Terpene Biosynthesis in Glandular Trichomesof Hop1,2[W][OA]
Guodong Wang3, Li Tian3,4, Naveed Aziz, Pierre Broun5, Xinbin Dai, Ji He, Andrew King,Patrick X. Zhao, and Richard A. Dixon*
Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73410 (G.W., L.T., X.D.,J.H., P.X.Z., R.A.D.); and Center for Novel Agricultural Products, Department of Biology, University of York,York YO10 5YW, United Kingdom (N.A., P.B., A.K.)
Hop (Humulus lupulus L. Cannabaceae) is an economically important crop for the brewing industry, where it is used to impartflavor and aroma to beer, and has also drawn attention in recent years due to its potential pharmaceutical applications.Essential oils (mono- and sesquiterpenes), bitter acids (prenylated polyketides), and prenylflavonoids are the primaryphytochemical components that account for these traits, and all accumulate at high concentrations in glandular trichomes ofhop cones. To understand the molecular basis for terpene accumulation in hop trichomes, a trichome cDNA library wasconstructed and 9,816 cleansed expressed sequence tag (EST) sequences were obtained from random sequencing of 16,152cDNA clones. The ESTs were assembled into 3,619 unigenes (1,101 contigs and 2,518 singletons). Putative functions wereassigned to the unigenes based on their homology to annotated sequences in the GenBank database. Two mono- and twosesquiterpene synthases identified from the EST collection were expressed in Escherichia coli. Hop MONOTERPENESYNTHASE2 formed the linear monterpene myrcene from geranyl pyrophosphate, whereas hop SESQUITERPENE SYN-THASE1 (HlSTS1) formed both caryophyllene and humulene from farnesyl pyrophosphate. Together, these enzymes accountfor the production of the major terpene constituents of the hop trichomes. HlSTS2 formed the minor sesquiterpene constituentgermacrene A, which was converted to b-elemene on chromatography at elevated temperature. We discuss potential functionsfor other genes expressed at high levels in developing hop trichomes.
Hop (Humulus lupulus) is a perennial, dioeciousplantthat belongs to the Cannabaceae family. “Hops” is thecommon term for the female inflorescences of hopplants,well known for their use in beer flavoring. Theseinflorescences develop into cones upon maturation.The lower parts of the inner surface of the bracts ofmature female hop cones are covered with glandulartrichomes, termed lupulin glands (Fig. 1). Glandular
trichomes, also referred to as secretory or peltate tri-chomes, are lipophilic glands comprising a group ofsecretory cells and a cuticle-enclosed cavity that fillswith the secreted compounds (Oliveira and Pais, 1990;Saito et al., 1995). The plastids in glandular trichomeshave less-defined membrane structures than chloro-plasts and may be associated with synthesis and/orsecretion of secondary metabolites, such as terpenoidsand flavonoids (Oliveira and Pais, 1990).
Three major classes of secondary metabolites aresynthesized and accumulated in hop lupulin glands;essential oils, bitter acids, and prenylflavonoids. Com-mercial hop varieties often differ in the content ofthese components, which determines their use inbittering and finishing (adding flavor and aroma) ofbeer. Essential oils are the principal aroma compo-nents of hops. Essential oils make up 0.5% to 3% (v/w)of the whole hop cone, and terpenoids are abundant inthis fraction (Eri et al., 2000), accounting for up to 90%of the oil. The composition of essential oils is charac-teristic of the hop genotype and, together with thatof bitter acids and flavonoids, has been used fordistinguishing different hop varieties. In addition tohydrocarbon compounds, which are predominantlyterpenes, oxygenated compounds and small amountsof sulfur-containing compounds are also found. Themajor monoterpene and sesquiterpene componentsof hop essential oils are myrcene, a-humulene, andb-caryophyllene (Bernotiene et al., 2004). Most studiesof hop terpenes have analyzed whole hop cones and
1 This work was supported by the National Science FoundationPlant Genome Program (grant no. DBI–0605033 to R.A.D.) and by theSamuel Roberts Noble Foundation.
2 Any opinions, findings, and conclusions or recommendationsexpressed in this article are those of the authors and do notnecessarily reflect the views of the National Science Foundation.
3 These authors contributed equally to this article.4 Present address: Department of Plant Sciences, Asmundson
Hall, Room 221, University of California, 1 Shield Avenue, Davis, CA95616.
5 Present address: Nestle R & D Center Tours, Plant Science andTechnology, 101 Avenue Gustave Eiffel, 7390 Notre-Dame D’Oe,France.
* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Richard A. Dixon ([email protected]).
[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-
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there is little information on the content of thesecompounds in other tissues. It is also not clear whetherthe trichome is the exclusive organ for their biosyn-thesis and storage.The bittering agents in beer are called bitter acids,
which account for 10% to 20% of the hop cone by dryweight. The two representative bitter acids, a-acid andb-acid (humulone and lupulone), are prenylated poly-ketide derivatives. Prenylated flavonoids have alsobeen identified from hops and beer and include thetwo prenylchalcones xanthohumol and desmethylxan-thohumol and the three prenylflavanones isoxantho-humol, 8-prenylnaringenin, and 6-prenylnaringenin.Prenylchalcones and bitter acids accumulate at lowlevels at the onset of flowering and their concentra-tions gradually increase during the development offemale hop cones (De Keukelere et al., 2003). The invitro prenylation of the aromatic intermediates in thebiosynthesis of bitter acids has been described using acrude hop extract (Zuurbier et al., 1998). In contrast tomembrane-bound (iso)flavonoid prenyltransferases(Welle and Grisebach, 1991; LaFlamme et al., 1993;Sasaki et al., 2008), the hop bitter acid prenyltransfer-ase activities were in the soluble fraction of the proteinextract (Zuurbier et al., 1998). However, the bitter acidprenyltransferase remains to be characterized and it isnot clear whether prenylflavonoids and bitter acids aresynthesized by the same soluble prenyltransferase orwhether polyketide prenylation is functionally relatedto other areas of terpene metabolism in hop trichomes.In one of the first examples of applying genomics
approaches to plant secondary metabolism, sequenc-ing of a peppermint (Mentha piperita) oil gland cDNAlibrary proved highly effective for studying essentialoil biosynthesis in peppermint glandular trichomes(Lange et al., 2000). A similar approach has beenreported recently with hop trichomes, leading to iden-tification of an O-methyltransferase active in the bio-synthesis of prenyl chalcone (Nagel et al., 2008).Essential oils, bitter acids, and prenylflavonoids areall derived from pathways of terpene metabolism, butthe enzymes responsible for these processes have yetto be identified in hops. To facilitate gene discovery inhop natural product biosynthesis, a cDNA library was
constructed from total RNA isolated from lupulinglands, and candidates for diverse terpene biosyn-thetic enzymes identified based on sequence similar-ities to previously known genes and direct functionalidentification through expression of recombinant ter-pene synthases in Escherichia coli. We here identify themono- and sesquiterpene synthases involved in theformation of myrcene, humulene, and caryophyllene,and also describe the most highly expressed genes inthe metabolically specialized hop trichomes.
RESULTS
Terpene Content and Composition of HopGlandular Trichomes
Hexane extracts of different tissues fromhop cultivarNugget were collected and analyzed by gas chroma-tography (GC)-mass spectrometry (MS). This revealedthat the linear monoterpene myrcene and an unidenti-fied compound of retention time 10.491 min werefound exclusively in trichomes. MS analysis wasmost consistent with a structure of 2,7-dimethyl-1,6-octadiene (C10H18) for the unidentified compound,whichaccounted for approximately 15%(n=3biologicalreplicates) of the total monoterpenes in the trichomes.The levels of both compounds increased during tri-chome development. In the trichomes of 4-week-oldhop cones, myrcene comprised about 80% (n = 3) ofthe monoterpenes based on the area of peaks withmonoterpene-specific fragments (10–15 min, m/z 136,121, 93, 69) resolvedbyGC (Fig. 2). Othermonoterpenes,suchas linalool (retention time14.007min),werepresentin trace amountsandaccumulatedmainly infloral tissue(data not shown).
Humulene and caryophyllene were the two majorsesquiterpenes in isolated hop trichomes. Together,they account for approximately 85% (n = 3) of the totalsesquiterpenes in the trichomes (16.5–24 min, peakswith sesquiterpene-specific fragments of m/z 204,161, 93, 69; Fig. 2). However, unlike myrcene and2,7-dimethyl-1,6-octadiene (tentative), humulene andcaryophyllene were not specific to trichomes and werealso found in other tissues, such as leaves and flowers.Moreover, the ratios of humulene to caryophyllene indifferent tissues were almost identical, at about 3.0based on peak areas (Fig. 2). This suggests the possi-bility that they may be formed by the same terpenecyclase enzyme (see below).
We also extracted the terpenoids from hop trichomeswith ethyl acetate in place of hexane. This resulted inoverall extraction of more terpenoids as determined byGC-MS and comparison to the internal standard (10%moremyrceneandabout35%morehumuleneandcaryo-phyllene), but the profiles included some nonterpenoidcompounds eluting at higher retention times. Impor-tantly, the patterns of terpenoid compounds were verysimilar using the two different extraction protocols andthe ratio of humulene to caryophyllene was the same.
Figure 1. Isolation of glandular trichomes from female hop bracts. A,Glandular trichomes at the base of the female bracteole. B, Femalebracteole after trichome isolation. C, Isolated glandular trichomes.
Terpene Biosynthesis in Hop Trichomes
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Construction, Annotation, and Functional Classificationof a Hop Glandular Trichome EST Library Unigene Set
The Phoenix variety used for generation of thecDNA library has high essential oil and a-acid contentand was developed in the UK as a dual-purpose hopfor both bittering and finishing. It has 8% to 12%a-acids, 4.2% to 5.5% b-acids, 0.54% xanthohumol, anda total essential oil percentage of 1.2% to 2.5%.
Glandular trichomes were collected from femalebracts (Fig. 1). The conventional method for cDNAlibrary construction requires impractically large
amounts of trichomes to extract sufficient quantitiesof mRNA. We therefore employed a PCR-based cDNAlibrary constructionmethod that uses small amounts oftotal RNA as starting material; 12,665 single-pass ESTsequences were generated by random sequencing of16,152 cDNA clones. Vector, low-quality, and shortsequences (less than 100 bp) were excluded by usingthe cross-match program (http://www.phrap.org/phredphrapconsed.html) along with manual curation.A high percentage of ribosomal RNA contaminationwas observed and the corresponding sequences were
Figure 2. Accumulation of mono-and sesquiterpenes in trichomesand other tissues of hop. A, Devel-opmental sequence of developinghop flowers and trichomes. Bars ontrichome photographs are 200 mm.WAF,Weeks after flowering. B, GC-MS analysis of terpenoids from ma-ture trichomes from female bractsof hop cultivar Nugget. IS, Internalstandard (toluene). C, Levels of un-identified compound (retentiontime 10.491 min; see B), myrcene,caryophyllene, and humulene indifferent tissue and in trichomes atdifferent stages of development.
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removed during postsequencing cleansing of theESTs, resulting in 9,816 cleansed high-quality EST se-quences.An EST database was generated containing the 9,816
sequences assembled into 1,101 contigs and 2,518singletons. The contigs and singletons are collectivelyreferred to as unigenes. The unigenes were searchedagainst the National Center for Biotechnology Infor-mation (NCBI) nonredundant protein database (re-leased on 9/30/07) using the BLASTX algorithm, andannotated according to their homologous sequences inthe GenBank database. The most abundant unigenesare shown in Table I, and unigenes encoding enzymesof terpene and prenylflavonoid biosynthesis are listedin Table II and their frequencies depicted diagram-matically on metabolic pathways in Figure 3.In addition to annotation based on sequence similar-
ities to NCBI database entries, the unigenes werealso classified into three major functional categories—cellular component, molecular function, and biologicalprocess—according to the standardGeneOntology (GO;www.geneontology.org) terms, by searching the GOstandard protein database released in August 2007(BLASTX, e-value ,1 e-04; Supplemental Fig. S1). Ofthe cellular component GO terms, 44% and 34% of hopunigenes were related to cell part and organelle, respec-tively. In the category of molecular function, 47% of hopunigenes were involved in catalytic activity and 26% inbinding activity. Under the category of biological pro-cess, 31% and 30%were involved in cellular process andmetabolic process, respectively, indicating that the hopglandular trichomes are highlymetabolically active. Thehop glandular trichome unigene sequences and GOclassificationsweredeposited in theTrichOMEdatabasealong with trichome EST sequences from other speciesand are publicly available for viewing and searching(http://trichome.noble.org/trichomedb).
The Most Abundant Hop Trichome Unigenes
The 15 most highly represented unigenes in the hopglandular trichome cDNA library are listed in Table I.The abundance of their transcripts suggests criticalroles in secondary metabolite synthesis and transportand in glandular trichome development. Indeed, fourof these highly represented unigenes encoded en-zymes involved in secondary metabolite synthesis,namely, valerophenone synthase (VPS), chalcone syn-thase (CHS), chalcone isomerase (CHI)-like protein,and isopentenyl diphosphate (IPP) isomerase. UnigeneTCHL10255 encoded an AMP-dependent synthetaseand ligase family protein (also known as acyl-activatingenzyme; Shockey et al., 2003). Acyl-activating en-zymes convert carboxylic acids to acyl-AMP interme-diates and then to acyl-CoAs, and could potentially beinvolved in the formation of branched-chain acyl-CoAs as substrates for VPS.
ESTs homologous to food allergens and nonspecificlipid transfer proteins (LTPs) were also highly ex-pressed in hop glandular trichomes. Trichomes arealso known for their roles in storage and secretion ofheavy metals and in defense (Kupper et al., 2000; Choiet al., 2001), and unigene TCHL10880 (containing 120ESTs) was similar to metallothioneins and TCHL10947(composed of 137 ESTs) was homologous to cystatin.Metallothioneins are heavy-metal binding proteinsthat play dual roles in heavy-metal detoxificationand metal ion uptake/transport, and cystatin is aCys protease inhibitor that is induced by biotic andabiotic stresses. TCHL10134 showed strong sequencesimilarity to a tobacco (Nicotiana tabacum) senescence-related protein, the expression of which was tran-siently increased upon bacterial (Rhodococcus fascians)infection (Simon-Mateo et al., 2006), suggesting apotential role in pathogen defense.
Table I. The top 15 most abundant unigenes in the hop glandular trichome cDNA library
Hop bitter acids and prenylflavonoids are formedby the transfer of one or more single prenyl (dimethyl-allyl diphosphate; DMAPP) groups to a polyketideacceptor molecule, and mono- and sesquiterpenes arederived from longer chain allylic pyrophosphatesformed from DMAPP/IPP. Both the cytosolic mevalo-nate (MVA) pathway and the plastidial 2-C-methyl-D-erythritol-4-P (MEP) pathway can produce IPP, whichis then converted to its allylic isomer, DMAPP, throughthe action of IPP isomerase (Lichtenthaler, 1999). Basedon the stable isotope-labeling pattern of dimethylallylgroups in humulone, it was concluded that hop bitteracids are derived from the MEP pathway (Goese et al.,1999).Consistentwith these labeling results, unigenes en-coding the MEP pathway enzymes 1-deoxy-D-xylulose-5-P synthase, 1-deoxy-D-xylulose-5-P reductoisomerase,2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate syn-thase, and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphos-phate reductase, were present in the hop glandulartrichome cDNA library (Fig. 3; Table II). ESTs encoding2-C-methyl-D-erythritol-4-P cytidylyltransferase and4-diphosphocytidyl-2-C-methyl-D-erythritol kinase werenot identified, indicating that, although abundant inbiosynthetic genes, our hop unigene set did not con-tain all of the transcripts for secondary metabolite syn-thesis in glandular trichomes. In a recent report on ahop trichome EST collection from cultivars Taurusand Nugget, both of these enzymes were identified,
although the libraries in this case were a mixture ofnormalized and non-normalized (Nagel et al., 2008). Itshould, however, be noted that no ESTs correspondingto any of the enzymes of the MVA pathway wererepresented in the present collection.
Hop essential oils are synthesized from geranyldiphosphate (GPP) and farnesyl diphosphate (FPP).A cDNA encoding FPP synthase (FPPS) was clonedand characterized from hop that showed both GPPsynthase and FPPS activities, but not prenyltransferaseactivity toward phlorisovalerophenone (PIVP) in bitteracid synthesis (Okada et al., 2001). We could notidentify this FPPS gene or its homolog in our glandu-lar trichome cDNA library. Unigenes homologous toGPP synthase large and small subunits were, however,present in the EST database (data not shown), and thebiochemistry of the heterodimeric hop GPP synthasewill be described elsewhere.
Functional Identification of Hop Monoterpene Synthases
A search of the hop trichome EST database forpotential terpene synthases revealed three differentunigenes with high protein sequence identity to func-tionally identified monoterpene synthases (designatedas HlMTS1, HlMTS2, and HlMTS3). The EST clones ofHlMTS1 and HlMTS2 were found to be truncated attheir N termini. 5#-RACE was therefore used to clonethe corresponding full-length cDNAs for these twoputative monoterpene synthases. The full-length
Table II. Hop unigenes encoding enzymes of terpene and prenylflavonoid/bitter acid biosynthesis
cDNA of HlMTS1 (2,095 bp) encodes a peptide se-quence of 585 amino acids with a calculated molecularmass of 67,510 D and a pI of 4.9. The full-length cDNAof HlMTS2 (1,953 bp) contains an open reading frameof 1,842 nucleotides that encode a predicted protein of613 amino acids with a pI of 5.68. HlMTS1 andHlMTS2 share 46.7% amino acid identity to each other.Both have a plastid-targeting peptide at the N termi-nus (the first 31 and 46 amino acids for MTS1 andMTS2, respectively; predicted with TargetP 1.1 soft-ware). The deduced proteins of HlMTS1 and HlMTS2show 48.9 and 52% amino acid identity with Vitisvinifera (2)-a-terpineol synthase (Martin and Bohlmann,2004), respectively (Fig. 4A). According to BLAST re-sults, HlMTS3 is an ortholog of linalool synthase, andwas highly expressed in flower tissues where linaloolaccumulation was highest. We did not attempt tofurther characterize HlMTS3.Truncated forms of HlMTS1 and HlMTS2 with the
predicted plastid-targeting peptides removed wereexpressed in E. coli. After induction of protein expres-sion with isopropylthio-b-D-galactoside at 16�C for16 h, crude bacterial extracts were used for terpenesynthase assays using GPP (for monoterpene syn-
thase), FPP (for sesquiterpene synthase), and geranyl-geranyl diphosphate (GGPP; for diterpene synthase)as substrates. GC-MS analysis showed that myrcenewas the only product when the extract containingHlMTS2 was incubated with GPP (Fig. 5A). No prod-uct could be detected with FPP or GGPP as substrates.These results therefore indicate that HlMTS2 activityleads to the formation of myrcene in hop trichomes.We were unable to detect monoterpene, sesquiterpene,or diterpene synthase activity in extracts containingHlMTS1.
Kinetic analysis of purified His-tagged recombinantHlMTS2 revealed a Km value for geranyl pyrophos-phate of 7.65 6 2.40 mM (n = 3).
Functional Identification of HopSesquiterpene Synthases
ESTs corresponding to two different sesquiterpenesynthase-like genes (designated as HlSTS1 andHlSTS2) were also were identified from the hop tri-chome library. The full-length cDNAs of HlSTS1 andHlSTS2 were obtained using 5#-RACE. The full-lengthcDNA of HlSTS1 (1,842 bp) encodes a peptide se-
Figure 3. Biosynthetic pathways forterpene-derived natural productsfound in hop trichomes, showingESTabundance in the hop glandulartrichome cDNA library. A, The MEPpathway leading to DMAPP biosyn-thesis. B, General reactions ofmono- and sesquiterpene biosyn-thesis. C, Prenylflavonoid and bitteracid biosynthesis. Chemical struc-tures of selected compounds areshown. The abundance of ESTs cor-responding to each biosyntheticgene in the hop glandular trichomecDNA library is indicated inparentheses. DXPS, 1-Deoxy-D-xylulose-5-P synthase;DXR,1-deoxy-D-xylulose-5-P reductoisomerase;MCT, 2-C-methyl-D-erythritol-4-Pcytidylyltransferase; CMK,4-diphos-phocytidyl-2-C-methyl-D-erythritolkinase; MCS, 2-C-methyl-D-ery-thritol-2,4-cyclodiphosphatesynthase;HDS, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase;HDR, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase;IPPI, isopentenyl diphosphate/dimethylallyl diphosphate isomer-ase; OMT,O-methyltransferase; PT,prenyltransferase. Numbers in pa-rentheses represent the number ofoccurrences of an EST correspond-ing to the particular enzyme in the9,816 cleansed hop ESTs.
Terpene Biosynthesis in Hop Trichomes
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quence of 563 amino acids with a calculated molecularmass of 66,218 D and a pI of 5.1. The full-length cDNAof HlSTS2 (1,863 bp) contains an open reading frame of1,692 nucleotides that encode a predicted protein of563 amino acids with a pI of 5.44. HlSTS1 and HlSTS2share 92.4% identity to each other at the protein level(Fig. 4B) and 50% amino acid identity with a function-
ally characterized V. vinifera (2)-germacrene D syn-thase (Lucker et al., 2004).
HlMTS1, HlMTS2, HlSTS1, and HlSTS2 all containthe RR(P)X8W, RXR, and DDXXD (X is any amino acid)motifs, which are key features of most angiospermterpene synthases (Keeling and Bohlmann, 2006;Fig. 4).
Figure 4. Alignments of hop MTS (A) and STS (B) sequences. Other MTS sequences are from Citrus unshiu (accession no.BAD27259) and V. vinifera (AAS79352). Identical amino acids are shown as white letters on a black background. The RR(P)X8W,RXR, and DDXXD motifs are underlined.
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HlSTS1 and H1STS2 were expressed in E. coli usingthe same strategy as employed for the monoterpenesynthases. Both enzymes were active with FPP assubstrate, but not with GPP or GGPP. Terpenoid prod-ucts were identified by GC-MS. The major sesquiter-pene products of HlSTS1 were identified as humulene(70% of total products) and caryophyllene (25%; Fig.5B). This ratio of humulene to caryophyllene wassimilar to the value in the essential oil of the hoptrichomes.In spite of the very close sequence identity between
HlSTS1 and HlSTS2, the products of HlSTS2 are nothumulene and caryophyllene (Fig. 5B). The majorproduct of HlSTS2 was shown by GC-MS analysis tobe b-elemene, a relatively minor component of hopessential oil. However, b-elemene can be formed invitro by the rearrangement of germacrene A at hightemperatures, such as used in the present GC-MSanalysis. We therefore analyzed the product of the
STS2 enzymatic reaction by GC-MS using lower injec-tion temperatures (150�C and 180�C instead of theusual 280�C). b-Elemene was no longer observed andwas replaced by a broad peak corresponding togermacrene A).
Kinetic analysis of purified His-tagged recombinantHlSTS1 and H1STS2 revealed Km values for farnesylpyrophosphate of 0.70 6 0.07 (n = 3) and 0.49 6 0.04(n = 3) mM, respectively.
Tissue-Specific and Developmental Expression of HopTerpene Synthases
To test whether the patterns of terpene metaboliteproduction in hops can be explained by the expressionof the above-characterized terpene synthases, real-time PCR was first performed to examine the expres-sion of HlMTS1 and HlMTS2 in different tissues and intrichomes at different developmental stages. HlMTS1
Figure 5. GC-MS analysis of enzymatic products from recombinant hop mono- and sesquiterpene synthases. A, Portion ofchromatogram showing the product of the reaction of GPP with HlMTS1 and its corresponding mass spectrum (inset). Bottom,Same for an authentic standard of myrcene. B, Portion of chromatogram showing the two products of the reaction of FPP withHlSTS1 and their corresponding mass spectra (inset). Bottom, Same for HlSTS2.
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and HlMTS2 transcript levels followed the same de-velopmental pattern as the monoterpene metabolites(Fig. 6, A and B). Both HlMTS1 and HlMTS2 transcriptlevels were highest in trichomes from cones at 4 weeksafter flowering (Fig. 6, A and B).
Because of the high sequence identity betweenHlSTS1 and HlSTS2, we could not find good primersfor real-time reverse transcription (RT)-PCR to distin-guish between the two genes. Semiquantitative RT-PCR was therefore used to analyze the expressionpatterns of HlSTS1 and HlSTS2. HlSTS1 transcriptswere abundant in those tissues with high levels ofhumulene and caryophyllene, and also paralleled thelevels of these compounds in hop trichomes duringdevelopment. HlSTS2 transcripts were abundant inyoung leaf tissue, although they were also detected inother tissues (Fig. 6C).
Polyketide Biosynthesis and Prenylation
Hop prenylflavonoids are formed by transfer of a5-carbon prenyl group to a chalcone precursor, itselfformed by the condensation of malonyl-CoA and4-coumaroyl-CoA by CHS. 4-Coumaroyl-CoA is de-rived from L-Phe by the sequential actions of L-Pheammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H),and 4-coumarate:CoA ligase (4CL). Four PAL gene ho-mologs were present in the hop glandular trichomecDNA library (Table II), and three unigenes were simi-lar to C4H from different plant species; TCHL10606matched to the N terminus of the enzyme, whereas theclosely related TCHL10666 and the singleton ES652343matched to the C terminus. Arabidopsis (Arabidopsisthaliana) 4CL-like protein homologs were also found inthe cDNA library.
Figure 6. Tissue-specific expression of hopterpene synthases. A, Quantitative real-timePCR analysis of HlMTS1 transcript levels indifferent hop tissues and different develop-mental stages of cones and trichomes. Notrichome = mature cones with trichomes re-moved. B, As above, for HlMTS2 transcripts.C, Semiquantitative RT-PCR analysis ofHlSTS1 and H1STS2 transcripts in differenthop tissues and different developmentalstages of cones and trichomes.
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Bitter acid and prenylflavonoid biosynthesis shareseveral features, particularly the involvement of apolyketide synthase and subsequent prenyltransfer-ases. The polyketide synthase for bitter acid biosyn-thesis, VPS, is related to CHS, and produces PIVP frommalonyl-CoA and isovaleryl-CoA. VPS and CHS fromhop have been cloned and functionally characterized(Paniego et al., 1999; Okada and Ito, 2001; Okada et al.,2004). In contrast to a single VPS gene in hops, CHSconstitutes a family of four genes, CHS2, CHS3, CHS4,and CHS_H1. VPS and CHS2 were among the mostabundant unigenes in the present hop glandular tri-chome cDNA library, represented by 184 and 58 ESTs,respectively (Table I). CHS4 and CHS_H1 were alsopresent, whereas CHS3 was not identified (Table II).Plant CHIs are classified into four subfamilies ac-
cording to their phylogenetic relationship (Ralstonet al., 2005). Proteins in the CHI3 and CHI4 subfamiliesshow sequence similarity to previously characterizedCHIs, but have not been biochemically characterized todate and are therefore described as CHI-like proteins.HlCHI-like 1, which was among the most abundantunigenes in the glandular trichome cDNA library (Ta-ble I), was homologous to a tomato (Solanum lycopersi-cum) CHI-like protein (LeCHI-like) that belongs to theCHI3 subfamily, and HlCHI-like 2 was similar to anArabidopsis CHI-like protein (At5g05270) that belongsto the CHI4 subfamily. Neither HlCHI-like 1 norHlCHI-like 2 exhibited CHI activity when expressedas recombinant proteins in E. coli (data not shown).Transfer of DMAPP to polyketide acceptor mole-
cules is likely catalyzed by aromatic prenyltransfer-ases in hops (Stevens and Page, 2004). Aromaticprenyltransferases have been isolated and character-ized from bacteria and plants and can be grouped intotwo classes: the soluble bacterial prenyltransferasesand the membrane-bound bacterial and plant UbiAfamily prenyltransferases (Sasaki et al., 2008; Tianet al., 2008). The UbiA family prenyltransferases sharethe common motif (N/D)DXXD for prenyl diphos-phate binding (Brauer et al., 2004). By searching hopunigenes with the prenyl diphosphate-binding motif,a single unigene was identified that showed strongsequence similarity to a membrane-bound prenyl-transferase involved in ubiquinone synthesis (datanot shown).
DISCUSSION
The generation and sequencing of an EST libraryfrom hop lupulin glands has enabled us to identify andcharacterize the enzymes involved in the biosynthesisof the major mono- and sesquiterpene aroma com-pounds produced in these trichomes. As in a previousstudy (Nagel et al., 2008), ESTs corresponding to MEPpathway enzymes were highly abundant as comparedwithMVApathway transcripts. Although this suggeststhat theDMAPP/IPP for all classes of terpene synthesisin the hop trichomes originates primarily from theMEP
pathway, it is perhaps dangerous to equate ESTcountsto expressed enzymatic activities. Nevertheless, theMEP pathway has previously been shown to provideprecursors for both mono- and sesquiterpene biosyn-thesis in snapdragon (Antirrhinum majus) flowers(Dudareva et al., 2005).
On comparing the overall transcript abundances inour EST library with those in the two non-normalizedlibraries previously generated from trichomes of hopcultivars Taurus and Nuggett (Nagel et al., 2008),47.5% of the ESTs (1,087 of 2,290) in the Taurus/Nuggett libraries could hit targets in the cultivarPhoenix library described in this article, and 33.4% ofthe Phoenix ESTs could hit targets in the Taurus/Nuggett libraries. The lack of closer coincidence couldarise from differences in both hop cultivar and devel-opmental stage. However, all of the terpene synthasesidentified in this article could be found in the Taurus/Nuggett libraries, and two of the three O-methyltrans-ferases previously identified (Nagel et al., 2008) werealso represented in the Phoenix library.
Myrcene, caryophyllene, and humulene representthe bulk of the terepene component of hop essentialoil. The relative proportions of caryophyllene andhumulene were very similar in all tissues in whichthese compounds were produced and the same asproduced by recombinant HlSTS1 in vitro. Further-more, we did not observe one of the compoundswithout the other in any of the hop tissues analyzed.
H1STS1 and HlSTS2 are 92.4% identical at the aminoacid level, but make different products. b-Elemene, theinitially measured product of HlSTS2, has been previ-ously described as a minor component of hop essentialoils (Katsiotis et al., 1989), although, as shown here, itmay be derived nonenzymatically from germacrene A.It is likely that the active-site cavities of HlSTS1 andHlSTS2 differ in size, shape, or polarity as a result ofthe few amino acids that are different between the twoenzymes, leading to different modes of cyclization.
The previously described myrcene synthase fromgrand fir (Abies grandis) is more closely related tosesquiterpene and diterpene synthases from conifersthan it is to monoterpene synthases from angiosperms(Bohlmann et al., 1997). The hop myrcene synthaseexhibited 52% amino acid identity with V. vinifera(2)-a-terpineol synthase (Martin and Bohlmann,2004), but only 29% identity to grand fir myrcenesynthase. However, the hop myrcene synthase is alsoonly 29.8% identical to myrcene synthase from snap-dragon (Dudareva et al., 2003); it is more closelyrelated (40.8%) to Arabidopsis myrcene synthase(At2g24210), although this enzyme, unlike that fromhop, can also produce ocimene from GPP (Bohlmannet al., 2000).
It is well known that sesquiterpene synthases withthe ability to form humulene can also form caryophyl-lene. Thus, rice (Oryza sativa) caryophyllene synthaseproduces caryophyllene as the major product, al-though several other products are made with FPP assubstrate, including humulene and b-elemene (Cheng
Terpene Biosynthesis in Hop Trichomes
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et al., 2007). It is interesting that this latter product isproduced by a separate enzyme (HlSTS2) in hop.Similar product complexity to that observed withrice sesquiterpene synthases is seen in Arabidopsis,where genetic evidence has shown that only twoenzymes account for most of the complex mixture ofover 20 sesquiterpenes in the floral scent (Tholl et al.,2005). HlSTS1 appears to be a less promiscuous ses-quiterpene synthase than those of rice and Arabidop-sis, and its dual product specificity in vitro correlateswith the humulene to caryophyllene ratio in the dif-ferent hop tissues in which this enzyme is expressed,suggesting that it is the major determinant of the levelsof these two compounds.
Although the two monoterpene synthases describedin this article have clear N-terminal plastid-targetingsequences, we could not detect similar sequences inthe two sesquiterpene synthases using TargetP 1.1software. If these latter enzymes are truly cytoplasmicand if, as we suggest, the plastidial MEP pathway isresponsible for most, if not all, of the formation of thebuilding blocks for GPP and FPP, there would need tobe transport of IPP/DMAPP between plastid andcytosol in hop trichomes, as occurs in snapdragonflowers (Dudareva et al., 2005).
Although the genomics approach applied in thisarticle successfully identified the genes involved inmono- and sesquiterpene biosynthesis in hop tri-chomes, the prenylation step in the formation of bitteracids and prenylflavonoids still requires elucidation.Recently, the first plant flavonoid prenyltransferasewas described, an enzyme from Sophora flavescens thatprenylates the flavanone naringenin (Sasaki et al.,2008). This enzyme is a member of the membrane-associated UbiA family of plant prenyltransferases.However, preliminary biochemical evidence suggeststhat the hop bitter acid prenyltransferase is a solubleenzyme (Zuurbier et al., 1998). It is not clear whetherthe same or different enzymes catalyze the prenylationof the different polyketide (naringenin chalcone andPIVP) intermediates in prenylflavonoid and bitter acidbiosynthesis. We could only identify a single ESTrepresenting a plant UbiA family prenyltransferasein our EST collection. In contrast, 23 hop ESTs anno-tated as potential aromatic prenyltransferases werereported from an EST collection derived from normal-ized and non-normalized libraries, although none wasfunctionally identified (Nagel et al., 2008). This sug-gests that the aromatic prenyltransferases of bitter acidand prenyl flavonoid biosynthesis in hop are, if similarto currently known prenyltransferases, expressed atrelatively low levels in comparison to the correspond-ing polyketide synthases.
Bacterial prenyltransferases involved in antibioticsynthesis were identified recently and are solubleproteins that contain a conserved protein fold but sharelow similarity at the primary sequence level (Kuzuyamaet al., 2005). The bacterial prenyltransferase sequenceswere also used to search against our hop unigenes, butno homologous sequences were identified.
Several previous studies have revealed a high pro-portion of LTP transcripts in plant trichomes (Langeet al., 2000; Aziz et al., 2005). LTPs are small basicpolypeptides that bind to fatty acid derivatives and aresecreted to the cell walls in plants (Kader, 1996). Some ofthe food allergens belong to the LTP family (Pastorelloet al., 1999). The function of LTPs in trichomes is un-known, although it has been suggested that LTPs mayplay a role in plant defense against pathogens (Garcıa-Olmedo et al., 1998). It has also been suggested that theLTPs inpeppermint glandular trichomes are involved inintracellular trafficking and secretion of essential oils(Lange et al., 2000).
In conclusion, we have described the constructionand analysis of a hop glandular trichomeEST database.Mining the sequences in the database resulted in theidentification of many of the genes involved in terpenenatural product biosynthesis, and mono- and sesqui-terpene synthases responsible for formation of themajor hop essential oil components were functionallycharacterized. The database, which is publicly avail-able as a part of the Noble Foundation’s TrichOMEdatabase (http://trichome.noble.org/trichomedb), pro-vides a resource for further characterization of themolecular basis of hop trichome development and me-tabolism, aswell as being apotential sourceofmolecularmarkers to facilitate hop breeding.
MATERIALS AND METHODS
Plant Material
For cDNA library construction, mid-developmental stage female cones
were collected from hop (Humulus lupulus ‘Phoenix’) plants, grown at the Hop
Research Institute, Wye, Kent, UK. The large cones (unlikely to develop much
further) and the small cones (containing few trichomes) were discarded and
only the medium-sized cones were used.
Rhizomes of hops of cultivar Nugget were purchased from Northern
Brewer Company and grown in the greenhouse (after flowering, the day-
length was reduced from 16 to 14.5 h to initiate production of cones). Young
leaves (1–2 cm in diameter), old leaves (8–10 cm in diameter), cones, and
glandular trichomes (see below) were collected and stored at280�C until used
for chemical extraction or analysis of transcript levels.
Preparation of Trichomes
A total of 10 female cones were used for each batch of RNA isolated. The
cones were frozen in liquid nitrogen and kept on ice while each bract was
removed from the cone using a fine tip forceps. The bracts were transferred to
a chilled 50-mL falcon tube and cold diethyl pyrocarbonate-treated water was
added to submerge all the plant material. Approximately 2 g of glass beads
(Sigma glass beads; 600 mm; acid washed) were added and the tube firmly
capped. Glandular trichomes were separated by vortexing the tube for 1 to
2 min while keeping the tube in a horizontal position. Trichomes were sifted
through a 500-mmmetal mesh followed by low-speed centrifugation to collect
trichomes in the bottom of the tube. The trichomes were used immediately for
total RNA extraction.
RNA Isolation, cDNA Library Construction, and
EST Sequencing
Total RNA was isolated from trichomes using the cold-phenol method as
described (Carpenter and Simon, 1998). RNA concentration and quality were
determined with a Nanodrop spectrophotometer (Thermo Fisher Scientific)
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