Abstract
Main conclusion
Using RNA-Seq, we cloned and characterized a unique monoterpene synthase responsible for the formation of a scent-determining S-linalool constituent of lavender oils from Lavandula × intermedia.
Several species of Lavandula produce essential oils (EOs) consisting mainly of monoterpenes including linalool, one of the most abundant and scent-determining oil constituents. Although R-linalool dominates the EOs of lavenders, varying amounts (depending on the species) of the S-linalool enantiomer can also be found in these plants. Despite its relatively low abundance, S-linalool contributes a sweet, pleasant scent and is an important constituent of lavender EOs. While several terpene synthase genes including R-linalool synthase have been cloned from lavenders many important terpene synthases including S-linalool synthase have not been described from these plants. In this study, we employed RNA-Seq and other complementary sequencing data to clone and functionally characterize the sparsely expressed S-linalool synthase cDNA (LiS-LINS) from Lavandula × intermedia. Recombinant LiS-LINS catalyzed the conversion of the universal monoterpene precursor geranyl diphosphate to S-linalool as the sole product. Intriguingly, LiS-LINS exhibited very low (~ 30%) sequence similarity to other Lavandula terpene synthases, including R-linalool synthase. However, the predicted 3D structure of this protein, including the composition and arrangement of amino acids at the active site, is highly homologous to known terpene synthase proteins. LiS-LINS transcripts were detected in flowers, but were much less abundant than those corresponding to LiR-LINS, paralleling enantiomeric composition of linalool in L. × intermedia oils. These data indicate that production of S-linalool is at least partially controlled at the level of transcription from LiS-LINS. The cloned LiS-LINS cDNA may be used to enhance oil composition in lavenders and other plants through metabolic engineering.
This is a preview of subscription content, log in via an institution to check access.
Abbreviations
- EOs:
-
Essential oils
- GPP:
-
Geranyl pyrophosphate
- mTPS:
-
Monoterpene synthase
- LiS-LINS:
-
L. × intermedia S-linalool synthase
- LiR-LINS:
-
L. × intermedia R-linalool synthase
- NPP:
-
Neryl pyrophosphate,
- qPCR:
-
Quantitative real-time PCR
- TPS:
-
Terpene synthase
References
Adal AM, Sarker LS, Lemke AD, Mahmoud SS (2017) Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula × intermedia. Plant Mol Biol. https://doi.org/10.1007/s11103-017-0588-6
Aharoni A, Giri AP, Verstappen FWA, Bertea CM, Sevenier R (2004) Gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species. Plant Cell 16:3110–3131. https://doi.org/10.1105/tpc.104.023895.native
Annadurai RS, Jayakumar V, Mugasimangalam RC, Katta MA, Anand S, Gopinathan S, Sarma SP, Fernandes SJ, Mullapudi N, Murugesan S, Rao SN (2012) Next generation sequencing and de novo transcriptome analysis of Costus pictus D. Don, a non-model plant with potent anti-diabetic properties. BMC Bioinf 13:1–15. https://doi.org/10.1186/1471-2164-13-663
Aprotosoaie AC, Hǎncianu M, Costache II, Miron A (2014) Linalool: a review on a key odorant molecule with valuable biological properties. Flavour Fragr J 29:193–219. https://doi.org/10.1002/ffj.3197
Aprotosoaie AC, Gille E, Trifan A, Luca VS, Miron A (2017) Essential oils of Lavandula genus: a systematic review of their chemistry. Phytochem Rev 16:761–799. https://doi.org/10.1007/s11101-017-9517-1
Baser KHC, Özek T, Konakchiev A (2005) Enantiomeric distribution of linalool, linalyl acetate and camphor in bulgarian lavender oil. J Essent Oil Res 17:135–136. https://doi.org/10.1080/10412905.2005.9698855
Benabdelkader T, Guitton Y, Pasquier B, Magnard JL, Jullien F, Kameli A, Legendre L (2015) Functional characterization of terpene synthases and chemotypic variation in three lavender species of section Stoechas. Physiol Plant 153:43–57. https://doi.org/10.1111/ppl.12241
Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA 95:4126–4133. https://doi.org/10.1073/pnas.95.8.4126
Cavanagh HMA, Wilkinson JM (2002) Biological activities of lavender essential oil. Phytother Res 16:301–308. https://doi.org/10.1002/ptr.1103
Chen F, Tholl D, D’Auria JC, Farooq A, Pichersky E, Gershenzonb J (2003) Biosynthesis and emission of terpenoid volatiles from arabidopsis flowers. Plant Cell 15:481–494. https://doi.org/10.1105/tpc.007989.2
Chen F, Tholl D, Bohlmann J, Pichersky E (2011) The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J 66:212–229. https://doi.org/10.1111/j.1365-313X.2011.04520.x
Christianson DW (2006) Structural biology and chemistry of the terpenoid cyclases. Chem Rev. https://doi.org/10.1021/cr050286w
Degenhardt J, Köllner TG, Gershenzon J (2009) Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 70:1621–1637. https://doi.org/10.1016/j.phytochem.2009.07.030
Demissie ZA, Sarker LS, Mahmoud SS (2011) Cloning and functional characterization of β-phellandrene synthase from Lavandula angustifolia. Planta 233:685–696. https://doi.org/10.1007/s00425-010-1332-5
Demissie ZA, Cella MA, Sarker LS, Thompson TJ, Rheault MR, Mahmoud SS (2012) Cloning, functional characterization and genomic organization of 1,8-cineole synthases from Lavandula. Plant Mol Biol 79:393–411. https://doi.org/10.1007/s11103-012-9920-3
Demissie ZA, Erland LAE, Rheault MR, Mahmoud SS (2013) The biosynthetic origin of irregular monoterpenes in Lavandula: isolation and biochemical characterization of a novel cis-prenyl diphosphate synthase gene, lavandulyl diphosphate synthase. J Biol Chem 288:6333–6341. https://doi.org/10.1074/jbc.M112.431171
Denner SS (2009) Lavandula angustifolia Miller: english lavender. Holist Nurs Pr 23:57–64. https://doi.org/10.1097/01.HNP.0000343210.56710.fc
Despinasse Y, Fiorucci S, Antonczak S, Moja S, Bony A, Nicolè F, Baudino S, Magnard JL, Jullien F (2017) Bornyl-diphosphate synthase from Lavandula angustifolia: a major monoterpene synthase involved in essential oil quality. Phytochemistry 137:24–33. https://doi.org/10.1016/j.phytochem.2017.01.015
Do TKT, Hadji-Minaglou F, Antoniotti S, Fernandez X (2015) Authenticity of essential oils. Trends Anal Chem 66:146–157. https://doi.org/10.1016/j.trac.2014.10.007
Dudareva N, Martin D, Kish CM, Kolosova N, Gorenstein N, Fäldt J, Miller B, Bohlmann J (2003) (E)-Beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily. Plant Cell 15:1227–1241. https://doi.org/10.1105/tpc.011015
Erland LAE, Mahmoud SS (2015) Lavender (Lavandula angustifolia) oils. Essent Oils Food Preserv Flavor Saf. https://doi.org/10.1016/B978-0-12-416641-7.00057-2
Flores G, Blanch GP, del Castillo MLR, Herraiz M (2005) Enantiomeric composition studies in Lavandula species using supercritical fluids. J Sep Sci 28:2333–2338. https://doi.org/10.1002/jssc.200500124
Galata M, Sarker LS, Mahmoud SS (2014) Transcriptome profiling, and cloning and characterization of the main monoterpene synthases of Coriandrum sativum L. Phytochemistry 102:64–73. https://doi.org/10.1016/j.phytochem.2014.02.016
Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellsten U, Putnam N, Rokhsar DS (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res 40:1178–1186. https://doi.org/10.1093/nar/gkr944
Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652. https://doi.org/10.1038/nbt.1883
Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, MacManes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, LeDuc RD, Friedman N, Regev A (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc. https://doi.org/10.1038/nprot.2013.084
Han Y, Gao S, Muegge K, Zhang W, Zhou B (2015) Advanced applications of RNA sequencing and challenges. Bioinform Biol Insights 9:29–46. https://doi.org/10.4137/BBI.S28991
Hrdlickova R, Toloue M, Tian B (2017) RNA-Seq methods for transcriptome analysis. Wiley Interdiscip Rev RNA. https://doi.org/10.1002/wrna.1364
Hu B, Jin J, Guo AY, Zhang H, Luo J, Gao G (2015) GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics 31:1296–1297. https://doi.org/10.1093/bioinformatics/btu817
Hyatt DC, Youn B, Zhao Y, Santhamma B, Coates RM, Croteau RB, Kang C (2007) Structure of limonene synthase, a simple model for terpenoid cyclase catalysis. Proc Natl Acad Sci USA 104:5360–5365. https://doi.org/10.1073/pnas.0700915104
Iriti M, Colnaghi G, Chemat F, Smadja J, Faoro F, Visinoni FA (2006) Histo-cytochemistry and scanning electron microscopy of lavander glandular trichomes following conventional and microwave-assisted hydrodistillation of essential oils: a comparative study. Flavour Fragr J 21:704–712. https://doi.org/10.1002/ffj.1692
Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C, McWilliam H, Maslen J, Mitchell A, Nuka G, Pesseat S, Quinn AF, Sangrador-Vegas A, Scheremetjew M, Yong SY, Lopez R, Hunter S (2014) InterProScan 5: genome-scale protein function classification. Bioinformatics 30:1236–1240. https://doi.org/10.1093/bioinformatics/btu031
Jullien F, Moja S, Bony A, Legrand S, Petit C, Benabdelkader T, Poirot K, Fiorucci S, Guitton Y, Nicolè F, Baudino S, Magnard JL (2014) Isolation and functional characterization of a τ-cadinol synthase, a new sesquiterpene synthase from Lavandula angustifolia. Plant Mol Biol 84:227–241. https://doi.org/10.1007/s11103-013-0131-3
Källberg M, Wang H, Wang S, Peng J, Wang Z, Lu H, Xu J (2012) Template-based protein structure modeling using the RaptorX web server. Nat Protoc 7:1511–1522. https://doi.org/10.1038/nprot.2012.085
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:msw054. https://doi.org/10.1093/molbev/msw054
Landmann C, Fink B, Festner M, Dregus M, Engel KH, Schwab W (2007) Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia). Arch Biochem Biophys 465:417–429. https://doi.org/10.1016/j.abb.2007.06.011
Lane A, Boecklemann A, Woronuk GN, Sarker L, Mahmoud SS (2010) A genomics resource for investigating regulation of essential oil production in Lavandula angustifolia. Planta 231:835–845. https://doi.org/10.1007/s00425-009-1090-4
Li W, Godzik A (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658–1659. https://doi.org/10.1093/bioinformatics/btl158
Li W, Liu B, Yu L, Feng D, Wang H, Wang J (2009) Phylogenetic analysis, structural evolution and functional divergence of the 12-oxo-phytodienoate acid reductase gene family in plants. BMC Evol Biol 9:1–19. https://doi.org/10.1186/1471-2148-9-90
Li W-Y, Wang X, Li R, Li W-Q, Chen K-M (2014) Genome-wide analysis of the NADK gene family in plants. PLoS ONE 9(6):e101051
Liao P, Hemmerlin A, Bach TJ, Chye ML (2016) The potential of the mevalonate pathway for enhanced isoprenoid production. Biotechnol Adv 34:697–713. https://doi.org/10.1016/j.biotechadv.2016.03.005
Lis-Balchin M (2002) Chemical composition of essential oils from different species, hybrids and cultivars of Lavandula. Lavender: the genus Lavandula. Taylor & Francis, London, pp 251–262
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−ΔΔCT) method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T (2017) Transcriptomics technologies. PLoS Comput Biol 13:1–23. https://doi.org/10.1371/journal.pcbi.1005457
Martin DM, Aubourg S, Schouwey MB, Daviet L, Schalk M, Toub O, Lund ST, Bohlmann J (2010) Functional annotation, genome organization and phylogeny of the grapevine (Vitis vinifera) terpene synthase gene family based on genome assembly, FLcDNA cloning, and enzyme assays. BMC Plant Biol 10:226. https://doi.org/10.1186/1471-2229-10-226
Mendoza-Poudereux I, Muñoz-Bertomeu J, Navarro A, Arrillaga I, Segura J (2014) Enhanced levels of S-linalool by metabolic engineering of the terpenoid pathway in spike lavender leaves. Metab Eng 23:136–144. https://doi.org/10.1016/j.ymben.2014.03.003
Mendoza-Poudereux I, Kutzner E, Huber C, Segura J, Eisenreich W, Arrillaga I (2015) Metabolic cross-talk between pathways of terpenoid backbone biosynthesis in spike lavender. Plant Physiol Biochem 95:113–120. https://doi.org/10.1016/j.plaphy.2015.07.029
Mi H, Huang X, Muruganujan A, Tang H, Mills C, Kang D, Thomas PD (2017) PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Res 45:D183–D189. https://doi.org/10.1093/nar/gkw1138
Nagegowda DA, Gutensohn M, Wilkerson CG, Dudareva N (2008) Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers. Plant J 55:224–239. https://doi.org/10.1111/j.1365-313x.2008.03496.x
Özek T, Tabanca N, Demirci F, Wedge DF, Hüsnü Can Başer K (2010) Enantiomeric distribution of some linalool containing essential oils and their biological activities. Rec Nat Prod 4:180–192
Pragadheesh VS, Chanotiya CS, Rastogi S, Shasany AK (2017) Scent from Jasminum grandiflorum flowers: investigation of the change in linalool enantiomers at various developmental stages using chemical and molecular methods. Phytochemistry 140:83–94. https://doi.org/10.1016/j.phytochem.2017.04.018
Prasath D, Karthika R, Habeeba NT, Suraby EJ, Rosana OB, Shaji A, Eapen SJ, Deshpande U, Anandaraj M (2014) Comparison of the transcriptomes of ginger (Zingiber officinale Rosc.) and mango ginger (Curcuma amada Roxb.) in response to the bacterial wilt infection. PLoS One. https://doi.org/10.1371/journal.pone.0099731
Rehman R, Hanif MA, Mustaq Z, Al-Sadi AM (2016) Biosynthesis of essential oils in aromatic plants: a review. Food Rev Int 32:117–160
Renaud ENC, Charles DJ, Simon JE (2001) Essential oil quantity and composition from 10 cultivars of organically grown lavender and lavandin. J Essent Oil Res 13:269–273. https://doi.org/10.1080/10412905.2001.9699691
Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, Hoff MJB, Den Van, Moorman AFM (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. https://doi.org/10.1093/nar/gkp045
Sarker LS, Mahmoud SS (2015) Cloning and functional characterization of two monoterpene acetyltransferases from glandular trichomes of L. × intermedia. Planta 242:709–719. https://doi.org/10.1007/s00425-015-2325-1
Sarker LS, Galata M, Demissie ZA, Mahmoud SS (2012) Molecular cloning and functional characterization of borneol dehydrogenase from the glandular trichomes of Lavandula × intermedia. Arch Biochem Biophys 528:163–170. https://doi.org/10.1016/j.abb.2012.09.013
Sarker LS, Demissie ZA, Mahmoud SS (2013) Cloning of a sesquiterpene synthase from Lavandula × intermedia glandular trichomes. Planta 238:983–989. https://doi.org/10.1007/s00425-013-1937-6
Satyal P, Pappas RS (2016) Antique lavender essential oil from 1945, its chemical composition and enantiomeric distribution. Nat Volat Essent Oils 3:20–25
Sievers F, Wilm A, Dineen D (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. https://doi.org/10.1038/msb.2011.75
Trapp SC, Croteau RB (2001) Genomic organization of plant terpene synthases and molecular evolutionary implications. Genetics 158:811–832
Turner GW, Gershenzon J, Croteau RB (2000) Development of peltate glandular trichomes of peppermint. Plant Physiol 124:665–679. https://doi.org/10.1104/pp.124.2.665
Vranová E, Coman D, Gruissem W (2012) Structure and dynamics of the isoprenoid pathway network. Mol Plant 5:318–333. https://doi.org/10.1093/mp/sss015
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63
Wang S, Ma J, Peng J, Xu J (2013) Protein structure alignment beyond spatial proximity. Sci Rep. https://doi.org/10.1038/srep01448
Whittington DA, Wise ML, Urbansky M, Coates RM, Croteau RB, Christianson DW (2002) Bornyl diphosphate synthase: structure and strategy for carbocation manipulation by a terpenoid cyclase. Proc Natl Acad Sci USA 99:15375–15380. https://doi.org/10.1073/pnas.232591099
Wiederstein M, Sippl MJ (2007) ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 35:407–410. https://doi.org/10.1093/nar/gkm290
Xu G, Guo C, Shan H, Kong H (2012) Divergence of duplicate genes in exon-intron structure. Proc Natl Acad Sci 109:1187–1192. https://doi.org/10.1073/pnas.1109047109
Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572. https://doi.org/10.1038/nprot.2007.199
Yue Y, Yu R, Fan Y (2015) Transcriptome profiling provides new insights into the formation of floral scent in Hedychium coronarium. BMC Genomics 16:470. https://doi.org/10.1186/s12864-015-1653-7
Zeng G (1998) Sticky-end PCR: new method for subcloning. Biotechniques 25:206–208
Zuzarte MR, Dinis AM, Cavaleiro C, Salgueiro LR, Canhoto JM (2010) Trichomes, essential oils and in vitro propagation of Lavandula pedunculata (Lamiaceae). Ind Crops Prod 32:580–587. https://doi.org/10.1016/j.indcrop.2010.07.010
Acknowledgements
This work was supported through Grants and/or in-kind contributions to SSM by UBC, and Natural Sciences and Engineering Research Council of Canada. We thank Dr I. Hwang, Pohang University of Science and Technology, Korea for kindly providing p326::sGFP construct DNA; Aaron Johnstone, UBC, Dr. Phillip Barker’s lab for assisting on inverted fluorescent microscopy and Joan Chisholm, Dr. Helene Sanfacon’s Lab, Summerland Agriculture and Agri-Food Canada for assisting on N. benthamiana protoplasts isolation and transfection.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflicts of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Appendix S1
: Annotated transcriptome sequences from Lavandula (XLSX 5066 kb)
Appendix S2:
KEGG pathway distribution using KO annotation (XLSX 19 kb)
Table S1
. TPSs clustered under TPS-g subfamily, and previously known from Lavandula, with the corresponding GenBank/ UniProt accession numbers that are incorporated in the phylogenetic analysis of LiS-LINS (DOCX 13 kb)
Table S2
. Species distribution of the top BLAST hits of Lavandula transcriptome unigenes. The transcriptomic sequences from Lavandula were highly homologous to Solanum lycopersicum (23.59 %) and Vitis vinifera (15.45 %) compared to transcriptome sequences from any of other plant species (< 8 %) (DOCX 12 kb)
Table S3
. Summary of the most common protein families and domains found in the Lavandula transcriptome database. Of the total protein classes and domains detected from Lavandula transcriptome sequences, protein kinases, nucleoside triphosphate hydrolase and Leucine-rich repeat domain are the most abundant containing over 1000 copy numbers (DOCX 16 kb)
Rights and permissions
About this article
Cite this article
Adal, A.M., Sarker, L.S., Malli, R.P.N. et al. RNA-Seq in the discovery of a sparsely expressed scent-determining monoterpene synthase in lavender (Lavandula). Planta 249, 271–290 (2019). https://doi.org/10.1007/s00425-018-2935-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-018-2935-5