Abstract
Hedychium coronarium, a perennial herb belonging to the family Zingiberaceae, is cultivated as a garden plant or cut flower as well as for medicine and aromatic oil. Its flowers emit a fresh and inviting scent, which is mainly because of monoterpenes present in the profile of the floral volatiles. However, fragrance produced as a result of monoterpenes has not been well studied. In the present study, two novel terpene synthase (TPS) genes (HcTPS7 and HcTPS8) were isolated to study the biosynthesis of monoterpenes in H. coronarium. In vitro characterization showed that the recombinant HcTPS7 was capable of generating sabinene as its main product, in addition to nine sub-products from geranyl diphosphate (GPP). Recombinant HcTPS8 almost specifically catalyzed the formation of linalool from GPP, while it converted farnesyl diphosphate (FPP) to α-bergamotene, cis-α-bisabolene, β-farnesene and other ten sesquiterpenes. Subcellular localization experiments revealed that HcTPS7 and HcTPS8 were located in plastids. Real-time PCR analyses showed that HcTPS7 and HcTPS8 genes were highly expressed in petals and sepals, but were almost undetectable in vegetative organs. The changes of their expression levels in petals were positively correlated with the emission patterns of sabinene and linalool, respectively, during flower development. The results indicated that HcTPS7 and HcTPS8 were involved in the biosynthesis of sabinene and linalool in H. coronarium flowers. Results on these two TPSs first characterized from H. coronarium provide new insights into molecular mechanisms of terpene biosynthesis in this species and also lay the basis for biotechnological modification of floral scent profile in Hedychium.
Similar content being viewed by others
Abbreviations
- DMAPP:
-
Dimethylallyl pyrophosphate
- FPP:
-
Farnesyl diphosphate
- GC-MS:
-
Gas chromatography-mass spectrometry
- GFP:
-
Green fluorescence protein
- GGPP:
-
Geranylgeranyl diphosphate
- GPP:
-
Geranyl diphosphate
- IPP:
-
Isopentenyl pyrophosphate
- LB:
-
Luria–Bertani
- ORF:
-
Open reading frame
- PDMS:
-
Polydimethylsiloxane
- RACE:
-
Rapid amplification of cDNA ends
- RT-PCR:
-
Reverse transcription PCR
- TPS:
-
Terpene synthase
References
Aharoni A, Giri AP, Deuerlein S, Griepink F, de Kogel WJ, Verstappen FW, Verhoeven HA, Jongsma MA, Schwab W, Bouwmeester HJ (2003) Terpenoid metabolism in wild-type and transgenic Arabidopsis plants. Plant Cell 15:2866–2884
Aharoni A, Giri AP, Verstappen F, Bertea CM, Sevenier R, Sun ZK, Jongsma MA, Schwab W, Bouwmeester HJ (2004) Gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species. Plant Cell 16:3110–3131
Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250
Aros D, Gonzalez V, Allemann RK, Muller CT, Rosati C, Rogers HJ (2012) Volatile emissions of scented Alstroemeria genotypes are dominated by terpenes, and a myrcene synthase gene is highly expressed in scented Alstroemeria flowers. J Exp Bot 63:2739–2752
Aubourg S, Lecharny A, Bohlmann J (2002) Genomic analysis of the terpenoid synthase (AtTPS) gene family of Arabidopsis thaliana. Mol Genet Genomics 267:730–745
Baez D, Pino JA, Morales D (2011) Floral scent composition in Hedychium coronarium J. Koenig analyzed by SPME. J Essent Oil Res 23:64–67
Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. P Natl Acad Sci USA 95:4126–4133
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Chen F, Tholl D, D’Auria JC, Farooq A, Pichersky E, Gershenzon J (2003) Biosynthesis and emission of terpenoid volatiles from Arabidopsis flowers. Plant Cell 15:481–494
Chen F, Tholl D, Bohlmann J, Pichersky E (2011a) 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
Chen L, Zhong H, Kuang J, Li J, Lu W, Chen J (2011b) Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions. Planta 234:377–390
Christianson DW (2006) Structural biology and chemistry of the terpenoid cyclases. Chem Rev 106:3412–3442
Crowell AL, Williams DC, Davis EM, Wildung MR, Croteau R (2002) Molecular cloning and characterization of a new linalool synthase. Arch Biochem Biophys 405:112–121
Cseke L, Dudareva N, Pichersky E (1998) Structure and evolution of linalool synthase. Mol Biol Evol 15:1491–1498
Danner H, Boeckler GA, Irmisch S, Yuan JS, Chen F, Gershenzon J, Unsicker SB, Koellner TG (2011) Four terpene synthases produce major compounds of the gypsy moth feeding-induced volatile blend of Populus trichocarpa. Phytochemistry 72:897–908
Davidovich-Rikanati R, Lewinsohn E, Bar E, Iijima Y, Pichersky E, Sitrit Y (2008) Overexpression of the lemon basil alpha-zingiberene synthase gene increases both mono- and sesquiterpene contents in tomato fruit. Plant J 56:228–238
Degenhardt J, Kollner TG, Gershenzon J (2009) Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 70:1621–1637
Dos Santos BCB, Barata LES, Marques FA, Baroni ACM, Karnos BAC, de Oliveira PR, Guerrero PG Jr (2010) Composition of leaf and rhizome essential oils of Hedychium coronarium Koen. from Brazil. J Essent Oil Res 22:305–306
Dudareva N, Pichersky E (2000) Biochemical and molecular genetic aspects of floral scents. Plant Physiol 122:627–633
Dudareva N, Cseke L, Blanc VM, Pichersky E (1996) Evolution of floral scent in Clarkia: Novel patterns of S-linalool synthase gene expression in the C. breweri flower. Plant Cell 8:1137–1148
Dudareva N, Martin D, Kish CM, Kolosova N, Gorenstein N, Faldt 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
Dudareva N, Pichersky E, Gershenzon J (2004) Biochemistry of plant volatiles. Plant Physiol 135:1893–1902
Exposito-Rodriguez M, Borges AA, Borges-Perez A, Perez JA (2008) Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol 8:131
Faehnrich A, Krause K, Piechulla B (2011) Product variability of the ‘Cineole Cassette’ monoterpene synthases of related Nicotiana Species. Mol Plant 4:965–984
Falara V, Akhtar TA, Nguyen TTH, Spyropoulou EA, Bleeker PM, Schauvinhold I, Matsuba Y, Bonini ME, Schilmiller AL, Last RL, Schuurink RC, Pichersky E (2011) The tomato terpene synthase gene family. Plant Physiol 157:770–789
Fan YP, Yu RC, Huang Y, Chen YF (2003) Studies on the essential constituent of Hedychium flavum and H. coronarium. Acta Horticulturae Sinica 30:475 (in Chinese)
Fan YP, Wang XR, Yu RC, Yang P (2007) Analysis on the aroma components in several species of Hedychium. Acta Horticulturae Sinica 34:231–234 (in Chinese)
Green S, Baker EN, Laing W (2011) A non-synonymous nucleotide substitution can account for one evolutionary route to sesquiterpene synthase activity in the TPS-b subgroup. FEBS Lett 585:1841–1846
Green SA, Chen X, Nieuwenhuizen NJ, Matich AJ, Wang MY, Bunn BJ, Yauk Y, Atkinson RG (2012) Identification, functional characterization, and regulation of the enzyme responsible for floral (E)-nerolidol biosynthesis in kiwifruit (Actinidia chinensis). J Exp Bot 63:1951–1967
Gutensohn M, Orlova I, Nguyen TTH, Davidovich-Rikanati R, Ferruzzi MG, Sitrit Y, Lewinsohn E, Pichersky E, Dudareva N (2013) Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits. Plant J 75:351–363
Guterman I, Shalit M, Menda N, Piestun D, Dafny-Yelin M, Shalev G, Bar E, Davydov O, Ovadis M, Emanuel M, Wang J, Adam Z, Pichersky E, Lewinsohn E, Zamir D, Vainstein A, Weiss D (2002) Rose scent: genomics approach to discovering novel floral fragrance-related genes. Plant Cell 14:2325–2338
Gutierrez L, Mauriat M, Guenin S, Pelloux J, Lefebvre J, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Van Wuytswinkel O (2008) The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol J 6:609–618
Hillwig ML, Xu M, Toyomasu T, Tiernan MS, Wei G, Cui G, Huang L, Peters RJ (2011) Domain loss has independently occurred multiple times in plant terpene synthase evolution. Plant J 68:1051–1060
Hsiao Y, Jeng M, Tsai W, Chuang Y, Li C, Wu T, Kuoh C, Chen W, Chen H (2008) A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)(2-4)D motif. Plant J 55:719–733
Jaakola L, Pirttila AM, Halonen M, Hohtola A (2001) Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit. Mol Biotechnol 19:201–203
Jones CG, Moniodis J, Zulak KG, Scaffidi A, Plummer JA, Ghisalberti EL, Barbour EL, Bohlmann J (2011) Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases. J Biol Chem 286:17445–17454
Joy B, Rajan A, Abraham E (2007) Antimicrobial activity and chemical composition of essential oil from Hedychium coronarium. Phytother Res 21:439–443
Kampranis SC, Ioannidis D, Purvis A, Mahrez W, Ninga E, Katerelos NA, Anssour S, Dunwell JM, Degenhardt J, Makris AM, Goodenough PW, Johnson CB (2007) Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function. Plant Cell 19:1994–2005
Kohzaki K, Gomi K, Yamasaki-Kokudo Y, Ozawa R, Takabayashi J, Akimitsu K (2009) Characterization of a sabinene synthase gene from rough lemon (Citrus jambhiri). J Plant Physiol 166:1700–1704
Koo HJ, Gang DR (2012) Suites of terpene synthases explain differential terpenoid production in ginger and turmeric tissues. PLoS One 7:e51481
Lan JB, Yu RC, Yu YY, Fan YP (2013) Molecular cloning and expression of Hedychium coronarium farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral and wounding/herbivory induced leaf volatile sesquiterpenoids. Gene 518:360–367
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
Lee S, Chappell J (2008) Biochemical and genomic characterization of terpene synthases in Magnolia grandiflora. Plant Physiol 147:1017–1033
Li R, Fan Y (2011) Molecular cloning and expression analysis of a terpene synthase gene, HcTPS2, in Hedychium coronarium. Plant Mol Biol Rep 29:35–42
Li G, Koellner TG, Yin Y, Jiang Y, Chen H, Xu Y, Gershenzon J, Pichersky E, Chen F (2012) Nonseed plant Selaginella moellendorffii has both seed plant and microbial types of terpene synthases. P Natl Acad Sci USA 109:14711–14715
Martin DM, Faldt J, Bohlmann J (2004) Functional characterization of nine Norway spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily. Plant Physiol 135:1908–1927
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
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
Pichersky E, Dudareva N (2007) Scent engineering: toward the goal of controlling how flowers smell. Trends Biotechnol 25:105–110
Pichersky E, Lewinsohn E, Croteau R (1995) Purification and characterization of S-linalool synthase, an enzyme involved in the production of floral scent in Clarkia breweri. Arch Biochem Biophys 316:803–807
Pichersky E, Noel JP, Dudareva N (2006) Biosynthesis of plant volatiles: nature’s diversity and ingenuity. Science 311:808–811
Raguso RA (2008) Wake up and smell the roses: the ecology and evolution of floral scent. Annu Rev Ecol Evol Syst 39:549–569
Schnee C, Kollner TG, Gershenzon J, Degenhardt J (2002) The maize gene terpene synthase 1 encodes a sesquiterpene synthase catalyzing the formation of (E)-beta-farnesene, (E)-nerolidol, and (E, E)-farnesol after herbivore damage. Plant Physiol 130:2049–2060
Tholl D (2006) Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol 9:297–304
Tholl D, Chen F, Petri J, Gershenzon J, Pichersky E (2005) Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers. Plant J 42:757–771
Trapp SC, Croteau RB (2001) Genomic organization of plant terpene synthases and molecular evolutionary implications. Genetics 158:811–832
Vainstein A, Lewinsohn E, Pichersky E, Weiss D (2001) Floral fragrance. New inroads into an old commodity. Plant Physiol 127:1383–1389
van Schie C, Haring MA, Schuurink RC (2007) Tomato linalool synthase is induced in trichomes by jasmonic acid. Plant Mol Biol 64:251–263
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: research0034.1–0034.11. doi:10.1186/gb-2002-3-7-research0034
Wang W, Zhou W (2008) Efficient extraction and application of total RNA in sisal. Chin J Trop Crops 29:725–729
Williams DC, McGarvey DJ, Katahira EJ, Croteau R (1998) Truncation of limonene synthase preprotein provides a fully active ‘Pseudomature’ form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair. Biochemistry 37:12213–12220
Wise ML, Savage TJ, Katahira E, Croteau R (1998) Monoterpene synthases from common sage (Salvia officinalis): cDNA isolation, characterization, and functional expression of (+)-sabinene synthase, 1,8-cineole synthase, and (+)-bornyl diphosphate synthase. J Biol Chem 273:14891–14899
Wu ZY, Raven HP (2001) Flora of China 24. Missouri Botanical Garden Press, Saint Louis, pp 370–377
Wu SQ, Schalk M, Clark A, Miles RB, Coates R, Chappell J (2006) Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants. Nat Biotechnol 24:1441–1447
Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protoc 2:1565–1572
Yu F, Utsumi R (2009) Diversity, regulation, and genetic manipulation of plant mono- and sesquiterpenoid biosynthesis. Cell Mol Life Sci 66:3043–3052
Zhuang X, Koellner TG, Zhao N, Li G, Jiang Y, Zhu L, Ma J, Degenhardt J, Chen F (2012) Dynamic evolution of herbivore-induced sesquiterpene biosynthesis in sorghum and related grass crops. Plant J 69:70–80
Acknowledgments
This work was supported in part by the National Natural Science Foundation of China (Grant No. 30972026 and 31370694), the Guangdong-industry Technology Research System (Grant No. 2009-356), Key Laboratory of Innovation and Utilization for Germplasm Resources in Horticultural Crops in Southern China of Guangdong Higher Education Institutes, South China Agricultural University (No. KBL11008), and a Specialized Research Fund for the Doctoral Program of Higher Education of China to Yanping Fan (Grant No. 20134404110016).
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Yue, Y., Yu, R. & Fan, Y. Characterization of two monoterpene synthases involved in floral scent formation in Hedychium coronarium . Planta 240, 745–762 (2014). https://doi.org/10.1007/s00425-014-2127-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-014-2127-x