Research Article Functional Characterization of ...downloads.hindawi.com/journals/tswj/2014/840592.pdf · Research Article Functional Characterization of Sesquiterpene Synthase from

Research ArticleFunctional Characterization ofSesquiterpene Synthase from Polygonum minus

Su-Fang Ee12 Zeti-Azura Mohamed-Hussein12 Roohaida Othman12

Noor Azmi Shaharuddin3 Ismanizan Ismail12 and Zamri Zainal12

1 School of Biosciences and Biotechnology Faculty of Science and Technology Universiti Kebangsaan Malaysia43600 Bangi Selangor Malaysia

2 Institute of Systems Biology (INBIOSIS) Universiti Kebangsaan Malaysia 43600 Bangi Selangor Malaysia3 Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia 43400 Serdang Selangor Malaysia

Correspondence should be addressed to Zamri Zainal zzukmmy

Received 25 October 2013 Accepted 24 December 2013 Published 11 February 2014

Academic Editors Y H Cheong and H Verhoeven

Copyright copy 2014 Su-Fang Ee et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Polygonum minus is an aromatic plant which contains high abundance of terpenoids especially the sesquiterpenes C15H24

Sesquiterpenes were believed to contribute to the many useful biological properties in plants This study aimed to functionallycharacterize a full length sesquiterpene synthase gene from P minus P minus sesquiterpene synthase (PmSTS) has a completeopen reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein Similar to other sesquiterpene synthases PmSTShas two large domains the N-terminal domain and the C-terminal metal-binding domain It also consists of three conservedmotifs the DDXXD NSEDTE and RXR A three-dimensional protein model for PmSTS built clearly distinguished the two maindomains where conserved motifs were highlightedWe also constructed a phylogenetic tree which showed that PmSTS belongs tothe angiosperm sesquiterpene synthase subfamily Tps-a To examine the function of PmSTS we expressed this gene in Arabidopsisthaliana Two transgenic lines designated as OE3 and OE7 were further characterized both molecularly and functionally Thetransgenic plants demonstrated smaller basal rosette leaves shorter and fewer flowering stems and fewer seeds compared to wildtype plants Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for theproduction of 120573-sesquiphellandrene

1 Introduction

Plants have developed a range of strategies to survive andadapt to their environment One such strategy is to produce alarge variety of secondarymetabolites [1] To date there are anestimated 200000 secondary metabolites that are producedby plants [2] Polygonum minus an aromatic plant that isindigenous to Malaysia produces large number of secondarymetabolites TraditionallyPminus is used to treat indigestionand dandruff problems and as a postnatal tonic [3] Thisplant has a unique sweet and pleasant flavour and aroma andthus is commonly used in local cuisine Its unique flavour ismainly due to the secondary metabolites present in the plant[4] The secondary metabolites present in P minus are alsoresponsible for its useful biological properties such as itsantioxidant antiulcer antiviral antimicrobial and antifungalactivities [5 6]

Secondary metabolites are divided into three majorgroups terpenoids alkaloids and phenylpropanoids [7]Terpenoids are the largest andmost structurally diverse classDifferent terpenoids are distributed unevenlywithin the plantkingdom [8] Some terpenoids are restricted to one species orone genus [8] The essential oils of P minus have been shownto contain a high abundance of terpenoids especially thesesquiterpenes [4] Twenty-four different types of sesquiter-penes including 120572-humulene 120572-farnesene 120573-farnesenevalencene 120572-panasinsene 120572-bergamotene 120573-caryophyllene120575-cadinene and 120572-curcumene have been identified to date[4] This diversity suggests that P minus is a good source ofsesquiterpenes for research on secondary metabolites par-ticularly the sesquiterpenes Previously most studies focusedon a metabolomic approach to examine this plant whereasthose adopting a molecular approach for characterizing and

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 840592 11 pageshttpdxdoiorg1011552014840592

2 The Scientific World Journal

understanding the biosynthetic regulation of sesquiterpenerelated genes are still lacking

Based on our previously constructed cDNA-AFLP tran-scriptome profiles a sesquiterpene synthase gene (GenBankHO079100 and HO079108) was shown to be highly upregu-lated upon stress induced by salicylic acid [9] Although tran-scriptomic data are available the major challenge for studiesof P minus is the lack of a transformation and regenerationsystem for the functional study of its genes To address thisconcern the model plant A thaliana was used in this workA thaliana is the first angiosperm to have had its completegenome sequenced Since then it has been well studiedIts short life cycle of only 3 months and its ability to producea large number of progeny seeds have encouraged manyresearchers to use A thaliana for functional studies [10]The well-established floral-dip transformation system alsoprovides a fast and efficient method to transfer genes fromAgrobacterium into A thaliana [11] This method involvesonly a simple immersion of the floral buds in an Agrobac-terium suspension

PmSTS was previously cloned and expressed in Lactococ-cus lactis [12] That study was aimed solely at maximizingmetabolite production in a bacterial system and did notstudy how the gene is regulated in plants In addition thePmSTS gene introduced into L lactis contained two incorrectnucleotides that resulted in a single amino acid change [12]calling into question the true function of this protein

In this study we expressed the correct sequence of PmSTSto determine the function of its protein product in a modelplant system As there is no established transformation andregeneration system available in P minus the model plant Athaliana was used to investigate the role of PmSTS in plantsThe overexpression of PmSTS inA thaliana provides a betterunderstanding of the gene function The results of this studyfurther enhance our understanding of the biosynthesis andregulation of secondary metabolites in P minus particularlythe sesquiterpenes

2 Materials and Methods

21 Plant Material and Growth Conditions A thaliana eco-type Columbia-0was grown in a growth chamber (Conviron)at a temperature of 22∘Cday20∘Cnight and relative humidityof 50ndash70Thephotoperiodwas set at 16 h day8 hnight witha light intensity of 100ndash150 120583molesmminus2 sminus1 using fluorescentbulbsTheplants were ready for floral-dip transformation oneweek after the primary inflorescences were clipped Wateringwas stopped three days prior to transformation to increase thetransformation efficiency

22 In Silico Analysis of PmSTS The nucleotide sequenceof PmSTS was retrieved from the NCBI database withGenBank ID of JX025008 The physiochemical properties ofthe PmSTS were determined using PROTPARAM software(httpwebexpasyorgprotparam) The presence of signalpeptide was predicted using SignalP 41 software(httpwwwcbsdtudkservicesSignalP) [13] Comparativesequence analysis of PmSTS was performed using NCBI

BLAST against the protein database (httpblastncbinlmnihgov) Multiple sequence alignment was done withBIOEDIT software using the default parameters (httpwwwmbioncsuedubioeditbioedithtml) The three-dimen-sional (3D) protein structure homology-modelling ofthe PmSTS was generated using I-TASSER software [14]The stereochemical quality of the predicted 3D proteinstructure was examined through PROCHECK analysis(httpwwwebiacukthornton-srvsoftwarePROCHECK)Phylogenetic tree was built using MEGA5 software withneighbour joining method Bootstrap of 1000 replicates wasdone Terpene synthases from seven previously recognizedTPS subfamilies Tps-a to Tps-g were retrieved from the NCBIGenBank database according to Bohlmann et al [15] andDanner et al [16] The Tps-c and Tps-e subfamilies whichare composed of the copalyl diphosphate (cdp) synthases andkaurene synthases and are involved in primary metabolismwere chosen as outgroups

23 Gene Amplification and Construction of the pCAMSSoverexpression Vector PmSTS (GenBank JX025008)was amplified by standard PCR methods using thePmSTS specific forward primer 51015840-GGGCAGATCTTATGTATTCCATGATC-31015840 and reverseprimer 51015840-GGCTGGTGACCTTATATCAGTATGGG-31015840 Tofacilitate the cloning process restriction enzymes (RE) sitesfor BglII and BstEII (underlined) were attached to the 51015840ends of the forward and reverse primers respectively Thenucleotides in bold type are the start (ATG) and termination(TTA) codons in the open reading frame of the PmSTS gene

The vector pCAMBIA1301 (Centre for the Applicationof Molecular Biology of International Agriculture BlackMountain Australia) was used as the backbone for the con-struction of the plant transformation vector pCAMSS whichharboured thePmSTS gene To construct the pCAMSS vectorthe 120573-glucuronidase (GUS) reporter gene was first excisedfrom the pCAMBIA1301 vector and then replaced with thePCR-amplified PmSTS gene Both the pCAMBIA1301 vectorand the PmSTS gene were digested with the BglII andBstEII restriction enzymes to generate complementary stickyends for ligation The digested fragment of the PmSTS genewas ligated into the corresponding sites of pCAMBIA1301yielding the pCAMSS vector (Figure 1) The pCAMSS vectorwas then transformed into Agrobacterium tumefaciens strainGV3101 The cloned pCAMSS vector was sent for sequencingand RE digestion to confirm the integration of the PmSTSgene in the correct orientation

24 Agrobacterium-Mediated Floral-Dip Plant Transforma-tion A thaliana was transformed using the Agrobacterium-mediated floral dip method [11] Agrobacterium cells weregrown to an OD

600of 07 The floral-dip inoculation medium

contained harvested cells that were resuspended in 5sucrose and 005 Silwet The secondary inflorescences wereimmersed in the inoculation medium and swirled gently toallow the intake of Agrobacterium harbouring the pCAMSSvector into the flower gynoecium The transformed plantswere kept in the dark and wrapped with plastic overnight to

The Scientific World Journal 3

CaMV35S promoterHygromycinCaMV35S

polyALacZ alpha

CaMV35S promoter PmSTS

T border (L)

T border (R)

Multiple cloning sites

XhoI XhoI BstXI BgIII BstEII

Nos polyA

Figure 1 Schematic diagram of the T-DNA region of pCAMSS vector The pCAMSS recombinant vector contains the full length PmSTSgene which is expressed under the control of the constitutive CaMV35S promoter

maintain humidity The next day the plants were returnedto their normal growth conditions The transformation wasrepeated after a week to increase the transformation effi-ciency Plants were grown for additional 4-5 weeks until all ofthe siliques became brown and dryThe seeds were harvestedand stored at 4∘C under desiccation

25 Selection of Transgenic A thaliana Seeds were surfacesterilized with 50 Clorox containing 005 Tween-20 for10min followed by 80 ethanol for 2min and the seeds werethen rinsed three times with distilled water before platingTo select the transformed plants approximately 100 sterilizedseeds were screened on Murashige and Skoog (MS) solidmedia containing 25mg Lminus1 hygromycin The seeds werecultivated following the standard method of Harrison et al[17] The plated seeds were stratified at 4∘C for 2 daysThe seeds were then placed under light for 6 h to inducegermination followed by 2 days of incubation in the darkand then returned to normal growth conditionsThe putativetransgenic plants were selected by two weeks of growth onhygromycin plates The putative transformants grown onselection media had long hypocotyls green leaves and longroots These putative transformants were transferred to potswith soil and grown under normal growth conditions Theseeds from themature plants were harvested after onemonth

To verify the presence of the PmSTS gene DNA fromthe putative transformants and wild type A thaliana wasextracted using the standard CTAB extraction method [18]Wild type A thaliana were used as the negative controlfor the PCR amplification Genomic PCR was performedusing a forward primer containing a region of the CaMV35Spromoter (51015840-TCCCACTATCCTTCGCAAGACCC-31015840) anda reverse primer containing a PmSTS gene-specific sequence(51015840-AGTGATAGGCAACTCCAAGC-31015840)

26 Semiquantitative RT-PCR Analysis of the Transgenic Athaliana Semiquantitative RT-PCR was conducted to com-pare the expression of the PmSTS gene in the T

2transgenicA

thaliana and wild type A thaliana Total RNA was extractedfrom the leaves using the TRI Reagent (Molecular ResearchCentre Inc Cincinnati OH USA) according to the man-ufacturerrsquos instructions First strand cDNA was synthesizedwith the Maxima First Strand cDNA Synthesis Kit (Thermo

Fisher ScientificUSA) using total RNAas the template Semi-quantitative RT-PCR analysis was performed using standardPCR methods with 500 ng of cDNA template The forwardprimer (51015840-CCATGATGCAGCCAACCGAGAT-31015840) corre-sponded to nucleotide 1523ndash1544 of the PmSTS gene whilethe reverse primer (51015840-AATCCATCCTCTCCGGCGTCAT-31015840) corresponded to nucleotide 1622ndash1643 As a con-trol a PCR with the housekeeping gene 4HPPD (Gen-Bank AT1G065701NM 100536) was performed in paral-lel using 51015840-GCGCTTCCATCACATCGAGTTC-31015840 and 51015840-AATCCAATGGGAACGACGACGC-31015840 as the forward andreverse primers respectively

27 GC-MS Analysis of The Transgenic A thaliana Thevolatiles emitted from the leaf samples were extractedusing the headspace solid-phasemicroextraction (HS-SPME)method A polydimethylsiloxane-(PDMS-) coated fibre wasexposed to the headspace of the sample vial containing 2 gof leaves for 30min at 55ndash60∘C before injection into thegas chromatograph mass spectrometer (GC-MS) The GC-MS analysis was performed on an Agilent 7890A gas chro-matograph (GC) that was directly coupled to the mass spec-trometer system (MS) of an Agilent 5975C inert MSD witha triple-axis detector Separation was achieved with a 5phenyl methylpolysiloxane column (model AB-5MS AbelIndustries) that was 30m long and 025mm in diameter andhad a film thickness of 025 120583m Helium was used as thecarrier gas with a flow rate of 13mLminminus1 A splitless injec-tion was set at 50∘C hold for 3min increased to 250∘C at arate of 6∘Cminminus1 and hold at 250∘C for 5minThepeakswereidentified by searching the NISTEPANIH mass spectrallibrary (version 20) and the results were combined in a GC-MS chromatogram

3 Results and Discussion

Sesquiterpene synthases broadly refer to enzymes that con-vert farnesyl diphosphate (FPP) into various sesquiter-penes Previous studies of sesquiterpenes often commenton the structural complexity and diversity of sesquiterpenemetabolism The main causes of sesquiterpene diversity arethe large number of different sesquiterpene synthases that are


MSVQSSVVLLAPSKNLSPEVGRRCANYHPSIWGDHFLSYAS-EFTNTDDHLKQHVQQLKE 59-MATYLQASSGPCSTIVPEITRRSANYHPNIWGDQFLKYNSFDLSKTDANTKEHFRQLKE 59---MSVPVSQIPSLKAKGVIMRRNANYHPNIWGDRFINYVPVDKMNHTCHLQA-IEELKD 56---MSIQVSTCPLVQIPKPEHRPMAEFHPSIWGDQFIAYTPEDEDTRACKEKQ-VEDLKA 56--------------MSSGETFRPTADFHPSLWRNHFLKGASDFKTVDHTATQERHEALKE 46--------------MSSGETFRPTADFHPSLWRNHFLKGASDFKTVDHTATQERHEALKE 46MYSMIMTSAAPNPLPLSKPAPREVAGFKPCPWGDRFLDYTPADNKVIEAQEKM-AKELKE 59

EVRKMLMAADDD-SVQKLLLIDAIQRLGVAYHFESEIDEALKHMFD--GSVASAEEDVYT 116EVKKMLVDAGPN---QQLNLIDDIQRLGVAYQFEAEIDAALQRMN---VIFQGNDDDLHT 113AVRRELLTATG---LSQLNLIDAIQRLGVGYHFERELEEALQHVYHKNHYHDDTEDNLYI 113EVRRELMAAAGN-PAQLLNFIDAVQRLGVAYHFEREIEESLQHIYDRFHDADDTEDDLYN 115EVRRMITDAEDK-PVQKLRLIDEVQRLGVAYHFEKEIEDAIQKLCP--IYIDSNRADLHT 103EVRRMITDAEDK-PVQKLRLIDEVQRLGVAYHFEKEIGDAIQKLCP--IYIDSNRADLHT 103ELKKELQSIGDKPLIEQLRLIDSIERLGVAYHFEQEIEDALRRIHE--TSTNTSFDDLAL 117

176ASLRFRLLRQQGYHVSCDLFNNFKDNEGNFKESLSSDVRGMLSLYEATHLRVHGEDILDE

173ISLRFRLLRQHGYNVSSDVFRKFMDNNGKFKECLISDLRGVLSLYEATHFRVHGEDILED

173ISLRFRLLRQHGYYVSCDILNKFKDEKDNFKESLTTDVPGMLSLYEAAHLGAHGEDILDE

175IALQFRLLRQQGYNISCGIFNKFKDEKGSFKEDLISNVQGMLGLYEAAHLRVHGEDTLEE

163VSLHFRLLRQQGIKISCDVFEKFKDDEGRFKSSLINDVQGMLSLYEAAYMAVRGEHILDE


TSLFFRLLRQHGYRVSSDVFKEFIGSNGNLKDEVTNDVHALLSFYEACHLRLHGEGVLDE 177

ALAFTTTHLQ-SAAKYSLNP--LAEQVVHALKQPIRKGLPRLEARHYFSIY--QADDSHH 231ALEFTTSHLE-RLKSHLKNP--LAAQVIRALKCPIHKGLNRLEAKHYISIYQ-QEDDSHN 229AIAFTTTHLKSLAIDHLRNPS-LASQVIHALRQPLHRGVPRLENRRYISIY--QDEVSHN 230ALAFTTTHLK-ATVESLGYP--LAEQVAHALKHPIRKGLERLEARWYISLY--QDEASHD 230AIAFTTTHLKSLVAQDHVTPK-LAEQINHALYRPLRKTLPRLEARYFMSMINSTSDHLYN 222AIAFTTTHLKSLVAQDHVTPK-LAEQINHALYRPLRKTLPRLEARYFMSMINSTSDHLCN 222ALSSTESNLIKLVSDPNPLSEALEERVKHALHKPLNKRLVRVESVRYMQVYE-EDDPLHN 236

KALLKLAKLDFNLLQKLHQKELSDISAWWKDLDFAHKLPFARDRVVECYFWILGVYFEPQ 291KVLLNFAKLDFNLLQKMHQGELSHITRWWKELNFAKKLPFARDRVVECYFWILGVYFEPQ 289KALVKLFKLDFNLVQSLHKKELSEISKWWKELDLANKLPFARDRLVECYFWIIGVYYEPQ 290KTLLKLAKLDFNLVQSLHKEELSNLARWWKELDFATKLPFARDRFVEGYFWTLGVYFEPQ 290KTLLNFAKLDFNILLELHKEELNELTKWWKDLDFTTKLPYARDRLVELYFWDLGTYFEPQ 282KTLLNFAKLDFNILLELHKEELNELTKWWKDLDFTTKLPYARDRLVELYFWDLGTYFEPQ 282EALLKFAKLDFNLLQVLHKQELSEMCRWWKKVNMTKKMEL-RDRMVESYFWGSAVYYEPQ 295

FFLARRILTKVITMTSTIDDIYDVYGTLEELELFTEAVERWDISVIDQLPEYMRVCYRAL 351YLIARRFLTKIIAMASVADDIYDVYGTLEELVILTDAIERWDMGALDQIPECMRVYHRAL 349YSLARKILTKIIAMASIIDDIYDVYGTREELNLFTDAIERWDASCMDLLPEYMQIFYEAL 350YSRARRILTKLFAMASIIDDIYDAYGTLEELQPFTEAIERWDIKSIDHLPEYMKLFYVTL 350YAFGRKIMTQLNYILSIIDDIYDAYGTLEELSLFTEAVQRWNIEAVDMLPEYMKLIYRTL 342YAFGRKIMTQLNYILSIIDDIYDAYGTLEELSLFTEAVQRWNIEAVDMLPEYMKLIYRTL 342FSLARKCNVPGCQILTTLDDIYDAYGTLEELHTFTDAVDKWDKSCLDQLPGDLRWTYQTL 355

-LDVYSEIEEEMAKEGR-SYRFYYAKEAMKKQVRAYYEEAQWLQAQQIPTMEEYMPVASA 409-LDVYTEMEEEMAKTGRPSYRVHYAKEAYKELVRQYLAEAKWFQEDYDPTLEEYLPVALI 408-LDLYNEIEKEIAKEGW-SYRVHYAKEAMKILVRGYHDESKWFHNNYIPTMEEYMRVALV 408-LDLYKEIDQELEKYGN-QYRVYYAKEVLKSQVRAYFAEAKWSHEGYIPTIEEYMLVALV 408-LDAFNEIEEDMAKQGR-SHCVRYAKEENQKVIGAYSVQAKWFSEGYVPTIEEYMPIALT 400-LDAFNEIEEDMAKQGR-SHCVRYAKEENQKVIGAYSVQAKWFSEGYVPTIEEYMPIALT 400ILDAHEEMERDFAPKGG-SRYVYYAREELKALCHSYLTEAKWRHEKYIPTLDEYMEFVHR 414

RXR

DDXXD

VvBCSEtACSTsSTSVvGASCsTPS1CsVSPmSTS







lowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowast lowast

lowast

lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast


lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast

lowastlowastlowastlowast lowast

(a)

Figure 2 Continued


TSGYPMLATTSFIAMGDVVTKETFDWVFSEPKIVRASATVSRLMDDMVSHKFEQKRGHVA 469SGGYKMLATHSFVGMGDLATKEAFDWVSNNPLIVKASSVICRLSDDMVGHEVEHERGDVA 468TSAYTMLTTSSFLGMDNIVTKETFDWVFSGPKIIRASETISRLMDDVKSHKFEQERGHAA 468TSGSCILATWSFIGMGEIMTKEAFDWVISDPKIITASTVIFRLMDDITTHKFEQKRGHVA 468SCAYTFVITNSFLGMGDFATKEVFEWISNNPKVVKAASVICRLMDDMQGHEFEQKRGHVA 460SCAYTFVITNSFLGMGDFATKEVFEWISNNPKVVKAASVICRLMDDMQGHEFEQKRGHVA 460NMAYVPAVVYSFLGM-DMATEAEFKWVSSLPKPIKSLCVLLRILNDIGGNMLVENREHPC 473

SAVECYMKQHGASEQETRDEFKKQVRDAWKDINQECLM-PTAVPMTVLMRILNLARVMDV 528SAVECYMKQYGVTKQEVYIEFQKQISNAWKDMNQECLH-PTTVTMPLLTVIFNMTRVINL 527SAVECYMEQHGVSEQEVCKEFYQQVGNAWKDINQDFLK-PTDVPMTILMRVLNLARVIDV 527SGIECYMKQYGVSEEQVYSEFHKQVENAWLDINQECLK-PTAVPMPLLTRVVNLSRVMDV 527

520SAIECYTKQHGVSKEEAIKMFEEEVANAWKDINEELMMKPTVVARPLLGTILNLARAIDF


LILECYKKQYGLSHEGACKRLIEEVDELWKDLNEAMMQ-PTEIPMPLVKCILNLARAVEA 532

VYKH-EDGYTHSGTFLKDLVTSLLIDSVPI 557LYDE-EDGYTNSNTRTKDFITSVLIDPVQI 556VYKE-GDGYTHVGKVMKENVASLLIDPIPV 556IYKE-GDGYTHVGKVMKDNIGSVLIDPIV- 555IYKE-DDGYTHS-YLIKDQIASVLGDHVPF 548IYKE-DDGYTHS-YLIKDQIASVLGDHVPF 548

562IYDAGEDGFTTVNRPMKDNIRSLYIDPILI

NSEDTE




lowast

lowast


lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowastlowastlowastlowastlowast lowast

(b)

Figure 2 Multiple sequence alignment The deduced amino acid sequence of PmSTS was aligned with homologues identified from theBLASTX analysis Sequences highlighted in black indicate identical residues while those in grey indicate similar residues The conservedmotifs RXR DDXXD and NSEDTE are marked PmSTS sesquiterpene synthase [P minus] TsSTS sesquiterpene synthase [Toona sinensis]CsTPS1 terpene synthase 1 [Citrus sinensis]CsVS valencene synthase [Citrus sinensis]VvGAS germacreneA synthase [Vitis vinifera]VvBCS(E-) beta-caryophyllene synthase [Vitis vinifera] EtACS alpha-copaene synthase [Eleutherococcus trifoliatus]

DDXXDmotif

NSEDTEmotif

Figure 3 Predicted 3D model of PmSTS generated by the I-TASSER server The two conserved domains are highlighted The N-terminaldomain is in blue and the C-terminal domain is in green Both aspartate-rich motifs are displayed using ball and stick representation withCPK colour The wire bonds represent the rest of the protein

expressed in plants and the ability of some sesquiterpene syn-thases to form multiple products from a single FPP substrate[19] The PmSTS gene has an open reading frame of 1689 bpwhich encodes a 562 amino acid protein with a calculatedmolecular mass of 651 kDa and a theoretical isoelectric point(pI) of 529 The deduced amino acid sequence of PmSTSshowed no signal peptide which is consistent with other

sesquiterpene synthases of 550ndash580 amino acids and isshorter than monoterpene synthases (600ndash650 amino acids)The absence of an N-terminal plastid targeting signal peptidesuggests that PmSTS is localized to the cytosol where FPP isfound and where sesquiterpene biosynthesis takes place

Based on the BLASTX analysis (Table 1) the closesthomologue to PmSTS is the sesquiterpene synthase from


Limonene synthase [Lavandula angustifolia]

Linalool synthase [Lavandula latifolia]

Alpha-farnesene synthase [Pyrus communis]

Pinene synthase [Rosmarinus officinalis]Cineole synthase [Salvia fruticosa]

(+)-A-terpineol synthase [Vitis vinifera]Pinene synthase [Quercus ilex]

(minus)-Limonene synthase [Cannabis sativa](+)-Limonene synthase 1 [Citrus limon]

(E E)-Alpha-farnesene synthase [Malus x domestica]

Linalool synthase [Mentha aquatica]

Linalool synthase [Arabidopsis lyrata subsp lyrata]

Linalool synthase [Arabidopsis thaliana]

(3S)-Nerolidol(3S)-linalool synthase [Fragaria x ananassa]Nerolidollinalool synthase 1 [Antirrhinum majus]

Myrcene synthase [Antirrhinum majus](E)-B-ocimene synthase [Antirrhinum majus]

Alpha-humulene synthase [Zingiber zerumbet]Germacrene D synthase [Zingiber officinale]

(E)-Beta-farnesene synthase [Mentha x piperita]

PmSTSGermacrene D synthase [Solidago canadensis]

(minus)-Germacrene D synthase [Populus trichocarpa x Populus deltoides](minus)-Germacrene D synthase [Vitis vinifera]

Germacrene C synthase [Solanum lycopersicum]

(+)-Delta-cadinene synthase [Gossypium hirsutum]E E-Alpha-farnesene synthase [Picea abies]

Alpha-farnesene synthase [Pinus taeda]

(minus)-Linalool synthase [Picea abies](+)-Alpha-pinene synthase [Pinus taeda]

(minus)-Camphene synthase [Abies grandis](minus)-Limonene synthase [Picea sitchensis]

Linalool synthase-like protein [Oenothera californica subsp arizonica]

Linalool synthase 2 [Clarkia breweri]

Linalool synthase [Clarkia concinna]Ent-kaurene synthase [Arabidopsis thaliana]

Ent-kaurene synthase [Populus trichocarpa]

Ent-kaurene synthase B [Cucurbita maxima]Ent-copalyl diphosphate synthase [Arabidopsis thaliana]

Copalyl diphosphate synthase [Populus trichocarpa]

Ent-copalyl diphosphate synthase [Zea mays]

02

Angiosperm monoterpene

synthases (Tps-b)

synthase cluster

Angiosperm acyclic

monoterpene synthases (Tps-g)

Angiosperm sesquiterpene

synthases (Tps-a)

Gymnosperm terpene

synthases (Tps-d)

Linalool synthases (Tps-f)

Kaurene synthases (Tps-e)

Cdp synthases (Tps-c)

100100

7097

53

9995

6497

100

100

65

96

10063

67 100100

100

100

100

33

3599

99

8245

10062

100

4122

100

994949

100

81

100

88

Linalool synthase [Actinidia polygama]

120572-farnesene

Figure 4 Phylogenetic tree of PmSTSwith selected terpene synthases fromother plants Seven previously identifiedTPS subfamilies (Tps-a toTps-g) were chosen based on Bohlmann et al [15] and Danner et al [16]The Tps-c and Tps-e subfamilies which are composed of the copalyldiphosphate (cdp) synthases and kaurene synthases and are involved in primary metabolism were chosen as outgroups The alignment wasperformed using the Clustal Omega algorithmThe tree was built using the neighbour joining method and 1000 replicates for bootstrappingThe numbers indicated are the actual bootstrap values of the branches


Table 1 BLASTX analysis PmSTS was compared with the NCBIprotein database for gene identification purposes

Descriptiona Organism 119864 value Identity()

Sesquiterpene synthase Toona sinensis 5119890minus147 43Terpene synthase 1 Citrus sinensis 2119890minus135 42Valencene synthase Citrus sinensis 2119890minus133 42Germacrene A synthase Vitis vinifera 4119890minus141 42(119864)-Beta-caryophyllenesynthase Vitis vinifera 2119890minus136 42

Alpha-copaene synthase Eleutherococcustrifoliatus 2119890minus136 42

Germacrene D synthase Actinidia deliciosa 6119890minus135 41

Germacrene B synthase Cistus creticus subspcreticus 8119890minus130 41

(+)-Delta-cadinenesynthase Gossypium arboreum 4119890minus136 41

Terpene synthase Camellia sinensis 6119890minus137 41Beta-curcumenesynthase Vitis vinifera 2119890minus130 41aDescriptionmdashhomology search using BLASTX

Toona sinensis with which it shares 43 identity Althoughthe level of amino acid sequence similarity between PmSTSand the other homologueswas relatively low (le43)multiplesequence alignment identified several conserved motifs thatare found in typical terpene synthases (Figure 2) The twohighly conserved aspartate-rich motifs DDXXD (residues314ndash318) and NSEDTE (residues 465ndash473) which are foundin most of the sesquiterpene synthases were highlighted inFigure 2 together with the other commonly conserved RXRmotif (residues 277ndash279) regionTheDDXXD and NSEDTEmotifs have been reported to flank the entrance of the activesite [20] They are involved in binding a trinuclear magne-sium cluster with DDXXD binding twomagnesium ions andNSEDTE binding one magnesium ion [21] Catalysis of theFPP substrate occurs when it reaches the hydrophobic activesite where the diphosphate moiety of FPP interacts with themagnesium ions [22]Thus this magnesium cluster is impor-tant for the positioning of FPP in the hydrophobic substratebinding pocket of PmSTS [23]

Similar to other sesquiterpene synthases PmSTS containstwo large conserved domains which were identified in aPFAM search PF01397 corresponds to the terpene synthasefamily N-terminal domain and PF03936 corresponds to theterpene synthase family C-terminal metal-binding domainThese domains are shown in the 3D model built using I-TASSER in Figure 3 The 3D protein model was constructedusing 5-epi-aristolochene synthase (TEAS) [PDB accession1HXG] as a template The quality of the PmSTS 3D proteinmodel was checked using a Ramachandran plot analysis [24]The PmSTS 3D protein model exhibited a good fit with thereference geometry with 904 of nonglycine and nonprolineresidues in the most favoured regions From the 3D modelPmSTS was shown to consist entirely of 120572-helices and short

05 cm

Figure 5 Comparison of hygromycin-sensitive and hygromycin-resistant (shown in arrow) seedlings The seedlings were screenedfor 20 days on selection plate containing MS media supplementedwith 25mg Lminus1 hygromycin

connecting loops and turns Both of the conserved aspartate-rich regions the DDXXD and NSEDTE motifs were foundin the C-terminal domain demonstrating the importance ofthe C-terminal domain which contains the active site forthe catalysis of the substrate FPP [25] While the actual func-tion of the N-terminal domain remains unknown it has beensuggested to be involved in facilitating the proper folding ofthe catalytically active C-terminal domain [26] Phylogeneticanalysis of the deduced amino acid sequence of PmSTSshowed that it belongs to the Tps-a subfamily of angiospermsesquiterpene synthases (Figure 4) [15 16]

The role and product specificity of PmSTS weredetermined by generating transgenic A thalianaOverexpression of PmSTS in A thaliana was accomplishedusing Agrobacterium harbouring the transformation vectorpCAMSS Using the Agrobacterium-mediated floral-diptransformation method ten hygromycin-resistant transgenicA thaliana were successfully generated These plants hadlong hypocotyls green leaves and long main roots withthe formation of lateral roots (Figure 5) In contrast thenontransformants showed short hypocotyls bleached outleaves and no lateral root formation (Figure 5) The putativetransformants were further verified using PCR amplificationof the plant genomic DNA Fully mature leaves from ten60-day-old putative transgenic plants and one wild typeplant were collected for DNA extraction All the putativetransformants gave rise to a band of the expected size of365 bp while the wild type plant showed no amplificationThis result confirmed the presence of the PmSTS gene in thegenomes of the transgenic plants

Two of the transgenic plants designated as OE3 andOE7were selected for further analysis Semiquantitative RT-PCRanalysis was performed Both theOE3 andOE7 plants showedhigh expression of the PmSTS gene by the amplificationof a distinct band at 100 bp which was absent in the wildtype plants (Figure 6) Meanwhile from the morphologicalanalysis both plants showed delayed growth compared to the


Sesquiterpene synthase

4HPPD control gene

1000 bp

500bp 500bp

100 bp

M WT1WT2 OE3 OE7 N M WT1WT2 OE3 OE7 N

Figure 6 Semiquantitative RT-PCR for sesquiterpene synthase (PmSTS) PCR for the 4HPPD gene was performed in parallel as a positivecontrol M 100 bp DNA ladder WT1 and WT2 wild type A thaliana plants OE3 OE3 transgenic plant OE7 OE7 transgenic plant N notemplate control

OE3

OE7

Transgenic Wild type

(a)

OE3 OE7 Wild typeWild type

(b)

Figure 7 Comparison of the phenotypes of the transgenic A thaliana and wild type A thaliana (a) Plants (2-month-old) were grown onsoil for 1 month Upper panel OE3 transgenic plant and wild type plant Lower panel OE7 transgenic plant and wild type plant (b) Two4-month-old OE3 and OE7 T

2plants compared with a 3-month-old wild type plant

wild type plants (Figure 7) The OE3 and OE7 plants took anadditionalmonth to reach the seedmaturation stepThis phe-notype was inheritable as the T

2plants of these two lines also

showed similar growth retardation The plants also demon-strated smaller basal rosette leaves and shorter and fewerflowering stems Although flowers and viable seeds werestill produced from these plants the number of seedsobtained was halved compared to the wild type plants

It has been suggested that the overexpression of thePmSTS gene in transgenic plants interferes with the IPPsubstrate pool in the cytosol (Figure 8) This overexpressioncauses the channelling of more isopentyl pyrophosphate(IPP) the building block for terpenes and FPP to the over-expressed PmSTSThis certainly lowers the flux of IPP to theplastids for the synthesis of other essential and larger isopreneproducts that are important for the plant growth such asgibberellins (GA) [27 28] This model was further sup-ported when many of the gene modifications involvingterpenoid biosynthesis such as the overexpression of thestrawberry linaloolnerolidol synthase (monoterpene) andtaxadiene synthase in A thaliana also resulted in a dwarfphenotype due to a decrease in the level of GA [27 29]The GA-deficient A thaliana mutants designated as dwarf

and delayed-flowering 1 (ddf1) also demonstrated a similarreduction in plant size and other similar phenotypes [30]

A GC-MS analysis was performed to identify the specificproduct produced by transformation with the PmSTS geneIn this analysis material extracted from the A thalianaleaf samples was examined using the HS-SPME method Byusing leaf samples we were able to reduce the detection ofbackground terpenes as A thaliana leaves were previouslyreported to not emit or to only emit traces of terpenevolatiles [33] In addition GC-MS analysis was performedwith wild type plants as a control The GC-MS analysisyielded two chromatograms with similar patterns (Figure 9)However a very clear difference was observed for the trans-genic plant OE3 as an additional peak was present at theretention time of 20834 This peak was identified as 120573-sesquiphellandrene based on the closest hit from a search ofthe NISTEPANIH library (version 20)Themass spectrumof the 120573-sesquiphellandrene peak compared with that of thehighest hit from the library was shown in Figure 10 Theproduction of 120573-sesquiphellandrene by PmSTS was in agree-ment with the findings from Song et al [12] This result alsoshowed that the point mutation introduced by Song et al[12] at K266E does not affect the product specificity of


Cytosolmevalonate

pathway (MVA) Acetyl CoA

HMG-CoA

MVA

IPPDMAPP

2x

2x

FPP (C15)

Squalene (C30) Sesquiterpenes (C15)

Cholesterolbrassinosteroids

+

+

+

minus

minus

minus

minus

DXR

MEP

IPP DMAPP (C5)

GPP (C10)2x IPP

Monoterpenes (C10)

GGPP (C20) Phytolchlorophylls

Carotenoids

PlastidMEP pathway

Pyruvate + GA3P

Diterpenes (C20)gibberellins

Figure 8 A simplified version of the sesquiterpene biosynthetic pathwayThis pathway shows the predicted flux of substrate (IPP and FPP) toproduce more sesquiterpenes (C

15) in transgenic plants overexpressing the PmSTS gene (+) indicates the increased flux of substrate and (minus)

indicates the decreased flux of substrate GA3P glyceraldehyde 3-phosphate HMG-CoA 3-hydroxy-3-methylglutaryl-CoA DXR 1-deoxy-D-xylulose 5-phosphate reductoisomerase MVA mevalonate acid MEP methyl erythritol phosphate IPP isopentyl pyrophosphate and itsisomer DMAPP dimethylallyl diphosphate FPP farnesyl diphosphate GGPP geranylgeranyl diphosphate (modified from Okada et al [31]and Vickers et al [32])

Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)

Wild type Arabidopsis leaftimes105

(a)

20834

OE3 Arabidopsis leaf

Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)

times105

(b)

Figure 9 GC-MS chromatograms of the transgenic OE3 (b) and a wild type plant (a) The arrow (darr) indicates the peak at the retention timeof 20834 that shows high match with 120573-sesquiphellandrene in the NISTEPANIH library

Abun

danc

e

5000

0

411

551

691

931

106

1 12011331

1471

1611

175

118

92 2042

2809

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz

(a)

Abun

danc

e

5000

0270

410

690

550930

1090 13301470

1610

1750 189

0

2040

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz

120573-Sesquiphellandrene

(b)

Figure 10Mass spectra of 120573-sesquiphellandrene (a) shows themass spectrum for the sample compound while (b) shows themass spectrumof the highest hit in the NISTEPANIH library


PmSTS in producing 120573-sesquiphellandrene Having obtainedthe same product in both L lactis and A thaliana wehave showed that PmSTS was responsible for the productionof 120573-sesquiphellandrene through the common mevalonatepathway in sesquiterpene biosynthesis

4 Conclusion

The diversity of the sesquiterpenes found in P minus rendersthis plant a major resource for research related to sesquiter-pene biosynthesis In this study we applied transgenic tech-nology by overexpressing PmSTS in A thaliana Growthretardation was clearly observed in the transgenic lines OE3and OE7 These two plants showed a high expression ofthe PmSTS gene which resulted in the production of 120573-sesquiphellandrene The 120573-sesquiphellandrene produced intheseA thaliana transgenic plants indicated the effectivenessof A thaliana in synthesizing the same product as in pre-viously expressed L lactis through the common mevalonatepathway of sesquiterpene biosynthesisThis research stronglysuggests the potential of the A thaliana plant system for thestudy of the sesquiterpene synthase genes in P minus

Conflict of Interests

All authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was funded by Universiti Kebangsaan Malaysiathrough University Research Grants (UKM-GUP-KPB-08-33-135) awarded to Professor Dr Zamri Zainal The prepara-tion of this paper was supported by Grant UKM-DLP-2013-004 funded by Universiti Kebangsaan Malaysia The authorsthank Ministry of Science Technology and Innovation inMalaysia for the National Science Fellowship (NSF) awardedto Su-Fang EeThey also thank Dr Md Imtiaz Uddin and DrNurul Hikma Md Isa for proofreading this paper

References

[1] J Zhao L C Davis and R Verpoorte ldquoElicitor signal trans-duction leading to production of plant secondary metabolitesrdquoBiotechnology Advances vol 23 no 4 pp 283ndash333 2005

[2] K Yonekura-Sakakibara and K Saito ldquoFunctional genomics forplant natural product biosynthesisrdquo Natural Product Reportsvol 26 no 11 pp 1466ndash1487 2009

[3] C Wiart Medicinal Plants of Asia and the Pacific CRC Press2006

[4] S N Baharum H Bunawan M A Ghani W A W Mustaphaand N M Noor ldquoAnalysis of the chemical composition of theessential oil of Polygonum minusHuds Using two-dimensionalgas chromatography-time-of-flight mass spectrometry (GC-TOF MS)rdquoMolecules vol 15 no 10 pp 7006ndash7015 2010

[5] A A Almey A Jalal S I Zahir K M Suleiman M R Aisyahand K Rahim ldquoTotal phenolic content and primary antioxidantactivity of methanolic and ethanolic extracts of aromatic plantsrsquo

leavesrdquo International Food Research Journal vol 17 no 4 pp1077ndash1084 2010

[6] SWQaderM A Abdulla S C Lee and S Hamdan ldquoPotentialbioactive property of Polygonum minus Huds (kesum) reviewrdquoScientific Research and Essays vol 7 pp 90ndash93 2012

[7] L Taiz and E Zeiger ldquoSecondary metabolites and plantdefenserdquo Plant Physiology vol 4 pp 315ndash344 2006

[8] B BuchananW Gruissem and R Jones Biochemistry ampMolec-ular Biology of Plants John Wiley amp Sons Rockville Md USA2002

[9] S F Ee J M Oh N Mohd Noor T R Kwon Z A Mohamed-Hussein et al ldquoTranscriptome profiling of genes induced bysalicylic acid and methyl jasmonate in Polygonum minusrdquoMolecular Biology Reports vol 40 pp 2231ndash2241 2013

[10] J Salinas and J J Sanchez-Serrano Arabidopsis ProtocolsHumana Press Totowa NJ USA 2006

[11] S J Clough and A F Bent ldquoFloral dip a simplified methodfor Agrobacterium-mediated transformation of Arabidopsisthalianardquo Plant Journal vol 16 no 6 pp 735ndash743 1998

[12] A A L Song J O Abdullah M P Abdullah N ShafeeR Othman et al ldquoOverexpressing 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGR) in the Lactococcal mevalonatepathway for heterologous plant sesquiterpene productionrdquo PloSONE vol 7 no 12 Article ID e52444 2012

[13] T N Petersen S Brunak G Von Heijne and H Nielsen ldquoSig-nalP 40 discriminating signal peptides from transmembraneregionsrdquo Nature Methods vol 8 no 10 pp 785ndash786 2011

[14] Y Zhang ldquoI-TASSER server for protein 3D structure predic-tionrdquo BMC Bioinformatics vol 9 article 40 2008

[15] J Bohlmann G Meyer-Gauen and R Croteau ldquoPlant ter-penoid synthasesmolecular biology and phylogenetic analysisrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 95 no 8 pp 4126ndash4133 1998

[16] H Danner G A Boeckler S Irmisch et al ldquoFour terpene syn-thases produce major compounds of the gypsy moth feeding-induced volatile blend of Populus trichocarpardquo Phytochemistryvol 72 no 9 pp 897ndash908 2011

[17] S J Harrison E K Mott K Parsley S Aspinall J C Gray etal ldquoA rapid and robust method of identifying transformedAra-bidopsis thaliana seedlings following floral dip transformationrdquoPlant Methods vol 2 p 19 2006

[18] J D Clarke ldquoCetyltrimethyl ammonium bromide (CTAB)DNA miniprep for plant DNA isolationrdquo Cold Spring HarborProtocols vol 4 no 3 2009

[19] T G Kollner P E OrsquoMaille N GattoW Boland J Gershenzonand J Degenhardt ldquoTwo pockets in the active site of maizesesquiterpene synthase TPS4 carry out sequential parts of thereaction scheme resulting in multiple productsrdquo Archives ofBiochemistry and Biophysics vol 448 no 1-2 pp 83ndash92 2006

[20] F Lopez-Gallego G T Wawrzyn and C Schmidt-DannertldquoSelectivity of fungal sesquiterpene synthases role of the activesitersquos H-1120572 loop in catalysisrdquo Applied and Environmental Micro-biology vol 76 no 23 pp 7723ndash7733 2010

[21] D W Christianson ldquoStructural biology and chemistry of theterpenoid cyclasesrdquo Chemical Reviews vol 106 no 8 pp 3412ndash3442 2006

[22] J Degenhardt T G Kollner and J Gershenzon ldquoMonoterpeneand sesquiterpene synthases and the origin of terpene skeletaldiversity in plantsrdquo Phytochemistry vol 70 no 15-16 pp 1621ndash1637 2009


[23] EMDavis andR Croteau ldquoCyclization enzymes in the biosyn-thesis of monoterpenes sesquiterpenes and diterpenesrdquo inBiosynthesis Aromatic Polyketides Isoprenoids Alkaloids J CVederas and F J Leeper Eds pp 53ndash95 Springer BerlinGermany 2000

[24] R A Laskowski M W MacArthur D S Moss and J MThornton ldquoPROCHECK a program to check the stereochemi-cal quality of protein structuresrdquo Journal of Applied Crystallog-raphy vol 26 pp 283ndash291 1993

[25] T G Kollner M Held C Lenk et al ldquoA maize (E)-120573-caryophyllene synthase implicated in indirect defense responsesagainst herbivores is not expressed in most American maizevarietiesrdquo Plant Cell vol 20 no 2 pp 482ndash494 2008

[26] T G Kollner C Schnee J Gershenzon and J Degenhardt ldquoThevariability of sesquiterpenes emitted from two Zea mays culti-vars is controlled by allelic variation of two terpene synthasegenes encoding stereoselective multiple product enzymesrdquoPlant Cell vol 16 no 5 pp 1115ndash1131 2004

[27] A Aharoni A P Giri S Deuerlein et al ldquoTerpenoidMetabolism in Wild-Type and Transgenic Arabidopsis PlantsrdquoPlant Cell vol 15 no 12 pp 2866ndash2884 2003

[28] J D Newman and J Chappell ldquoIsoprenoid biosynthesis inplants carbon partitioning within the cytoplasmic pathwayrdquoCritical Reviews in Biochemistry and Molecular Biology vol 34no 2 pp 95ndash106 1999

[29] O Besumbes S Sauret-Gueto M A Phillips S Imperial MRodrıguez-Concepcion and A Boronat ldquoMetabolic engineer-ing of isoprenoid biosynthesis inArabidopsis for the productionof taxadiene the first committed precursor of taxolrdquo Biotechnol-ogy and Bioengineering vol 88 no 2 pp 168ndash175 2004

[30] H Magome S Yamaguchi A Hanada Y Kamiya and K OdaldquoDwarf and delayed-flowering 1 a novel Arabidopsis mutantdeficient in gibberellin biosynthesis because of overexpressionof a putative AP2 transcription factorrdquo Plant Journal vol 37 no5 pp 720ndash729 2004

[31] K Okada H Kasahara S Yamaguchi et al ldquoGenetic evidencefor the role of isopentenyl diphosphate isomerases in themevalonate pathway and plant development in ArabidopsisrdquoPlant and Cell Physiology vol 49 no 4 pp 604ndash616 2008

[32] C E Vickers J GershenzonM T Lerdau and F Loreto ldquoA uni-fiedmechanismof action for volatile isoprenoids in plant abioticstressrdquo Nature Chemical Biology vol 5 no 5 pp 283ndash2912009

[33] M Huang C Abel R Sohrabi et al ldquoVariation of herbivore-induced volatile terpenes among Arabidopsis ecotypes dependson allelic differences and subcellular targeting of two terpenesynthases TPS02 and TPS03rdquo Plant Physiology vol 153 no 3pp 1293ndash1310 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


understanding the biosynthetic regulation of sesquiterpenerelated genes are still lacking

Based on our previously constructed cDNA-AFLP tran-scriptome profiles a sesquiterpene synthase gene (GenBankHO079100 and HO079108) was shown to be highly upregu-lated upon stress induced by salicylic acid [9] Although tran-scriptomic data are available the major challenge for studiesof P minus is the lack of a transformation and regenerationsystem for the functional study of its genes To address thisconcern the model plant A thaliana was used in this workA thaliana is the first angiosperm to have had its completegenome sequenced Since then it has been well studiedIts short life cycle of only 3 months and its ability to producea large number of progeny seeds have encouraged manyresearchers to use A thaliana for functional studies [10]The well-established floral-dip transformation system alsoprovides a fast and efficient method to transfer genes fromAgrobacterium into A thaliana [11] This method involvesonly a simple immersion of the floral buds in an Agrobac-terium suspension

PmSTS was previously cloned and expressed in Lactococ-cus lactis [12] That study was aimed solely at maximizingmetabolite production in a bacterial system and did notstudy how the gene is regulated in plants In addition thePmSTS gene introduced into L lactis contained two incorrectnucleotides that resulted in a single amino acid change [12]calling into question the true function of this protein

In this study we expressed the correct sequence of PmSTSto determine the function of its protein product in a modelplant system As there is no established transformation andregeneration system available in P minus the model plant Athaliana was used to investigate the role of PmSTS in plantsThe overexpression of PmSTS inA thaliana provides a betterunderstanding of the gene function The results of this studyfurther enhance our understanding of the biosynthesis andregulation of secondary metabolites in P minus particularlythe sesquiterpenes

2 Materials and Methods

21 Plant Material and Growth Conditions A thaliana eco-type Columbia-0was grown in a growth chamber (Conviron)at a temperature of 22∘Cday20∘Cnight and relative humidityof 50ndash70Thephotoperiodwas set at 16 h day8 hnight witha light intensity of 100ndash150 120583molesmminus2 sminus1 using fluorescentbulbsTheplants were ready for floral-dip transformation oneweek after the primary inflorescences were clipped Wateringwas stopped three days prior to transformation to increase thetransformation efficiency

22 In Silico Analysis of PmSTS The nucleotide sequenceof PmSTS was retrieved from the NCBI database withGenBank ID of JX025008 The physiochemical properties ofthe PmSTS were determined using PROTPARAM software(httpwebexpasyorgprotparam) The presence of signalpeptide was predicted using SignalP 41 software(httpwwwcbsdtudkservicesSignalP) [13] Comparativesequence analysis of PmSTS was performed using NCBI

BLAST against the protein database (httpblastncbinlmnihgov) Multiple sequence alignment was done withBIOEDIT software using the default parameters (httpwwwmbioncsuedubioeditbioedithtml) The three-dimen-sional (3D) protein structure homology-modelling ofthe PmSTS was generated using I-TASSER software [14]The stereochemical quality of the predicted 3D proteinstructure was examined through PROCHECK analysis(httpwwwebiacukthornton-srvsoftwarePROCHECK)Phylogenetic tree was built using MEGA5 software withneighbour joining method Bootstrap of 1000 replicates wasdone Terpene synthases from seven previously recognizedTPS subfamilies Tps-a to Tps-g were retrieved from the NCBIGenBank database according to Bohlmann et al [15] andDanner et al [16] The Tps-c and Tps-e subfamilies whichare composed of the copalyl diphosphate (cdp) synthases andkaurene synthases and are involved in primary metabolismwere chosen as outgroups

23 Gene Amplification and Construction of the pCAMSSoverexpression Vector PmSTS (GenBank JX025008)was amplified by standard PCR methods using thePmSTS specific forward primer 51015840-GGGCAGATCTTATGTATTCCATGATC-31015840 and reverseprimer 51015840-GGCTGGTGACCTTATATCAGTATGGG-31015840 Tofacilitate the cloning process restriction enzymes (RE) sitesfor BglII and BstEII (underlined) were attached to the 51015840ends of the forward and reverse primers respectively Thenucleotides in bold type are the start (ATG) and termination(TTA) codons in the open reading frame of the PmSTS gene

The vector pCAMBIA1301 (Centre for the Applicationof Molecular Biology of International Agriculture BlackMountain Australia) was used as the backbone for the con-struction of the plant transformation vector pCAMSS whichharboured thePmSTS gene To construct the pCAMSS vectorthe 120573-glucuronidase (GUS) reporter gene was first excisedfrom the pCAMBIA1301 vector and then replaced with thePCR-amplified PmSTS gene Both the pCAMBIA1301 vectorand the PmSTS gene were digested with the BglII andBstEII restriction enzymes to generate complementary stickyends for ligation The digested fragment of the PmSTS genewas ligated into the corresponding sites of pCAMBIA1301yielding the pCAMSS vector (Figure 1) The pCAMSS vectorwas then transformed into Agrobacterium tumefaciens strainGV3101 The cloned pCAMSS vector was sent for sequencingand RE digestion to confirm the integration of the PmSTSgene in the correct orientation

24 Agrobacterium-Mediated Floral-Dip Plant Transforma-tion A thaliana was transformed using the Agrobacterium-mediated floral dip method [11] Agrobacterium cells weregrown to an OD

600of 07 The floral-dip inoculation medium

contained harvested cells that were resuspended in 5sucrose and 005 Silwet The secondary inflorescences wereimmersed in the inoculation medium and swirled gently toallow the intake of Agrobacterium harbouring the pCAMSSvector into the flower gynoecium The transformed plantswere kept in the dark and wrapped with plastic overnight to



polyALacZ alpha


T border (L)

T border (R)



Nos polyA






2transgenicA




















RXR

DDXXD








lowastlowast


lowast

lowast


lowast






lowast


(a)

Figure 2 Continued









NSEDTE




lowast

lowast




(b)


DDXXDmotif

NSEDTEmotif


































02


synthases (Tps-b)

synthase cluster

Angiosperm acyclic



synthases (Tps-a)

Gymnosperm terpene

synthases (Tps-d)




100100

7097

53

9995

6497

100

100

65

96

10063

67 100100

100

100

100

33

3599

99

8245

10062

100

4122

100

994949

100

81

100

88


120572-farnesene













05 cm







4HPPD control gene

1000 bp

500bp 500bp

100 bp



OE3

OE7


(a)


(b)










Cytosolmevalonate


HMG-CoA

MVA

IPPDMAPP

2x

2x

FPP (C15)



+

+

+

minus

minus

minus

minus

DXR

MEP

IPP DMAPP (C5)

GPP (C10)2x IPP

Monoterpenes (C10)


Carotenoids

PlastidMEP pathway

Pyruvate + GA3P





Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)


(a)

20834


Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)

times105

(b)


Abun

danc

e

5000

0

411

551

691

931

106

1 12011331

1471

1611

175

118

92 2042

2809

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz

(a)

Abun

danc

e

5000

0270

410

690

550930

1090 13301470

1610

1750 189

0

2040

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz


(b)




4 Conclusion




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



polyALacZ alpha


T border (L)

T border (R)



Nos polyA






2transgenicA




















RXR

DDXXD








lowastlowast


lowast

lowast


lowast






lowast


(a)

Figure 2 Continued









NSEDTE




lowast

lowast




(b)


DDXXDmotif

NSEDTEmotif


































02


synthases (Tps-b)

synthase cluster

Angiosperm acyclic



synthases (Tps-a)

Gymnosperm terpene

synthases (Tps-d)




100100

7097

53

9995

6497

100

100

65

96

10063

67 100100

100

100

100

33

3599

99

8245

10062

100

4122

100

994949

100

81

100

88


120572-farnesene













05 cm







4HPPD control gene

1000 bp

500bp 500bp

100 bp



OE3

OE7


(a)


(b)










Cytosolmevalonate


HMG-CoA

MVA

IPPDMAPP

2x

2x

FPP (C15)



+

+

+

minus

minus

minus

minus

DXR

MEP

IPP DMAPP (C5)

GPP (C10)2x IPP

Monoterpenes (C10)


Carotenoids

PlastidMEP pathway

Pyruvate + GA3P





Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)


(a)

20834


Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)

times105

(b)


Abun

danc

e

5000

0

411

551

691

931

106

1 12011331

1471

1611

175

118

92 2042

2809

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz

(a)

Abun

danc

e

5000

0270

410

690

550930

1090 13301470

1610

1750 189

0

2040

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz


(b)




4 Conclusion




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















RXR

DDXXD








lowastlowast


lowast

lowast


lowast






lowast


(a)

Figure 2 Continued









NSEDTE




lowast

lowast




(b)


DDXXDmotif

NSEDTEmotif


































02


synthases (Tps-b)

synthase cluster

Angiosperm acyclic



synthases (Tps-a)

Gymnosperm terpene

synthases (Tps-d)




100100

7097

53

9995

6497

100

100

65

96

10063

67 100100

100

100

100

33

3599

99

8245

10062

100

4122

100

994949

100

81

100

88


120572-farnesene













05 cm







4HPPD control gene

1000 bp

500bp 500bp

100 bp



OE3

OE7


(a)


(b)










Cytosolmevalonate


HMG-CoA

MVA

IPPDMAPP

2x

2x

FPP (C15)



+

+

+

minus

minus

minus

minus

DXR

MEP

IPP DMAPP (C5)

GPP (C10)2x IPP

Monoterpenes (C10)


Carotenoids

PlastidMEP pathway

Pyruvate + GA3P





Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)


(a)

20834


Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)

times105

(b)


Abun

danc

e

5000

0

411

551

691

931

106

1 12011331

1471

1611

175

118

92 2042

2809

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz

(a)

Abun

danc

e

5000

0270

410

690

550930

1090 13301470

1610

1750 189

0

2040

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz


(b)




4 Conclusion




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









NSEDTE




lowast

lowast




(b)


DDXXDmotif

NSEDTEmotif


































02


synthases (Tps-b)

synthase cluster

Angiosperm acyclic



synthases (Tps-a)

Gymnosperm terpene

synthases (Tps-d)




100100

7097

53

9995

6497

100

100

65

96

10063

67 100100

100

100

100

33

3599

99

8245

10062

100

4122

100

994949

100

81

100

88


120572-farnesene













05 cm







4HPPD control gene

1000 bp

500bp 500bp

100 bp



OE3

OE7


(a)


(b)










Cytosolmevalonate


HMG-CoA

MVA

IPPDMAPP

2x

2x

FPP (C15)



+

+

+

minus

minus

minus

minus

DXR

MEP

IPP DMAPP (C5)

GPP (C10)2x IPP

Monoterpenes (C10)


Carotenoids

PlastidMEP pathway

Pyruvate + GA3P





Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)


(a)

20834


Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)

times105

(b)


Abun

danc

e

5000

0

411

551

691

931

106

1 12011331

1471

1611

175

118

92 2042

2809

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz

(a)

Abun

danc

e

5000

0270

410

690

550930

1090 13301470

1610

1750 189

0

2040

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz


(b)




4 Conclusion




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























02


synthases (Tps-b)

synthase cluster

Angiosperm acyclic



synthases (Tps-a)

Gymnosperm terpene

synthases (Tps-d)




100100

7097

53

9995

6497

100

100

65

96

10063

67 100100

100

100

100

33

3599

99

8245

10062

100

4122

100

994949

100

81

100

88


120572-farnesene













05 cm







4HPPD control gene

1000 bp

500bp 500bp

100 bp



OE3

OE7


(a)


(b)










Cytosolmevalonate


HMG-CoA

MVA

IPPDMAPP

2x

2x

FPP (C15)



+

+

+

minus

minus

minus

minus

DXR

MEP

IPP DMAPP (C5)

GPP (C10)2x IPP

Monoterpenes (C10)


Carotenoids

PlastidMEP pathway

Pyruvate + GA3P





Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)


(a)

20834


Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)

times105

(b)


Abun

danc

e

5000

0

411

551

691

931

106

1 12011331

1471

1611

175

118

92 2042

2809

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz

(a)

Abun

danc

e

5000

0270

410

690

550930

1090 13301470

1610

1750 189

0

2040

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz


(b)




4 Conclusion




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












05 cm







4HPPD control gene

1000 bp

500bp 500bp

100 bp



OE3

OE7


(a)


(b)










Cytosolmevalonate


HMG-CoA

MVA

IPPDMAPP

2x

2x

FPP (C15)



+

+

+

minus

minus

minus

minus

DXR

MEP

IPP DMAPP (C5)

GPP (C10)2x IPP

Monoterpenes (C10)


Carotenoids

PlastidMEP pathway

Pyruvate + GA3P





Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)


(a)

20834


Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)

times105

(b)


Abun

danc

e

5000

0

411

551

691

931

106

1 12011331

1471

1611

175

118

92 2042

2809

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz

(a)

Abun

danc

e

5000

0270

410

690

550930

1090 13301470

1610

1750 189

0

2040

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz


(b)




4 Conclusion




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4HPPD control gene

1000 bp

500bp 500bp

100 bp



OE3

OE7


(a)


(b)










Cytosolmevalonate


HMG-CoA

MVA

IPPDMAPP

2x

2x

FPP (C15)



+

+

+

minus

minus

minus

minus

DXR

MEP

IPP DMAPP (C5)

GPP (C10)2x IPP

Monoterpenes (C10)


Carotenoids

PlastidMEP pathway

Pyruvate + GA3P





Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)


(a)

20834


Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)

times105

(b)


Abun

danc

e

5000

0

411

551

691

931

106

1 12011331

1471

1611

175

118

92 2042

2809

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz

(a)

Abun

danc

e

5000

0270

410

690

550930

1090 13301470

1610

1750 189

0

2040

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz


(b)




4 Conclusion




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Cytosolmevalonate


HMG-CoA

MVA

IPPDMAPP

2x

2x

FPP (C15)



+

+

+

minus

minus

minus

minus

DXR

MEP

IPP DMAPP (C5)

GPP (C10)2x IPP

Monoterpenes (C10)


Carotenoids

PlastidMEP pathway

Pyruvate + GA3P





Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)


(a)

20834


Abun

danc

e

50

40

30

20

10

1500 1600 1700 1800 1900 2000 2100 2200 2300 2400

Time (min)

times105

(b)


Abun

danc

e

5000

0

411

551

691

931

106

1 12011331

1471

1611

175

118

92 2042

2809

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz

(a)

Abun

danc

e

5000

0270

410

690

550930

1090 13301470

1610

1750 189

0

2040

20 40 60 80 100 120 140 160 180 200 220 240 260 280

mz


(b)




4 Conclusion




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Conclusion




Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Functional Characterization of ...downloads.hindawi.com/journals/tswj/2014/840592.pdf · Research Article Functional Characterization of Sesquiterpene Synthase from