Volatile compounds emitted by fully open flower of
1. Introduction
1.1. Orchidaceae
Orchidaceae is the largest family of angiosperms with an estimation of 17000 to 35000 species in 880 genera (Chai & Yu, 2007). In Malaysia, more than 230 orchid genera and 4000 species had been discovered (Go et al., 2012). In Penisular Malaysia, a total of 898 species in 143 genera are currently recognised (Go et al., 2010). The amazing vast diversity of types and forms enable the Orchidaceae to be successfully distributed and colonised almost every habitats worldwide (Arditti, 1992). As a result of selective forces from evolution, orchids are found to be evolved from its ancestral forms and adapted well to their present habitats (Aceto & Gaudio, 2011). Associated with their diverse floral morphology and physiology properties, they have drawn the attention of botanists and scientists for centuries. There are orchids which resemble moths (
Similar to other angiosperms, two whorls of perianth segment can also be found in orchids. The outer and inner whorls of the orchid flowers consist of three petals and three sepals. The labellum or lip (one of the petals), is distinctly evolved from the other two morphologically and physically (Arditti, 1992). The lifespan of opened-orchid flowers can range from as short as one day to as long as 270 days (Micheneau et al., 2008).
Most orchids are epiphytic that obtain their support from trees but not for nutrition while the rest are terrestrial plants (Rada & Jaimez, 1992). Orchids’ cultivation was famous since 5000 years ago in China where
Orchid hybrids cultivation started since 1856 by John Dominy (http://www/ionopsis.com/hybridization.htm).
The export of orchids from Malaysia, Thailand and Singapore contributed RM200 million annually in the world floral (Ooi, 2005). According to the Japan Florists’ Telegraph Delivery Association, cut flower in Japan constituted around 13.3% of the imported market in the year 2010 with Malaysia having the largest market share of imports, which accounted for 23.4% (7,648 million yen), followed by Columbia at 19.2% and China at 10.4%. Malaysian orchids consisted of 8.1% of the imported cut flower orchid to Japan.
1.2. Vandaceous orchids
Tropical Asia is the native home for approximately 50 vandaceous orchids species. They are distributed in Sri Lanka and southern India to New Guinea, northern Australia and Solomon Islands, and north to China, Taiwan and the Philippines. Thailand was found to be predominated with 11 vanda species. In Thailand, vanda is a vital commercial orchid. Most of them exhibit monopodial growth where their leaves are varies according to habitat. Vandas have many different colours, and majority are yellowish-brown with dark brown spots.
The vanda has been designated as the ‘Queen Orchid of the East’ due to its robust and large rounded flowers (Teo, 1981). Most of the vandaceous orchids prefer sunlight but some are well adapted to shady areas. Like any other tropical orchids, they require warm temperature with good aeration. The vandaceous orchids take around three and half to 10 years to become mature flowering plants (Kishor et al., 2008). Once matured, this orchid genus blooms every two to three months with the flowers lasting two to three weeks. As a result of land development, 28 orchid species have been listed as endangered species on Appendix II of the Convention on International Trade in Endangered Species (CITES) and prohibited from worldwide export. Among those orchids, two belong to vandas (
It is impossible to differentiate or identify an orchid species based on the vegetative parts of the plant alone. Hence, a convenient and flower-independent method to allow quick assessment of a given orchid vegetative specimen for species confirmation can be achieved with the help of molecular markers. To date, approximately 50 species are registered under vandas in the Royal Horticultural Society (RHS) database due to their commercial importance.
1.3. Vanda Mimi Palmer
Orchids cultivation entails hard work as the orchids can be easily infected by orchid-infecting viruses. More than 50 orchid-infecting viruses have been detected worldwide, with cymbidium mosaic virus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) infections being the most prevalent (Sherpa et al., 2007). Infected orchid cultivars usually exhibit blossoms with brown necrotic streaks and other necrosis symptoms. Infected flowers are smaller in size and poorer in quality. This has caused severe economic damages in the cut flower and potted plant industry (Ajjikuttira et al., 2002; Sherpa et al., 2007; Vejaratpimol et al., 1999). CymMV infection is dominant and extremely stable in Orchidaceae, and it was found to be prevalent in VMP. A screen of our VMP cDNA library revealed a 1.6% contamination rate with CymMV genes (Teh et al., 2011). Markers might be useful in facilitating the screening of virus-infected stock plants to minimise losses incurred in the floral industry. So far, the identification of CymMV infection is done through serological, bioassay or electron microscopy. Those techniques include enzyme-linked immunosorbent assays (ELISA), dot-blot immunoassay (DBIA), rapid immunofilter paper assay (RIPA), immunosorbent electron microscope (ISEM), DIG-labelled cRNA probes, reverse transcription polymerase chain reaction (RT-PCR), quartz crystal microbalance-based DNA biosensors and TaqMan real-time quantitative RT-PCR (Eun et al., 2002; Eun & Wong,2000; Hsu et al., 1992; Hu
2. Importance of floral fragrance
Floral fragrance plays various functions in both the floral and vegetative organs. Fragrance is defined as a highly complex component of floral phenotype for its dynamic patterns of emission and chemical composition (Raguso, 2008). Due to their restriction to specific lineages and interactions in species-specific ecology, these have led to their designation as specialized or secondary metabolites (Pichersky et al., 2006).
Floral fragrance has a significant impact in plant reproduction as it is a selective attractant in a variety of animal pollinators especially insects. Pollinators such as bees, butterflies and moths are able to discriminate visitation on plants based on the compositions of the floral scent. This plant-insect interaction has led to many successful pollinations and development of fruits in many crop species (Majetic et al., 2009; Shuttleworth & Johnson, 2009).
Anti-microbial or anti-herbivore properties of floral volatiles could be used by plants to protect their vital floral reproductive parts from potential predators. Two types of plant defences can be characterised based on floral volatiles property, that are direct and indirect defences such as herbivore-induced volatiles signals, and visual and olfactory floral signals to attract pollinators (Schiestl, 2010). Indirect plant defences protect the plants by minimizing damage to plant tissues through attracting arthropods that prey or parasitize the herbivores. This general property has been reported in cabbage (Park et al., 2005),
Through the discovery of pollinator-attracting floral scents as the source of olfactory pleasure since ancient times, humans had figured out unique values in certain types of floral scents and exploited them to cultivate and propagate specific plant species. For centuries, flowers with vibrant colours and scents have been used by people to enhance their beauty and this was seen in almost all major civilizations. Large numbers of aromatic plants have been used as flavourings, preservatives and herbal remedies (Pichersky et al., 2006). Their economic importance also relies on their petals which are found to be the main site of natural fragrances and flavourings in most of the plants (Baudino et al., 2007).
2.1. Fragrance biosynthesis pathways
Plants are known with the capability of synthesizing many volatile metabolites, either primary or secondary metabolites with variety of functions (Pichersky et al., 2006). However, volatile esters formation is not restricted to plant kingdom but also in yeast and fungi especially in the fermentation industry (Beekwilder et al., 2004). Floral scent is made up of a complex mixture of low-molecular-weight lipophilic compounds which are typically liquids with high vapour pressures (Vainstein et al., 2001). With the discovery of novel techniques including gas chromatography-nuclear magnetic resonance (GC-NMR), gas chromatography-mass spectrometry (GC-MS), headspace based techniques in volatiles detection and analyses, the number of identified volatile compounds has increased tremendously (Gonzalez-Mas et al., 2008; Mohd-Hairul et al., 2010; Nojima et al., 2011).
Most of the volatile compounds are derived from three major biosynthesis pathways: phenylpropanoids, fatty acid derivatives and terpenoids. They are thus classified into three major categories: terpenes, lipid derivatives and aromatic compounds. The terpenes are the largest class of plant volatiles, which consist of monoterpene alcohols and sesquiterpenes. There are also other terpene derivatives like ketones that are present in low quantities but have significant contributions to the floral fragrance. The second largest family of plant volatiles is the aromatic compounds. Most of them are derived from the intermediates in the benzenoid biosynthesis pathway that resulted in the synthesis of phenylalanine from the shikimate pathway, followed by a wide range of primary metabolites (eugenol, a lignin precursor, is one of them) and secondary non-volatile compounds (this was well reviewed in Bick & Lange, 2003; Pichersky & Dudareva, 2007).
The third category of plant volatiles is the lipid derivatives from the oxidative cleavage and decarboxylation of a variety of fatty acids which lead to shorter-chain volatiles with aldehyde and ketone moieties formation (reviewed in Baysal & Demirdoven, 2007). There are also other plant volatiles especially those with nitrogen or sulfur, which are produced through the cleavage of modified amino acids or their precursors (Cherri et al., 2007; Pichersky et al., 2006).
2.2. Discovery of fragrance-related genes
Due to the invisibility of floral scents and its dynamic nature, the study on flower scent is limited. There is no convenient plant model system that allows chemical and biochemical studies on floral scents. The well-established
‘Scent’ enzymes can be identified through
For more than 400 orchids (including both species and hybrids) that were discovered to emit fragrance (Frowine, 2005), in-depth scientific studies on orchid fragrance barely covered 2 percents of the fragrant orchids. Sadly, fragrance study in orchid is not as well established as in other flowers such as rose (Guterman et al., 2002), petunia (Verdonk
3. Expressed sequence tags (ESTs)
Expressed Sequence Tags (ESTs) are partial sequences generated from single-pass sequencing (5’- or 3’- end) from a reverse transcription of mRNA representing tissue of interest or a particular developmental stage of an organism (Adam et al., 1991). In plant, EST approach was first used in the model plant
EST libraries for many plant species such as
3.1. Importance of ESTs
EST is a fast, efficient and valuable tool for gene expression, genome annotation and evolutional studies. Analysis of ESTs provides a platform for functional genomics study as well as uncovering the potentially novel genes, and poses an avenue for genome sequencing projects (Ayeh, 2008; Kisiel & Podkowinski, 2005; Li et al., 2010; Lindqvist et al., 2006). Early EST projects were focused mostly on economically important plants and crop plants. In subsequent years, more EST projects on plants yet-to-achieve high economical impact started to materialise. The development of ESTs for some of the plants species from the early years until now is summarised in Table 2.
EST also proves to be a beneficial resource for comparative genomic studies in plant. Hsiao et al. (2006) deduced monoterpene biosynthesis pathway and identified a few fragrance-related genes in
Monoterpene 1 2 3 | 8.636 9.147 9.592 | 36,41,53,67,79,93,105,121 41,43,59,81,93,112 41,43,69,71,93,107,121,136 | Ocimene Linalool oxide Linalool |
Sesquiterpene 10 | 16.492 | 41,43,69,71,93,107,123,136, 162 | Nerolidol |
Benzenoid 4 5 | 9.702 10.030 | 51,77,105,136 39,51,65,78,91,105,122 | Methylbenzo- ate Benzyl acetate |
Phenylpropanoid 6 8 | 10.783 11.926 | 39,43,65,79,91,108,150 39,43,65,78,91,104 | Phenylethanol Phenylethyl acetate |
Indole 9 | 13.084 | 39,50,63,74,90,117 | Indole |
Formanilide 7 | 12.260 | 39,52,65,76,93,161 | Formanilide |
Organism | ESTs |
2,019,114 | |
1,529,700 | |
1,461,624 | |
1,252,989 | |
1,073,877 | |
720,590 | |
643,874 | |
501,838 | |
446,639 | |
334,384 | |
328,662 | |
324,742 | |
313,110 | |
297,239 | |
297,142 | |
269,238 | |
249,761 | |
242,432 | |
231,095 | |
110,006 | |
50,705 | |
38,190 | |
40,737 | |
11,078 | |
5,604 | |
5,565 | |
2,359 | |
800 | |
280 | |
103 |
3.2. Discovery of fragrance-related genes from VMPESTs
Expressed sequence tags (ESTs) have been developed from many monocot plant species including
A VMP floral cDNA library was previously constructed from opened flowers at different developmental stages and time-points (Chan et al., 2009). All the cDNA clones with the inserts sizes of 0.5 kb to 1.6 kb were mass excised and single-pass 5’-sequenced. From our attempt, a total of 2,132 ESTs was generated. This VMP dbEST (designated as VMPEST) was clustered, annotated and further classified with Gene Ontology (GO) identifier into three categories: Molecular Functions (51.2%), Cellular Components (16.4%) and Biological Processes (24.6%). Around 3.1% of the VMPEST had hits with other orchid species such as dendrobium, phalaenopsis, oncidium,
From the VMPEST, several fragrance-related transcripts were selected for full-length isolation and expression analysis using real-time quantitative RT-PCR. They were
GW392501 | 68671343 | 2.00E-108 | 358 | |
GW392566 | 68671408 | 1.00E-50 | 203 | |
GW392695 | 68671537 | 9.00E-19 | 97.8 | |
GW392657 | 68671499 | 2.00E-52 | 209 | |
GW392731 | 68671573 | 2.00E-15 | 85.9 | |
GW392740 | 68671582 | 3.00E-70 | 268 | |
GW392813 | 68671655 | 2.00E-73 | 279 | |
GW393688 | 68672530 | 5.00E-58 | 228 | |
GW392895 | 68671737 | 2.00E-46 | 189 | |
GW392922 | 68671764 | 6.00E-62 | 241 | |
GW393960 | 68672802 | 4.00E-103 | 378 | |
GW393331 | 68672173 | 4.00E-27 | 125 | |
GW393619 | 68672461 | 8.00E-56 | 221 | |
GW393628 | 68672470 | 2.00E-59 | 233 | |
GW394168 | 68673010 | 6.00E-41 | 171 | |
GW393499 | 68672341 | 1.00E-136 | 488 |
Among all the transcripts analysed,
4. Simple sequence repeat and its importance
Simple Sequence Repeat (SSR) or microsatellite is a short tandem repeats of a unique DNA sequence with one to six nucleotides motif (Jacob et al., 1991). SSR is a famous molecular marker because of its hyper variability, relative abundance, highly reproducible, multiallelic diversity, co-dominantly inherited and extensive coverage of the genome (Mohan et al., 1997).
Owing to its desirable genetic attributes, SSRs have been utilized in genetic and genomic analyses including genetic mapping, marker assisted plant breeding, development of linkage map, and ecology studies (Kalia et al., 2011; Sonah et al., 2011; Yue et al., 2006). Yue et al. (2006) reported the usage of SSRs in the protection of new
To date, the used of SSRs markers have been reported in several monocot and dicot species including raspberry and blackberry (Stafne et al., 2005), rice (Chakravarthi & Naravaneni, 2006), common bean (Yu et al., 2000),
So far, the reported SSRs generated from vandaceous orchids were used as selective marker only. Phuekvilai et al. (2009) generated SSRs from 33 vandas species for the sole purpose of identifying and evaluating the purity of cultivar in commercial samples. However, the identification and development of SSRs for VMP will be channelled towards facilitating the screening of any potential fragrance-related transcripts from closely related species. Besides, it will be used to determine the extent of inter-species transferability of genes, which had been reported in many plant species (Chapman et al., 2009; Stafne et al., 2005; Wang et al., 2004).
4.1. Data mining of VMPEST-SSR
In recent years, genic microsatellite or EST-SSRs which is less time consuming and relatively easy to develop has replaced the genomics SSRs (Sharma et al., 2007). The publicly available ESTs sequences facilitate the development of SSRs by using the SSR identification tools. These search tools include MISA (MIcroSAtellite), SSRIT (SSR Identification Tool), SciRoKo, TRF (Tandem Repeat Finder), Sputnik, SSRfinder, SAT (SSR Analysis Tool), Poly and SSR Primer. It is deemed important to choose a search tool which is user-friendly and has unlimited access to a non-redundant database (this was well reviewed by Kalia et al., 2011). The first attempt to develop such EST-SSR marker was in rice by Miyao et al. (1996).
SSRIT which is accessible at URL (http://www.gramene.org) was used to identify the SSR motifs in our VMPEST. The script assessed the sequences uploaded in FASTA-formatted files and detected the SSR motifs, the number of repeats as well as identified the sequence corresponded to the SSRs. A total of 98 (9.4%) unigenes containing 112 SSRs with motifs length ranging from two to six nucleotides were detected from VMPEST.
The VMPEST-SSRs were classified into 2 groups with 88.4% belonging to Class I (
Sharma et al. (2009) stressed that the length of repeats and the tools used in the EST-SSRs mining play a significant role in EST-SSR occurrences. The di-nucleotide motif (AT/TA) was present with the most abundance (33.9%) in our VMPEST-SSR. Such observation of A- and T-rich signatures being the most common repeat motifs in the VMPEST-SSRs is also reflected in the early findings of Chagne et al. (2004), Lagercrantz et al. (1993), and Morgante & Olivieri (1993). Interestingly, Blair et al. (2009) found that this motif occurred mainly in the 3’-end of the common bean cDNA clones.
Nevertheless, whatever mined results we obtained from our study, each SSR needs to be validated with further analyses such as selection of SSR primers and screening for utility in vandaceous orchids.
Besides EST-SSR, there are other alternative data mining techniques such as expressed sequence tag-single nucleotide polymorphism (EST-SNP) in maize (Batley et al., 2003), barley
5. Conclusion
The VMPEST dataset is a potential asset in facilitating the molecular biology and cloning of more genes involved in the fragrance biosynthesis pathway(s). Several fragrance-related transcripts were identified from our VMPEST including
Acknowledgement
Authors would like to thank the Ministry of Higher Education Malaysia and Universiti Putra Malaysia for financial support through the Fundamental Research Grant Scheme (02-12-10-1002FR) and Research University Grant Scheme (05-04-08-0551RU), respectively.
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