Planta (2010) 231:835–845

DOI 10.1007/s00425-009-1090-4

ORIGINAL ARTICLE

A genomics resource for investigating regulation of essential oil production in Lavandula angustifolia

Alexander Lane · Astrid Boecklemann · Grant N. Woronuk · Lukman Sarker · Soheil S. Mahmoud

Received: 27 October 2009 / Accepted: 12 December 2009 / Published online: 31 December 2009© Springer-Verlag 2009

Abstract We are developing Lavandula angustifolia(lavender) as a model system for investigating molecularregulation of essential oil (a mixture of mono- and sesqui-terpenes) production in plants. As an initial step towardbuilding the necessary ‘genomics toolbox’ for this species,we constructed two cDNA libraries from lavender leavesand Xowers, and obtained sequence information for 14,213high-quality expressed sequence tags (ESTs). Based onhomology to sequences present in GenBank, our ESTcollection contains orthologs for genes involved in the1-deoxy-D-xylulose-5-phosphate (DXP) and the mevalonicacid (MVA) pathways of terpenoid biosynthesis, and forknown terpene synthases and prenyl transferases. To gaininsight into the regulation of terpene metabolism in laven-der Xowers, we evaluated the transcriptional activity ofthe genes encoding for 1-deoxy-D-xylulose-5-phosphatesynthase (DXS) and HMG-CoA reductase (HMGR), whichrepresent regulatory steps of the DXP and MVA pathways,respectively, in glandular trichomes (oil glands) by real-time PCR. While HMGR transcripts were barely detectable,DXS was heavily expressed in this tissue, indicating thatessential oil constituents are predominantly producedthrough the DXP pathway in lavender glandular trichomes.As anticipated, the linalool synthase (LinS)—the generesponsible for the production of linalool, a major constituent

of lavender essential oil—was also strongly expressed inglands. Surprisingly, the most abundant transcript in Xoralglandular trichomes corresponded to a sesquiterpene syn-thase (cadinene synthase, CadS), although sesquiterpenesare minor constituents of lavender essential oils. Thisresult, coupled to the weak activity of the MVA pathway(the main route for sesquiterpene production) in trichomes,indicates that precursor supply may represent a bottleneckin the biosynthesis of sesquiterpenes in lavender Xowers.

Keywords Lavandula · Essential oil · Isoprenoids · EST library · Genomics · Glandular trichome

AbbreviationsEST Expressed sequence tagDXS 1-Deoxy-D-xylulose-5-phosphate synthaseHMGR 3-Hydroxy-3-methylglutartl-CoA reductaseLinS Linalool synthaseFarS [E]-�-Farnesene synthaseDXP 1-Deoxy-D-xylulose-5-phosphateMVA Mevalonic acidIPP Isopentenyl diphosphateDMAPP Dimethylallyl diphosphateHMGS 3-Hydroxy-3-methylglutartl-CoA synthaseGO Gene ontologyqRT-PCR Quantitative real-time polymerase chain reactionPCR Polymerase chain reactionContig Contiguous sequence

Introduction

The isoprenoids (or terpenoids) comprise a family of over25,000 structurally and functionally diverse secondarymetabolites with ecological and physiological functions in

A. Lane · G. N. Woronuk · L. Sarker · S. S. Mahmoud (&)Biology and Physical Geography, Irving K. Barber School of Arts and Sciences, University of British Columbia Okanagan, 3333 University Way, Kelowna, BC V1V 1V7, Canadae-mail: soheil.mahmoud@ubc.ca

A. BoecklemannBoucher Institute of Naturopathic Medicine, New Westminster, BC V3M 5Y6, Canada

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plant–environment interactions (e.g., pollinator attractionand defense) and in growth and development (e.g., as plantgrowth regulators) (Bohlmann et al. 1998; Chen et al. 2003;Gershenzon and Dudareva 2007; Grote and Niinemets2008). They are also industrially important, having a sig-niWcant impact on almost all aspects of human life. Inparticular, these compounds are extensively used in foods,cosmetics, and pharmaceuticals (Trapp and Croteau 2001;Cavanagh and Wilkinson 2002; Lund and Bohlmann 2006).Currently, tremendous resources are focused on under-standing the biosynthesis and regulation of these com-pounds in economically important plants. Notable examplesinclude Humulus, Artimesia, Picea, and Taxus (Hudginset al. 2006; Teoh et al. 2006; Nagel et al. 2008).

The mono- and sesquiterpenes (the C10 and C15 isopre-noids, respectively) are produced by all plants, at least insmall quantities, as the major contributors to the aroma andscent of fruits and Xowers (Dudareva et al. 1996; Dudarevaand Pichersky 2000; Dudareva et al. 2003). They are partic-ularly abundant in several species of the Lamiacea (themint family, e.g., lavenders) as the main constituents ofessential oils. In these plants, mono- and sesquiterpenes areproduced in secretory cells, and stored in subcuticular stor-age cavity of the glandular trichomes (or oil glands) presenton the surfaces of leaves and Xoral tissues (McCaskill et al.1992; Lange et al. 2000; Turner et al. 2000) (Fig. 1a, b).

Like other isoprenoids, the mono- and sesquiterpenes arederived through the condensation of the universal isopren-oid precursor isopentenyl diphosphate (IPP) and its allylicisomer, dimethyl allyl diphosphate (DMAPP) (Fig. 2). Inplants, IPP and DMAPP are produced via two independentpathways. One pathway [the 1-deoxy-D-xylulose-5-phos-phate (DXP) pathway] is localized in plastids, and suppliesIPP and DMAPP for the production of monoterpenes, diter-penes, and tetraterpenes. Flux through this pathway is con-trolled in part by 1-deoxy-D-xylulose-5-phosphate synthase(DXS), the Wrst enzyme of the pathway (Lois et al. 2000;Turner et al. 2000). In this context, overexpression of DXSresults in elevated production of total terpenoids in plants,including spike lavender (Munoz-Bertomeu et al. 2006).The other pathway—the classical mevalonic acid (MVA)pathway—operates in the cytosol and produces precursorfor the biosynthesis of sesqui- and triterpenes (Laule et al.2003). The Wrst and second steps of the MVA pathwayare catalyzed by 3-hydroxy-3-methylglutartl-CoA synthase(HMGS) and 3-hydroxy-3-methylglutartl-CoA reductase(HMGR), respectively (Fig. 2). HMGR catalyzes the keyregulatory step of the pathway in plants and in mammals. Inaddition, HMGS activity has been positively correlatedwith rubber production in Hevea brasiliensis, suggesting aregulatory role for this enzyme as well (Suvachittanont andWititsuwannakul 1995; Nagegowda et al. 2004). It has longbeen assumed that the DXP and MVA pathways are

independently regulated. However, recent studies suggestthat exchange of metabolites between the two pathwaysis possible (Bick and Lange 2003; Hemmerlin et al. 2003;Schuhr et al. 2003; Dudareva et al. 2005; Cusidó et al. 2007).

Lavandula angustifolia and up to 25 related lavenderspecies are cultivated worldwide both as ornamental peren-nials and as crops. These plants are grown primarily fortheir essential oils, which are produced commercially atover 2,000 metric tons per year augmenting a multimilliondollar essential oil industry (Castle and Lis-Balchin 2002;Upson and Andrews 2004). Lavender oils are primarilyused in cosmetic and food products. However, they alsoWnd increasing applications as natural medicines, nutraceu-ticals (Upson and Andrews 2004), and pesticides(Cavanagh and Wilkinson 2002).

The essential oil of lavender is a complex mixture ofmainly monoterpenes (e.g., linalool, camphor, 1,8-cineole,

Fig. 1 Essential oil-producing tissues of L. angustifolia. a Leaf andglandular trichomes viewed under light microscopy (scale bar»0.25 cm). b Scanning electron microscopy image of lavenderglandular trichomes (scale bar »50 �m). c Flower stage ‘Bud I’.d Flower stage ‘Bud II’. e Flower stage ‘Anthesis’. f Flower stage 30%,where 30% of the spike is in bloom. g Flower stage 70%, where 70%of the spike is in bloom. h Flower stage Bloomed Out, were the entirespike is in bloom. Scale bar »1 cm

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and borneol) and a few sesquiterpenes (e.g., [E]-�-farne-sene) as minor oil constituents (ISO 8902 1999; ISO 35152002; Lawrence 2004). Linalool and linalool acetate are themost abundant monoterpenes in popular varieties and areconsidered to be the most desired components of the Xoraloil, while camphor generally contributes an undesirableodor, diminishing oil quality. Some of the Wnest oils are

extracted from L. angustifolia, which contain the highestratio of linalool to camphor. However, L. angustifolia isrelatively small in size, diYcult to propagate, and low inoverall oil yield. In contrast, certain L. intermedia hybrids(lavandins; e.g., Grosso lavender), which accumulate rela-tively high levels of camphor, are more productive in termsof essential oil yield. A detailed understanding of isopren-oid metabolism will open several avenues for improvingthe quality of the essential oil in these plants. It will alsolead to the discovery of technologies for altering Xoralaroma proWle and for production of industrially signiWcantterpenes in plants.

Several terpene synthases have been isolated and clonedfrom lavender relatives including mint and sage (Darshanand Doreswamy 2004; Huber et al. 2004). Despite theseimpressive advancements, our current understanding ofessential oil and scent formation and secretion in aromaticplants such as lavender is very limited. A nucleotide searchof GenBank reveals 27 Lavandula-derived sequences; how-ever, only linalool synthase (LinS), limonene synthase, andtrans-� �-bergamotene synthase from lavender have beenreported (Landmann et al. 2007). The regulation of expres-sion of these genes is not understood. We have adopted agenomics approach to improve our understanding of theregulation of mono- and sesquiterpene synthesis in laven-der. We constructed two cDNA libraries from Xowers andleaves of L. angustifolia, sequenced approximately 15,0005�-expressed sequence tags (ESTs), obtained orthologs forLinS, FarS, CadS, HMGR, and DXS, and studied theexpression pattern for these genes in whole Xower tissueand in isolated secretory cells of lavender oil glands. Ourresults demonstrated that lavender glandular trichomes pre-dominantly utilize the DXP pathway for the production ofessential oil constituents. They also indicated that precursorsupply might limit sesquiterpene production in lavender oilglands.

Materials and methods

Construction of cDNA libraries

Leaf and Xower tissues used for cDNA library constructionwere harvested from Weld-grown L. angustifolia plantsfarmed at the Kelowna campus of the University of BritishColumbia. All tissues were immediately Xash frozen in liq-uid N2 and kept at ¡80°C until needed. RNA isolation, aswell as subsequent steps of cDNA library construction,insert sequencing, bioinformatics, and clone archiving wereperformed at the Plant Biotechnology Institute’s NaturalProducts Genomic laboratory (PBI-NAPGEN). In brief,total RNA was isolated from the respective tissues usingthe RNeasy Midi kit (Qiagen). The quality of all RNA

Fig. 2 The MVA and DXP pathways of isoprenoid synthesis inplants. AACT acetoacetyl-coenzyme A thiolase; CDP-ME 4-(cytidine5�-diphospho)-2-C-methyl-D-erythritol; CDP-MEP CDP-ME-2-phos-phate; cMEPP 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; CMK4-(cytidine 5�-diphospho) 2-C-methyl-D-erythritol kinase; CMS 2-C-methyl-D-erythritol 4-phosphate transferase; DMAPP dimethylallyldiphosphate; DXP 1-deoxy-D-xylulose 5-phosphate; DXR DXP reduc-toisomerase; DXS DXP synthase; GAP glyceraldehyde 3-phosphate;GPP geranyl diphosphate; GPPS GPP synthase; FPP farnesyl diphos-phate; FPPS FPP synthase; HDS 1-hydroxy-2-methyl-2-(E)-butenyl4-diphosphate; HMBPP 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphos-phate synthase; HMG-CoA 3-hydroxy-3-methylglutaryl coenzymeA; HMGR HMG-CoA reductase; HMGS HMG-CoA synthase; IDSisopentenyl diphosphate/dimethylallyl diphosphate synthase; IPP iso-pentenyl diphosphate; IPPi IPP isomerase; MCS 2-C-methyl-D-eryth-ritol-2,4-cyclodiphosphate synthase; MDD mevalonate diphosphatedecarboxylase; MEP 2-C-methyl-D-erythritol 4-phosphate; MK MVAkinase; MPK mevalonate-5-phosphate kinase; MTS monoterpene syn-thase; MVA mevalonate; SES sesquiterpene synthase

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samples was conWrmed using an Agilent 2100 Bioanalyzer.Messenger RNA was puriWed from total RNA using a Dyn-abeads® mRNA puriWcation kit (Invitrogen). The cDNAlibrary was constructed with a ZAP Express® cDNA Syn-thesis Kit (Stratagene) as prescribed by the manufacturer.The respective libraries were then cloned into the pBlueScriptSK§ vector (Stratagene), and transformed into ElectroMAXDH10B T1 Phage Resistant Cells (Invitrogen). Aliquots ofeach library were plated on LB growth medium containing100 �g/ml of carbenicillin. A total of 15,000 colonies(5,000 from the leaf library and 10,000 from the Xowerlibrary) were randomly selected for insert sequencing.These colonies were then cultured and archived at ¡80°Cin 96 well micro-titer plates as 10% glycerol stocks. Plas-mid DNA was isolated from the 15,000 cultured coloniesand sequenced using the 5� T3 vector speciWc primer.

Bioinformatics

Raw sequence Wles were produced using the ‘phred’ soft-ware package (Ewing and Green 1998; Ewing et al. 1998).These sequences were then vector-trimmed and low-qualitytrimmed using ‘crossmatch’ (Chou and Holmes 2001;Phrap 2008) and ‘lucy’ (Ewing and Green 1998; Ewinget al. 1998) software, respectively. Poly-AT regions werethen removed using custom scripts and low-complexityregions were masked with ‘mdust’ (The ComputationalBiology and Functional Genomic Laboratory 2008). Allgood quality sequences were then BLASTX searchedagainst NCBI non-redundant (nr) database. Sequence clus-tering was performed using ‘tgicl’ with parameters set asfollows: tgicl sequences -p 94 -v 1000 -O ‘-p 98 -o 49 -t10000’ (The Computational Biology and Functional Geno-mic Laboratory 2008). Gene ontology (GO) terms wereassigned to the ESTs by transferring the GO terms from theTAIR database (TAIR 2008) assigned to their top BLASThit.

Evaluation of gene expression in glandular trichomes

Lavender Xowers were collected directly in ice-cold meth-ylcellulose buVer (200 mM sorbitol, 10 mM sucrose,25 mM MOPSO, 0.5 mM PO4 buVer, 10 mM sodium bisul-Wte, 10 mM ascorbic acid, 1 mM EDTA, 1% PVP-40, and0.6% methylcellulose) containing 2 mM aurinticarboxylicacid, 5 mM thiourea, and 2 mM DTT at pH 6.6, and soakedfor 1 h in ice. Secretory cells were abraded in methylcellu-lose buVer using glass beads as previously reported (Langeet al. 2000), and rinsed with wash buVer (10% glycerol,25 mM PO4 buVer, 1 mM EDTA, 2 mM aurinticarboxylicacid, 5 mM thiourea, and 2 mM DTT).

Total RNA was isolated from 0.1 g (fresh weight) ofXower tissue or isolated glandular trichome cells using the

RNeasy Plant Mini Kit (Qiagen), and treated with DNaseusing the Qiagen on-column DNase digestion kit. RNA wasthen checked for integrity by agarose gel electrophoresisand quantiWed spectrophotometrically. cDNA was pro-duced by reverse transcribing 2 �g of total RNA using theiScript™ cDNA Synthesis Kit (Bio-Rad Laboratories)according to the manufacturer’s directions. qRT-PCR anal-yses (n = 3) were carried out using the GeneJET™ FastPCR Master Mix (Fermentas, ON, Canada), approximately150 ng of the cDNA template and 5 pmol of each primer ina total volume of 25 �l on a Bio-Rad CFX96TM instrument(Bio-Rad). Gene-speciWc primers used in quantitative real-time PCR experiments were designed to amplify 100–150 bp products using primer sets 2–7 (Table 1). Thefollowing program was used for all of these PCR: 95°C for10 min followed by 40 cycles of 30 s at 95°C, 30 s at 55°C,and 30 s at 72°C. Starting quantities of cDNA for each genewas derived from a standard curve of six standards(R2 = 0.982) using CFX96TM Real-Time PCR DetectionSystem software, and normalized to �-actin as a referencegene.

Evaluation of linalool content and LinS expression in developing Xowers

Flower spikes were harvested from Weld-grown L. angusti-folia (cv. Munstead) plants, and assorted according to size(length of the spike in cm) and number of open Xowers perspike (Fig. 1c–h). Buds I and II included Xower buds ·1and 1–2 cm in length, respectively. At Anthesis stage, all

Table 1 Primer sequences used in qRT-PCR experiments

F forward, R reverse

Primer set Target gene

Primer sequence

1 Actin F-5�-AGGCCAATCGTGAGAAGATG-3�

R-5�-AAGGATTGCATGAGGGAGTG-3�

2 Actin F-5�-TGTGGATTGCCAAGGCAGAGT-3�

R-5�-AATGAGCAGGCAGCAACAGCA-3�

3 HMGR F-5�-TTAACGCCGAGTTCCCAGACA-3�

R-5�-TGATTTGCCACGGCCTTCGAT-3�

4 DXS F-5�-CCAACTCCGTGAAGCAGCAAA-3�

R-5�-TTGCCCGCGAATCCTTTCAGA-3�

5 FarS F-5�-TTGTTGCAAGAGGCTGCGAGA-3�

R-5�-TGCTGCAAAGCTCGCTGTACT-3�

6 CadS F-5�-ACTCGGAGATCACAAGCTGCAT-3�

R-5�-ACTGCGAAGTTTGGCTCGTT-3�

7 LinS F-5�-ACACGCACGACAATTTGCCA-3�

R-5�-AGCCCTCCAATGAAGTGGGAT-3�

8 LinS F-5�-ATGTCGATCAATATCAACATGCC-3�

R-5�-TCATGCGTACGGCTCGAACAGC-3�

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individual Xowers were closed, and at the 30 and 70%stages, 30 or 70% of individual Xowers per Xower spikewere opened, respectively. Finally, at the Bloomed Outstage, all Xowers had senesced. Tissues were Xash frozen inliquid N2 and stored at ¡80°C until used.

Essential oil was extracted from 5 to 10 g of Xoral tissue(pooled from three to four plants) by simultaneous steamdistillation/solvent extraction as previously described (Falket al. 2009; Mahmoud and Croteau 2002; Mahmoud et al.2004). Prior to extraction, 1 mg of menthol was added toeach sample as an internal standard for quantiWcationpurposes. Essential oil samples were diluted 100-fold inanalytical grade pentane (Sigma) prior to analysis of 1 �laliquots by a Varian GC 3800 gas chromatographer cou-pled to a Saturn 2200 Ion Trap mass detector. The gas chro-matography mass spectrometry (GC/MS) system wasequipped with a 30 m £ 0.25 mm capillary column coatedwith a 0.25 �m Wlm of acid-modiWed polyethylene glycol(ECTM 1000, Alltech). The oven temperature program wasinitiated at 40°C (held for 3 min), increased to 170°C at7°C/min, and then to 230°C at the rate of 30°C/min. Thecarrier gas (Helium) Xow rate was set to 1 ml/min. Majoressential oil constituents (including linalool, linalool ace-tate, 1,8-cineole, camphor, and borneol) were identiWed bycomparing their retention times and mass spectra to thoseof pure authentic standards, and to the mass spectra cata-loged by the National Institute of Standards and Technol-ogy (NIST). Linalool content (in percent total oil) for eachessential oil sample was estimated by dividing the area ofthe peak corresponding to linalool by the sum of all peaksdetected on the GC chromatogram [(peak area for linalool/sum of all peak areas) £ 100 = percentage of linalool in thesample)]. Reported values represent the mean and standarddeviation for four replicates (n = 4; two GC runs £ twoessential oil extracts for each Xoral stage).

To study the expression of the linalool synthase gene(LinS), total RNA was isolated from Xower tissue using theRNeasy Plant Mini Kit (Qiagen) according to the manufac-turer’s instructions. Messenger RNA was reverse tran-scribed into Wrst strand cDNA with Superscript II reversetranscriptase (Invitrogen) and an oligo-dT 20-mer (Fermen-tas). LinS transcript abundance was estimated using theApplied Biosystems 7500 real-time PCR system and Quan-tiTect SYBR Green PCR kit (Qiagen). Approximately250 ng of the Wrst strand cDNA was combined with 2£SYBR Green master mix and 0.3 �M forward and reverseprimer in a total volume of 25 �l as recommended by thesupplier (Qiagen). LinS was ampliWed using primer set 8(Table 1). All PCR were activated at 95°C for 15 min, fol-lowed by 40 cycles at 94°C for 15 s, 64°C for 30 s, and72°C for 2 min. The reaction was terminated by a Wnalextension for 5 min at 72°C. A melting curve analysis(5 min at 58°C) of the PCR product(s) was/were performed

subsequently to verify product speciWcity and identity.Transcript abundances were estimated based on a Wve-pointcalibration curve generated for a 191 bp portion of L.angustifolia �-actin using primer set 1 (Table 1). Transcriptlevels for LinS were normalized to those for �-actin.Reported values represent mean and standard deviation forthree reactions.

Result and discussion

Construction of cDNA libraries

Two cDNA libraries were constructed for L. angustifolia,one using mRNA extracted from young leaves (the leaflibrary), and the other from Xowers (the Xower library).A total of 15,000 clones (10,000 from the Xower and 5,000from the leaf library) were randomly isolated for furtheranalysis. The average length of the cloned transcripts wasapproximately 1.5 kb, based on gel electrophoresis analysisof PCR-ampliWed inserts from randomly selected clones.All ESTs were 5� sequenced, with average sequence readlengths of 723 and 670 bp for the leaf and Xower libraries,respectively. A total of 4,866 leaf ESTs and 9,447 XowerESTs were determined to be of high quality. In total, 9,453unique transcripts were identiWed, with both the leaf andXower libraries producing similar proportions of unigenesand hypothetical contiguous sequences (contigs). A largeproportion of genes were represented by a single EST(Fig. 3a, b). Inversely, a relatively small proportion of con-tigs were composed of more than Wve ESTs, suggesting thatour libraries are non-redundant. Within both libraries, 85–87% of the ESTs were signiWcantly similar (E-value <10¡6)to nucleotide entries in GenBank. Approximately 49% ofall ESTs were signiWcantly similar (E-value <10¡6) to thesequences found in the Arabidopsis genome and 34% werehomologous to those found in the rice genome.

All ESTs with signiWcant annotations (E-value <10¡6)were classiWed according to their predicted molecular func-tions, such as catalytic activity or transferase activity(Fig. 4). Categories constituting <1% of the total EST col-lection were amalgamated into the category labeled ‘other’.These included those with receptor activity, enzyme regula-tor activity, signal transducer activity, translation factoractivity, nucleic acid binding, nuclease activity, receptorbinding, motor activity, and carbohydrate binding. A rela-tively large proportion of transcripts encode proteins withpresumed (homology-based) functions in trichome andXower development, and in isoprenoid metabolism. Thisgroup includes sequences with signiWcant homology toMYB, WRKY, basic helix-loop-helix, MADS box-typetranscription factors, cytochrome P450 hydroxylases, andABC transporters (Table 2). Interestingly, the proportion of

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transcripts with putative transcription factor activity (8%)or transporter activity (12–13%) is particularly high, incomparison to the proportion of these categories in the Ara-bidopsis genome (5 and 4.9%, respectively) (TAIR 2008).Given that there is little known about the regulation of iso-prenoid metabolism or the shuttling of metabolites, this

presents an important resource for future studies aimed atelucidating regulation of terpene synthase gene expressionin plants.

The two cDNA libraries were very similar in containinga similar proportion of sequences related to isoprenoidmetabolism. Both libraries contained numerous clones withhomology to HMG-CoA synthase (gi|7799986|), HMG-CoAreductase (gi|25990288|), mevalonate kinase (gi|92874540|),mevalonate-5-phosphate kinase (gi|16417948|), mevalonatediphosphate decarboxylase (gi|21593243|), 1-deoxy-D-xylulose 5-phosphate synthase (gi|30315812|), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (gi|116175431|),and IPP isomerase (gi|13603408|). They also containedgenes encoding geranyl diphosphate (gi|6449050|) and far-nesyl diphosphate (gi|14488053|) synthases. Also, 155 ESTsequences were homologous to known terpene synthases,such as linalool synthase (gi|82408413|), limonene synthase(gi|82408411|), 1,8-cineole synthase (gb|AAC26016.1|),�-myrcene synthase (gi|55740207|), menthofuran synthase(gi|15723953|), pulegone reductase (gi|34559418|), [E]-�-farnesene synthase (gi|2754818|), taxadiene 5-�-hydrox-ylase (gi|86279656|), and lupeol synthase (gi|6456434|)(Table 3). The expression of most of these sequences inlavender was anticipated as the presumed terpene productfor the encoded enzymes (as determined based on thehomology of the gene to known terpene synthases) can befound in lavender essential oil. In addition, the seeminglyunexpected detection of transcripts for an enzyme such asmenthofuran synthase, which catalyzes formation of ment-hofuran in peppermint (Bertea et al. 2001), but not in laven-der, is not surprising either, as such phenomenon haspreviously been reported. For example, spearmint plants

Fig. 3 Number of ESTs contributing to contigs. a L. angustifoliaXower library. b L. angustifolia leaf library

Fig. 4 Molecular function Gene Ontology classiWcations. Blackbars represent the L. angustifolia Xower library and gray bars theL. angustifolia leaf library

Table 2 Selection of gene families represented in the L. angustifoliaEST collection

a Number of ESTs predicted to have the molecular function indicatedb Number of unique transcripts found for each gene family

Predicted function Number of ESTsa Unique genesb

Terpene synthase-like 155 25

Prenyl transferase 21 4

Myb-like 98 61

WRKY-like 13 9

ABC transporter-like 52 21

Cytochrome p450 71 30

MADS box-like 14 7

WD40 repeat 31 21

Basic helix-loop-helix 3 2

GL1-like 41 5

Transcription related 8% of total

Transporter related 12% of total

Catalytic activity 18% of total

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have been shown to express a full complement of genesrequired for the production of menthol (the main compo-nent of peppermint essential oil), although the plant doesnot produce detectable quantities of this monoterpene(Croteau et al. 1991).

Tissue-speciWc regulation of terpene synthesis

The biochemical steps involved in the biosynthesis of theisoprenoids can be classiWed into four general stages(Mahmoud and Croteau 2002). Stage 1 includes reactionsthat lead to the production of IPP and DMAPP. In stage 2,IPP and DMAPP are condensed to produce linear precur-sors for diVerent classes of isoprenoids, including geranyldiphosphate (GPP) and farnesyl diphosphate (FPP), therespective precursors for mono- and sesquiterpene biosyn-thesis (Fig. 2). In stage 3, the linear precursors are con-verted to individual terpenes through the catalytic action ofspeciWc terpene synthases, and stage 4 includes enzyme-mediated transformation of individual terpenes to otherrelated metabolites. As an example of the latter in laven-ders, linalool generated during stage 3 is acetylated to pro-duce linalool acetate during stage 4 (Dudareva et al. 1996;

Cseke et al. 1998; Crowell et al. 2002). The conversion of3-hydroxy-3-methylglutaryl coenzyme A to 3R-mevalonicacid catalyzed by HMGR, and the condensation of pyruvatewith glyceraldehydes 3-phosphate to produce 1-deoxy-D-xylulose-5-phosphate catalyzed by DXS are consideredtranscriptionally controlled regulatory steps (Rodriguez-Concepcion et al. 2001; Munoz-Bertomeu et al. 2006). Thetransformation of linear precursors to speciWc terpenes cat-alyzed by speciWc terpene synthases (TPS) is also consid-ered to be a regulatory step, as the precursors may bedirectly converted to numerous distinct terpenes (Bohl-mann et al. 1998, 2000; Tholl 2006; Cheng et al. 2007). Forexample, in lavenders GPP may be converted to linalool,geraniol, 1,8-cineole, or many other monoterpenes.

To gain insight into the regulation of mono- and sesqui-terpene production in lavender oil glands, we examined theexpression of HMGR, DXS, linalool synthase (LinS), [E]-�-farnesene synthase (FarS), and cadinene synthase (CadS).LinS catalyzes the transformation of GPP to the monoter-pene linalool, while FarS and CadS convert FPP to [E]-�-farnesene and cadinene (two sesquiterpenes), respectively.These genes have been previously characterized fromlavenders, or the closely related peppermint species

Table 3 Putative terpene synthase-like sequences found in L. angustifolia EST collection

Putative ID Accession E-value

Limonene synthase (L. angustifolia) gi|82408411| 1.0E-133

trans-�-Bergamotene synthase (L. angustifolia) gi|82408415| 1.0E-116

(+)-Sabinene synthase (Salvia oYcinalis) gi|62900766| 1.0E-108

cis-Muuroladiene synthase (Mentha £ piperita) gi|54969666| 1.0E-107

(+)-Pulegone reductase (Mentha £ piperita) gi|34559418| 1.0E-106

(E)-�-farnesene synthase (Mentha £ piperita) gi|2754818| 1.0E-106

�-Myrcene synthase (Ocimum basilicum) gi|55740207| 1.0E-106

Taxadiene 5-�-hydroylase cytochrome P450 (Artemisia annua) gi|86279656| 6.0E-99

(+)-Bornyl diphosphate synthase (Salvia oYcinalis) gi|62899675| 1.0E-93

ent-Kaurene synthase No1 (Lactuca sativa) gi|9971225| 2.0E-80

Lupeol synthase (Olea europaea) gi|6456434| 2.0E-80

ent-Kaurene synthase B (Cucurbita maxima) gi|62900385| 3.0E-78

Terpenoid synthase (Medicago truncatula) gi|92885651| 2.0E-77

Terpenoid synthase (Vitis vinifera) gi|45934584| 2.00E-76

1,8-Cineole synthase (Salvia oYcinalis) gi|62900763| 4.0E¡75

Chloroplast terpene synthase (Quercus ilex) gi|14331015| 1.0E-71

Taxane 13-�-hydroxylase cytochrome P450 (Artemisia annua) gi|86279654| 1.0E-71

3-Carene synthase (Salvia stenophylla) gi|22023928| 1.0E-66

Epidermal germacrene C synthase (Lycopersicon esculentum) gi|11934931| 5.0E-60

Terpene synthase domain (Arabidopsis thaliana) gi|18401414| 8.0E-56

(+)-4R-Limonene synthase (Schizonepeta tenuifolia) gi|14717383| 5.0E-53

Menthofuran synthase (Mentha £ piperita) gi|15723953| 1.0E-51

Pinene synthase (Quercus ilex) gi|121490210| 8.0E-50

D-Limonene synthase (Agastache rugosa) gi|16506632| 1.0E-37

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842 Planta (2010) 231:835–845

(Landmann et al. 2007; Crock et al. 1997). While linaloolcan make up to 40% of lavender essential oil, cadinene and[E]-�-farnesene are sesquiterpenes detected at trace quanti-ties in lavender oils (Lawrence 2004). We hypothesizedthat (a) the DXP pathway provides the bulk of the precursorfor essential oil synthesis, as is the case for other mints(Gershenzon et al. 2000; Rodriguez-Concepcion et al.2001; Liu et al. 2005), and hence DXS is strongly expressedin lavender oil gland; (b) the MVA pathway does not con-tribute to essential oil biosynthesis, and thus HMGR ispoorly expressed in this tissue; (c) lavender oil glands pro-duce high levels of mRNA for LinS, but not for FarS andCadS. To test these hypotheses, we measured the steadystate transcript levels for the above genes by real-time PCRin whole Xower tissue and in isolated Xoral glandulartrichomes. The Xoral tissue was selected, as the bulk of thelavender essential oils (including commercial products) areproduced in this tissue.

As predicted, DXS and LinS were strongly expressed inglandular trichomes, as transcripts for these genes wereheavily concentrated in glands compared to whole Xowertissue (Fig. 5). Meanwhile, transcripts for HMGR werebarely detectable in whole Xower tissue or in isolatedglands, indicating that the MVA pathway is not a majorcontributor to essential oil production in lavender Xowers(Fig. 5).

We also studied the transcription pattern of LinS in rela-tion to linalool accumulation in developing L. angustifolia

Xowers (Fig. 1c–h), where the bulk of linalool synthesisoccurs. While the youngest Xower buds contained little lin-alool (1.7% of total oil), linalool abundance increased grad-ually (Bud II stage 4.5%, Anthesis 16.8%, 30%-stage17.5%) and accounted for 30% of the total oil by the timeof Xower maturation (Fig. 6). Similarly, the expression lev-els of the LinS transcripts (standardized against �-actinmRNA) increased steadily as the Xower matured, peakingwhen 70% of the Xower spikes were in bloom (Fig. 6).Therefore, LinS transcription closely paralleled linaloolaccumulation during Xower development, suggesting thatlinalool production in lavender Xowers may be, at least inpart, controlled through transcriptional regulation of LinS(Fig. 6). The direct correlation between LinS transcriptabundance and linalool concentration in an organ-speciWcmanner conWrms previous Wndings in Clarkia breweri(snapdragon) Xowers, in which a positive correlationbetween terpenoid synthesis and enzyme activity was dem-onstrated (Cseke et al. 1998; Raguso and Pichersky 1999;Negre et al. 2003). Similarly, it was previously shown thatthe production of the monoterpene menthofuran in pepper-mint is regulated primarily at the level of transcription fromthe menthofuran synthase gene (Mahmoud and Croteau2003). In lavender, LinS transcription decreased toward theend of Xower development while linalool levels remainedhigh. Consistent with previous observations in C. breweriXowers (Pichersky et al. 1994; Dudareva et al. 1996), thisdata suggests that LinS transcription preceded linalool pro-duction and that linalool was stored beyond the time ofpeak linalool synthesis.

Our results clearly conWrm that the monoterpene constit-uents of lavender essential oil are chieXy produced through

Fig. 5 Expressions of HMGR (3-hydroxy-3-methylglutaryl CoAreductase), DXS (1-deoxy-D-xylulose-5-phosphate synthase), LinS(linalool synthase), FarS (farnesene synthase), and CadS (cadinenesynthase) in L. angustifolia Xower (white boxes) and isolated glandtissue (gray boxes). Transcript levels were normalized to �-actin anderror bars indicate standard deviation (n = 3)

Rel

ativ

e ex

pres

sion

3500

3000

2500

2000

10

5

1.0

0.5

0.10

0.05

0HMGR DXS LinS FarS CadS

Fig. 6 Linalool content (dark boxes) and LinS expression (light boxes)during Xower development in L. angustifolia. Expression of LinS wasnormalized to �-actin and error bars indicate standard deviation(n = 3). Flower developmental stages correspond to those shown inFig. 1

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the DXP pathway. This conclusion is supported by previ-ous Wndings demonstrating that overexpression of DXS—aregulatory step in the DXP pathway—can enhance essentialoil production in transgenic spike lavender (Munoz-Ber-tomeu et al. 2006). Further, our expression data suggeststhat overexpression of LinS should lead to increased linal-ool production in transgenic lavender plants.

A surprising outcome of this study was that FarS andCadS were also strongly expressed in lavender oilglands, although the products for the encoded enzymesare minor sesquiterpene constituents of lavender essen-tial oils. In particular, transcripts for CadS representedthe most abundant transcript species among the three ter-pene synthases tested (Fig. 5). These observations indi-cate that precursor (FPP) supply may be a limiting factorin the production of sesquiterpenes in lavender glandu-lar trichomes. This conclusion is supported by poorexpression of the MVA pathway, which provides precur-sor for sesquiterpene synthesis in plants (Steliopouloset al. 2002; Hampel et al. 2005, 2006).

Conclusions

As part of an eVort to build the ‘genomics toolbox’ for lav-enders, a substantive genomics resource has been devel-oped. Over 14,200 high-quality ESTs from leaf and Xoraltissues have been sequenced and archived. This collectionincludes sequences representing all stages of isoprenoidsynthesis, and has laid the groundwork for comprehensivegenomics studies.

As an initial study into the regulation of mono- and ses-quiterpene synthesis in lavender, we evaluated transcrip-tional expression of LinS, CadS, FarS, DXS, and HMGR inglandular trichomes. Our results conWrm that the expressionof essential oil-related biosynthetic genes is virtuallyrestricted to glandular trichomes. Both DXS and LinS wereheavily expressed in glandular trichomes, where essentialoil constituents are produced and stored (Fig. 5). The lowlevel of LinS and DXS transcripts detected in whole Xowersamples undoubtedly correspond to the glandular trichomespresent on the Xoral tissue. Further, our data indicates thatoil yield and composition in lavender is regulated, at leastin part, through the transcriptional control of the relatedgenes, including LinS and DXS. We also suggest that ses-quiterpene compounds make up a very small proportion oflavender essential oil due to limited precursor (FPP) avail-ability in the glandular trichomes, and that eVorts toincrease sesquiterpene production in oil glands of lavenders(and perhaps other plants) should focus on increasing theoutput of the MVA pathway in this tissue. ConWrming thisproposition, the overexpression of a truncated ArabidopsisHMGR was recently shown to increase mono- and sesqui-

terpene production in spike lavender (Munoz-Bertomeuet al. 2007).

Although our studies suggest that the production of linal-ool in lavender Xowers is regulated at the level of transcrip-tion of LinS, the involvement of other regulatorymechanisms in the process cannot be ruled out. For exam-ple, transcriptional upregulation of other genes, along withLinS (e.g., transporters) may be required for linalool bio-synthesis. In addition, post-transcriptional regulatory mech-anisms, which appear to control production of menthone inmints (Mahmoud and Croteau 2003), may be involved.

Acknowledgments This work was supported by grants from NaturalSciences and Engineering Research Council of Canada, InvestmentAgriculture Foundation of British Columbia, Canada Foundation forInnovation, British Columbia Knowledge Development Fund, andUBC Okanagan. We would like to thank Michael Weis (PaciWc Agri-Food Research Centre, Summerland, Canada) for his assistance withelectron microscopy. We also thank NAPGEN (Plant BiotechnologyInstitute; Saskatoon, Canada) for supporting the research. In particular,we are grateful to Rong Li and Dustin Cram for construction of thecDNA libraries and related bioinformatics analyses. Finally, we aregrateful to Dr. Mark Rheault of UBC Okanagan for his assistance withreal-time PCR.

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