Terpenoid, Benzenoid, and Phenylpropanoid Compounds in the Floral Scent of Vanda Mimi Palmer
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- Mohd-Hairul, A., Namasivayam, P., Cheng Lian, G.E. et al. J. Plant Biol. (2010) 53: 358. doi:10.1007/s12374-010-9123-x
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Vanda Mimi Palmer is the product of a cross between Vanda Tan Chay Yan and Vanda tessellata. The flower of this hybrid produces a sweet-smelling fragrance during day time at the open-flower stage. This study aimed to investigate the floral scent constituents in Vanda Mimi Palmer. Scent emission analysis of this orchid was carried out at different time points in a 24-h cycle and also at different floral developmental stages. A comparison was also made on the volatiles emitted by Vanda Mimi Palmer and both of its parents. Gas chromatography-mass spectrometry (GC-MS) analysis showed that the scent of Vanda Mimi Palmer was dominated by terpenoid, benzenoid, and phenylpropanoid compounds. The identified terpenoids were ocimene, linalool oxide, linalool, and nerolidol; while the benzenoid and phenylpropanoid compounds were methylbenzoate, benzyl acetate, phenylethanol, and phenylethyl acetate. The emission of terpenoid, benzenoid, and phenylpropanoid compounds was developmentally and temporally regulated. Comparison of the volatiles emitted by both of its parents showed that the scent of Vanda Mimi Palmer is dissimilar to that of its fragrant parent, V. tessellata.
KeywordsVanda Mimi PalmerScent emissionGas chromatography mass-spectrometry (GC-MS)TerpenoidsBenzenoid and phenylpropanoid compounds
Volatiles from a flower attract pollinators such as birds and insects to visit flowering plants and help in pollination (Knudsen et al. 1993; Schiestl et al. 1997; Miyake et al. 1998; Odell et al. 1999; Jurgens et al. 2000). More than 1,700 floral scent compounds covering 990 taxa have been identified using the floral headspace method (Knudsen and Gershenzon 2006). The scent of flowers varies between species due to the combination of different compositions and levels of each molecule of low molecular mass such as monoterpenes, seisquiterpenes, benzenoids, phenylpropanoids, and fatty acid derivatives (Knudsen et al. 1993; Dudareva and Pichersky 2000). The entire floral organs are involved in scent emission but the petal is the main source of floral scent in most flowers (Pichersky et al. 1994). Some floral scent compounds are stored in special oil glands such as trichome prior to release into the air as volatiles (Effmert et al. 2006).
In floral scent studies, more than 500 terpenoid compounds have been identified including monoterpenes (C10), seisquiterpenes (C15), diterpenes (C20), and irregular terpenes (Knudsen and Gershenzon 2006). Monoterpenoid compounds such as linalool, ocimene, mycrene, nerol, and geraniol have been identified in Antirrhinum majus (Dudareva et al. 2003; Nagegowda et al. 2008), Clarkia breweri (Raguso and Pichersky 1995), and Arabidopsis thaliana (Chen et al. 2003). While sesquiterpenes such as germacrene D, caryophyllene, and nerolidol were detected in Rosa hybrida (Hendel-Rahmanim et al. 2007), Petunia hybrida (Verdonk et al. 2003), A. thaliana (Chen et al. 2003), carnation (Dianthus caryophyllus) (Schade et al. 2001), and A. majus (Nagegowda et al. 2008). Another class that is highly distributed among scented flowers is benzenoid/phenylpropanoid compounds. More than 300 compounds of this class have been identified which includes methylbenzoate, methylsalicylate, phenylacetaldehyde, phenylethyl acetate, benzyl acetate, phenylethanol, eugenol, and isoeugenol (Knudsen and Gershenzon 2006).
In orchids, volatile compounds encompassing terpenoids, benzenoid/phenylpropanoid compounds, and also fatty acid derivatives have been reported from Aerangis confusa, Cattleya lawrenceana, Dendrobium trigonopus, Dendrochilum cobbianum, Platanthera chlorantha, Polystachya cultriformis, and Zygopetalum crinitum (Kaiser 1993). More recently, linalool, mycrene, nerol and 2-hexanol were detected in Phalaenopsis bellina using gas chromatography mass-spectrometry (GC-MS; Hsiao et al. 2006).
Materials and Methods
Orchid plants (Vanda Mimi Palmer and Vanda Tan Chay Yan) used in this study were purchased and maintained by the United Malaysian Orchids Sdn. Bhd., a nursery located in Rawang, Selangor, Malaysia. Both Vanda Mimi Palmer and Vanda Tan Chay Yan used in this study were grown separately in pots with charcoal under tropical climate (12 h in light followed by 12 h in dark). Fully open flowers used for this study were between 5 and 7 days old. Floral buds of 1.5 cm in length and 2 days old half-open flowers were used for the scent emission study at different flower developmental stages.
Volatiles Captured from Single Flower for Analysis
Analysis of Volatiles by GC-MS
The volatile compounds captured by SPME fiber was injected into the injector port of a Shimadzu Gas Chromatography-Mass Spectrometry (GC-17A/GCMS-QP5050) with a splitless injection mode and thermally desorbed for 1 min at 250°C. The volatile compounds were separated using a capillary HP-5 column (50 m × 0.32 mm, film thickness 1.05 μm) with helium (21 kPa) as a carrier gas. The GC oven was programmed at 45°C for 1 min followed by an increase of 10°C per minute to 280°C and a final extension at 280°C for 10 min. The mass spectra of eluted compounds were recorded for the m/z of 30–300. The spectrum of each compound was compared to the 2002 National Institute of Standards and Technology library (Scientific Instrument Services, USA).
Comparison of Volatiles Emitted by Vanda Mimi Palmer and Its Parents
A comparison was carried out between the volatiles emitted by Vanda Mimi Palmer and its parents (Vanda Tan Chay Yan and V. tessellata). Volatiles emitted by fully open flower of both Vanda Mimi Palmer and Vanda Tan Chay Yan were captured at 12:00 noon and analyzed by GC-MS as described above while the result of headspace analysis of volatiles emitted by V. tessellata reported by Kaiser (1993) was used in the comparison.
Floral Scent Constituents of Vanda Mimi Palmer
Volatile compounds emitted by fully open flower of Vanda Mimi Palmer with their relative retention time and fragments
Retention time (min)
Main fragments (m/z)
93, 41, 79, 53, 105, 67, 121, 36
43, 59, 41, 93, 81, 112
71, 41, 43, 93, 69, 121, 136, 107
41, 69, 43, 93, 71, 107, 136, 162, 123
105, 77, 136, 51
91, 122, 65, 39, 51, 78, 105
108, 43, 91, 150, 79, 65, 39
104, 43, 91, 65, 78, 39
117, 90, 63, 39, 50, 74
93, 161, 65, 39, 52, 76
Profiles of Vanda Mimi Palmer’s Scent Emission
Comparison of Volatiles of Vanda Mimi Palmer with Its Parents
Comparison of terpenoids and benzenoid/phenylpropanoid compounds emitted by Vanda Mimi Palmer, Vanda Tan Chay Yan, and Vanda tessellata
Vanda Mimi Palmer
Vanda Tan Chay Yan
V. tessellata (Kaiser 1993)
The GC-MS analyses of the volatile compounds of Vanda Mimi Palmer showed there were four candidates, ocimene, linalool oxide, linalool, and nerolidol, potentially derived from the terpenoid pathway. Ocimene, linalool oxide and linalool are classified as monoterpenes (Croteau and Karp 1991; Knudsen and Gershenzon 2006) while nerolidol is a sesquiterpene (Knudsen and Gershenzon 2006; Nagegowda et al. 2008). Monoterpenes and sesquiterpenes are common volatile compounds detected in scented orchids including P. bellina (Hsiao et al. 2006), Dendrobium beckleri, and Phalaenopsis violacea (Kaiser 1993). The terpenoid compounds emitted by Vanda Mimi Palmer flowers were also reported to be present in other flowers such as Anthirrhinum majus (linalool, ocimene and nerolidol) (Dudareva et al. 2003; Nagegowda et al. 2008) and C. breweri (linalool) (Pichersky et al. 1994). This implies that the terpenoid pathway in Vanda Mimi Palmer is possibly involved in the biosynthesis of linalool, ocimene, and nerolidol compounds, and similar to the terpenoid pathway reported in other well studied scented flowers.
Comparison of the volatiles emitted by Vanda Mimi Palmer and its parents, Vanda Tan Chay Yan (non-fragrant orchid) and V. tessellata (fragrant orchid; see Table 2) showed ocimene is the only common compound detected in all three orchids. An ocimene synthase from Vanda Mimi Palmer which has yet to be identified might be involved in the final step catalyzing the formation of ocimene from geranyl diphosphate, a precursor for monoterpenoids biosynthesis. For another monoterpene compound which is linalool, a linalool synthase which has been identified in the floral ESTs of Vanda Mimi Palmer (unpublished data) might be involved in catalyzing the formation of linalool from geranyl diphosphate. The linalool synthase gene might have been derived from V. tessellata since linalool compound was also reported in the scent of V. tessellata previously by Kaiser (1993) in his GC-MS analysis. Besides that, formation of nerolidol (a sesquiterpene compound) might be catalyzed by a sesquiterpene synthase. Since no sesquiterpene compound was identified in V. tessellata (Kaiser 1993), we assume that the level is too low to be detectable or the sesquiterpene synthase gene is not present in this orchid. The sesquiterpene synthase gene in Vanda Mimi Palmer might be derived from Vanda Tan Chay Yan since copaene (a sesquiterpene) was identified in its scent (see Fig. 7).
In summary, the scent emission of Vanda Mimi Palmer, which is dominated by terpenoids as well as benzenoid/phenylpropanoid compounds, is developmentally and temporally regulated and might be contributed by the genes pool from both parents, V. tessellata and Vanda Tan Chay Yan.
We thank Universiti Putra Malaysia for the Research University Grant Scheme (RUGS; Grant No. 05-04-08-0551RU) to support this project and also Department of Chemistry, Faculty of Science, Universiti Putra Malaysia for the use of Gas Chromatography-Mass Spectrometry.
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