds in the essential oil of

S. fruticosa is responsible for its anti-oxidative properties

(Exarchou et al. 2002; Matsingou et al. 2003; Ozcan 2003;

Papageorgiou et al. 2008; Pizzale et al. 2002).

The production, storage and secretion of the essential oil

takes place in unbranched epidermal appendages, called

glandular trichomes. All the aerial parts of S. fruticosa are

covered with trichomes, but their density is higher in young

leaves and at the lower leaf surface (Karousou et al. 2000).

Although the chemical composition of its essential oil can

vary depending on the season (Papageorgiou et al. 2008)

and the genetic diversity within the species (Bellomaria

et al. 1992; Pitarokili et al. 2003; Skoula et al. 2000), its

main components found in different studies are oxygenated

monoterpenes (1,8-cineole, camphor, borneol), monoter-

pene hydrocarbons (a-pinene, b-pinene, myrcene, camph-

ene), sesquiterpene hydrocarbons (b-caryophyllene) and

small amounts of diterpenes (manool and labd-7,13-dien-

15-ol) (Karioti et al. 2003; Langer et al. 1996; Papageor-

giou et al. 2008; Pitarokili et al. 2003; Putievsky et al.

1986; Skoula et al. 2000). Apart from terpenoids, other

secondary metabolites like phenylpropanoids (Gang et al.

2001) or flavonoids (Aziz et al. 2005) can accumulate in

the trichomes of different species. The functional roles of

secondary metabolites in trichomes include defence against

pathogens (Friedman et al. 2002), resistance to herbivores

(Chermenskaya et al. 2001; Mauricio and Rausher 1997)

and attraction of pollinators (Koeduka et al. 2006).

The biological and economical significance of second-

ary metabolites in glandular trichomes warrants the study

of their biosynthesis. For the biotechnological production

of a desirable compound in large scale or in a host species,

it is necessary to identify the genes participating in its

biosynthesis. A useful tool in that direction is the con-

struction of a cDNA library of the tissue producing the

compound of interest. In the last decade, a number of

cDNA libraries from glandular trichomes of different

species, for instance hop (Nagel et al. 2008; Wang et al.

2008), Cistus creticus (Falara et al. 2008), Artemisia annua

(Bertea et al. 2006), alfalfa (Aziz et al. 2005), tomato

(Fridman et al. 2005), Salvia stenophylla (Hoelscher et al.

2003), basil (Gang et al. 2001) and peppermint (Lange

et al. 2000), have been constructed and led to the identi-

fication of several genes participating in biosynthesis of

important secondary metabolites. To that end, we report

the analysis of a glandular trichomes cDNA library from

S. fruticosa in order to identify putative genes that are

involved in secondary metabolism pathways and especially

in terpenoid biosynthesis.

Materials and methods

Plant material

Glandular trichomes were isolated from young leaves using

a method modified from Gershenzon et al. (1992). In brief,

approx. 7 g of young, not fully expanded leaves were

harvested, soaked in ice-cold, distilled water containing

0.05% Tween 20 for 2 h. The water was then decanted and

the leaves were washed twice with ice-cold, distilled water,

and abraded using a cell disrupter (Bead Beater, Biospec

Products, Bartlesville, USA). The chamber was filled with

the plant material, 65 ml of glass beads (0.5–1.0 mm

diameter), XAD-4 resin (1 g/g plant material), and ice-cold

extraction buffer [25 mM HEPES pH 7.3, 12 mM KCl,

5 mM MgCl2, 0.5 mM K2HPO4, 0.1 mM Na4P2O7, 5 mM

DTT, 2.4 g l-1 sucrose, 26.4 g l-1D-sorbitol, 6 g l-1

methyl cellulose, and 10 g l-1 polyvinylpyrrolidone (PVP;

Mr 40,000)] to full volume. Glands were abraded from the

leaves with three pulses of 1 min at medium speed, with a

1-min rest between pulses. Following abrasion, the glands

were separated from leaf material, glass beads, and XAD-4

resin by passing the supernatant of the chamber through a

500-lm mesh cloth. The residual plant material and beads

were rinsed twice with 10 ml ice-cold isolation buffer

(extraction buffer without methylcellulose) and passed

through the 500-lm mesh. The combined 500-lm filtrates

were then consecutively filtered through membranes with

decreasing mesh size (350, 200, and 100 lm). Finally,

clusters of secretory cells (approx. 60 lm in diameter)

were collected by passing the 100-lm filtrate through a 20-

lm mesh. An aliquot of the isolated cell clusters were

checked for integrity and purity with a light-optical

microscope before being transferred to a 1.5 ml reaction

tube and frozen in liquid nitrogen prior to RNA extraction.

RNA isolation, cDNA library construction and EST

sequencing

Total RNA was extracted with Trizol in accordance to the

manufacturer’s instructions. The cDNA was prepared with

‘‘SMART’’ cDNA synthesis kit (Clontech) and was ligated

to the pDNRLib vector in the SfiIA and SfiIB restriction

sites. The single pass sequencing of 2,304 ESTs (using the

T7 primer) was carried out by the Purdue Genomics Core

Facility of the University of Purdue. The reads were then

processed (poly-A trimming and vector trimming) using

the pipeline Lucy (Chou and Holmes 2002) resulting in

1,615 high-quality sequences ([100 bases).

524 Plant Cell Rep (2010) 29:523–534

123

Contig assembly and sequence analysis

High-quality ESTs (1,615 sequences) were used for

assembling into clusters. The analysis was performed using

the Contig Assembly Program CAP3 (Huang and Madan

1999) (http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.

py?form=cap3) using default parameters. The resulting

unique ESTs (contigs and singletons) were then imported

into the bioinformatics tool Blast2GO (Conesa et al. 2005)

(http://www.blast2go.de/) and were compared against the

National Center for Biotechnology Information (NCBI)

non-redundant protein database BLASTX (E value \ 10-3)

(Altschul et al. 1997).

Furthermore, in order to identify similar sequences in

other trichome containing plant species, all ESTs were

compared against trichome-specific databases. Sequences

were downloaded from the TrichOME Database (http://

www.planttrichome.org/) and local BLAST searches were

performed.

Open reading frames (ORFs) were predicted by Orf-

Predictor (Min et al. 2005) (https://fungalgenome.concordia.

ca/tools/OrfPredictor.html). It should be noted that this site

has been recently moved to a new web address (http://

proteomics.ysu.edu/tools/OrfPredictor.html). The GC con-

tent of the ESTs was estimated using geecee provided

by the Institut Pasteur (http://mobyle.pasteur.fr/cgi-bin/

MobylePortal/portal.py?form=geecee). Signal peptides for

the subcellular localization of the predicted proteins were

identified using the software TargetP (Emanuelsson et al.

2000) (http://www.cbs.dtu.dk/services/TargetP/). TargetP

was used only for the full-length sequences or for those who

predicted to contain the N-terminal according to manual

BLASTX search. Its prognosis is based on the presence or

absent of secretory pathway signal peptide, chloroplast

transit peptide or finally mitochondrial targeting peptide.

In order to assign a putative function to the proteins, we

used the Blast2GO tool. The mapping and annotation of

the sequences according to gene ontology (GO) terms

(Ashburner et al. 2000) is based on sequence similarity and

therefore, sequences without BLAST hit were not anno-

tated. For the annotation configuration the default settings

were used (E value filter of 1E-6 and annotation cutoff of

55). Each sequence could have more than one GO terms,

either at the different GO categories (Biological Process,

Molecular Function and Cellular Component) or at the

same category. Furthermore, in order to improve annotat-

ability we used InterProScan, which searched the databases

BlastProDom, FPrintScan, HMMPIR, HMMPfam, HMM-

Smart, HMMTigr, ProfileScan, ScanRegExp and Super-

Family (Quevillon et al. 2005) (http://www.ebi.ac.uk/

interpro/index.html) provided by the EBI (Labarga et al.

2007) (http://www.ebi.ac.uk/) through Blast2GO. The

sequences are mapped according to their domain/motif

similarity and the GO results can be merged with the

remaining annotations. Furthermore, the assignment of the

peptides into metabolic pathways was done by the Kyoto

Encyclopedia of Genes and Genomes (KEGG, http://www.

genome.jp/kegg/kegg2.html).

Expression analysis

Total RNA from young and old leaves, glandular trichomes

and the remaining part of the leaves was extracted as

described. One microgram of total RNA from each tissue

was used in a reverse transcription reaction using 0.5 lg

30RACE Adapter Primer and SuperScript III-RT (Invitro-

gen, Carlsbad, CA, USA) following the manufacturer’s

protocol. Successful cDNA synthesis was monitored by

amplifying a fragment of the eIF4a gene. PCR was per-

formed using the Platinum SYBR Green qPCR SuperMix-

UDG (Invitrogen, Carlsbad, CA, USA) with 1/20 of the

synthesized cDNAs as template and the corresponding

primers at 0.4 lM. The cycling conditions were: 2 min/

50�C, 2 min/94�C, 40 cycles of 15 s/94�C, 20 s/58�C,

20 s/72�C and a final extension step of 10 min/72�C. The

primers used for the expression experiments are indicated

in Table 1S. Two different housekeeping genes to be used

as controls for the cDNA quantity, GAPDH and eIF4a,

were tested. The eIF4a presented more stable expression

levels in all the examined tissues and was used as internal

control for cDNA normalization in the expression analysis.

Quantitative expression analysis of the genes was per-

formed in a real-time PCR, using the RG6000 (Corbett Life

Science, Sydney, AU) Real-Time PCR system. For identi-

fication of the PCR products a melting curve was performed

from 65 to 95�C with read every 0.2�C and 5 s hold between

reads. The whole experimental procedure was performed at

least three times starting from cDNA synthesis. The thresh-

old cycle (Ct) values of the triplicate PCRs were averaged

and relative quantification of the transcript levels was per-

formed using the comparative Ct method (Livak and Sch-

mittgen 2001). The fold change in the target gene was

determined with the following formula: fold change =

E-DDCT, where DDCT = (Ct target gene - Ct Ref) at Point

X - (Ct target gene - Ct Ref) Point Y and E the efficiency

of the reaction calculated using the LinRegPCR software

(Ramakers et al. 2003). The results were expressed as rela-

tive values to the gene expression levels in leaves.

Results and discussion

Generation of ESTs and contig assembly

A total of 2,304 clones were sequenced from a cDNA

library constructed from total leaf trichome total RNA of

Plant Cell Rep (2010) 29:523–534 525

123

S. fruticosa. Using the vector/quality clipping program

Lucy (Chou and Holmes 2002), clones with no insert or

sequences shorter than 100 bp were excluded, resulting in

1,615 high-quality ESTs with a mean length of 592 bp

(Table 1) (Fig. 1S). All edited EST sequences have been

submitted to the GenBank dbEST and are accessible under

the accession numbers from FE535951 to FE537409.

Cluster analysis with CAP3 (Huang and Madan 1999)

indicated the presence of 197 contigs consisting of 908

ESTs and 707 singletons. The transcript redundancy of the

EST library was 56% (number of clustered ESTs/total

ESTs), that means that the gene discovery rate was 44%.

According to the clustering, a total of 904 unique

sequences (UniESTs) were identified (Table 1), of which

878 (97%) had open reading frames (ORFs) (data not

shown). The GC content distribution of the coding

sequences is presented in Fig. 1 with an average GC con-

tent of about 43%.

The most highly expressed ESTs (C10 ESTs) are ranked

according to the number of contributing ESTs (Table 2).

The abundance of these ESTs suggests an important role of

their transcripts in the metabolism of these tissues. Seven

of them were detected over 1% of the total sequences. Of

these, the most numerous cluster was 94, which is com-

posed of 71 ESTs and bears homology to a non-specific

lipid transfer protein (LTPs) precursor (Table 2). Plant

LTPs transfer phospholipids as well as glycolipids, fatty

acids and sterols across membranes (Guerbette et al. 1999;

Kader et al. 1984). Other functions for LTPs have been

proposed, such as participation in the protection against

pathogens and the wax or cutin deposition in the cell walls

of expanding epidermal cells and certain secretory tissues

(Lange et al. 2000; Maldonado et al. 2002; Pyee et al.

1994; Regente and De La Canal 2003; Sterk et al. 1991).

Plant trichomes are rich in LTPs as judged by the EST

analysis of a number of trichome-specific cDNA libraries

such as hop (Wang et al. 2008), Cistus creticus (Falara

et al. 2008), alfalfa (Aziz et al. 2005), basil (Gang et al.

2001) and peppermint (Lange et al. 2000). The increased

presence of LTP transcripts in the leaf trichome cDNA

library of S. fruticosa indicates its involvement in plant

defence (Maldonado et al. 2002; Molina et al. 1993). This

hypothesis is also supported by the higher expression in

trichomes of a S. fruticosa LTP (GenBank: FE537054)

(Fig. 4). The second most abundant cluster was the 151

with 60 ESTs (Table 2). This cluster revealed homology to

a hypothetical protein (NitaMp027) from Nicotiana taba-

cum with unknown function (Sugiyama et al. 2005).

However, most of the remaining contigs contain 2 or 3

ESTs, a reflection of an effective representation of genes

with low abundance within the library (Fig. 2S). The

redundancy of the clusters is similar to other trichomes’

cDNA libraries recently evaluated (Falara et al. 2008;

Lange et al. 2000).

Among the most abundant ESTs, several secondary

metabolism-related ESTs were identified (Table 2). More

specifically, clusters 14, 53 and 180 reveal homology to

phenylcoumaran benzylic ether reductase homolog Fi1

(PCBER), germacrene D synthase and caffeic acid 3-O-

methyltransferase (COMT), respectively (Table 2).

PCBERs are involved in lignan biosynthesis, which play an

important role in plant defence (Gang et al. 1999; Min et al.

2003; Vander Mijnsbrugge et al. 2000), while COMT is a

key enzyme in lignin and flavonoid biosynthesis which

converts caffeic acid to ferulic acid or 5-hydroxyferulate to

sinapate (Anterola and Lewis 2002; Guo et al. 2001).

COMT is induced by mechanical wounding and water

stress, but it is not affected by insect feeding (Reymond

et al. 2000). Germacrene D synthase catalyzes the forma-

tion of germacrene D from farnesyl diphosphate (FPP)

(Dudareva and Pichersky 2006) and it was recently found

to exhibit a trichome-specific expression (Falara et al.

2008). Germacrene D is a sesquiterpene abundant in many

essential oils (Flamini et al. 2007; Ogutcu et al. 2008;

Pitarokili et al. 2002). As germacrene D is a volatile

hydrocarbon, it contributes to plant scents (Guterman et al.

2002) and can play a significant role in the attraction of

pollinating insects (Buttery et al. 1986; MacFarlane et al.

2003). In addition, a monoterpene synthase (cluster 61)

Table 1 Summary statistics of the ESTs generated from leaves’

trichomes of Salvia fruticosa

Feature Value

Total number of clones sequenced 2,304

Number of high-quality sequences 1,615 (70%)

Average length of high-quality ESTs (bp) 592 ± 5.8

Number of contigs 197

Number of ESTs in contigs 908 (56%)

Number of singletons 707 (44%)

Number of UniESTs 904

Fig. 1 GC content distribution. The GC content of each UniEST is

plotted against their abundance (average GC content = 42.5%)

526 Plant Cell Rep (2010) 29:523–534

123

with high similarity to a S. fruticosa cineole synthase

(SfCinS1) has been identified (Kampranis et al. 2007). 1,8-

Cineole synthase was found to be either a single product

enzyme (Shimada et al. 2005) or a multi-product enzyme

that forms of a terpenoid mixture with 1,8-cineole being

the major product (Kampranis et al. 2007; Roeder et al.

2007; Wise et al. 1998). With up to 74% of the total

content, this monoterpene was the dominant component in

the essential oil (Karousou et al. 2000; Langer et al. 1996;

Papageorgiou et al. 2008; Putievsky et al. 1986; Sivro-

poulou et al. 1997; Skoula et al. 2000, 1999; Sokovic et al.

2002) and showed antifungal (Pitarokili et al. 2003;

Sokovic et al. 2002), antimicrobial and antiviral activity

(Sivropoulou et al. 1997).

In order to ascribe a putative function to each unique

EST, a similarity search against the NCBI non-redundant

protein database BLASTX (E value \ 10-3) (Altschul

et al. 1997) using the Blast2GO analysis tool (Conesa et al.

2005) was performed. Of the 904 unique ESTs, 628

(69.5%) revealed at least one significant match, while the

remaining 276 had no significant similarity (26.9%) or

were similar to unknown proteins (3.6%). The majority of

the annotated sequences had top BLAST hits to transcripts

from Oryza sativa (20%), followed by Arabidopsis thali-

ana (16%) and Vitis vinifera (14%) (Fig. 3S). Taxonomi-

cally, the vast majority of the top BLAST hits belonged to

the asterids subclass (64%) which includes the Solanaceae

and Lamiaceae families. Two other subclasses, namely

rosids (24%; Fabaceae, Brassicaceae and Salicaceae) and

commelinids (12%; Poaceae and Arecaceae), were less

represented.

Putative subcellular localization

The amino acid sequences of the full-length UniESTs or

those who had the N-terminus were used for the prediction

of the subcellular localization of the proteins. The analysis

was performed using the software TargetP (Emanuelsson

et al. 2000). Of the 239 sequences analyzed, 38 (15.9%)

contained secretory pathway signal peptide, 33 (13.8%)

contained a chloroplast transit peptide and 27 (11.3%)

contained a mitochondrial targeting peptide, while for the

remaining sequences (141, 58.9%) no prediction was

available (Table 2S).

Functional analysis of the unigenes

Gene ontology annotation

Of the 904 UniESTs, 628 (69.5%) had significant hits in

the non-redundant protein database and were annotated in

order to retrieve a putative function. A total of 517

(82.3%) unique sequences were functionally classified in

one or more ontologies [GO categories: biological process

(P), molecular function (F) and cellular component (C)]

(Ashburner et al. 2000) (Fig. 4S). For the functional

annotation, the automated software Blast2GO was used.

However, it must be taken into account that many

Table 2 The most abundant ESTs detected from the sequencing

Cluster ID No. of ESTs/

contig

Description from nr hit (BlastX) % identity E value % of total

ESTs

94 71 Non-specific lipid transfer protein precursor (Fragaria 9 ananassa) 53 1E-19 4.40

151 60 Hypothetical protein NitaMp027 (Nicotiana tabacum) 98 2E-41 3.72

47 38 H ? -transporting two-sector ATPase chain 9.1 (Raphanus sativus) 91 3E-09 2.35

58 38 No significant similarity – – 2.35

14 24 Phenylcoumaran benzylic ether reductase homolog Fi1

(Forsythia 9 intermedia)

73 7E-122 1.49

68 23 Putative stress-responsive protein [Oryza sativa(japonica cultivar-group)]

50 4E-21 1.42

86 17 No significant similarity – – 1.05

37 15 Unknown protein (Vitis vinifera) 42 5E-22 0.93

69 15 Cytochrome P450 monooxygenase isoform I (Sesamum indicum) 48 4E-69 0.93

53 13 Germacrene D synthase (Ocimum basilicum) 53 1E-163 0.80

111 12 Pathogenesis-related protein 10 (Vitis vinifera) 60 1E-51 0.74

61 12 Cineole synthase (Salvia fruticosa) 99 0.0 0.74

3 11 Unknown protein (Arabidopsis thaliana) 25 0.005 0.68

159 10 ATPase subunit 1 (Beta vulgaris subsp. vulgaris) 95 0.0 0.62

180 10 Caffeic acid 3-O-methyltransferase (Catharanthus roseus) 64 1E-58 0.62

Contigs with more than 10 EST members are presented

Plant Cell Rep (2010) 29:523–534 527

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sequences had more than one assignment within a GO

category, resulted in 1,915 GO terms in total and at a

mean GO level of 4.92 (Fig. 5S).

In the biological class (second level GO terms, Fig. 2a)

the majority of the GO terms were grouped into two cate-

gories, namely, cellular (GO:0009987, 32%) and metabolic

(GO:0008152, 31%) processes. The secondary metabolite

processes were included in metabolic processes (third level

GO terms) and represented the 1.2% of the GO terms on that

level (data not shown). Considering the molecular function

class (second level GO terms, Fig. 2b), the vast majority

were involved in the catalytic activity (GO:0003824, 40%)

and the binding activity (GO:0005488, 39%). Furthermore,

for the cellular component class (second level GO terms,

Fig. 2c) the assignments were mostly given to cell

(GO:0005623, 29%), cell part (GO:0044464, 29%) and

organelle (GO:0043226, 23%).

Annotation augmentation using InterProScan

The number of the Blast-based annotated sequences with

GO terms can be increased using InterProScan (Quevillon

et al. 2005). The search for additional databases for motif/

domain similarities resulted in 132 new annotations (2,047

in total) with a slight difference in the mean GO level

(4.90). The most common InterPro families are presented

in Table 3. There are 422 InterPro families recognized,

with the most frequent family being the NAD(P)-binding

with 11 members, followed by the thioredoxin fold

(9 members). The terpene synthase, metal-binding and the

terpenoid synthase families consist of 7 and 6 members,

respectively (Table 3).

Pathway analysis

For the establishment of pathway association, the Kyoto

Encyclopedia of Genes and Genomes (KEGG) within

Blast2GO were used. The mapping process of GO terms

allows the recovery of the Enzyme Commission numbers

(EC number) and their classification in KEGG pathways. A

total of 220 (24.3%) UniESTs disposed one or more EC

numbers providing 263 ECs. These EC numbers were

mapped to 121 KEGG pathways (Table 3S). The 25 most

represented pathways (C7 UniESTs) are presented in

Table 4. Among the metabolic pathways identified, six

secondary metabolism-related pathways were included and

concerned terpenoid (ko00900), monoterpenoid (ko00902),

phenylpropanoid (ko00940), flavonoid (ko00941) and

alkaloid I (ko00950) and II biosynthesis (ko0090) (Table 5).

Five enzymes participating either in the cytosolic (MVA

pathway), or in the plastidic (MEP pathway) terpenoid bio-

synthesis pathways have been mapped into two KEGG

pathway categories, namely terpene biosynthesis and

especially monoterpene biosynthesis pathways (Table 5).

Several other enzymes contributing to phenylpropanoid,

flavonoid and alkaloid biosynthesis have been recognized

(Table 5).

Genes participating in biosynthesis of secondary

metabolites in Salvia fruticosa leaf trichomes

The trichomes of S. fruticosa are the ‘‘factories’’ where the

biosynthesis of a wide range of secondary metabolites,

such as terpenoids (mono-, di- and sesqui-terpenoids),

flavonoids and phenylpropanoids takes place (Exarchou

et al. 2002; Karousou et al. 2000; Pizzale et al. 2002;

Sivropoulou et al. 1997). Although a number of alkaloids

have been isolated from roots of Salvia species, like

S. yunnanensis and S. prionitis (Li et al. 2000; Lin et al.

2006), there is no references for their presence in S. fruti-

cosa essential oil. The presence of transcript of putative

alkaloid biosynthesis enzymes needs further investigation.

The cDNAs of these genes will provide a useful tool for the

elucidation and genetic manipulation of the pathways.

Several sequences in the trichomes EST library of

S. fruticosa revealed homology to genes participating in

biosynthesis of secondary metabolites. A complete list of

the putative genes is presented in Table 4S. Most of the

secondary metabolism-related ESTs had high similarity

with genes from the terpenoid biosynthetic pathway

(44.3%), followed by the phenylpropanoid pathway

(24.7%) (Table 4S). The genes contributing to flavonoid

and alkaloid biosynthesis represent the 10.3 and the 5.7%,

respectively. In addition, a number of putative P450s

(14.9%) that might participate in secondary metabolites’

biosynthesis have been recognized (Table 4S).

Concerning the terpenoid biosynthesis, genes partici-

pating in both the plastidic and cytosolic metabolic path-

ways have been identified (Table 4S). In particular,

nine out of ten genes (1-deoxyxylulose-5-phosphate syn-

thase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase,

2-C-methyl-D-erythritol 4-phosphate cytidyltransferase,

4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, 2C-

methyl-D-erythritol 2,4-cyclodiphosphate synthase, 1-hydroxy-

2-methyl-butenyl 4-diphosphate reductase, isopentenyl

pyrophosphate isomerase, geranyl diphosphate synthase

and geranylgeranyl diphosphate synthase) are involved in

the formation of the geranyl diphosphate and geranylger-

anyl diphosphate, the substrates of mono- and diterpene

biosynthases, respectively. Also identified in the trichome

library were several monoterpene synthases (cineole syn-

thase 1, cineole synthase 2, bornyl diphosphate synthase

homologue) and diterpene synthases (copalyldiphosphate

synthase, ent-kaurene synthase homologue) (Table 4S).

Furthermore, most of the genes (five out of eight) acting in

the cytosol for the biosynthesis of the sesquiterpene

528 Plant Cell Rep (2010) 29:523–534

123

precursor FPP have been recognized (acetoacetyl-

CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase,

3-hydroxy-3-methylglutaryl-CoA reductase, isopentenyl

pyrophosphate isomerase, farnesyl diphosphate synthase),

as well as some sesquiterpene synthases (germacrene D

synthases, cis-muuroladiene synthase) (Table 4S). A subset

Fig. 2 Gene Ontology (GO)

assignment (2nd level GO

terms) of 904 Salvia fruticosaannotated UniESTs using the

Blast2GO software. The three

GO categories, biological

process (a), molecular function

(b) and cellular component (c)

are presented

Plant Cell Rep (2010) 29:523–534 529

123

of six predicted genes involved in different predicted

metabolic pathways, was selected for expression analysis

(Figs. 3, 4).

To validate the tissue specificity of the analyzed

S. fruticosa glandular trichome library we selected a rep-

resentative set of genes for expression analysis using real-

time PCR. The EST of SfLTP (GenBank: FE537054)

identified as the most numerous gene encountered, the

monoterpene cineole synthase 1 encountered in 12 EST

SfCinSin1 (GenBank: DQ785793), the second cineole

synthase SfCinSin2 (GenBank: FJ618810) encountered in 2

EST, the monoterpene bornyl diphosphate synthase

homologue SfBPPS1 (GenBank: FE537328) with 4 EST,

the phenylcoumaran benzylic ether reductase homologue

Fi1 SfIFR (GenBank: GU479926) representing the most

abundant EST from the flavonoid biosynthetic pathway

with 24 EST identified and the triose-phosphate isomerase

homologue SfTPI (GenBank: FE536366) a core biosyn-

thetic gene. Two housekeeping genes were chosen as

controls for the cDNA quantity, the SfGAPDH (GenBank:

FE536708) and the SfeIF4a (GenBank: FE536666). Among

them, SfeIF4a was found to have the most stable expression

levels in all examined tissues and was subsequently

selected for normalization. Initially, the expression levels

of SfGAPDH, SfTPI and SfIFR were tested in old and

young leaves. The results indicated a lower expression of

approx. 25–30% in older leaves of all three genes

examined. Interestingly also the SfGAPDH presents a

decreased expression levels using eIF4a as internal control

(Fig. 3) probably due to a decrease of the overall metab-

olism in old leaves.

Quantitative expression analysis in whole leaves, iso-

lated trichomes and leaves without trichomes revealed a

higher expression of SfLTP, SfCinSin1, SfCinSin2,

SfBPPS1 and SfIFR in isolated trichomes compared to

leaves without trichomes or to whole leaves. More spe-

cifically, SfLTP showed a 20- to 25-fold induction, the

SfCinSin1, which is the main cineole synthase of Salvia

fruticosa exhibited[100-fold induction, the second cineole

synthase SfCinSin2 with [30-fold increase, the SfBPPS1

homologue with[30-fold induction and SfIFR with 18-fold

induction. No significant difference for SfTPI was detected

(Fig. 4). The expression data confirmed the tissue speci-

ficity of the library analyzed. Their relative transcript

abundance corresponds quite well with the number of EST

identified for each transcript but they are not identical. The

Table 3 The most frequent InterPro families found in Salvia fruti-cosa EST library

InterPro No. Description UniESTs

sequence

count

IPR016040 NAD(P)-binding 11

IPR012335 Thioredoxin fold 9

IPR005630 Terpene synthase, metal-binding 7

IPR008949 Terpenoid synthase 6

IPR001806 Ras GTPase 6

IPR000719 Protein kinase, core 6

IPR012340 Nucleic acid-binding, OB-fold 5

IPR000886 Endoplasmic reticulum,

targeting sequence

5

IPR005225 Small GTP-binding protein 5

IPR000916 Bet v I allergen 5

IPR012677 Nucleotide-binding, alpha–beta plait 5

IPR013785 Aldolase-type TIM barrel 4

IPR001199 Cytochrome b5 4

IPR001023 Heat shock protein Hsp70 4

IPR001128 Cytochrome P450 4

In the list are presented the families with more than 4 UniEST

members

Table 4 The 25 most represented KEGG pathways

KEGG pathway UniESTs

sequence

count

Enzymes

Oxidative phosphorylation 19 6

Carbon fixation 17 11

Glycolysis/gluconeogenesis 16 9

Biosynthesis of steroids 13 9

Beta-Alanine metabolism 12 6

Urea cycle and metabolism

of amino groups

12 6

Methionine metabolism 11 6

Nucleotide sugars metabolism 10 6

Ubiquitin mediated proteolysis 10 2

Purine metabolism 9 4

Alanine and aspartate metabolism 9 6

Fructose and mannose metabolism 9 4

Glutathione metabolism 8 6

Calcium signaling pathway 8 2

Pantothenate and CoA biosynthesis 8 2

Inositol metabolism 8 4

Citrate cycle (TCA cycle) 8 6

Propanoate metabolism 8 8

Selenoamino acid metabolism 8 4

Terpenoid biosynthesis 8 4

Phenylalanine metabolism 7 3

Pentose phosphate pathway 7 5

Thiamine metabolism 7 2

Pyrimidine metabolism 7 3

Reductive carboxylate cycle (CO2 fixation) 7 5

Metabolic pathway with more than 7 UniESTs are presented

530 Plant Cell Rep (2010) 29:523–534

123

tissues used for the library preparation were harvested in

March at the first strong onset of essential oil production,

whereas the tissues used for the real-time PCR analysis

were harvested in mid-June which is the peak production

period for essential oils. As such, the expression levels of

SfCinS1, SfCinS2 and SfBPPS were probably overrepre-

sented in the June tissues. It should also be noted that the

SfCinS2 has not yet been identified from the other well

studied related Salvia species S. officinalis. Its contribution

to cineole production although less than enzyme 1, is

expected to be significant, as it possesses equivalent cata-

lytic activity by in vitro assays (S. Kampranis, personal

communication). Taken together our results indicate that

the S. fruticosa glandular trichome library is highly rich in

tissue-specific transcripts and complex enough to contain

a series of novel genes associated with secondary

Table 5 The six KEGG pathways related to biosynthesis of secondary metabolites

Biosynthesis of secondary metabolites

EC Name of enzyme UniESTs

KEGG pathway: terpenoid biosynthesis

2.5.1.1 Dimethylallyltranstransferase Contig 40, Contig 88, Salviafruticosa_01_H10_T7

2.5.1.29 Farnesyltranstransferase Contig 88

5.3.3.2 Isopentenyl-diphosphate delta-isomerase Salviafruticosa_05_B01_T7

KEGG pathway: monoterpenoid biosynthesis

4.2.3.11 Sabinene-hydrate synthase Salviafruticosa_06_L11_T7

5.5.1.8 Bornyl diphosphate synthase Contig 92, Salviafruticosa_06_L11_T7

KEGG pathway: phenylpropanoid biosynthesis

2.1.1.68 Caffeate O-methyltransferase Contig 180, Salviafruticosa_04_E11_T7

1.11.1.7 Peroxidase Contig 55, Salviafruticosa_02_J23_T7,

Salviafruticosa_05_M23_T7

1.1.1.195 Cinnamyl-alcohol dehydrogenase Salviafruticosa_04_O24_T7

KEGG pathway: flavonoid biosynthesis

2.1.1.68 Caffeate O-methyltransferase Contig 180, Salviafruticosa_04_E11_T7

KEGG pathway: alkaloid biosynthesis I

1.3.3.8 Tetrahydroberberine oxidase Contig 105

2.6.1.1 Aspartate transaminase Salviafruticosa_04_N15_T7

KEGG pathway: alkaloid biosynthesis II

3.1.1.1 Carboxylesterase Contig 35, Salviafruticosa_03_H03_T7,

Salviafruticosa_03_M01_T7,

Salviafruticosa_04_E03_T7,

Salviafruticosa_04_J22_T7

The enzyme names, the EC numbers and the UniESTs in each pathway are given

Fig. 3 Expression of SfGAPDH

(GenBank: FE536708), SfTPI

(GenBank: FE536366) and

SfIFR (GenBank:GU479926)

transcripts in new and old

leaves. The values are the

mean ± SD of at least three

independent experiments

relative to the fold change of the

transcripts in new leaves

Plant Cell Rep (2010) 29:523–534 531

123

metabolism. Further analysis should enable us to identify

additional minor EST that contributes to natural product

biosynthesis in S. fruticosa.

Acknowledgments We thank M. Georgiadou for technical assis-

tance and N. Dudareva for fruitful colaboration. This work is part of

the 875-03ED research project implemented within the framework of

the ‘‘Reinforcement Programme of Human Research Manpower’’

(PENED) 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)

and EU-FP7-227448 TERPMED.

References

Abou-Jawdah Y, Sobh H, Salameh A (2002) Antimycotic activities of

selected plant flora, growing wild in Lebanon, against phyto-

pathogenic fungi. J Agric Food Chem 50:3208–3213

Adam K, Sivropoulou A, Kokkini S, Lanaras T, Arsenakis M (1998)

Antifungal activities of Origanum vulgare subsp. hirtum,

Mentha spicata, Lavandula angustifolia, and Salvia fruticosaessential oils against human pathogenic fungi. J Agric Food

Chem 46:1739–1745

Ali-Shtayeh MS, Abu Ghdeib SI (1999) Antifungal activity of plant

extracts against dermatophytes. Mycoses 42:665–672

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,

Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new

generation of protein database search programs. Nucleic Acids

Res 25:3389–3402

Anterola AM, Lewis NG (2002) Trends in lignin modification: a

comprehensive analysis of the effects of genetic manipulations/

mutations on lignification and vascular integrity. Phytochemistry

61:221–294

Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM,

Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill

DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richard-

son JE, Ringwald M, Rubin GM, Sherlock G (2000) Gene

ontology: tool for the unification of biology. Nat Genet 25:25–29

Aziz N, Paiva N, May G, Dixon R (2005) Transcriptome analysis of

alfalfa glandular trichomes. Planta 221:28–38

Bellomaria B, Arnold N, Valentini G (1992) Contribution to the study

of the essential oils from three species of Salvia growing wild in

the Eastern Mediterranean region. J Essent Oil Res 4:607–614

Fig. 4 Expression of SfTPI

(GenBank: FE536366), SfLTP

(GenBank: FE537054),

SfCinSin1 (GenBank.

DQ785793), SfCinSin2

(GenBank: FJ618810), SfBPPS1

(GenBank: FE537328) and

SfIFR (GenBank:GU479926)

transcripts in whole leaves,

leaves without trichomes and

trichomes. The values are the

mean ± SD of at least three

independent experiments

relative to the fold change of the

transcripts in new leaves

532 Plant Cell Rep (2010) 29:523–534

123

Bertea CM, Voster A, Verstappen FWA, Maffei M, Beekwilder J,

Bouwmeester HJ (2006) Isoprenoid biosynthesis in Artemisiaannua: cloning and heterologous expression of a germacrene A

synthase from a glandular trichome cDNA library. Arch

Biochem Biophys 448:3–12

Buttery RG, Maddox DM, Light DM, Ling LC (1986) Volatile

components of yellow starthistle. J Agric Food Chem 34:786–788

Chermenskaya TD, Burov VN, Maniar SP, Pow EM, Roditakis N,

Selytskaya OG, Shamshev IV, Wadhams LJ, Woodcock CM

(2001) Behavioural responses of western flower thrips, Frank-liniella occidentals (Pergande), to volatiles from three aromatic

plants. Insect Sci Appl 21:67–72

Chou HH, Holmes MH (2002) DNA sequence quality trimming and

vector removal. Bioinformatics 17:1093–1104

Conesa A, Gotz S, Garcıa-Gomez JM, Terol J, Talon M, Robles M

(2005) Blast2GO: a universal tool for annotation, visualization

and analysis in functional genomics research. Bioinformatics

21:3674–3676

Daferera DJ, Ziogas BN, Polissiou MG (2000) GC–MS analysis of

essential oils from some Greek aromatic plants and their

fungitoxicity on Penicillium digitatum. J Agric Food Chem

48:2576–2581

Dudareva N, Pichersky E (2006) Floral scent metabolic pathways:

their regulation and evolution. In: Dudareva N, Pichersky E (eds)

Biology of floral scent. CRC Press by Taylor and Francis Group,

Boca Raton, FL, pp 55–71

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

Exarchou V, Nenadis N, Tsimidou M, Gerothanassis IP, Troganis A,

Boskou D (2002) Antioxidant activities and phenolic composi-

tion of extracts from Greek oregano, Greek sage, and summer

savory. J Agric Food Chem 50:5294–5299

Falara V, Fotopoulos V, Margaritis T, Anastasaki T, Pateraki I,

Bosabalidis A, 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

Flamini G, Cioni PL, Maccioni S, Baldini R (2007) Composition of

the essential oil of Daucus gingidium L. ssp. gingidium. Food

Chem 103:1237–1240

Fridman E, Wang J, Iijima Y, Froehlich JE, Gang DR, Ohlrogge J,

Pichersky E (2005) Metabolic, genomic, and biochemical

analyses of glandular trichomes from the wild tomato species

Lycopersicon hirsutum identify a key enzyme in the biosynthesis

of methylketones. Plant Cell 17:1252–1267

Friedman M, Henika PR, Mandrell RE (2002) Bactericidal activities

of plant essential oils and some of their isolated constituents

against Campylobacter jejuni, Escherichia coli, Listeria mono-cytogenes, and Salmonella enterica. J Food Prot 65:1545–1560

Gang DR, Kasahara H, Xia ZQ, Mijnsbrugge KV, Bauw G, Boerjan

W, Van Montagu M, Davin LB, Lewis NG (1999) Evolution of

plant defense mechanisms: relationships of phenylcoumaran

benzylic ether reductases to pinoresinol-lariciresinol and iso-

flavone reductases. J Biol Chem 274:7516–7527

Gang DR, Wang J, Dudareva N, Nam KH, Simon JE, Lewinsohn E,

Pichersky E (2001) An investigation of the storage and

biosynthesis of phenylpropenes in sweet basil. Plant Physiol

125:539–555

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

Guerbette F, Grosbois M, Jolliot-Croquin A, Kader J-C, Zachowski A

(1999) Lipid-transfer proteins from plants: structure and binding

properties. Mol Cell Biochem 192:157–161

Guo D, Chen F, Inoue K, Blount JW, Dixon RA (2001) Downreg-

ulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA

3-O-methyltransferase in transgenic alfalfa: impacts on lignin

structure and implications for the biosynthesis of G and S lignin.

Plant Cell 13:73–88

Guterman I, Shalit M, Menda N, Piestun D, Dafny-Yelin M, Shalev

G, Bar E, Davydov O, Ovadis M, Emanuel M, Wang J, Adam Z,

Pichersky E, Lewinsohn E, Zamir D, Vainstein A, Weiss D

(2002) Rose scent: genomics approach to discovering novel

floral fragrance-related genes. Plant Cell 14:2325–2338

Hoelscher DJ, Williams DC, Wildung MR, Croteau R (2003) A

cDNA clone for 3-carene synthase from Salvia stenophylla.

Phytochemistry 62:1081–1086

Huang X, Madan A (1999) CAP3: a DNA sequence assembly

program. Genome Res 9:868–877

Kader JC, Julienne M, Vergnolle C (1984) Purification and charac-

terization of a spinach-leaf protein capable of transferring

phospholipids from liposomes to mitochondria or chloroplasts.

Eur J Biochem 139:411–416

Kaileh M, Berghe WV, Boone E, Essawi T, Haegeman G (2007)

Screening of indigenous Palestinian medicinal plants for

potential anti-inflammatory and cytotoxic activity. J Ethnophar-

macol 113:510–516

Kampranis SC, Ioannidis D, Purvis A, Mahrez W, Ninga E, Katerelos

NA, Anssour S, Dunwell JM, Degenhardt J, Makris AM,

Goodenough PW, Johnson CB (2007) Rational conversion of

substrate and product specificity in a Salvia monoterpene

synthase: structural insights into the evolution of terpene

synthase function. Plant Cell 19:1994–2005

Karioti A, Skaltsa H, Demetzos C, Perdetzoglou D, Economakis CD,

Salem AB (2003) Effect of nitrogen concentration of the nutrient

solution on the volatile constituents of leaves of Salvia fruticosaMill. in solution culture. J Agric Food Chem 51:6505–6508

Karousou R, Hanlidou E, Kokkini S (2000) The sage plants of

Greece: distribution and infraspecific variation. In: Kintzios S

(ed) Sage: the genus Salvia. Harwood Academic Publishers, The

Netherlands, pp 27–53

Koeduka T, Fridman E, Gang DR, Vassao DG, Jackson BL, Kish CM,

Orlova I, Spassova SM, Lewis NG, Noel JP, Baiga TJ, Dudareva

N, Pichersky E (2006) Eugenol and isoeugenol, characteristic

aromatic constituents of spices, are biosynthesized via reduction

of a coniferyl alcohol ester. Proc Natl Acad Sci USA 103:10128–

10133

Labarga A, Valentin F, Anderson M, Lopez R (2007) Web services at

the European bioinformatics institute. Nucleic Acids Res

35:W6–W11

Lange BM, Wildung MR, Stauber EJ, Sanchez C, Pouchnik D,

Croteau R (2000) Probing essential oil biosynthesis and

secretion by functional evaluation of expressed sequence tags

from mint glandular trichomes. Proc Natl Acad Sci USA

97:2934–2939

Langer R, Mechtler C, Jurenitsch J (1996) Composition of the

essential oils of commercial samples of Salvia officinalis L. and

S. fruticosa Miller: a comparison of oils obtained by extraction

and steam distillation. Phytochem Anal 7:289–293

Li M, Zhang JS, Ye YM, Fang JN (2000) Constituents of the roots of

Salvia prionitis. J Nat Prod 63:139–141

Lin FW, Damu AG, Wu TS (2006) Abietane diterpene alkaloids from

Salvia yunnanensis. J Nat Prod 69:93–96

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression

data using real-time quantitative PCR and the 2(-Delta Delta

C(T)) method. Methods 25:402–408

Longaray Delamare AP, Moschen-Pistorello IT, Artico L, Atti-

Serafini L, Echeverrigaray S (2007) Antibacterial activity of the

essential oils of Salvia officinalis L. and Salvia triloba L.

cultivated in South Brazil. Food Chem 100:603–608

Plant Cell Rep (2010) 29:523–534 533

123

MacFarlane AT, Mori SA, Purzycki K (2003) Notes on Eriothecalongitubulosa (Bombacaceae), a rare, putatively hawkmoth-

pollinated species new to the Guianas. Brittonia 55:305–316

Maldonado AM, Doerner P, Dixon RA, Lamb CJ, Cameron RK

(2002) A putative lipid transfer protein involved in systemic

resistance signalling in Arabidopsis. Nature 419:399–403

Matsingou TC, Petrakis N, Kapsokefalou M, Salifoglou A (2003)

Antioxidant activity of organic extracts from aqueous infusions

of sage. J Agric Food Chem 51:6696–6701

Mauricio R, Rausher MD (1997) Experimental manipulation of

putative selective agents provides evidence for the role of natural

enemies in the evolution of plant defence. Evolution 51:1435–

1444

Min T, Kasahara H, Bedgar DL, Youn B, Lawrence PK, Gang DR,

Halls SC, Park H, Hilsenbeck JL, Davin LB, Lewis NG, Kang C

(2003) Crystal structures of pinoresinol-lariciresinol and phe-

nylcoumaran benzylic ether reductases and their relationship to

isoflavone reductases. J Biol Chem 278:50714–50723

Min XJ, Butler G, Storms R, Tsang A (2005) OrfPredictor: predicting

protein-coding regions in EST-derived sequences. Nucleic Acids

Res 33:W677–W680

Molina A, Segura A, Garcia-Olmedo F (1993) Lipid transfer proteins

(nsLTPs) from barley and maize leaves are potent inhibitors of

bacterial and fungal plant pathogens. FEBS Lett 316:119–122

Nagel J, Culley LK, Lu Y, Liu E, Matthews PD, Stevens JF, Page JE

(2008) EST analysis of hop glandular trichomes identifies an

O-methyltransferase that catalyzes the biosynthesis of xanthohu-

mol. Plant Cell 20:186–200

Ogutcu H, Sokmen A, Sokmen M, Polissiou M, Serkedjieva J,

Daferera D, Sahin F, Baris O, Gulluce M (2008) Bioactivities of

the various extracts and essential oils of Salvia limbataC.A.Mey. and Salvia sclarea L. Turk J Biol 32:181–192

Ozcan M (2003) Antioxidant activities of rosemary, sage, and sumac

extracts and their combinations on stability of natural peanut oil.

J Med Food 6:267–270

Papageorgiou V, Gardeli C, Mallouchos A, Papaioannou M, Komaitis

M (2008) Variation of the chemical profile and antioxidant

behavior of Rosmarinus officinalis L. and Salvia fruticosa Miller

grown in Greece. J Agric Food Chem 56:7254–7264

Perfumi M, Arnold N, Tacconi R (1991) Hypoglycemic activity of

Salvia fruticosa Mill. from Cyprus. J Ethnopharmacol 34:135–140

Pitarokili D, Couladis M, Petsikos-Panayotarou N, Tzakou O (2002)

Composition and antifungal activity on soil-borne pathogens of

the essential oil of Salvia sclarea from Greece. J Agric Food

Chem 50:6688–6691

Pitarokili D, Tzakou O, Loukis A, Harvala C (2003) Volatile

metabolites from Salvia fruticosa as antifungal agents in

soilborne pathogens. J Agric Food Chem 51:3294–3301

Pizzale L, Bortolomeazzi R, Vichi S, Uberegger E, Conte LS (2002)

Antioxidant activity of sage (Salvia officinalis and S. fruticosa)

and oregano (Origanum onites and O. indercedens) extracts

related to their phenolic compound content. J Sci Food Agric

82:1645–1651

Putievsky E, Ravid U, Dudai N (1986) The essential oil and yield

components from various plant parts of Salvia fruticosa. J Nat

Prod 49:1015–1017

Pyee J, Yu H, Kolattukudy PE (1994) Identification of a lipid transfer

protein as the major protein in the surface wax of broccoli

(Brassica oleracea) leaves. Arch Biochem Biophys 311:460–468

Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, ApweilerR, Lopez R (2005) InterProScan: protein domains identifier.

Nucleic Acids Res 33:W116–W120

Ramakers C, Ruijter JM, Deprez RH, Moorman AF (2003) Assump-

tion-free analysis of quantitative real-time polymerase chain

reaction (PCR) data. Neurosci Lett 339:62–66

Regente M, De La Canal L (2003) A cDNA encoding a putative lipid

transfer protein expressed in sunflower seeds. J Plant Physiol

160:201–203

Reymond P, Weber H, Damond M, Farmer EE (2000) Differential

gene expression in response to mechanical wounding and insect

feeding in Arabidopsis. Plant Cell 12:707–719

Rivera D, Obon C, Cano F (1994) The botany, history and traditional

uses of three-lobed sage (Salvia fruticosa MIller) (Labiatae).

Econ Bot 48:190–195

Roeder S, Hartmann AM, Effmert U, Piechulla B (2007) Regulation

of simultaneous synthesis of floral scent terpenoids by the

1,8-cineole synthase of Nicotiana suaveolens. Plant Mol Biol

65:107–124

Savelev SU, Okello EJ, Perry EK (2004) Butyryl- and acetyl-

cholinesterase inhibitory activities in essential oils of Salviaspecies and their constituents. Phytother Res 18:315–324

Shimada T, Endo T, Fujii H, Hara M, Omura M (2005) Isolation and

characterization of (E)-beta-ocimene and 1,8 cineole synthases

in Citrus unshiu Marc. Plant Sci 168:987–995

Sivropoulou A, Nikolaou C, Papanikolaou E, Kokkini S, Lanaras T,

Arsenakis M (1997) Antimicrobial, cytotoxic, and antiviral

activities of Salvia fruticosa essential oil. J Agric Food Chem

45:3197–3201

Skoula M, El Halali E, Makris AM (1999) Evaluation of the genetic

diversity of Salvia fruticosa Mill. clones using RAPD markers

and comparison with the essential oil profiles. Biochem Syst

Ecol 27:559–568

Skoula M, Abbes JE, Johnson CB (2000) Genetic variation of

volatiles and rosmarinic acid in populations of Salvia fruticosaMill growing in Crete. Biochem Syst Ecol 28:551–561

Sokovic M, Tzakou O, Pitarokili D, Couladis M (2002) Antifungal

activities of selected aromatic plants growing wild in Greece.

Die Nahrung 46:317–320

Sterk P, Booij H, Schellekens GA, Van Kammen A, De Vries SC

(1991) Cell-specific expression of the carrot EP2 lipid transfer

protein gene. Plant Cell 3:907–921

Sugiyama Y, Watase Y, Nagase M, Makita N, Yagura S, Hirai A,

Sugiura M (2005) The complete nucleotide sequence and

multipartite organization of the tobacco mitochondrial genome:

comparative analysis of mitochondrial genomes in higher plants.

Mol Genet Genomics 272:603–615

Vander Mijnsbrugge K, Beeckman H, De Rycke R, Van Montagu M,

Engler G, Boerjan W (2000) Phenylcoumaran benzylic ether

reductase, a prominent poplar xylem protein, is strongly

associated with phenylpropanoid biosynthesis in lignifying cells.

Planta 211:502–509

Wang G, Tian L, Aziz N, Broun P, Dai X, He J, King A, Zhao PX,

Dixon RA (2008) Terpene biosynthesis in glandular trichomes of

hop (Humulus lupulus L.). Plant Physiol 148:1254–1266

Wise ML, Savage TJ, Katahira E, Croteau R (1998) Monoterpene

synthases from common sage (Salvia officinalis). cDna isolation,

characterization, and functional expression of (?)-sabinene

synthase, 1,8-cineole synthase, and (?)-bornyl diphosphate

synthase. J Biol Chem 273:14891–14899

Yaniv Z, Dafni A, Friedman J, Palevitch D (1987) Plants used for the

treatment of diabetes in Israel. J Ethnopharmacol 19:145–151

534 Plant Cell Rep (2010) 29:523–534

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