Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master

of Science CHEMICAL COMPOSITION OF FLORAL VOLATILES AND EXPRESSION OF SCENT-RELATED GENES IN VANDA MIMI PALMER

By MOHD HAIRUL AB. RAHIM November 2010

Chairman: Parameswari a/p Namasivayam, PhD Faculty: Biotechnology and Biomolecular Sciences

Vanda Mimi Palmer is an orchid hybrid of Vanda Tan Chay Yan and Vanda tessellata. The flower of this orchid produces a sweet fragrance during daylight hours at the openflower stage. Lately, a lot of effort has been channeled into understanding the fragrance pathway in scented flowers but none in Vandaceous orchids. This study aims to investigate on the molecular and biochemical aspects of the fragrance in Vanda Mimi Palmer. Scent emission analysis of this orchid was carried out at different developmental stages and at different time points in a 24-hour cycle. Gas chromatography mass spectrometry (GC-MS) analysis has shown that the scent of Vanda Mimi Palmer is dominated by metabolites from the terpenoid, benzenoid and phenylpropanoid pathways. Identified volatile compounds derived from terpenoid pathway are linalool, ocimene and nerolidol. Meanwhile, methylbenzoate, phenylethanol, benzyl acetate and phenylethyl acetate are the metabolites identified from the benzenoid and phenylpropanoid pathways. Scent emission of Vanda Mimi Palmer is also developmentally and temporally regulated.

On the molecular biology aspect, fragrance-related cDNA transcripts have been identified by a differential screening of the Vanda Mimi Palmers floral cDNA library. ReverseNorthern analysis was carried out by hybridizing the putative positive clones with two cDNA probes representing mRNA transcripts of bud and fully-open flower during the daylight hour separately. The clones that showed up-regulated expression in fully-open flowers were selected for sequencing. From the sequencing results, putative 4-(cytidine 5-diphospho)-2-C-methyl-D-erythritol kinase (VMPCMEK), putative cytochrome P450 (VMPCyP450), and an unknown protein (VMPA28) were selected for molecular characterization. The three transcripts with a putative phenylacetaldehyde synthase (VMPPAAS), a previously isolated transcript from Expressed Sequence-Tags (ESTs), were subjected to full-length cDNA isolation and expression analyses by real-time RTPCR. Expression analyses of these transcripts were investigated in different tissues, at different developmental stages, and time points in a 24-hour cycle using real-time RTPCR. The transcripts are highly expressed in floral tissues compared to vegetative tissues as well as developmentally and temporally regulated. In conclusion, from the biochemical and molecular work on the fragrance, there are two putative biochemical pathways which might be involved in fragrance biosynthesis in Vanda Mimi Palmer that are the terpenoid, and also the benzenoid and phenylpropanoid pathways.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains KOMPOSISI KIMIA HARUMAN DAN EKSPRESI GEN-GEN BERKAITAN WANGIAN DALAM VANDA MIMI PALMER Oleh MOHD HAIRUL AB. RAHIM November 2010

Pengerusi: Fakulti:

Parameswari a/p Namasivayam, PhD Bioteknologi dan Sains Biomolekul

Vanda Mimi Palmer ialah orkid kacukan di antara Vanda Tan Chay Yan dan Vanda tesellata. Bunga orkid ini yang telah berkembang sepenuhnya mengeluarkan bau yang harum pada waktu siang. Kebelakangan ini, perhatian diberikan terhadap tapak jalan biokimia penghasilan wangian bagi bunga wangi selain daripada orkid Vanda. Kajian ini bertujuan untuk mengkaji wangian Vanda Mimi Palmer yang merangkumi aspek-aspek biokimia dan biologi molekul. Analisis bagi wangian yang dihasilkan oleh orkid ini dijalankan pada setiap peringkat perkembangan bunga dan juga masa yang berbeza dalam kitaran 24 jam sehari. Analisis dijalankan menggunakan alat kromatografi gasspectrometrik jisim (GC-MS). Analisis GC-MS tersebut menunjukkan wangian Vanda Mimi Palmer didominasi oleh metabolit daripada tapak jalan terpenoid, dan juga, benzenoid dan phenylpropanoid. Pengeluaran wangian Vanda Mimi Palmer juga didapati dikawalatur mengikut peringkat perkembangan bunga dan juga peredaran masa. Dalam aspek biologi molekul, transkrip cDNA berkaitan penghasilan wangian telah dikenalpasti melalui penyaringan pembezaan perpustakaan cDNA bunga (floral cDNA library) Vanda

Mimi Palmer. Analysis reverse-Northern dijalankan

dengan menghibridkan secara

berasingan klon-klon positif bersama dua cDNA probe yang berbeza mewakili transkrip mRNA bagi peringkat kudup dan juga bunga kembang penuh. Klon-klon yang menunjukkan ekspresi yang lebih tinggi bagi peringkat bunga kembang penuh berbanding kudup dipilih untuk jujukan. Daripada keputusan analisis jujukan, 4-(cytidine 5-diphospho)-2-C-methyl-D-erythritol kinase putatif (VMPCMEK), cytochrome P450 protein putatif (VMPCyP450), dan transkrip protein yang belum dikenalpasti (VMPA28) dipilih untuk pencirian biologi molekul. Ketiga-tiga transkrip tersebut bersama transkrip phenylacetaldehyde synthase putatif (VMPPAAS) yang dipencilkan daripada Expressed Sequence-Tags (ESTs) bunga Vanda Mimi Palmer dipilih untuk pencirian termasuk pemencilan cDNA lengkap dan juga analisis ekspresi menggunakan tindakbalas rantaian polimerase masa nyata (RT-PCR). Analisis ekspresi tersebut dijalankan bagi tisu yang berbeza, peringkat perkembangan bunga yang berbeza dan masa yang berbeza dalam kitaran 24 jam sehari menggunakan RT-PCR. Transkrip tersebut menunjukkan ekspresi yang tinggi pada tisu bunga berbanding tisu vegetatif, dan dikawalatur oleh peringkat perkembangan bunga dan juga peredaran masa. Kesimpulannya, daripada hasil kajian biokimia dan biologi molekul, dua tapak jalan biokimia telah dikenalpasti berkemungkinan terlibat bagi penghasilan wangian dalam Vanda Mimi Palmer iaitu tapak jalan terpenoid dan juga tapak jalan benzenoid dan phenylpropanoid.

ACKNOWLEDGEMENTS

I would like to express my utmost gratitude to my supervisor, Dr. Parameswari a/p Namasivayam, for her patience, encouragement, time as well as precious advice and guidance, leading me throughout this research project. My sincere appreciation is also extended to my co-supervisors, Assoc. Prof. Dr. Janna Ong Abdullah and Prof. Dr. Gwendoline Ee Cheng Lian for their guidance, support and technical advice.

Special thanks to Malaysia Toray Science Foundation (MTSF) for giving me a research grant for the screening and isolation of putative fragrance-related cDNAs. Another special thanks to Universiti Putra Malaysia for supporting my work on biochemical and molecular characterization of the fragrance of Vanda Mimi Palmer through Research University Grant Scheme (RUGS) and also for providing me my stipend for the last two years through Graduate Research Fellowship (GRF). My deepest appreciation also goes to Chemistry Department, Faculty of Science UPM for giving me permission to use GCMS for my biochemical analysis of the scent of Vanda Mimi Palmer and also to En. Zainal Abidin Kasim for his help in my biochemical analysis with GC-MS.

I would like to thank all my lab mates in Molecular Biology Laboratory, Biotech 3, UPM as well as all of the laboratory staff in the laboratory, for their technical guidance, support, care, forgiveness, valuable ideas and experience. My deepest appreciation is extended to family for their endless support, care and love, accompanying me through all the happiness and sadness in my study.

CHAPTER 1 INTRODUCTION

Floral scent or floral fragrance is an important constituent for perfume and food industries. The extracts from flowers including jasmine and rose have been used extensively in fragrance and flavour industries. Besides that, there is always a high demand for scented flowers from aromatherapy industries especially in Malaysia and Thailand. In horticultural and agricultural industries, floral scent is very important for pollination of crops. Floral scent studies have been well established in some scented flowers including Clarkia breweri, Antirrhinum majus, Rosa hybrida and Petunia hybrida in both biochemical and molecular aspects covering three fragrance biosynthetic pathways that are terpenoid, lipoxygenase-catalyzed fatty acid derivatives, and also benzenoid and phenylpropanoid pathways (Pichersky and Dudareva, 2007).

Orchids with fragrance have higher demand in the orchid industry and fetching higher prices compared to non-fragrance orchids (Eric Kok, Manager, Malaysian Orchids Sdn. Bhd., pers. comm. on 20th May 2008). In orchid industry, extensive work has been focused on hybridizing scented orchid with non-scented orchid in order to produce flower with attractive colour appearances. Most of the progenies produced have diluted scent or no scent at all. In orchids, floral scent identification started in the early 1990s (Kaiser, 1993) but the knowledge on fragrance biosynthetic pathways is still far from being understood. More recently, a few fragrance-related cDNAs were identified from expressed sequence-tags (ESTs) of Phalaenopsis bellina (Hsiao et al., 2006) and the only

cDNA that has been well characterized is geranyldiphosphate synthase that is involved in the biosynthesis of geranyl diphosphate, a precursor for monoterpenes biosynthesis (Hsiao et al., 2008).

Besides Phalaenopsis bellina there are a lot of fragrance orchids which are still not well studied for their fragrance characteristics including Vanda Mimi Palmer. Vanda Mimi Palmer is a well-known commercial orchid hybrid especially in Malaysia and Thailand. This orchid produces a sweet smelling fragrance during day time in fully-open flower stage (Janna et al., 2005). This orchid had won several awards for its sweet smelling fragrance including the Champion Award for Fragrant Orchid organized by the Royal Horticultural Society of Thailand in 1993 and the Best Orchid Fragrance in the 17th World Orchid Conference in 2002 (Nair and Arditti, 2002). Thus, Vanda Mimi Palmer with its fragrance emission characteristic was chosen for this study in order to understand its fragrance biosynthetic pathways.

The knowledge on the sequences of fragrance-related cDNAs isolated from Vanda Mimi Palmer can be used for transformation into non-scented orchids and other non-scented flowers in order to increase the commercial value of the orchids and other ornamental flowers. Besides that, understanding on the fragrance biosynthetic pathways of Vanda Mimi Palmer will assist in the cloning of fragrance-related cDNAs into bacterial and yeast expression vector for production of fragrance compounds in bulk. The knowledge of the proportion of each compound in the fragrance of Vanda Mimi Palmer will facilitate

the production of custom-made perfume of the same smell as Vanda Mimi Palmer either biologically or chemically synthesized.

The specific objectives for this study were:

1) to determine the constituents of the scent of Vanda Mimi Palmer in comparison to its parents, 2) to isolate and characterize selected putative fragrance-related transcripts of Vanda Mimi Palmer, and 3) to analyze the expression profile of the selected putative fragrance-related cDNAs of Vanda Mimi Palmer

CHAPTER 2 LITERATURE REVIEW

2.1 Orchid An Introduction

Orchids are classified under the Orchidaceae, one of the largest families of flowering plants with an estimated population of 20,000 to 35,000 species (Dressler, 1993; Mabberly, 1997). More than 800 orchid genera have been identified from the entire world including Aranda, Aranthera, Cattleya, Dendrobium, Oncidium, Phalaenopsis, Paphiopedilum and Vanda. In Malaysia, there are more than 120 genera and 2000 species that have been discovered (Hamdan, 2008). An orchid flower consists of three sepals and three petals. The petals and sepals are usually nearly alike where petals are located in the first whorl while sepals in the second whorl of the flower. One of the petals is often highly modified to form the lip or labellum, and is complicated in shape (Seidenfaden and Wood, 1992).

The habitats of orchids vary such as mountainous forests, highlands, tropical mountain forests and also lowlands (Fadelah et al., 2001). In nature, there are epiphytic orchids which grow on branches and trunks of trees, terrestrial orchids which grow on soil and lithophyte orchids which grow on rocks (Hamdan, 2008). The epiphytic orchids use branches and trunks of trees only for support purpose without taking anything from the trees. Living up on the trees helps the orchids to get away from competition with other plants on the forest floor and escape from mineral contaminants on soil (Rittershausen

and Rittershausen, 2008). The source of nutrients for epiphyte and lithophyte are from organic substances of dead leaves, mosses and insects meanwhile for terrestrial orchids, the nutrients for growth are directly from the soil (Hamdan, 2008).

To date, more than 100,000 orchid hybrids have been established in the world either by crossing between the same genera (interspecific hybrid) or by crossing with different genera (intergeneric hybrid) (Hands, 2006). The first orchid hybrid in the world is Calanthe Dominiyi produced in 1856, a cross of Calanthe masuca and Chalanthe furcata (Sheela, 2008). The list of new orchid hybrids is now controlled by The Royal Horticultural Society, England (RHS). Lately, a few hundreds orchid hybrids with commercial values are established annually for orchid industry (Hamdan, 2008). In Malaysia, the Vandaceous hybrids like Dendrobiums and Oncidiums are the most popular cut-flower cultivated since they are easily grown and cultivated in Malaysias climate (Fadelah et al., 2001).

In the floriculture industry, the demand on orchid species and orchid hybrids is very high from all over the world. The demand on orchids is high due to their aesthetic values (exotic and limited sources) including colour, scent appearance and morphology. In orchid industry, the price of scented orchids is often higher compared to scentless orchids (Eric Kok, Manager, Malaysian Orchids Sdn. Bhd., pers. comm. on 20th May 2008).

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2.2 Vanda Mimi Palmer and Its Parents

Vanda Mimi Palmer is a cross between Vanda tessellata and Vanda Tan Chay Yan (Motes, 1997) (Figure 1). The special characteristic of Vanda Mimi Palmer compared to other orchid hybrids is the fragrance characteristic. Vanda Mimi Palmer has won a few international awards for its strong sweet fragrance such as the Champion Award for Fragrant Orchid organized by the Royal Horticultural Society of Thailand in 1993 and the Best Orchid Fragrance in the 17th World Orchid Conference in 2002 (Nair and Arditti, 2002).

Vanda Mimi Palmer could have inherited its fragrance and colour characteristics from Vanda tessellata, an epiphytic orchid from Sri Lanka, India and Burma (Kaiser, 1993; Motes, 1997). Vanda tessellata has inflorescences of 25cm to 30cm long and grey-greenbrown flowers. The lip is white at the side and violet purple in the middle. The shape of Vanda Mimi Palmers flower closely resembles Vanda Tan Chay Yans which is a hybrid of Dutch Vanda Josephine and Vanda dearei (Yeoh, 1978). Vanda Tan Chay Yan has round and flat petals and sepals. This hybrid has won many awards such as First Class certificate (the highest award of the Royal Horticultural Society) in 1954, the Trophy for The Best Vanda at the Second World Conference in Hawaii and numerous Singapore and Malayan Prizes. In 1960s, this hybrid lost its popularity as commercial cut orchid due to its disability to flower more than twice a year (Yeoh, 1978).

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Vanda tessellata

Vanda Tan Chay Yan

Vanda Mimi Palmer

Figure 1: Vanda Mimi Palmer and Its Parents. Vanda Mimi Palmer is a hybrid of Vanda tessellata (adapted from Hamdan, 2008) and Vanda Tan Chay Yan.

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In Malaysia, the demand for Vanda Mimi Palmer is high due to the fragrance emitted by its flower rather than its beautiful colour and structure (Eric Kok, Manager, Malaysian Orchids Sdn. Bhd., pers. comm. on 20th May 2008). Floral extracts from Vanda tessellata (one of the parents of Vanda Mimi Palmer) have been used in some local traditional practices for medicinal purposes in India, such as treatment for inflammatory conditions and instilled into the ear as remedy for otitis (Chopra et al., 1956). Besides that, the extract from the leaves in the form of paste is applied to the human body for cooling down a fever (Chopra et al., 1956; Basu et al., 1971). Root extract from Vanda tessellata had also been used for rheumatism treatment, fever, dyspepsia, bronchitis and also nervous problem (Kirtikar and Basu, 1975). A scientific study on the extracts of Vanda tessellata has shown inflammatory property against acute inflammation induced by carrageenan, serotonin and formaldehyde (Suresh Kumar et al., 2000). Besides that, alcohol extract from Vanda tessellata has shown an enhancement of male sexual activity in normal mice (Suresh Kumar et al., 2000). Thus, Vanda Mimi Palmer might have some medicinal properties as Vanda tessellata since half of the gene pool of Vanda Mimi Palmer was derived from Vanda tessellata.

2.3 The Biological Importance of Floral Scent

In general, floral buds do not have scent, and the fragrance characteristic of a flower appears during anthesis as the petals open (Schade et al., 2001). Floral scent emission patterns vary among species. Some flowering plants such as Citrus medica and Odontoglossum constrictum emit scent primarily during day time meanwhile some other

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plants such as Petunia hybrida and Clarkia breweri emit their scent at the highest level during night time (Altenburger and Matile, 1988). Floral scent emission patterns are different among species due to the control of cicardian clock, photoperiod and also adaptation to specific pollinators active time (Verdonk et al., 2003).

Floral scent is one of the factors that attract pollinators to help in pollination. The pollinator varies among plant species including birds, insects and bats. Some flowering plants need very specific pollinators for their pollination (Dobson, 1994) as they are attracted to specific odors or scent emitted by flowering plants. For example, beetles are attracted to flowers that have musty, spicy and fruity odors (Kaiser, 1993; Frowine, 2005) while bees and flies are attracted and help in pollination of sweet scented flowers that can be detected by human nose (Reinhard et al., 2004).

Besides pollination purpose, some plants emit volatiles such as monoterpenes, sesquiterpenes and hormones such as salicylic acid, jasmonic acid and ethylene from their vegetative tissues to defend themselves against pathogenic microorganisms and insects attack (Arimura et al., 2005; Wei et al., 2007). Volatiles produced by some green leaves have been reported to reduce bioactivity and performance of herbivores and sometimes have antifungal activity (Kaori et al., 2006).

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2.4 The Economic Importance of Floral Scent

Floral scent or flower fragrance is very important in perfumery, cosmetic, agricultural and cut flower industries. Flower fragrance produced by flowering plants such as jasmine, roses, and lavender are pleasant to human sensory system and have potential application as perfume ingredients (Rees, 1991). The high demand on floral fragrances for perfumery and food industries has caused researchers to focus on fragrance-related biochemical compounds and their biosyntheses (Knudsen et al., 1993). The knowledge on natural floral fragrances and their specific components are used in perfume production to produce synthetic perfumes and mimic the natural floral fragrance (Verdonk et al., 2003). An example of the highly commercialized floral fragrances in perfumery industry are rose (Rosa hybrida) (Zuker et al., 1998; Guterman et al., 2002) and jasmine (Jasminum grandiflorum) (Kaiser, 1993).

In agricultural industry, pollination of crops is very important for fruit development. The highest yield can be obtained whenever the highest pollination occurs in field with the help of pollinators. Domesticated crops from other parts of the world might not be suitable for local pollinators due to drastic changes of morphology and biochemistry of the plants (Pichersky and Dudareva, 2007). Commercialization of the plants might also be prevented by the lack of natural pollinators. Domestication of natural pollinators of the plant into other territory is also not usually successful due to the lack of ability of the pollinators to adapt to the new environment (Buchmann and Nabhan, 1996). Scent

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engineering of local and new introduced plant species into new territory might enhance pollination by local pollinators (Pichersky and Dudareva, 2007).

In cut-flower industry which is known as a multi-billion dollar industry, extensive work on breeding of cultivated flowers to improve their vase life, shipping characteristics, and visual aesthetic values such as shape and colour has contributed to the lost of their original scent (Vainstein et al., 2001). Genetic engineering approach by transformation of selected genes for the selected traits might restore the original scent in the plants. Besides that, the production of scent in scentless flowering plants or modification of floral scent can also be done by genetic engineering to increase the commercial values of the flower in cut-flower industry (Pichersky and Dudareva, 2007).

2.5 Floral Scent and Its Volatile Compounds

The scent of scented flowers varies between species due to the combination of the compositional and the level of each compound (Knudsen et al., 1993; Dudareva et al., 2000). The entire floral organs are involved in floral scent emission but petal is the main source of floral scent in most flowers (Pichersky et al., 1994). Floral scents are stored in special oil glands such as trichome before released to the air as volatiles (Effmert et al., 2006). Analysis on volatile compounds in floral scent by headspace with gas chromatography-mass spectrometry (GC-MS) method has led to the discovery of more than 1700 chemical structures (Knudsen and Gershenzon, 2006). Floral scent is a complex mixture of low molecular mass molecules such as monoterpenes,

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sesquiterpenes, benzenoids, phenylpropanoids and fatty acid derivatives (Knudsen et al., 1993). Besides that, there are also other compounds in floral scents such as nitrogen and sulfur containing compounds including indole, a compound from amino acid metabolism (Knudsen and Gershenzon, 2006).

In floral scent studies, more than 500 terpenoid compounds have been identified including monoterpene (C10), sesquiterpene (C15), diterpenes (C20) and irregular terpenes (Knudsen and Gershenzon, 2006). Examples of monoterpenoid compounds identified in floral scent studies are linalool, ocimene, mycrene, nerol, citranellol and geraniol. Linalool compound has been identified in floral scent of snapdragon (Antirrhinum majus) (Nagegowda et. al, 2008), Clarkia breweri (Raguso and Pichersky, 1995), Arabidopsis thaliana (Chen et al., 2003) and a lot of orchid species such as Dendobium beckleri, Dendobium brymerianum, and Phalaenopsis violacea (Kaiser, 1993). Other

monoterpenoid compounds such as mycrene and ocimene were detected in the scent of Anthirrinum majus (Dudareva et al., 2003), Nicotiana suaveolens (tobacco) (Raguso et al., 2003), Arabidopsis thaliana (Chen et al., 2003) and also in some orchids such as Platanthera chlorantha, Polystachya cultriformis and Zygopetalum crinitum (Kaiser, 1993).

Besides monoterpenoid compounds, sesquiterpenoids such as germacrene D, farnesene, caryophyllene, copaene and nerolidol were also detected in floral scent of many plant species. Germacrene D has been detected in the scent of Rosa hybrida (HendelRahmanim et al., 2007), Petunia hybrida (Verdonk et. al, 2003), and some orchids such

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as Aerangis confusa, Aerangis biloba, and Dendrochilum cobbianum (Kaiser, 1993). Caryophyllene, another volatile sesquiterpene compound, was also detected in many scented flowers including Arabidopsis thaliana (Chen et al., 2003), carnation (Dianthus caryophyllus) (Schade et al., 2001), and some orchids such as, Cattleya lawrenceana, Cattleya percivaliana, Dendrobium trigonopus and Dendrochilum cobbianum (Kaiser, 1993). Besides germacrene D and caryophyllene, nerolidol is another compound of sesquiterpene found in scented flowers including Antirrhinum majus (Nagegowda et. al, 2008), Nicotiana suaveolens (tobacco) (Raguso et al., 2003) and also in some orchids such as Diaphananthe pulchella, Epidendrum ciliare, Masdevallia estradea and Zygopetalum crinitum (Kaiser, 1993).

The other class of volatile compounds that is highly distributed among scented flowers is benzenoids and phenylpropanoids. So far, more than 300 volatile compounds of this class have been identified in floral scent of plant species which include methylbenzoate, methylsalicylate, phenylacetaldehyde, phenylethyl acetate, benzyl acetate, phenylethanol, eugenol and isoeugenol (Knudsen and Gershenzon, 2006). Methylbenzoate has been detected in some scented flowers such as Petunia hybrida (Verdonk et. al, 2003), Antirrhinum majus (Nagegowda et. al, 2008), Stephanotis floribunda (Pott et al., 2002) and also in some orchids such as Dendrobium trigonopus, Encyclia baculus and Laelia perinii (Kaiser, 1993). Benzyl benzoate has been identified to be emitted by floral organs of some scented flowers including Petunia hybrida (Verdonk et. al, 2003), Nicotiana suaveolens (Raguso et al., 2003), Stephanotis floribunda (Pott et al., 2002) and also in some orchids such as Dendrobium moniliforme, Dendobium monophyllum, Dendrobium

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williamsonii, and Dendrochilum cobbianum (Kaiser, 1993). Eugenol and isoeugenol are compounds of benzenoid and phenylpropanoid classes which also contribute to the floral scent of scented flowers including Petunia hybrida (Verdonk et. al, 2003), Clarkia breweri (Raguso and Pichersky, 1995), Stephanotis floribunda (Pott et al., 2002) and also some orchids such as Angraecum bosseri, Himantoglossum hircinum, Lycaste aromatica, Phalaenopsis violancea, and Platanthera bifolia (Kaiser, 1993).

Other important class of floral scent compounds is fatty acid derivatives which are derived from lipoxygenase pathway such as hexanol, hexanal, nonanal, pentadecane, decanal and dodecanal (Knudsen and Gershenzon, 2006). Volatile of fatty acid derivatives are normally detected in vegetative tissues and play an important role in plant defense. Traces of fatty acid derived compounds have been detected in floral scent of some scented flowers (Knudsen and Gershenzon, 2006). In the floral scent of Petunia hybrida, some of the fatty acid derivatives that were detected included decanal, dodecanal, 3-hexenal and 2-hexenal (Verdonk et al., 2003). Besides that, some orchids were also reported to emit fatty acid derivatives such as octanal, 2-heptanol, nonanal, decanal, methyl decanoate and ethyl decanoate (Kaiser, 1993; Hsiao et al., 2006).

2.6 The Fragrance Biosynthetic Pathway and Molecular Biology of Floral Scent

To date, many volatile compounds have been identified but only few enzymes and genes involved in the fragrance biosynthetic pathways have been characterized. Therefore, the mechanisms of fragrance formation are still not fully understood (Dudareva et al., 2000).

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Most of the volatile compound analyses have been done by using gas chromatographymass spectrometry (Guterman, 2002). For many years, research on floral scent was focused on its chemical rather than biological aspects due to the complexity of the studies involved in volatile emission (Vainstein et al., 2001). There are three major biosynthetic pathways involved in floral scent production which are terpenoid, benzenoid/ phenylpropanoid and lipoxygenase pathways (Croteau and Karp, 1991). Common modifications such as hydroxylation, acetylation and methylation have been described even though the pathways leading to the final products have not been fully characterized (Guterman et al., 2002).

Terpenoid compounds are synthesized via the terpenoid pathway (Figure 2) which is localized in both the plastid and cytosol (McCaskill and Croteau, 1995; Lichtenthaler, 1999; Rohmer, 1999). There are two initial pathways in the terpenoid pathway; Methylerythritol Phosphate (MEP) Pathway in the plastid (Lichtenthaler, 1999; Rohmer, 1999) and Mevalonic Acid (MVA) Pathway in the cytosol (McCaskill and Croteau, 1995). Both pathways play an important role in the production of dimethylallyl diphosphate (DMAPP) and its isomer isopentenyl diphosphate (IPP) (McCaskill and Croteau, 1995; Lichtenthaler, 1999; Rohmer, 1999). Condensation of one DMAPP molecule with one IPP molecule generates the production of geranyl diphosphate (GPP) which is the main precursor for monoterpenes such as linalool, ocimene, and mycrene (Ogura and Koyama, 1998; Poulter and Rilling, 1981) meanwhile condensation of a DMAPP with two IPP molecules generates the production of farnesyl diphosphate (FPP) which is the main precursor for sesquiterpenes such as caryophyllene, germacrene D and

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Figure 2: Terpenoid Biosynthetic Pathway in Plastid and Cytosol. This diagram was adopted from Nagegowda et al., 2008. Abbreviations: DMAPP, dimethylallyl diphosphate; GA-3P, glyceraldehydes-3phosphate; DXP, 1-deoxy-D-xylulose-5-phosphate; DXR, DXP reductoisomerase; MEP, 2-C-methyl-D-erythritol-4-phosphate; DXS, DXP synthase; FPP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; GPP, geranyl diphosphate; GPPS, geranyl diphosphate synthase; GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl diphosphate synthase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGR, 3hydroxy-3-methylglutaryl-CoA reductase; IPP, isopentenyl diphosphate; MVA, mevalonic acid.

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nerolidol (McGarvey and Croteau, 1995). At present, several fragrance-related genes, cDNAs and enzymes responsible for the production of terpenoid have been isolated and characterized from several plants such as linalool synthase from Clarkia breweri (Pichersky et. al, 1995), Arabidopsis thaliana (Chen et. al, 2003), Antirrhinum majus (Nagegowda et. al, 2008), ocimene synthase from Antirrhinum majus (Dudareva et. al, 2003), mycrene synthase from Antirrhinum majus (Dudareva et. al, 2003) and germacrene D synthase from Rosa hybrida (Guterman et al., 2002).

Benzenoid and phenylpropanoid pathway (Figure 3) is another fragrance biosynthetic pathway identified in the plant system besides the terpenoid pathway. Benzenoids and phenylpropanoids are derived from phenylalanine as the main precursor (Gang et al., 2001). The pathway starts with the deamination of L-phenylalanine to trans-cinnamic acid catalyzed by L-phenylalanine ammonia lyase (PAL), followed by shortening of the C2 unit of the side chain of cinnamic acid for formation of benzaldehyde compound via a few proposed pathways such as CoA-dependent -oxidative, CoA-independent-non-oxidative pathway or a combination of these two pathways (Boatright et al., 2004). The benzenoid pathway is proceeded with the oxidation of benzaldehyde to benzoic acid which is the main precursor for the formation of volatile benzenoids and phenylpropanoids such as benzylbenzoate, benzylacetate, methybenzoate, and

phenylethyl acetate (Boatright et al., 2004). There are other phenylpropanoids emitted in scented plants such as eugenol, isoeugenol, and methyleugenol which are synthesized via other routes without going through benzoic acid as an intermediate. Coniferyl alcohol and coniferyl acetate play a role in this other routes as intermediates with cinnamic acid as the

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Figure 3: Benzenoid and Phenylpropanoid Biosynthetic Pathway. This chart was adopted from Pichersky and Dudareva, 2007. It represents a compilation of reactions and enzymes in several scented plants including Clarkia breweri and Petunia hybrida. Abbreviations: BEAT, acetyl-coenzyme A:benzylalcohol acetyltransferase; BPBT, benzoyl-CoA:benzylalcohol/2-phenylethanol benzoyltransferase; BSMT, benzoic acid/salicylic acid carboxylmethyltransferase; BZL, benzoate:CoA ligase; CFAT, coniferyl alcohol acyltransferase; EGS, eugenol synthase; IEMT, S-adenosyl-Lmethionine:(iso)eugenol O-methyltransferase; IGS, isoeugenol synthase; PAAS, phenylacetaldehyde synthase; PAL, phenylalanine ammonia-lyase; SAMT, salicylic acid carboxyl methyltransferase.

23

main precursor for the production of these compounds (Boatright et al., 2004: Pichersky and Dudareva, 2007).

There are other volatile phenylpropanoid compounds derived from phenylalanine without going through cinnamic acid such as phenylacetaldehyde and phenylethanol (Boatright et. al, 2004). The pathway starts with the transamination of phenylalanine to phenylpyruvate followed by decarboxylation to phenylacetaldehyde (Vuralhan et al., 2003; Boatright et al., 2004). Reduction of phenylacetaldehyde produces phenylethanol while oxidation of phenylacetaldehyde will lead to the production of an ester phenylacetate (Erlich 1907; Vuralhan et al., 2003). Phenylacetaldehyde synthase (PAAS) plays an important role in the decarboxylation of phenylalanine to phenylacetaldehyde (Boatright et al., 2004; Kaminaga et al., 2006). PAAS has been reported to be a cytosolic homotetradimeric enzyme that belongs to group II pyridoxal 5-phosphate-dependent amino acid decarboxylase (Sandmeier et al., 1994). In floral scent studies, two PAAS have been identified from Petunia hybrida (PhPAAS) and Rosa hybrida (RhPAAS) that share 64% identity (Kaminaga et al., 2006). PhPAAS and RhPAAS were reported to share ~50-60% identity with other plant decarboxylases such as tyrosine decarboxylases, tryptophan decarboxylases and aromatic amino acid decarboxylases (Kaminaga et al., 2006).

The third pathway related to fragrance biosynthesis is lipoxygenase pathway. The products of this pathway are derived from C18 fatty acids (linoleic and linoleic acids) which are cleaved into C6 and C12 components by hydroperoxide lyase (Feussner and

24

Wasternack, 1998). Hydroperoxide lyase produces either 3-cis hexenal or hexanal which are the common constituents of volatiles in green leaf or flower depending on the C18 substrate (Knudsen et al., 1993). This 3-cis hexenal or hexanal can be further converted to alcohols (3-cis-hexenol or hexanol) or 3-hexenyl acetate (DAuria et al., 2002).

2.7 Floral Scent Studies on Orchids

Floral scent studies on orchids was initiated in early 1980s. However, early work was focused on detection of volatile compounds emitted by orchid flowers using gas chromatography-mass spectrometry (GC-MS) method (Kaiser, 1993). Identification of floral scent compounds in orchids has led to extensive biochemical and molecular studies of other scented plants such as Clarkia breweri and Petunia hybrida. However, the floral scent biosynthetic pathways of orchids are still far from understood (Hsiao et al., 2006).

Identification of volatile compounds has been carried out on the scent of more than 180 orchid species and hybrids including Cattleya araguaiensis, Cymbidium formosanum, Dendrobium carniferum, Dendrobium superbum, Oncidium curcutum, Phalaenopsis violacea, Vanda tessellata (Kaiser, 1993) and Phalaenopsis bellina (Hsiao et al., 2006) by GC-MS. Based on the GC-MS analysis on the orchids scent, monoterpenoids and sesquiterpenoids are the highly distributed compounds among orchid species. Some of those include linalool, mycrene, ocimene, germacrene D and nerolidol. Benzenoids and phenylpropanoid compounds that are also found in scented orchids include methylbenzoate, benzyl benzoate, benzyl acetate, phenylethyl acetate, eugenol and

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isoeugenol. Besides that, there were traces of other compounds detected in scented orchids such as fatty acid derivatives, indole and formanilide (Kaiser, 1993; Hsiao et al., 2006).

In Vanda tessellata (one of the parents of Vanda Mimi Palmer), more than 20 volatile compounds have been identified in its scent including linalool, mycrene, ocimene, methyl benzoate, methyl isobutyrate, cinnamic aldehyde, cinnamic alcohol, methyl cinnamate, benzyl acetate, phenylethyl acetate and indole. Methylbenzoate was the highest volatile compound emitted by Vanda tesellata representing 61.5% of the total scent, followed by linalool (23%), cinnamic aldehyde (5.1%) and methyl cinnamate (4.6%). Other minor compounds identified in the scent of Vanda tessellata were methyl isobutyrate, methyl 2methylbutyrate, -pinene, mycrene, ocimene, benzyl acetate, methyl salicylate, 3phenylpropanoid, cinnamic aldehyde, -ionone, cinnamic alcohol and indole (Kaiser, 1993).

On the molecular biology aspect, only recently a group of researchers from Taiwan have reported expressed sequence-tags (ESTs) on a scented orchid species Phalaenopsis bellina (Hsiao et al., 2006). Isolation and identification of putative fragrance-related cDNAs was achieved by comparing floral ESTs sequences of Phalaenopsis bellina with ESTs of a non-scented orchid, Phalaenopsis equestris. From their work, the monoterpenes biosynthetic pathway of linalool, mycrene and geraniol was elucidated using the bioinformatics approach, Pathway and Literature (PAL) finder program. From the ESTs of Phalaenopsis bellina, several fragrance-related cDNAs that encode for

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geranyl diphosphate synthase, epimerase, lipoxygenase, diacylglycerol kinase, Omethytransferase, and cytochrome P450 monooxygenase were isolated. The only fragrance-related cDNA which has been well characterized in orchids is geranyl diphosphate synthase (PbGDPS) from Phalaenopsis bellina (Hsiao et al., 2008). Realtime RT-PCR analysis has shown that the expression of PbGDPS gene increased gradually once the bud open and reached the highest peak on the fifth-day after budopening. After that, the expression decreased gradually until the end of the flowers life. The same expression pattern was reported on the emission of monoterpene compounds such as linalool and geraniol. Protein characterization has shown the PbGDPS enzyme is a homodimeric enzyme which can catalyze the formation of both geranyl diphosphate (GDP) and farnesyl diphosphate (FPP) which are the main precursors for the production of monoterpenoids and sesquiterpenoids, respectively.

2.8 Floral Scent Studies in Vanda Mimi Palmer

Preliminary work on scent analysis of Vanda Mimi Palmer was carried out by nose detection during day time from 8.00am to 5.00pm and at different floral developmental stages (Janna et al., 2005). The study shows that emission was at the highest level between 12-2pm when the flower was fully-open. Subsequent molecular biology work on Vanda Mimi Palmer was focused on the construction of a floral cDNA library of Vanda Mimi Palmer representing almost all mRNA transcripts of fully-open flower at different time points in a 24-hour cycle and at different flower developmental stages such as early bud (green), mature bud (red), half-open flower and fully-open flower (Chan, 2009; Chan

27

et al., 2009).Two fragrance-related cDNAs were isolated from a preliminary sequencing of 100 clones from the floral cDNA library. The transcripts are 1-deoxy-D-xylulose 5phosphate reductoisomerase (accession number: EU145744) (Chan et al., 2009) and lipoxygenase (Chan, 2009). Besides that, a suppression subtraction hybridization (SHH) was also carried out to isolate more fragrance-related cDNAs by hybridizing the substracted open flower cDNA library with two different cDNA probes of open flower and bud stage during day time separately. From the SSH work, another two fragrancerelated cDNAs were successfully isolated which are sesquiterpene synthase (accession number: EU145743) and alcohol acyltransferase (accession number: EU145742) (Chan, 2009).

Molecular characterization has been carried out on the 1-deoxy-D-xylulose 5-phosphate reductoisomerase, by subjecting it to a full-length cDNA isolation and gene expression analysis by real-time RT-PCR. The expression studies were carried out in different tissues, at different floral developmental stages and at different time points in a 24-hour cycle. Besides that, other fragrance-related cDNAs such as sesquiterpene synthase and alcohol acyltransferase were also characterized in the same manner (Chan, 2009). Characterization of the fragrance-related transcripts showed upregulated expression in floral tissues especially in the petal and sepal compared to vegetative tissues such as leaf, shoot and root. Expression analyses of the fragrance-related transcripts at different developmental stages and different time points in a 24-hour cycle has shown that fragrance biosynthesis in Vanda Mimi Palmer is developmentally and rhythmically regulated (Chan, 2009; Chan et al., 2009).

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CHAPTER 3 MATERIALS AND METHODS

3.1 Plant Material

Orchid plants (Vanda Mimi Palmer and Vanda Tan Chay Yan) used for this study were purchased from the United Malaysian Orchids Sdn. Bhd., a nursery located in Rawang, Selangor. The purchased plants were maintained in the nursery by Mr. Eric Kok. Both Vanda Mimi Palmer and Vanda Tan Chay Yan used in this study were grown separately in pots with charcoal under tropical climate (12 hours in light followed by 12 hours in dark), temperature between 25-300C and exposed to 70-80% of sunlight. Samples for volatile analysis such as flowers and buds were directly captured from the orchid plants without detaching the flowers. For essential oil extraction, the flowers were directly processed after being detached from the mother plants without freezing in liquid nitrogen. Meanwhile, samples for RNA work such as flowers, buds, leaves, shoots and roots were detached from the mother plant, frozen in liquid nitrogen and stored in -800C before use. All of the plants used for volatile analyses, essential oil extraction as well as RNA extraction were brought to Universiti Putra Malaysia and placed outside of laboratory building with almost similar condition to the nursery at Rawang (not directly exposed to the sun) to ensure growth as well as scent biosynthesis and emission are similar to when the plants are grown in the nursery.

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3.2 Analysis of the Scent of Vanda Mimi Palmer by GC-MS

Determination of the constituents of the scent of Vanda Mimi Palmer was carried out by gas chromatography-mass spectrometry (GC-MS) to identify the volatile compounds emitted by Vanda Mimi Palmer at different developmental stages and also at different time points in a 24-hour cycle. Emission analysis of the floral scent of Vanda Mimi Palmer was carried out by GC-MS at three different floral developmental stages; bud, half-open flower and fully-open flower. Temporal emission analysis was carried out by GC-MS for every two hours interval (12am, 2am, 4am, 6am, 8am, 12pm, 2pm, 4pm, 6pm, 8pm and 10pm) in a 24-hour cycle to determine the emission pattern of each single compound. Besides that, GC-MS analysis on open flower of Vanda Tan Chay Yan was carried out in order to compare with the volatiles emitted by Vanda Mimi Palmer. All of the volatile emission analyses were carried out in three replicates using flowers from different mother plants. Average of the three replicates of the volatile analysis in a 24hour cycle was used to plot graphs with the error bar showing the standard error for the three replicates.

Volatiles emitted by single flower were captured by Solid Phase Micro-Extraction (SPME) (Supelco, USA). The SPME with silica fiber that was coated with 100m polydimethylsiloxane (PDMS) was used to absorb the volatiles emitted by the flowers of Vanda Mimi Palmer and Vanda Tan Chay Yan. A single flower was put into a modified funnel without detaching the flower from the flower stalk (Figure 4). The back of the funnel was covered with aluminum foil. The SPME holder was pressed to allow the silica

30

SPME holder

Funnel Aluminium foil Flower

Retort stand

Silica fiber of SPME

Figure 4: Solid Phase Micro Extraction (SPME) Used to Capture Volatile Compounds Emitted by the Flower of Vanda Mimi Palmer. The flower was trapped and captured by the SPME for 15 minutes.

31

fiber in the SPME emerge from the SPME syringe and captured volatiles produced by the flower for 15 minutes. After that, The SPME fiber was thermally desorbed for 1 minute at 2500C in an injector port of Shimadzu GC-MS (Shimadzu, Japan) with the port in splitless injector mode. Volatile compounds were separated by using a capillary HP-5 column (50m x 0.32mm, film thickness 1.05m) with helium (21 kPa) as a carrier gas. The GC oven was programmed 450C for 1 minute followed by an increase of 100C per minute to 2800C. The temperature 2800C was extended for 10 minutes. Mass spectra of the eluted compounds were recorded for the m/z value of 30-300. The spectrum given by each compound was compared to the National Institute of Standards and Technology (NIST) spectral library 2002 (Scientific Instrument Services, USA).

3.3 Extraction and Analysis of Essential Oil of Vanda Mimi Palmer

Essential oil was extracted from Vanda Mimi Palmer by soaking 100 grams of open flower of Vanda Mimi Palmer in 800ml hexane for 48 hours. After soaking, the debris was removed from hexane with essential oil by filtering with 1MM Whatman filter paper (GE healthcare, USA). The essential oil was recovered by evaporating in a 250 ml round bottom flask with a neck size of 24/29 using a rotary evaporator machine with water bath set at 450C. This step was repeated until all of the hexane was evaporated from the essential oil. The weight of the round bottom flask was recorded before and after the evaporation process by the evaporator machine. After that, the essential oil in paste form was dissolved in 5ml acetone and then adjusted to 10 part per million (ppm). A volume of 1.5l of the 10ppm essential oil was analyzed by GC-MS using the same protocol as

32

described in section 3.2. The same approach was applied to the flowers of Vanda Tan Chay Yan.

3.4 Isolation of Total RNA

The RNA extraction procedure was based on Yu and Goh method (2000) with minor modifications (Chan et al., 2009). One gram samples of Vanda Mimi Palmer from different tissues/organs (petal, sepal, leaf, root and shoot), at different floral developmental stages (young bud (green), mature bud (red), half-open flower, fully-open flower, and 14-day old flower) and at different time points in a 24 hour cycle (12am, 2am, 4am, 6am, 8am, 12pm, 2pm, 4pm, 6pm, 8pm and 10pm) were ground saperately into fine powder in liquid nitrogen. The ground samples were mixed thoroughly with 20ml of pre-warmed (650C) extraction buffer [100mM Tris-Cl (pH 7.5) containing 20mM EDTA (pH 8), 2M NaCl, 2% (w/v) hexadecyl (cetyl) trimetyl ammonium bromide (CTAB), 1% (w/v) polyvinylpyrrilidone (PVP), and 2% (v/v) -mercaptoethanol]. The mixture was then incubated at 650C for 15 minutes followed by centrifugation at 12,857 x g, 40C for 15 minutes to pellet the cellular debris. After the centrifugation, recovered supernatant was mixed thoroughly with an equal volume of chloroform:iso-amylalcohol (24:1). The mixture was shaken vigorously followed by centrifugation at 12,857 x g, 40C for 15 minutes. After the centrifugation, chloroform:iso-amylalcohol (24:1) extraction was carried out twice to recover the top aqueous phase. Lithium chloride (LiCl) was then added to the recovered top aqueous phase to a final concentration of 2M followed by incubation at 40C overnight. Recovered RNA was then pelleted by centrifugation at

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12,857 x g, 40C for 30 minutes. The RNA pellet was rinsed in cold 80% (v/v) ethanol and air-dried before dissolving in 100l diethyl pyrocarbonate (DEPC)-treated water. The RNA samples were then stored at -200C until further use but no longer than two months. The quantity and quality of the total RNA were determined by spectrophotometric readings at 230nm, 260nm, and 280nm using the Biophotometer (Eppendorf, Germany) and nanospectrophotometer (Implen, Germany). The integrity of RNA was checked by formaldehyde denaturing agarose gel electrophoresis (see section 3.4.1).

3.4.1 Formaldehyde Denaturing Agarose Gel Electrophoresis

Formaldehyde denaturing agarose gel of 1.2% (w/v) was prepared by melting 0.4 gram agarose powder in 30ml of 1X F buffer [20mM MOPS buffer (pH 7) containing 1mM EDTA and 5mM NaOAc]. The melted agarose was allowed to cool to 500C and formaldehyde was added to a final concentration of 6% (v/v). The formaldehyde agarose gel was then poured into a gel cast and allowed to solidify. While waiting for the gel to be solidified, 1g of total RNA was added into sample buffer [1X F buffer containing 50% (v/v) formamide and 6% formaldehyde (v/v)] followed by addition of a final concentration of 0.24X loading dye [0.25%(w/v) bromophenol blue, 0.25% xylene cyanol, 30% (v/v) glycerol] and 1g of ethidium bromide. The sample was then incubated at 650C for 10 minutes and placed on ice immediately before loading into the gel. Gel electrophoresis was carried out in a formaldehyde running buffer [1X F buffer containing 6% (v/v) formaldehyde] at 45 Volts for 1 hour. The agarose gel was destained

34

for 30 minutes in autoclaved DEPC-treated water and viewed using a gel documentation system (Bio-Rad, USA).

3.5 Isolation of PolyA+ mRNA

The PolyATract mRNA Isolation Systems III (Promega, USA) was used to isolate the polyA+ mRNA from total RNA. One miligram of total RNA was made up to a final volume of 500l by addition of nuclease-free water provided in the kit. The RNA sample was then incubated at 650C for 10 minutes. Annealing reaction was then carried out by adding 150pmol biotinylated oligo(dT) probe and Sodium Saline Citrate (SSC) buffer, pH 7 to a final concentration of 0.5X (diluted from 20X SSC provided in the kit) to the RNA solution for the oligomers to anneal with polyA+ mRNA. The mixture was then mixed gently and allowed to completely cool at room temperature.

While waiting for the RNA sample to completely cool, streptavidin-paramagnetic particles (SA-PMPs) (provided in the kit) were washed by gently flicking the bottom of the SA-PMPs tube until the particles were completely dispersed. The SA-PMPs were then captured by placing the tube in a specific magnetic stand (provided with the kit) for about 30 seconds until the SA-PMPs were completely accumulated on the wall of the tube. Recovered supernatant was carefully removed and the SA-PMPs particles were washed three times with 300l of 0.5X SSC buffer (diluted from 20X SSC provided in the kit), each time being captured by magnetic stand as described above. The washed SAPMPs particles were finally resuspended in 100l of 0.5X SSC buffer.

35

The total RNA from the annealing reaction was then added to the washed SA-PMPs. The mixture was then incubated at room temperature for 10 minutes and mixed gently for every 2 minutes. The SA-PMPs particles were then captured again as described above and the recovered supernatant was carefully removed without disturbing the SA-PMPs particles. The SA-PMPs particles were washed four times with 300l of 0.1X SSC buffer (diluted from 20X SSC provided in the kit). Each washing was carried out by gently flicking the bottom of the tube until the SA-PMPs particles were completely dispersed. The final supernatant was removed as much as possible without disturbing the SA-PMPs particles.

These steps were followed by elution of polyA+ mRNA from the recovered SA-PMPs particles. The SA-PMPs particles were resuspended by gently flicking in 100l of RNAse free water. The SA-PMPs particles were then magnetically captured and the eluted mRNA was transferred into a sterile RNAse free microcentrifuge tube. The elution step was repeated by resuspending the SA-PMPs pellet in 200l of RNAse free water. The recovered polyA+ mRNA was precipitated by addition of a final concentration of 0.3M sodium acetate (NaOac) pH 5.2 and 80% (v/v) isopropanol. The precipitation was carried out at -200C overnight. After the overnight incubation, the mixture was centrifuged at 12,857g at 40C for 20 minutes. Recovered mRNA pellet was washed with 75% (v/v) ethanol and air-dried. The mRNA pellet was then dissolved in 20l RNAse-free water and stored in -200C for subsequent use. The quantity and purity of the eluted polyA+ mRNA was determined by spectrophotometric readings using a Biophotometer (Eppendorf, Germany).

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3.6 Double-stranded cDNA Synthesis

Universal Riboclone cDNA synthesis system (Promega, USA) was used for doublestranded cDNA synthesis from mRNA isolated in section 3.5. There are two major parts involved in the cDNA synthesis which are first-strand cDNA synthesis and second-strand cDNA synthesis. First-strand cDNA synthesis was carried out by addition of 1g of oligo (dT) into 2g of mRNA sample. Nuclease-free water (provided in the kit) was added up to a total volume of 15l. The mixture was then incubated at 70 0C for 10 minutes and placed on ice immediately followed by addition of 40 units of RNAsin and 5l of 5X first-strand buffer (provided in the kit) into the mixture. The mixture was heated at 42 0C for 5 minutes followed by addition of 30 units of AMV reverse transcriptase and sodium pyrophosphate to a final concentration of 2mM. The mixture was then incubated at 420C for 60 minutes. The first-strand cDNA synthesized was then used as template for the synthesis of second-strand cDNA.

Second-strand cDNA synthesis was carried out by adding 40l of 2.5X second-strand buffer (provided in the kit), 5g of acetylated Bovine Serum Albumin (BSA), 25 units of DNA polymerase I, and 0.8 units of RNAse H into the synthesized first strand-cDNA. Nuclease-free water was then added up to a total volume of 100l followed by incubation at 140C for two hours. The double-stranded cDNA synthesized was then heated at 700C for 10 minutes to stop the cDNA synthesis. The double-stranded cDNA was then placed on ice and 0.2 unit of T4 DNA polymerase was added into the mixture followed by incubation at 370C for 10 minutes. After the incubation, EDTA with a final concentration

37

of 20mM was added to stop the reaction. Phenol-chloroform extraction was then carried out by addition of equal volume of phenol:chloroform:isoamyl-alcohol (25:24:1) into the synthesized double-stranded cDNA. The mixture was then vortexed briefly, followed by a centrifugation at 15,871 x g at room temperature for 1 minute. After the centrifugation, recovered aqueous phase was precipitated by adding sodium acetate to a final concentration of 0.3M (pH5.2) and two volumes of cold absolute ethanol (pre-chilled at 40C). The precipitation was carried out at -800C for 30 minutes. Centrifugation at 15,871 x g for 15 minutes was then carried out at 40C to pellet the pure double-stranded cDNA. After centrifugation, the supernatant was discarded and the recovered double-stranded cDNA in pellet form was rinsed with 70% (v/v) ethanol. The pellet was then air dried, dissolved in 20l of Tris-EDTA (TE) buffer pH 8 and then stored at -200C until further use.

3.6.1 Quantification of Double-stranded cDNA

The quantity of the double-stranded cDNA synthesized was determined by ethidium bromide plate assay. A volume of 30ml of 0.8% (w/v) agarose gel was prepared in 1X Tris-acetate-EDTA (TAE) buffer, pH 8. The agarose was melted and allowed to cool to ~500C. Ethidium bromide to a final concentration of 0.1g/ml was added into the molten agarose. The agarose mixture was mixed well by swirling and then poured into a 100 mm Petri dish. A few columns and lanes were plotted at the back of the Petri dish before the agarose solution containing ethidium bromide was poured into it. The agarose solution was allowed to solidify.

38

Standard DNA (Lambda DNA) (Fermentas, Canada) used in the ethidium bromide assay were diluted in nuclease-free water. The concentrations of standard used for the ethidium bromide plate assay were 200ng/l, 150ng/l, 100ng/l, 75ng/l, 50ng/l, 25ng/l, 10ng/l and 5ng/l. The standards and the synthesized double-stranded cDNA in section 3.6 were spotted onto the solidified ethidium bromide plate. Both standards and cDNA spotted on the plate were allowed to be absorbed by the agarose placed in a fume hood for about 20 minutes. The ethidium bromide assay was viewed using a gel documentation system (Bio-Rad, USA). The concentration of the cDNA sample was determined by comparing the signal intensity produced by the cDNA with the standard.

3.7 cDNA Library Screening

A floral cDNA library of Vanda Mimi Palmer previously constructed by Miss Chan Wai Sun (Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia) was used for this study. The cDNA library was constructed by using the Zap-cDNA Gigapack III Gold Cloning Kit (Stratagene, USA). The cDNA library represents all mRNAs expressed in Vanda Mimi Palmers flower at all different flowering stages in a 24-hour cycle. The titer of the cDNA library provided was 7.8 x 10 9 pfu/ml. In this study, the floral cDNA library was hybridized with fully-open flower cDNA probe of Vanda Mimi Palmer containing pool of mRNA transcripts expressed by fully-open flower during daylight hours (8am, 10am, 12pm, 2pm, 4pm and 6pm). The clones that gave positive signals were selected as putative fragrance-related cDNA candidates for further characterization.

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3.7.1 Probe Labeling

The double-stranded cDNAs synthesized in section 3.6 was used as templates for probe preparation. The double-stranded cDNA was labelled with biotin by using the NEBlot Phototape Kit (New England BioLabs Inc, USA) according to the manufacturers instruction. The protocol of probe labeling used was based on random priming labeling method. The probe labeling was carried out with 1g double-stranded cDNA as template and nuclease-free water was added to a total volume of 34l. A control reaction was prepared by using 1g of unbiotinylated lambda DNA (Hind III digested lambda DNA) (provided in the kit) as the template. The same condition was applied for the control reaction. Incubation at 970C for 5 minutes was then carried out to break the hydrogen bond between the double-stranded cDNA. The denatured double-stranded cDNA was then placed on ice for 5 minutes followed by the addition of 10l of 5X labelling buffer containing biotinylated random octamers (provided in the kit), 5 units of klenow fragment (3-5exo-) and dNTPs mix a with final concentration of 5mM of dTTP, 5mM dGTP, 5mM dCTP, and 5mM of the mixture of dATP and biotynilated-dATP (Biotin-14dATP) (provided in the kit) and the mixture was incubated at 37 0C for 20 hours. The reaction was then terminated by the addition of EDTA (pH 8) to a final concentration of 20mM. Purification was then carried out by adding two volumes of cold absolute ethanol and 10M lithium chloride (LiCl) to a final concentration of 0.14M and a precipitation step was then carried by incubation at -800C for 30 minutes. The mixture was centrifuged at 15,871 x g, at 40C for 15 minutes to pellet the synthesized probe. The pellet was then

40

washed with 70% (v/v) ethanol. After air dried, the pelleted probe was dissolved in 20l of TE buffer and stored at -200C for further use.

3.7.1.1 Detection of Labeling Efficiency

The probe synthesized was subjected to a serial dilution of 1/5, 1/52, 1/53, 1/54, and 1/55 in nuclease-free water in order to check the labeling efficiency of the probe. One microliter of each diluted probe was spotted onto a Hybond-N+ nylon membrane (Amersham Bioscience, UK) and air dried for 10 minutes. The membrane was then soaked in 0.4N NaOH for 2 minutes followed by three times agitation in 2X SSC buffer for 2 minutes. The membrane was then air dried before continuing with SDS detection method. The SDS detection method was initiated by agitating the membrane in a washing buffer [2.5mM sodium phosphate buffer, pH 7.2 containing 0.5% (w/v) sodium dodecyl sulphate (SDS) and 12.5mM NaCl] for 5 minutes. The membrane was then agitated in 10ml blocking solution [25mM sodium phosphate buffer, pH 7.2 containing 5% (w/v) SDS and 125mM NaCl] for 15 minutes at room temperature. The blocking solution was then added with streptavidin-alkaline phosphatase conjugate (Promega, USA) at a dilution factor of 1/10,000 and agitated for 10 minutes. The blocking solution was then discarded and the membrane was washed twice with washing buffer. The first wash was carried out for 5 minutes while the second wash for 1 hour. The washing buffer was then discarded and the membrane was equilibrated in 10ml of detection buffer [20mM Tris-Cl pH 9.5 containing 20mM NaCl and 2mM MgCl2] for 5 minutes. Then, the membrane was transferred into a transparent plastic bag and was spread evenly with 200l of ready to

41

use CDP-star (Roche, USA), a chemiluminescent substrate. The CDP-star was allowed to equilibrate for 2 minutes and the excess CDP-star and bubbles were removed by rolling a glass rod on the plastic bag. The plastic bag was then sealed, placed in an X-ray cassette followed by an exposure to an X-ray film (Kodak, USA) for 30 minutes. After the exposure, the film was developed in a developer solution (Kodak, USA). The X-ray film was rinsed in distilled water, followed by fixation in a fixer solution (Kodak, USA) in a dark room to visualize the signals on the X-ray film.

3.7.2 Primary Screening

In this study, 500,000 pfu of the floral cDNA library was plated onto 10 NZY (Appendix A) agar plates (150 mm petri dishes) with XL1-Blue MRF cells, a recombinant Escherichia coli host strain provided in the Zap-cDNA Gigapack III Gold Cloning Kit (Stratagene, USA). Plaques were allowed to grow to hairpin size for 8 hours at 37 0C. The plaques were then transferred onto a 147 mm diameter plaque lift nylon membrane (Amersham Biosciences, UK) and hybridized with the fully-open flower cDNA probe. The clones that give positive signals on X-ray film were cored out and used for secondary screening.

3.7.2.1 Preparation of Bacterial Culture for Infection.

A loop of the XL1-Blue MRF cells from a glycerol stock was streaked onto LB agar (Appendix A) containing 12.5g/ml tetracycline. The plate was incubated overnight at

42

370C. After overnight incubation, a colony of the XL1-Blue MRF was picked and inoculated into 5ml of LB broth (Appendix A) with supplements [10mM MgSO4, 0.2% (w/v) maltose]. The culture was incubated at 370C overnight in an incubator shaker, shaking at 200rpm. The overnight bacterial culture was then subcultured into 50ml LB broth with supplements and incubated at 370C in the incubator shaker, shaking at 200rpm until the OD600 reached ~0.5. The bacterial culture was then transferred into two tubes equally and centrifuged at 12,857 x g at room temperature for 2 minutes to pellet the bacterial cells. After the centrifugation, the bacterial pellet was resuspended in half of the original volume with 10mM MgSO4 and the OD600 was then adjusted to 0.5 for infection purpose.

3.7.2.2 Preparation of Filter for Primary Screening

A serial dilution of the floral cDNA library of Vanda Mimi Palmer was carried out in SM buffer [50mM Tris-Cl, pH 7.5 containing 100mM NaCl, 8mM MgSO4.7H2O and 0.002% (w/v) gelatin] from the original stock to produce a titer of 780pfu/l for plating purpose. The diluted cDNA library in a volume of 64.1l (~50,000 pfu) was mixed with 600 l of XL1-Blue MRF cells (OD600 = 0.5) and incubated at 370C for 15 minutes to allow phage particles in the cDNA library to attach to the XL1-Blue MRF cells. NZY top agarose (see Appendix A) at ~370C in a volume of 6.5ml was mixed immediately with the mixture in a 15ml sterile centrifuge tube and then poured onto a NZY plate. The plate was swirled quickly to spread the NZY top agar evenly on the NZY plate before it solidified. The

43

plate was then incubated at 370C in an incubator for 8 hours to allow the formation of plaques. The plate was then stored at 40C at least for 2 hours prior to plaque lift.

3.7.2.3 Transferring the Plaques onto Membrane (Plaque Lift)

Commercial plaque lift Hybond-N+, nylon membrane (Amersham Biosciences, UK) with a diameter of 147 mm was used to transfer the plaques formed on NZY agar plates. The membranes were marked by cutting at three sites asymmetrically. A cut membrane was initially placed onto the plate containing plaques for 2 minutes and the cut sites were marked at the bottom of the plate. A second plaque lift was repeated for the same plate by placing another membrane for 4 minutes, as a duplicate. The membranes were then air dried for 15 minutes. Each of the membrane (plaque side up) was placed onto a 3MM Whatman paper (GE healthcare, USA) pre-wetted with a denaturing solution [1.5M NaCl, 0.5M NaOH] for 2 minutes followed by a neutralization solution [0.5M Tris-Cl buffer, pH 8 containing 1.5M NaCl] for 5 minutes. The membranes were then rinsed in a rinsing solution [0.2M Tris-Cl buffer, pH 7.5 containing 2X SSC) for 25 seconds in the same condition. The membranes were then air-dried for 30 minutes and then baked at 800C for 2 hours. The NZY agar plates were kept at 40C up to two weeks for further use.

3.7.2.4 Pre-hybridization and Hybridization of Membrane

Hybridization was carried out based on the instruction manual of Phototape -Star Detection Kit (New England Biolabs Inc., USA) with modifications. Firstly, baked nylon

44

membranes were pre-wetted in a 2X SSC buffer for 2 minutes. The membranes were then transferred into 15ml of pre-warmed (650C) pre-hybridization buffer and pre-hybridized in a HB-1000 Hybridization machine (Techne, UK) for 30 minutes at 550C. While prehybridizing, a mixture of 1g of biotin-labeled open-flower cDNA probe, 80g of sheared salmon sperm DNA and 10X SSC buffer was denatured in boiling water for 10 minutes. After denaturing, the probe mixture was added into the pre-hybridization solution in a hybridization bottle and hybridization was carried out at 55 0C for 16 hours.

After 16 hours of hybridization, the membranes were washed twice in low stringency condition with 2X washing solution [2X SSC, 0.1% SDS] by agitation at room temperature for 5 minutes. The 2X washing solution was discarded, followed by high stringency wash with 0.5X washing solution [0.5X SSC, 0.1% (w/v) SDS] at 550C for 15 minutes. The high stringency wash was repeated for 30 minutes with fresh 0.5X washing solution in the same manner. After the high stringency wash, the membranes were equilibrated in washing buffer [2.5mM sodium phosphate buffer, pH 7.2 containing 0.5% (w/v) SDS and 12.5mM NaCl] for 5 minutes. Blocking step was then carried out by agitating the membranes in blocking solution [25mM sodium phosphate, pH 7.2 containing 5% (w/v) SDS and 125mM NaCl] for 15 minutes at room temperature. The membranes were then agitated in fresh blocking solution with streptavidin-alkaline phosphatase conjugate (Promega, USA) at a dilution factor of 1/10,000 for 15 minutes at room temperature. The membranes were then washed twice with washing buffer [2.5mM sodium phosphate, pH 7.2 containing 0.5% (w/v) SDS and 12.5mM NaCl]. The first wash was carried out for 5 minutes while the second wash for 60 minutes. The

45

membranes were then equilibrated in detection buffer [20mM Tris-Cl pH 9.5 containing 20mM NaCl and 2mM MgCl2] for 10 minutes. The membranes were then transferred into a transparent plastic bag and 500l of ready to use CDP-star (Roche, USA), a chemiluminescent substrate was added and spread evenly on the membrane. The CDPstar was allowed to equilibrate for 2 minutes and the excess CDP-star and bubbles were removed by rolling a glass rode on the plastic bag. The plastic bag was then sealed, placed in an X-ray cassette followed by an exposure to an X-ray film (Kodak, USA) overnight. After the exposure, the film was developed as described in section 3.7.1.1.

3.7.2.5 Coring of Positive Clones

A light box was used to align back the plates with the positive signals on X-ray film. Each of the positive plaques was cored out into a fresh 1.5ml microcentrifuge tube containing 500l SM buffer and 20l chloroform. The tubes were then vortexed briefly and incubated at room temperature for 30 minutes. After that, the tubes were stored at 40C for further use.

3.7.2.6 Secondary Screening

A set of serial dilution (10 -1, 10-2, 10-3, 10-4, and 10-5) was prepared for the cored out phages from primary screening in SM buffer. A volume of 20l of each phage dilution was mixed with 350l of XL1-Blue MRF cells (OD600=0.5) in a fresh 1.5ml sterile microcentrifuge tube separately. The mixtures were then incubated at 37 0C for 15

46

minutes to allow the phage particles to attach onto the bacterial cells. Subsequently, the mixtures were resuspended with 3.5ml of ~370C NZY agarose top agar and immediately poured onto NZY agar plates. The plates were swirled evenly before the agarose solidified, sealed with parafilm and incubated at 370C for 16 hours. The plates were then kept at 40C for at least 2 hours before transferring the plaques onto membrane. The dilution of 10-3 was chosen to be applied for all of the clones cored out in primary screening because this dilution gave 50 to 100 plaques on each NZY agar plate (100 mm petri dish).

Plaque lifting was carried out in the same manner as described in the primary screening in section 3.6.2.3 with 87mm diameter immobilon membranes (Millipore, USA). The subsequent steps such as pre-hybridization, hybridization, washing and detection were carried out in the same condition as mentioned in section 3.7.2.4. Positive plaque from each plate that represents each positive clone was cored out and resuspended with 500l SM buffer and 20l chloroform in a sterile 1.5ml microcentrifuge tube. The tube was then vortexed briefly and incubated at room temperature for at least 30 minutes before storing at 40C for further use.

3.8 Single Clone In Vivo Excision

Phagemids of the putative positive clones were subjected to in vivo excision. Initially, SOLR cells and XL1-Blue MRF cells were cultured in 5ml LB broth (see Appendix A) with supplement [10mM MgSO4, 0.2% (w/v) maltose] separately. The culture was

47

incubated at 300C with shaking at 200 rpm for 16 hours. The 5ml overnight culture was sub-cultured into 50ml of LB with supplement. The culture was incubated at 30 0C with shaking at 200rpm until the OD600 reached 0.5. The tube containing the culture was then centrifuged at 1000g for 10 minutes to pellet the bacterial cells. The pellet was then resuspended in 25ml of 10mM MgSO4. The OD600 of the resuspended bacterial cells was then adjusted to 1.0.

In vivo excision was initiated by addition of 200l of XL1-Blue MRF cells (OD600 =1.0), 250l of phage (stock from secondary screening in SM buffer with chloform) and 1l of helper phage into 1.5ml microcentrifuge tube followed by incubation at 37 0C for 15 minutes to allow attachment of phage to XL1-Blue MRF cells. The mixture was then added into 3ml of LB broth with supplements in a 15ml centrifuge tube followed by incubation at 370C with shaking at 200rpm for 16 hours. After the incubation period, the cultures were heated at 700C for 20 minutes followed by a centrifugation at 1000 x g for 15 minutes. After the centrifugation, recovered filamentous phages in supernatants were transferred into fresh microcentrifuge tubes and stored at 4 0C for further use.

Plating of the excised phagemid was carried out by mixing 100l of filamentous phages of each clone with 400l of SOLR cells (OD600 = 1.0) separately. The mixture was then incubated at 370C for 15 minutes followed by plating of 200l of the mixture on LB plate containing 100g/ml of ampicillin separately. The plate was incubated at 37 0C for 16 hours. After the incubation period, a single colony grown on the plate was re-streaked on a fresh LB plate containing 100g/ml of ampicillin and incubated at 370C for 16 hours.

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The single colony was used for colony PCR reaction, plasmid mini preparation and also for glycerol stock preparation. The same procedure was applied for all of the clones cored out in secondary screening.

3.9 Reverse-Northern Analysis

Reverse-Northern analysis was carried out in this study to select putative positive clones that show up-regulated expression in fully-open flower stage compared to bud stage of Vanda Mimi Palmer as candidates for fragrance-related cDNAs. Reverse-Northern applied Southern Blotting method whereby PCR product of cDNA inserts were used in the experiment instead of genomic DNA.

Firstly, colony PCR amplification of cDNA insert from each clone was performed. Single colony of each clone grown on LB-ampicillin (100g/ml) agar plates were picked out using a tooth pick and swirled into PCR mix [1X PCR buffer (Invitrogen, USA), 1.5mM MgCl2 (Invitrogen, USA), 0.2M T3 universal primer (5-ATTAACCCTCACTAAAG3), 0.2M T7 universal primer (5-AATACGACTCACTATAG-3), 0.2mM dNTPs (Bioron, Germany), 1 unit Taq DNA polymerase (Invitrogen, USA)]. The PCR conditions were as follow: 1 cycle of pre-denaturation at 950C for 5 minutes, followed by 35 cycles of denaturation at 950C for 15 seconds, annealing at 550C for 45 seconds and extension at 720C for 1 minute, followed by final extension at 72 0C for 5 minutes. The PCR amplification was carried out by using MJ Cycler and Master Cyler (Bio-Rad, USA). The PCR products were then loaded in duplicate on 1.5% (w/v) agarose gel and

49

electrophoresis was carried out at 80 Volts for 40 minutes. The gels were stained in 1g/ml ethidium bromide solution for 1 minute followed by de-staining in distilled water for 30 minutes. The gels were then viewed using a gel documentation system (Biorad, USA). After viewing using the gel documentation system, the gels were used for Southern Blotting (Sambrook and Russell, 2001).

In Southern Blotting, the gels were denatured in 0.4 N NaOH (denaturing solution) for 15 minutes and blotted onto a Hybond-N+ nylon membranes (Amersham Bioscience, UK) through gravitational transfer overnight in 0.4 N NaOH (Sambrook and Russell, 2001). After the overnight transfer, the wells position on agarose gels were marked on the membranes by using a pencil. The membranes were then air dried and baked at 80 0C for 2 hours. The baked membranes were then hybridized with two different probes separately: open flower cDNA probe and bud cDNA probe. The open flower and bud cDNA probes were synthesized separately using NEBlot Phototape Kit (New England BioLabs Inc, USA) as described in section 3.7.1. The hybridization, washing and detection steps were same as mentioned in section 3.7.2.4. The clones that showed stronger signals with open flower probe compared to bud probe of Vanda Mimi Palmer were selected for partial sequencing and further characterization.

3.10 Elimination of Cymbidium mosaic Virus Coat Protein cDNA Transcripts

In preliminary sequencing of 100 clones of the floral cDNA library by Miss Chan Wai Sun, half of the clones in the floral cDNA library were reported to be contaminated by

50

Cymbidium mosaic virus coat protein cDNA transcript. The clones that represent the Cymbidium mosaic virus coat protein cDNA transcript (see Appendix B) were eliminated from the screened clones (section 3.9) by hybridizing the PCR-amplified inserts of all the in vivo excised clones with Cymbidium mosaic coat protein cDNA probe. The membranes used in reverse-Northern analysis in section 3.9 were stripped off by using hot-SDS method as described in the manual of Hybond+ nylon membrane (Amersham Bioscience, UK). Boiled 0.1% SDS (w/v) was poured onto each membrane in a tray and was agitated for 10 minutes. The stripped membranes were re-hybridized with Cymbidium mosaic virus coat protein cDNA probe as described in section 3.7.1.

The template used for probe synthesis was PCR-amplified from plasmid containing Cymbidium mosaic virus coat protein cDNA transcript. The amplification was carried out using gene specific primers (CMV-F 5-AGAGCCCACTCCAACTCCA-3 and CMVR 5-GCAGGCAGAGCATAG AGACTG-3) that amplified a partial cDNA sequence

(650bp) of the Cymbidium mosaic virus coat protein. The PCR reaction was carried out in a final volume of 20l containing PCR mix [1X PCR buffer (Invitrogen, USA) containing 1.5mM MgCl2 (Invitrogen, USA), 0.2M CMV-F primer, 0.2M CMV-R primer, 0.2mM dNTPs (Bioron, Germany) and 1 unit Taq DNA polymerase (Invitrogen, USA)] and 100ng plasmid as initial template. The PCR conditions were as follow: 1 cycle of pre-denaturation at 940C for 3 minutes, followed by 35 cycles of denaturation at 940C for 30 seconds, annealing at 530C for 45 seconds and extension at 720C for 45 seconds, and a final extension at 720C for 5 minutes.

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3.11 Plasmid Mini Preparation

The clones that showed up-regulated expression in fully-open flower stage compared to bud stage of Vanda Mimi Palmer were subjected to plasmid mini preparation as described in Sambrook et al., 1989 with minor modifications. Initially, single colonies of in vivo excised clones were inoculated in 3ml LB broth containing 100g/ml ampicillin. The cultures were then incubated in an incubator shaker at 370C, shaking at 200rpm for 16 hours. After the incubation period, the cultures were centrifuged at 15,294 x g for 1 minute at room temperature. The supernatants were then discarded and the recovered pellets were resuspended in 200l of Solution I [25mM Tris-Cl buffer pH 8 containing 10mM EDTA] followed by incubation on ice for 10 minutes. After that, 400l of Solution II [0.2N NaOH, 0.5% (w/v) SDS] was added into each tube and mixed gently by inverting the tubes for several times followed by 10 minutes incubation on ice. After that, 300l of Solution III [3M sodium acetate pH 4.8] was added into the tubes and mixed completely. The tubes were then incubated on ice for 15 minutes followed by centrifugation at 15,294 x g for 15 minutes at 40C. The supernatant was transferred into fresh microcentrifuge tube and 50g RNAse A was added into the tube. Removal of RNA from plasmid DNA by RNAse A was carried out by incubation at 37 0C for 30 minutes.

After the incubation with RNAse A, 400l of phenol: chloroform: isoamylalcohol (25:24:1) was added into the tube. The mixture was then vortexed completely and centrifuged at 15,294 x g at room temperature for 10 minutes. After that, top aqueous

52

phase was transferred into new fresh microcentrifuge tube followed by precipitation. Plasmid DNA was precipitated by adding 0.1 volume of 3M NaOAc, pH 5.2 and 0.6 volume of isopropanol and this was followed by incubation at -200C for 1 hour. The plasmid DNA was pelleted by centrifugation at 15,294 x g at 40C for 20 minutes. The supernatant was discarded and the pellet was rinsed with 70% (v/v) ethanol. The pellet was air dried and dissolved in 20l Tris-EDTA (TE) buffer, pH 8.

3.12 DNA Sequencing and Sequence Analysis

All of the clones that showed up-regulated expression in open flower stage compared to bud stage of Vanda Mimi Palmer after the removal of Cymbidium mosaic virus coat protein transcripts were sequenced using T3 primer. The sequencing reaction was carried out by Medigene Sdn. Bhd. and First Base Sdn. Bhd., Kuala Lumpur, Malaysia. Vector and adaptor sequences were eliminated before proceeding with sequence analysis. All the sequences were subjected to contigs and singletons analyses using the CAP3 software (http://pbil.univ-lyon1.fr/cap3.php) (Huang and Madan, 1999). The tentative unique genes were compared against the GeneBank non-redundant database at the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) using BLASTX and BLASTN with a cut off value less than 1e -05 and score more than 80. The Expressed Sequence-tags (ESTs) were then classified into functional groups based on their putative functionality (Zhao et al., 2006; Lindqvist et al., 2006).

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3.13 Verification of Fragrance-related cDNA by RT-PCR Analysis

Total RNA samples for the verification of fragrance-related cDNA was extracted from flowers and buds collected at 12.00 noon separately using the same protocol as described in section 3.4. The total RNA samples were used as template for first strand cDNA synthesis. PolyA tail mRNAs in the total RNA were reverse-transcribed into first strand cDNA by using Quantitect reverse transcription kit (Qiagen, Germany) according to the manufacturers instruction. Initially, 1g of total RNA template and 2l of 7X of gDNA wipe buffer (provided in the kit) were transferred into a clean PCR tube followed by addition of RNAse-free water to a total volume of 14l. The mixture was then incubated at 420C for 2 minutes and chilled on ice quickly. The remaining components of the kit were then added into the mixture which include reverse transcriptase enzyme (1l), Quantiscript RT buffer (4l), and RT primer mix (1l). The mixture was then incubated at 420C for 30 minutes followed by incubation at 950C for 3 minutes to inactivate the reverse transcriptase enzyme. The first strand cDNA for each sample was aliquoted into a few microcentrifuge tubes and kept at -200C for storage.

Forward and reverse primers (Table 1) of each selected transcript were designed using Primer 3 software with advance setting including the size of primers between 18-22bp, melting temperature (Tm) of the primers were between 50-600C and the product size ranged between 150-250bp. The primers were synthesized by First Base Sdn. Bhd., Malaysia. Gradient RT-PCR was carried out for each primer set for each clone of interest containing cDNA insert to get the best annealing temperature. The gradient RT-PCR

54

Table 1: Characteristics of Primers Used in Verification. Primers for putative fragrance-related transcripts and endogenous control that were used in RT-PCR.

Target/ Amplicon Length (bp) VMPCyP450 (201bp)

Primers

Primer Sequences 5 GCTGTTTTCATGTCTGGAAGC 3 5 TCCTGTTTGTGACGGCTCTT 3 5 GCAACGCTCTCATGGTTTAT 3 5 AAAAGCCTCGAAAAATCTGA 3 5 GTACACGGAAACGATCACTG 3 5 AACATGCAAGCCAAACATT 3 5- CTCCCGCATTGACCATAAAT-3 5-GGAACCACACCCAAACTCTC-3 5-TTGGATGTCGTGAAGGCAAT-3 5- CAACACAAGAAGATAGCACAGCA-3 5-CGAGGAAGACGAAGAGGAAG-3 5-CGAAAAATAGAACAGAGCCATAG-3 5-GCTGGTTAGGGTGAAGCAT-3 5-AAAAACATAGACAAATGGAGACC-3 5-GGAAAGGAAGAAAAGCAGCA-3 5-CGACACCAAGAAACATCTCC-3 5-TCGCCTTCTCTCATCTCTGAA-3 5- CAAGCCCACGCATAAAAGTA-3 5-GTACACGGAAACGATCACTG-3 5-AACATGCAAGCCAAACATT-3

Optimal Annealling Temperature 530C

Forward Reverse

VMPEST (182 bp)

Forward Reverse

570C

VMPCMEK (202bp)

Forward Reverse

590C

VMP36 (227bp)

Forward Reverse

550C

VMP48 (200bp)

Forward Reverse

530C

VMP59 (221bp)

Forward Reverse

530C

VMP83 (202bp)

Forward Reverse

530C

VMP90 (243bp)

Forward Reverse

590C

VMP96 (231bp)

Forward Reverse

590C

VMPA28 (202bp)

Forward Reverse

590C

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Target/ Amplicon Length (bp) VMPA46 (182bp)

Primers

Primer Sequences 5-GGATGTTCTACGGGTGGAC-3 5-AGAGAGGAGCACAGCTTTATTT-3 5-AAAAGCAGCGGTTTATGAAG-3 5-CCAAACGAAAACTCAGGAAT-3 5 CCGACCGCAAGGAGAGTTAT 3 5 AAGCCACGGAACAAAAACAG 3

Optimal Annealling Temperature 590C

Forward Reverse

VMPA54 (180bp)

Forward Reverse

530C

Elongation factor (507bp) -endogenous control

Forward Reverse

550C

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conditions were as follow: 1 cycle of pre-denaturation at 940C for 3 minutes, followed by 35 cycles of denaturation at 940C for 30 seconds, annealing at 50-600C for 45 seconds and extension at 720C for 45 seconds, and a final extension at 720C for 5 minutes. The best annealing temperature for each primer set for each sequence of interest was chosen based on the highest amount (most intense band) of PCR-amplified products viewed using a gel documentation system (Biorad, USA) with minimum or without primer dimers.

After determining the optimum annealing temperature, RT-PCR was carried out using the same PCR protocol as described above with specific annealing temperature (see Table 1 for the annealing temperature used) for each sequence of interest. The RT-PCR products were analyzed by agarose gel electrophoresis separately and viewed using a gel documentation system (Biorad, USA). After viewing, the agarose gels with RT-PCR product for each clone were transferred onto nylon membranes separately by Southern blot method as described in section 3.9. The membranes were baked at 80 0C for 2 hours. The membranes were then hybridized with specific probe labeled with biotin representing each cDNA of gene of interest. The membranes were hybridized for 16 hours with specific probe separately followed by washing and detection steps as described in section 3.7.2.4. Putative elongation factor, a housekeeping gene cDNA transcript was obtained from Miss Chan Wai Sun and used as an endogenous control for the RT-PCR reaction.

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The probes used for the hybridization were synthesized using NEBlot phototape kit as described in section 3.7.1. The cDNA inserts of each clone were PCR-amplified using T3 (5-ATTAACCCTCACTAAAG-3) and T7 (5-AATACGACTCACTATAG-3)

universal primers prior to the probe synthesis. The PCR-amplified cDNA inserts of each clone were purified using SpinPrep PCR clean-up kit (Novagen, Germany) according to the manufacturers instruction. A volume of 100l of PCR product was mixed completely with 400l of SpinPrep bind buffer A (provided in the kit). The mixture was transferred into a SpinPrep PCR filter with a receiver tube and centrifuged at 15,294 x g for 1 minute. The flow through was discarded and 400l of the SpinPrep bind buffer A was transferred into the filter and centrifuged at 15,294 x g for 1 minute. The PCR-amplified insert at the surface of the filters membrane was washed with 500l of SpinPrep wash buffer B (provided in the kit). The wash step was carried out by a centrifugation at 15,294 x g for 1 minute. The flow through was discarded and the filter was centrifuged again for 2 minutes to remove excess of wash buffer. After that, 30l of pre-warmed (700C) SpinPrep elution buffer C (provided in the kit) was transferred into the filter and allowed to stand for 3 minutes at room temperature. The filter was transferred into a fresh 1.5ml microcentrifuge tube and centrifuged at 15,294 x g for 1 minute. The same procedure was applied to all the clones selected for the verification of putative fragrance-related transcripts.

3.14 Full-length cDNA Isolation of Fragrance-related Transcripts

Full-length cDNA isolation was carried out for three selected putative fragrance-related cDNAs from the verified transcripts in section 3.13 [putative 4-(cytidine 5-diphospho)-258

C-methyl-d-erythritol kinase (VMPCMEK), putative cytochrome p450 protein transcript (VMPCyP450) and an unknown protein transcript (VMPA28)] and a putative fragrancerelated cDNA transcript (putative phenylacetaldehyde synthase (VMPPAAS) isolated by Miss Chan Wai Sun from the floral cDNA library of Vanda Mimi Palmer.

The cDNA templates for full-length cDNA isolation were synthesized using the SMART RACE cDNA Amplification Kit (Clonetech, USA) according to the manufacturers instruction. The templates used for full-length cDNAs isolation were the 5- and 3RACE-Ready cDNAs synthesized from total RNA of Vanda Mimi Palmer at open flower stage collected during day time at 12.00 noon. PCR amplification of 5- region was carried out by using 5- RACE-Ready cDNA while 3- RACE-Ready cDNA was used as template for PCR amplification of 3- region. For 5-RACE-Ready cDNA, 1g of total RNA sample was mixed with 1.7 M of 5-CDS primer A (5'(T)25V N3' ) (N = A, C, G, or T; V = A, G, or C) and 1.7 M of SMART II A oligo (5' AAGCAGTGGTATCAACGCAGAGTACGCGGG3'). Meanwhile for 3-RACE-Ready cDNAs preparation, 1g of total RNA sample was mixed with 1.7 M of 3-CDS primer A (5'AAGCAGTGGTATCAACGCAGAGTAC(T)30V N3' ) (N = A, C, G, or T; V = A, G, or C). The tubes for 5-RACE-cDNA and 3-RACE cDNA were incubated at 700C for 2 minutes followed by chilling on ice before addition of 1X first-strand buffer (provided in the kit), 2mM of DTT (provided in the kit), 1mM of dNTP mix (provided in the kit) and 1l of MMLV reverse transcriptase (provided in the kit) for each tube. The tubes were incubated at 420C for 1.5 hours to synthesize first-stand cDNA. After the incubation period, 100l of tricine-EDTA buffer (provided in the kit) was added into

59

each of the 5-RACE and 3-RACE first-strand reaction products. The 5- and 3-RACEReady cDNAs were then heated at 720C for 7 minutes to inactivate the reversetranscriptase enzyme. The 5- and 3-RACE-Ready cDNAs were then stored at -200C.

PCR amplification of 5- and 3-regions of the selected putative fragrance-related cDNA transcripts was carried out by using the Advantage 2 Polymerase Mix (Clonetech, USA) with gene specific primer for each transcript (Table 2) and universal primer mix (UPM) provided in the SMART RACE cDNA amplification kit (Clonetech, USA) according to the manufacturers instruction with minor modifications. The 5 cDNA fragments of VMPCMEK, VMPPAAS, VMPCyP450 and VMPA28 were PCR-amplified in 50l PCR reaction separately with 1X Advantage 2 PCR buffer (provided in the kit), 2.5l of 5RACE-Ready cDNA, 1X Universal primer A mix (UPM) (see Table 2), 0.2 M of gene specific primer (listed in Table 2), 0.2mM of dNTP mix, and 1X Advantage 2 Polymerase Mix. The gene specific primers were designed manually at the conserved region of the partial sequence of the cDNA transcript by considering the high melting temperature (Tm) in the range of 65-720C with high GC content especially at the 3 end of the primer, the size of the primers designed ranged between 25-30bp and synthesized by Bioneer, Korea. The PCR amplification of 5- cDNA fragments were carried out by pre- denaturation at 940C for 1 minute followed by 35 cycles of denaturaton at 940C for 30 seconds, annealing at 680C for 30 seconds and extension at 720C for 3 minutes. Final extension was carried out at 720C for 5 minutes. The 3 cDNA fragment of VMPCyP450 and VMPA28 cDNAs were amplified by using the 3-RACE-Ready cDNA as the template in the same manner as the 5- RACE PCR.

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Table 2: Primers Sequences for the Isolation of Full-length Transcripts. Primers of fragrance-related transcripts were designed and used to isolate the 5-, 3-ends and ORF. All of the primers except for UPM primers were synthesized by Bioneer, Korea. Meanwhile UPM primers were provided in the Advantage2 polymerase mix (Clonetech, USA).

Target/ Amplicon Length (bp) VMPCMEK 5 region

Primers

Primer Sequences

Optimal Annealling Temperature 680C

VMPCMEK 5 GSP UPM (long primer)

5-GACCCTGTGGACGAAGTTGGCTCTG-3 5-CTAATACGACTCACTATAGGGCAAGCAGT GGTATCAACGCAGAGT-3 5-CTAATACGACTCACTATAGGGC-3 5- CGC TTC TCA GCT TTT CTC CTA ACA ATG GCC T -3 5- TTT TCC TGT TTG TGA CGG CTC TTC TCT GCT CA -3 5- AGCGTGAATCTTCTTGGTACAACCACCTC -3 5-CTAATACGACTCACTATAGGGCAAGCAGT GGTATCAACGCAGAGT-3

UPM (short primer) VMPCMEK ORF VMPCMEK ORF forward VMPCMEK ORF reverse VMPPAAS 5 region VMPPAAS 5 GSP UPM (long primer)

680C

680C

UPM (short primer) 5-CTAATACGACTCACTATAGGGC-3 VMPPAAS ORF VMPPAAS ORF forward VMPPAAS ORF reverse VMPCyP450 5 region VMPCYP450 5 GSP UPM (long primer) 5- GAACTCCAGAAAATGGGCAGCCTTCCCA C-3 5- CTCAGAGTGTTTTGAGTTTCCAACCCAGC T-3 5-CAGTAGTGTCCCTCCCTGCAATAACA-3 5- CTAATACGACTCACTATAGGGCAAGCAGT GGTATCAACGCAGAGT-3 5-CTAATACGACTCACTATAGGGC-3 5-TGAGAGCTAAACAAAACGGGCATCA 3 5- CTAATACGACTCACTATAGGGCAAGCAGT GGTATCAACGCAGAGT-3 5-CTAATACGACTCACTATAGGGC-3 680C 680C

680C

UPM (short primer) VMPCyP450 3 region VMPCYP450 5 GSP UPM (long primer) UPM (short primer)

61

Target/ Amplicon Length (bp) VMPCyP450 ORF

Primers

Primer Sequences

Optimal Annealling Temperature 680C

VMPCyP450 ORF forward VMPCyP450 ORF reverse

5-GCTGCCACTAATGTCTTCTTCCTCAAGCTC C-3 5- GTCTGCACTTCACTTATGGACAACAAACA GAC-3 5-AAATATCCCGCAACCTGTCCCACCT-3 5-CTAATACGACTCACTATAGGGCAAGCAGT GGTATCAACGCAGAGT-3 5-CTAATACGACTCACTATAGGGC-3 5-TGCCGAGCATTTGATGGACGAAAGT-3 5-CTAATACGACTCACTATAGGGCAAGCAGT GGTATCAACGCAGAGT-3 5-CTAATACGACTCACTATAGGGC-3 5-TGTGAGATTAGTTCAATTCTTAGGCACC CCAG-3 5-GCTTGTAGACAGCAACATGCAAGCCAAA CATT-3

VMPA28 5 region

VMPA28 5 GSP UPM (long primer)

680C

UPM (short primer) VMPA28 3 region VMPA28 3 GSP UPM (long primer)

680C

UPM (short primer) VMPA28 ORF VMPA28 ORF forward VMPA28 ORF reverse

680C

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3.14.1 Purification of RACE-PCR Products

The RACE-PCR products were purified by using GeneAll Exspin Combo GP (GeneAll Biotechnology, Korea) according to the manufacturers instruction. The RACE-PCR products were electrophoresed on 1.0% (w/v) agarose gel and in 1X TAE buffer, pH 8 (see Appendix A) at 80 Volts for 30 minutes. The gel was viewed using a gel documentation system (Bio-Rad, USA). The band that showed expected size of PCR product was excised using ethanol-cleaned razor blade. The excised gel slice was weighed and transferred into a new fresh tube and 300l of GB buffer (provided in the kit) was added for each 100mg of the agarose gel slice. The mixture was incubated at 500C for 10 minutes and vortexed for every 2-3 minutes. The mixture in a volume of 700l was then transferred into SV column (provided in the kit) and centrifuged at 15,294 x g for 1 minute. The flow through was discarded and the step was repeated until finished all the mixture. After that, 500l of fresh GB buffer was applied to the SV column and centrifuged at 15,294 x g for 30 seconds. A volume of 700l of washing buffer (Buffer NW) (provided in the kit) was applied into the SV column and centrifuged at 15,294 x g for 30 seconds. The flow through was discarded and the SV column was centrifuged at 15,294 x g for 2 minutes to remove the excess washing buffer. The SV column was then transferred into a fresh new 1.5ml microcentrifuge tube and 30l of elution buffer (Buffer EB) (Provided in the kit) was transferred into the SV column and allowed to stand for 1 minute. A final centrifugation was carried out at 15,294 x g for 1 minute to recover the purified RACE-PCR product. The same procedure was applied for purification of all RACE-PCR products in this study.

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3.14.2 Preparation of Competent Cells

Competent cells were prepared by calcium chloride treatment using Escherichia coli DH5 strain (Sambrook et al., 1989). A single colony of DH5 from an LB (see Appendix A) agar plate was inoculated into 5ml of LB broth (see Appendix A). The LB broth culture was incubated at 370C overnight in an incubator shaker with shaking at 200rpm. A volume of 2ml of the overnight culture was transferred into 20ml fresh LB broth in a 50ml centrifuge tube and incubated in the incubator shaker at 370C with shaking at 200rpm for 2 hours. After the incubation period, the tube was centrifuged at 1000 x g for 5 minutes at 40C. The pellet was resuspended in 10ml of 75mM cold calcium chloride (CaCl2) followed by incubation on ice for 20 minutes. After the incubation period, the bacterial cells were centrifuged at 1000 x g for 5 minutes at 40C. The pellet was resuspended in 2ml of 75mM cold CaCl2, followed by addition of 15% (v/v) glycerol. A volume of 100l of competent cells with 15% (v/v) glycerol was aliquoted into 1.5ml microcentrifuge tubes. The stocks were kept at -800C for future use.

3.14.3 Cloning and Transformation of RACE-PCR Product

The purified RACE-PCR product in section 3.14.1 was cloned into the yTA cloning vector (Yeastern, Taiwan) according to the manufacturers instruction. An amount of 150ng of the purified RACE-PCR product was transferred into a 0.2ml PCR tube followed by addition of ligation components provided in the kit; 1l of ligation buffer A, 1l of ligation buffer B, 50ng of yT&A cloning vector, and 1unit of yT 4 DNA ligase.

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Nuclease-free water was then added up to a total volume of 10l. The ligation reaction was carried out at 40C overnight.

The ligation product was transformed into Escherichia coli DH5 competent cells prepared in section 3.14.2 by heat shock transformation method (Sambrook et al., 1989). A volume of 2.5l of the ligation product was transferred into the 100l of competent cells followed by incubation on ice for 20 minutes. After the incubation period, a heatshock treatment was carried out by incubating the tube at 42 0C for 90 seconds followed by incubation on ice for 1 minute. A volume of 1ml of LB broth (see Appendix A) was added into the tube. The tube was then incubated at 37 0C with shaking at 250rpm for 45 minutes. After that, the tube was centifugated at 15,294 x g for 1 minute and 800l of supernatant was discarded. The pellet was then resuspended in the remaining supernatant. A volume of 100l of the resuspended DH5 cells was spreaded onto LB plate containing 2.5mg of ampicillin (see Appendix A) with 0.8mg of X-gal and 1.4mg of IPTG for blue and white screening. The plate was then incubated at 37 0C incubator oven for 16 hours.

After the 16 hours incubation period, the transformants (white colonies) were re-streaked onto a fresh LB plate containing 2.5mg of ampicillin. The plate was then incubated at 370C incubator oven for 16 hours. A single colony of each transformant was subjected for colony PCR reaction with M13 forward (5' GTAAAACGACGGCCAGT 3') and M13 reverse (5' GCGGATAACAATTTCACACAGG 3') universal primers to select for a transformant with the exact insert size. The colony PCR was carried out as described in

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section 3.8 except for the universal primers used were M13 forward and reverse instead of T3 and T7. The clone with the expected size of PCR product was subjected for plasmid mini preparation (see section 3.11) and sent for sequencing with both M13 forward and reverse primers. The same procedure was applied for all 5- and 3- RACEPCR products.

3.14.4 Isolation of Open Reading Frame (ORF) of the Putative Fragrance-related Transcripts

The open reading frame (ORF) of VMPCMEK, VMPPAAS, VMPCyP450 and VMPA28 were isolated by PCR-amplification using the gene specific primers (see Table 2). The amplifications were carried out in 50l reaction containing 0.2M forward primer (see Table 2), 0.2M reverse primer (see Table 2), 1mM of dNTP mix (Clonetech, USA), 2.5l of 5-RACE-Ready cDNA and 1X Advantage 2 Polymerase Mix (Clonetech, USA). The gene specific primers were designed manually at the conserved region of the partial sequence of the cDNA transcript by considering the high melting temperature (Tm) in the range of 65-720C with high GC content especially at the 3end of the primers. The size of the primers ranged between 30-34bp and synthesized by Bioneer, Korea. The PCR cycling parameters used were as follow; pre-denaturation at 940C for 1 minute followed by 35 cycles of denaturation at 940C for 30 seconds, annealing at 680C for 30 seconds and extension at 720C for 3 minutes. Final extension was carried out at 720C for 5 minutes. The PCR products were electrophoresed on 1.0% (w/v) agarose gel in 1X TAE buffer (see Appendix A) at 80 Volts for 30 minutes. The gel was viewed using a gel

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documentation system (Bio-Rad, USA). The band that showed the expected size of PCR product was excised using ethanol-cleaned razor blade. The PCR products were purified by using GeneAll Exspin Combo GP (GeneAll Biotechnology, Korea) according to the manufacturers instruction (see section 3.14.1). The purified PCR products were cloned into yT&A cloning vector (see section 3.14.3) and transformed into Escherichia coli DH5 competent cells (see section 3.14.3). A transformed colony was grown overnight for plasmid mini preparation (see section 3.11) and sent for sequencing. The sequencing was carried out by Macrogen Inc, Korea.

3.14.5 Sequence Analysis of the Cloned ORF

The ORF sequences of the putative fragrance-related cDNAs were analyzed by using BLASTX program (NCBI GeneBank) (http://www.ncbi.nlm.nih.gov) to search for the homologous sequences. Bioedit software version 7.0.1 (Hall, 1999) was used to translate the cDNA sequences of the putative fragrance-related transcripts into amino acid sequences. The amino acid sequences were then analyzed by BLASTP program (NCBI GeneBank) (http://www.ncbi.nlm.nih.gov). Clustal W multiple alignment program applying BLOSUM62 matrix in the Bioedit software was used to align the amino acid sequences of the putative fragrance-related transcripts with homologous sequences from other plants based on default setting. Expasy program was used to compute the theoretical pI and predict molecular weight (MW) of the proteins (Expasy) (http://br.expasy.org/tools/). Phylogenetic trees were constructed using the Mega version 4 software (Tamura et al., 2007) with Neighbour-Joining method to determine the genetic

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relationship between the deduced proteins of putative fragrance-related transcripts with closely related proteins available in NCBI GenBank database. Unrooted trees were produced with 1000 sample replication. Motifs search and signal peptide prediction was carried out by Localizome software (http://localodom.kobic. re.kr/LocaloDom/index.htm) (Lee et al., 2006), and Expasy tool (http://br.expasy. org/tools/).

3.15 Expression Studies of Fragrance-related Transcript by Real-time RT-PCR

Expression analysis of the putative fragrance-related transcripts (VMPPAAS, VMPCMEK, VMPCyP450 and VMPA28) was carried out by real-time RT-PCR in different tissues, at different developmental stages and different time points in a 24-hour cycle. For expression studies in different tissues, total RNA was isolated from both floral (bud, open-flower, petal, sepal and lip) and vegetative tissues (leaf, shoot, root and stalk). For expression studies at different flower developmental stages, total RNA was isolated from young bud (green), mature bud (red), half-open flower, fully-open flower and 14day old fully-open flower. All the samples for expression analysis on different tissues and developmental stages were collected at 12.00pm in the afternoon since the scent emission of Vanda Mimi Palmer was detected at high level at this time. For expression studies at different time points in a 24-hour cycle, fully open flowers were collected for every 2 hours starting at 8.00 am until 6.00 am on the next day. The procedure used for total RNA extraction from all the above mentioned samples was as described in section 3.4.

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For expression analysis, first-strand cDNA was synthesized from 1g of total RNA using Quantitect reverse transcription kit (Qiagen, Germany) as described in section 3.13. Realtime RT-PCR reaction was carried out by using Brilliant SYBR Green QPCR master mix (Stratagene, USA) with 1l of 10X diluted cDNA template with gene specific primers (150nM forward primer and 150nM reverse primer). The primers used for the real-time expression analysis were designed at the 3 end. The primers were designed using Primer 3 software with advanced setting including size of primers in the range of 20-25bp, melting temperature (Tm) between 55-650C and the product size between 150250bp. The primers were synthesized by Sigma-Aldrich, USA. The primers sequence used for expression studies of gene of interest VMPPAAS, VMPCMEK, VMPCyP450, and VMPA28 are listed in Table 3. Four replicates were prepared for each sample and they are known as technical replicates. Besides that, 4 replicates of negative control known as non-template control (NTC) were included for each real-time RT-PCR reaction using the same mastermix with the gene of interest without any template.

3.15.1 Optimization for Real-time RT-PCR

Gradient real-time RT-PCR was carried out to select the best annealing temperature for each putative fragrance-related transcript without any primer dimers. Gradient real-time RT-PCR was carried out by using iQ5 cycler (Biorad, USA) utilizing the following program: 950C for 10 min; 40 cycle of 950C for 30 sec, 55-650C for 30 sec (100C temperature range) and 720C for 30 sec; 81 cycles for melting curve analysis; 10 sec for each 0.50C (55-950C). Melt curve analyses were carried out for genes of interest and

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Table 3: Primers Sequences for the Real-time RT PCR. Primers were designed at 3 end of the gene of interest and were synthesized by Sigma-Aldrich, USA. The primers were used to analyze the expression of each putative fragrance-related transcript in different tissues, at different developmental stages and different time points in a 24-hour cycle.

Target/ Amplicon Length (bp) VMPCMEK (201bp)

Primers

Primer Sequences 5-GCTGTTTTCATGTCTGGAAGC-3 5-TCCTGTTTGTGACGGCTCTT-3 5-ACGAAATGTTGGGAAATGAATA-3 5-ACTTGCCTTCTCTTGAACCA-3 5-GCAACGCTCTCATGGTTTAT-3 5-AAAAGCCTCGAAAAATCTGA-3 5-GTACACGGAAACGATCACTG-3 5-AACATGCAAGCCAAACATT-3

Annealing Temperature 590C

Forward Reverse

VMPPAAS

Forward Reverse

590C

VMPCyP450 (182 bp)

Forward Reverse

570C

VMPA28 (202bp)

Forward Reverse

590C

Endogenous control / Amplicon length (bp) Actin (236bp) Forward Reverse Alpha tubulin (227bp) Forward Reverse Cyclophilin (200bp) Forward Reverse 5-CAGTGTTTGGATTGGAGGTTC-3 5- CCAGCAGCAGTCAGGAAAA-3 5- CTCCCGCATTGACCATAAAT-3 5-GGAACCACACCCAAACTCTC-3 5-TTGGATGTCGTGAAGGCAAT-3 5CAACACAAGAAGATAGCACAGCA-3 590C 560C 590C

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endogenous controls to check for sequence specificity amplification (see Appendix H). The gradient real-time RT-PCR reaction was also carried out for each reference genes (endogenous controls) using the same protocol as described above for the putative fragrance-related transcript. The endogenous controls selected for the expression studies were actin (Accession no: AF246716), alpha tubulin (Accession no: GW687608) and cyclophilin (Accession no: GU942927). The optimal annealing temperature for transcripts of interest and housekeeping genes are listed in Table 3.

PCR Amplification Efficiency Test

PCR amplification efficiency test was performed on all putative fragrance-related transcripts and reference genes as decribed in the Guide to Performing Relative Quantification of Gene Expression Using Real-time Quantive PCR (Applied Biosystems, 2004). First strand cDNA of fully-open flower of Vanda Mimi Palmer was used for PCR amplification efficiency test. The cDNA was diluted in a series of five fold (50, 5-1, 5-2, 5-3, and 5-4). The PCR amplification was carried out utilizing the following program: 950C for 10 min; 40 cycle of 950C for 30 sec, annealing for 30 sec (see Table 3) and extension at 720C for 30 sec; 81 cycles for melting curve analysis; 10 sec for each 0.5 0C (55-950C). A standard curve was plotted against the log input of template cDNA amount and the CT value of each dilution. The PCR efficiency was estimated by the slope of standard curve (Equation A, in Appendix C), calculated using Equation B (Appendix C). The accepted PCR efficiency is ranged between 90-110% and slope in the range of 3.1-

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3.6 as decribed in the Guide to Performing Relative Quantification of Gene Expression Using Real-time Quantive PCR (Applied Biosystems, 2004).

3.15.3 Expression Analysis of Putative Fragrance-related Transcripts

The relative quantity (Q) of cDNA transcript of each gene was calculated by the software using Equation C (see Appendix C). Normalization expression was carried out by normalizing the relative cDNA quantity of each gene of interest with cDNA of reference genes (endogenous control) using Equation D (see Appendix C) where the relative quantity of all reference genes was used for normalization factor (see Equation E in Appendix C). Finally, the normalized expression level of the target gene was rescaled with the calibrator using Equation F (see Appendix C).

For expression studies at different tissues and developmental stages, bud was used as calibrator while for relative expression studies at different time points, the relative quantity of the transcripts at 12.00am was used as calibrator. The calibrator for each expression analysis was selected as a reference tissue for comparative amount of transcript in each tissue studied whereby the expression of the calibrator is equal to 1. Up-regulated expression refers to the relative amount of the transcript in the tissue that is more than 1 compared to the calibrator while down-regulated expression refers to the relative amount of the transcript in the studied tissue that less than 1 in comparison to the calibrator.

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Expression of each putative fragrance-related transcript was analyzed using iQ5 software (Biorad, USA) by applying CT comparative method. The CT Comparative method was used to estimate the relative expression level of the fragrance-related transcripts. The normalized expression results were plotted in graphs for the relative quantity of the transcripts in different tissues, at different developmental stages and different time points in a 24-hour cycle compared to the calibrator.

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CHAPTER 4 RESULTS

4.1 Biochemical Analysis on Flowers of Vanda Mimi Palmer

4.1.1 GC-MS Analysis of the Scent of Vanda Mimi Palmer

Floral scent compounds of Vanda Mimi Palmer were captured by solid phase microextraction (SPME) and were determined by Gas chromatography-mass spectrometry (GC-MS). GC-MS analysis (Figure 5 and Table 4) shows that the scent of Vanda Mimi Palmer was dominated by terpenoids (linalool and ocimene) as well as benzenoids (methylbenzoate and benzyl acetate) and a phenylpropanoid (phenylethyl acetate). A few other compounds were also detected at low level such as linalool oxide, phenylethanol, nerolidol, indole and formanilide. Determination of the compounds was based on similarity of the mass spectra and the spectrum fragments of the compounds with the compounds in the NIST mass spectral library 2002 with the cut off value of Similarity Index (SI) equal to or more than 90%. Floral scent analyses in a 24-hour cycle in Figures 6 and 7 show that the emission of major terpenoids compounds (ocimene, linalool) as well as benzenoids (methylbenzoate and benzyl acetate) and the phenylpropanoid (phenylethyl acetate) started as early as 6.00am, and increased gradually until it reached the highest peak at 2.00 noon. After that, the emission of the compounds decreased gradually until 6.00pm. None of the volatile compounds was detected from the flowers by GC-MS at night (from 8.00pm until 4.00am). During the highest peak of the scent

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Figure 5: Gas Chromatogram of Volatile Compounds Emitted by Fully-open Flower of Vanda Mimi Palmer. The compounds detected were: (1) ocimene, (2) linalool oxide, (3) linalool, (4) methylbenzoate, (5) phenylethanol, (6) benzyl acetate, (7) formanilide, (8) phenylethyl acetate, (9) indole, and (10) nerolidol.

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Table 4: Volatile Compounds Emitted by Vanda Mimi Palmer with Their Relative Retention Time and m/z Fragments

Peak

Relative retention time (min)

Main spectrum fragments (m/z)

Compound name

Monoterpene 1 2 3 Sesquiterpene 10 Benzenoid 4 5 Phenylpropanoid 6 8 Indole 9 Formanilide 7 12.260 39,52,65,76,93,161 Formanilide 13.084 39,50,63,74,90,117 Indole 10.783 11.926 39,43,65,79,91,108,150 39,43,65,78,91,104 Phenylethanol Phenylethyl acetate 9.702 10.030 51,77,105,136 39,51,65,78,91,105,122 Methylbenzoate Benzyl acetate 16.492 41,43,69,71,93,107,123,136,162 Nerolidol 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

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Emission of Terpenoid Compounds in a 24-hour cycle

2500000 2250000 2000000 1750000
Abundance

ocimene linalool nerolidol linalool oxide

1500000 1250000 1000000 750000 500000 250000 0 12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm

8pm

10pm

Time

Figure 6: Emission of Terpenoids by Fully-open Flower of Vanda Mimi Palmer in a 24-hour Cycle: Ocimene, Linalool, Nerolidol and Linalool Oxide. The volatile compounds of fully-open flower of Vanda Mimi Palmer were captured by solid phase micro-extraction (SPME) before injection into GC-MS injector port. The experiment was carried out for 12 hours in light, followed by 12 hours in dark. The error bars represent the standard deviation of the relative amount of the volatile compounds detected in three replicates of scent analysis.

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Emission of Benzenoid/Phenylpropanoid Compounds in a 24-hour cycle

4000000 3750000 3500000 3250000 3000000 2750000 2500000
methylbenzoate benzyl acetate phenylethyl acetate phenylethanol

Abundance

2250000 2000000 1750000 1500000 1250000 1000000 750000 500000 250000 0 12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm

8pm

10pm

Time

Figure 7: Emission of Benzenoids and Phenylpropanoids of Vanda Mimi Palmer in a 24-hour Cycle: Methylbenzoate, Benzyl Acetate, Phenylethyl Acetate and Phenylethanol (Phenylethyl Alcohol). The volatile compounds of fully-open flower of Vanda Mimi Palmer were captured by solid phase micro-extraction (SPME) before injection into GC-MS injector port. The experiment was carried out for 12 hours in light, followed by 12 hours in dark. The error bars represent the standard deviation of the relative amount of the volatile compounds detected in three replicates of scent analysis.

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emission of Vanda Mimi Palmer (at 2.00pm), the scent of Vanda Mimi Palmer was represented by 34.8% of terpenoids, 62.6% of benzenoids and phenylpropanoids, and 2.6% traces of other compounds including indole and formanilide (Figure 8A). The major terpenoids detected in the scent of Vanda Mimi Palmer which were ocimene and linalool, representing 34% of the total scent (ocimene 16.3% and linalool 17.7%) (Figure 8B). Another terpenoids including linalool oxide (monoterpene) and nerolidol (seisquiterpene) were detected at trace levels during the highest peak of scent emission which represent 0.3% and 0.5%, respectively, of the total scent (see Figure 8B). While for benzenoids and phenylpropanoids, phenylethyl acetate was detected at the highest level representing 26.4% of the total scent of Vanda Mimi Palmer, followed by benzyl acetate (23.7%), methylbenzoate (11.1%) and phenylethyl alcohol (phenylethanol) (1.4%) (see Figure 8B).

Scent analyses of Vanda Mimi Palmer at three developmental stages (bud, half-open flower, and fully-open flower) in Figure 9 show no detectable volatile compounds emitted at bud stage. In the half-open flower stage, emission of terpenoids incuding linalool and ocimene was detected at a very high level compared to other compounds. In the fully-open flower stage, scent emission of Vanda Mimi Palmer is at the highest level. In contrast to half-open flower stage, the benzenoid and phenylpropanoid compounds including benzyl acetate and phenylethyl acetate were detected at the highest level from fully-open flower stage.

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other compounds 2.6%

terpenoids 34.8%

(A)
benzenoids and phenylpropanoids 62.6%

nerolidol 0.5%

(B)
phenylethyl acetate 26.4%

indole 2.2%

ocimene 16.3% linalool oxide 0.3%

linalool 17.7%

benzyl acetate 23.7%

methylbenzoate 11.1% phenylethyl alcohol 1.4% formanilide 0.4%

Figure 8: Percentage of (A) Terpenoid, Benzenoid and Phenylpropanoid, and other Compounds and (B) Each Compound in the Scent of Fully-open Flower of Vanda Mimi Palmer. The pie charts show the percentage of each individual compounds and class of compounds detected in the scent of Vanda Mimi Palmer during the highest peak of scent emission at 2.00pm.

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Figure 9: Comparison of Volatiles Emitted by Vanda Mimi Palmer at Three Different Flower Developmental Stages: (A) bud (B) half-open flower and (C) fullyopen flower. The compounds are; (1) ocimene, (2) linalool oxide, (3) linalool, (4) methylbenzoate, (5) phenylethanol, (6) benzyl acetate, (7) formanilide, (8) phenyletyl acetate, (9) indole and (10) nerolidol.

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The volatile compounds emitted by Vanda Mimi Palmer were compared with compounds emitted by both parents, Vanda Tan Chay Yan (non-scented orchid) and Vanda tessellata (scented orchid) in order to understand the fragrance characteristic of Vanda Mimi Palmer. Emission of some volatile compounds including ocimene, decane and copaene in Vanda Tan Chay Yan (Figure 10) were determined using GC-MS. The same GC-MS analysis was not carried out for Vanda tessellata due to inavailability of the sample source. Thus, a previously reported result by Kaiser (1993) on the scent of Vanda tessellata was used for comparison. In his study, fully-open flower of Vanda tessellata was trapped into a vessel for three hours (11.00 am-2.00 pm) and pumped into 10mg of pure activated charcoal and then dissolved in 10-50l of carbon disulphate followed by 10-30l of ethanol solution before injecting into GC-MS (GC- Carlo Erba FTV 4160 chromatograph, MS- Varian MAT Model 212/CH-5 mass spectrometer with Finnigan INCOS data system) (Kaiser, 1993). A comparison of the volatile compounds emitted by Vanda Mimi Palmer and both parents is shown in Table 5. From the comparison, ocimene was the only compound found to be emitted by Vanda Mimi Palmer and both of its parents. Some of the compounds that were detected in the scent of Vanda Mimi Palmer were also present in one of its parents (Vanda tessellata, a scented orchid) which included benzyl acetate, methylbenzoate and linalool (Kaiser, 1993). Meanwhile, other compounds emitted by Vanda Mimi Palmer included nerolidol, phenylethanol and phenylethyl acetate were not detected in both parents.

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Figure 10: Comparison of Volatiles Emitted by Fully-open Flower of (A) Vanda Mimi Palmer and (B) Vanda Tan Chay Yan. The compounds are: (1) ocimene, (2) linalool oxide, (3) linalool, (4) methylbenzoate, (5) phenylethanol, (6) benzyl acetate, (7) formanilide, (8) phenyletyl acetate, (9) indole and (10) nerolidol (11) decane and (12) copaene.

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Table 5: Comparison of Terpenoids, Benzenoids and Phenylpropanoids Emitted by Vanda Mimi Palmer, Vanda tessellata and Vanda Tan Chay Yan Compound Monoterpene -pinene Linalool Linalool oxide Mycrene Ocimene Sesquiterpene Copaene Nerolidol Vanda Mimi Palmer Vanda Tan Chay Yan Vanda tessellata (Kaiser, 1993)

Benzenoid Benzaldehyde Benzyl acetate Benzyl alcohol Cinnamyl alcohol Methyl benzoate Methyl cinnamate Methyl salicylate Phenylpropanoid Phenylethanol Phenylethyl acetate

Note: The indicates the presence of the compound in the volatiles of the flowers while the absence of the compound is indicated by - .

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4.1.2 Analysis of Essential Oil of Vanda Mimi Palmer and Vanda Tan Chay Yan

Essential oil extraction was carried out on fully-open flower of Vanda Mimi Palmer and Vanda Tan Chay Yan separately in order to understand the scent production and storage in Vanda Mimi Palmer. The essential oil was extracted by soaking the flowers for 48 hours in hexane and followed by GC-MS analysis. The GC-MS detected 63 compounds in the essential oil of Vanda Mimi Palmer and 63 compounds in the essential oil of Vanda Tan Chay Yan (Appendix D, subsection (c) and (d)). Comparison between the compounds detected in the essential oil of both flowers is presented in Table 6. Some of the volatile compounds detected in the scent of Vanda Mimi Palmer (Figure 4 in section 4.1.1) which included linalool, methylbenzoate, phenylethanol, benzyl acetate and phenylethyl acetate were also detected in the essential oil of Vanda Mimi Palmer (see Table 6). Besides that, other intermediate compounds such as benzyl alcohol and benzyl benzoate were also detected in the essential oil of Vanda Mimi Palmer. In addition, another two sesquiterpenes which were germacrene and copaene were detected in the essential oil of Vanda Mimi Palmer. Based on the comparison, phenylethanol compound which was detected in the scent of Vanda Mimi Palmer., was also detected in the essential oil of both Vanda Mimi Palmer and Vanda Tan Chay Yan. Interestingly, the presence of phenylethanol compound in the essential oil of Vanda Mimi Palmer was detected at high level representing 11.18% of the total essential oil (see Table 6). The essential oil analysis shows that the terpenoids represent 2.09% of while benzenoids and phenylpropanoids represent 26.51% of the total essential oil (Figure 11).

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Table 6: Comparison of Terpenoids, Benzenoids and Phenylpropanoids Detected in Essential Oils of Vanda Mimi Palmer and Vanda Tan Chay Yan

Compound

Vanda Mimi Palmer

Vanda Tan Chay Yan

Monoterpene 1-hydroxylinalool Linalool Sesquiterpene Copaene germacrene D (0.17 %) (0.13 %) (0.38 %)

(1.79 %)

Benzenoid Benzylacetaldehyde Benzyl acetate Benzyl alcohol Benzyl benzoate Methylbenzoate Naphthalene Phenylpropanoid Phenylethanol Phenylethyl acetate Vanillin (11.18 %) (0.85 %) (2.21 %) (2.00 %) (1.55 %) (2.02 %) (4.98 %) (0.20 %) (3.37 %) (0.15 %) (3.57 %)

Note: The indicates the presence of the compound in the essential oil of the flowers while the absence of the compound is indicated by - . The full list and percentage of volatiles, semi-volatiles and non-volatiles in the essential oil of both Vanda Mimi Palmer and Vanda Tan Chay Yan can be referred to Appendix D, in subsection (c) and (d).

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terpenoids 2.09% benzenoids and phenylpropanoids 26.51%

other compounds 71.40%

Figure 11: Composition of Terpenoids, Benzenoids and Phenylpropanoids, and Other Compounds in the Essential Oil of Vanda Mimi Palmer.

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4.2 Molecular Studies of Fragrance-related cDNA Transcripts of Vanda Mimi Palmer

4.2.1 Isolation of Putative Fragrance-related cDNAs

4.2.1.1 Floral cDNA Library Screening

Isolation of fragrance-related cDNAs from Vanda Mimi Palmer was carried out by screening the floral cDNA library representing all mRNA transcripts expressed in a 24hour cycle (12 hours in light and 12 hours in dark) and at different developmental stages including bud, half-open flower, and fully-open flower. In a primary screening, 500,000 phages (500,000 pfu) were screened by hybridizing the floral cDNA library with fullyopen flower cDNA probe of Vanda Mimi Palmer representing all mRNA transcripts expressed in fully-open flower between 8.00am to 6.00pm, a period when strong scent emission of Vanda Mimi Palmer was detected (refer Figures 6 and 7). From the primary screening (Figure 12a), 800 plaques showed positive signals on X-ray film by autoradiography detection method. The plaques with positive signals represent mRNA transcripts that are expressed between 8.00am to 6.00pm in fully-open flower of Vanda Mimi Palmer. Secondary screening was applied to all the putative positive plaques (Figure 12b) and the plaques with the strongest signal on X-ray film were cored out and selected for in vivo excision.

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Figure 12: Autoradiograph Containing Positive Signals (dark spot) of Putative Positive Plaques from (a) Primary Screening; (b) Secondary Screening. From the secondary screening, individual plaques with the strongest signal were cored out for in vivo excision.

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4.2.1.2 Reverse-Northern Analysis

All the clones were in vivo excised to phagemid form and used for reverse-Northern analysis. The reverse-Northern analysis was carried out by hybridizing PCR-amplified inserts (Figure 13a) with two different cDNA probes synthesized from mRNA transcripts of fully-open flower (scent emitting stage) and bud (non-scent emitting stage) of Vanda Mimi Palmer separately in order to isolate the clones that putatively represent fragrancerelated transcripts. From a total of 800 putative positive clones, 246 clones showed upregulated expression in fully-open flower stage during day time compared to the bud stage (Figures 13b and 13c). A previous study on floral cDNA library of Vanda Mimi Palmer has shown that half of the library was contaminated by transcripts encoding Cymbidium mosaic virus coat protein (Chan, 2009). Thus, clones containing Cymbidium mosaic virus coat protein transcripts (Figure 13d) were removed by hybridizing the PCRamplified cDNA inserts of the clones with probes representing the Cymbidium mosaic virus coat protein transcripts. Upon eliminating transcripts related to Cymbidium mosaic virus, only 62 clones were selected for plasmid isolation and sequenced as candidates for fragrance-related cDNAs.

4.2.1.3 Sequencing and Analysis

Single pass sequencing result of the 62 clones were assembled into 5 contigs and 52 singletons using the Cap3 sequence assembly program (Appendix E). All of the 62 sequences (100%) were readable sequences with more than 285bp. The tentative unique

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Figure 13: Hybridization of (a) PCR Amplified Inserts with Three Different Probes; (b) Fully-open Flower cDNA Probe, (c) Bud Stage cDNA Probe, and (d) Cymbidium mosaic Virus cDNA Probe. White-circles show selected clones that have up-regulated expression in fully-open flower of Vanda Mimi Palmer during day time compared to bud stage after removal of clones containing Cymbidium mosaic virus coat protein cDNA. Lane 1-25 represents PCR product of putative positive clones isolated from the screening of floral cDNA library.

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gene sequences (TUG) were aligned against the GeneBank nucleotide and protein database (BLASTX) to search for sequence similarity with the cut off value less than 1e05

and score more than 80. From the BLASTX search, fifty-seven tentative unique gene

sequences (TUG) were similar to the sequences in NCBI database (Appendix E). The other five sequences had e-value more than 1e-05 and were classified as sequences with no-significant hit due to no similarity sequence in the NCBI database. All the ESTs were classified into eleven groups based on their putative functionality (Zhao et al., 2006). The groups are metabolism (19%), unknown (16%), protein synthesis (11%), cellular transport (10%), cell cycle and DNA processing (10%), protein with binding function (8%), energy (8%), cell rescue, defense and virulence (6%), development (6%), biogenesis of cellular component (3%) and transcription (3%) (see Figure 14 and Appendix E). The unknown group represents ESTs with unknown function including the sequences that hit to hypothetical proteins and also with non-significant hit sequences.

4.2.1.4 Verification of Putative Fragrance-related cDNAs with Up-regulated Expression in Fully-open Flower Compared to Bud of Vanda Mimi Palmer

Based on BLASTX and literature search results, it was postulated that some cDNA transcripts classified in metabolism and unknown group might be potential putative fragrance-related cDNAs. Thus, a verification was carried out for thirteen selected clones (potential putative fragrance-related cDNA clones) by RT-PCR using two different

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transcription 3% biogenesis of cellular component 3% Development 6% Cell rescue, defence and virulence 6% energy 8% metabolism 19%

unknown 16%

protein with binding function 8% cell cycle and DNA processing 10% protein synthesis 11% cellular transport 10%

Figure 14: Classification of the Clones with Up-regulated Expression in Fully-open Flower Compared to Bud of Vanda Mimi Palmer. The 62 clones with up-regulated expression in fully-open flower compared to bud of Vanda Mimi Palmer were classified into eleven groups based on their putative functionalities.

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tissues which are fully-open flower and bud to choose for the cDNAs clones that have up- regulated expression in the fully-open flower. Three cDNA clones from the

metabolism group including putative esterase (VMPEST), putative 4-(cytidine 5diphospho)-2-C-methyl-d-erythritol kinase (VMPCMEK) and putative cytochrome p450 protein (VMPCyP450) were selected for the verification because the selected transcripts have been reported to be involved in fragrance biosynthesis in other plants. Besides that, another 10 putative fragrance-related transcripts from unknown group including hypothetical protein and no significant hit protein transcripts were selected for verification of their expression levels in the two different tissues. The cDNAs from this group could be novel transcripts involved in the fragrance biosynthetic pathway.

From the verification results, 5 clones showed up-regulated expression in fully-open flower compared to bud, 3 clones showed down-regulated expression in fully-open flower compared to bud and 6 clones showed equal expression in both fully-open flower and bud stages (see Table 7). Three putative fragrance-related cDNA clones which showed up-regulated expression in fully-open flower compared to bud of Vanda Mimi Palmer were selected for full-length cDNA isolation and expression analysis by real-time RT-PCR. The selected cDNA clones were putative 4-(cytidine 5-diphospho)-2-Cmethyl-d-erythritol kinase (hereafter referred as VMPCMEK), putative cytochrome p450 protein (hereafter referred as VMPCyP450) and an unknown protein cDNA transcript (hereafter referred as VMPA28).

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Table 7: Verification of Putative Fragrance-related cDNAs with Up-regulated Expression in Fully-open Flower Compared to Bud of Vanda Mimi Palmer by RTPCR; (a) cDNA transcripts of the selected clones were PCR-amplified using first-strand cDNA of the selected tissues. (b) The PCR products were hybridized with specific probe representing the cDNA sequence of each transcript (reverse Northern). Each lane representing different tissues selected for the verification; 1) bud of Vanda Mimi Palmer 2) fully-open flower of Vanda Mimi Palmer. No significant hit and hypothetical protein transcript sequences were named as unknown protein. Clone 51 Name Putative cytochrome P450 (VMPCyP450) (up-regulated in fullyopen flower stage) 31 Putative esterase (VMPEST) (down-regulated in fullyopen flower stage) 71 Putative 4-(cytidine 5'diphospho)-2-C-methylD-erythritol kinase (VMPCMEK) (up-regulated in fullyopen flower stage) 33 Hypothetical protein (up-regulated in fullyopen flower stage) A36 Unknown protein (equal expression) (a) (b) (a) (b) 1 2 (a) (b) 1 2 (a) (b) 1 2 1 2 PCR and reverse Northern 1 (a) (b) 1 (a) (b) 1 (a) (b) 2 (a) (b) 2 (a) (b) 1 2 2 (a) (b) 1 2 Housekeeping gene (elongation factor) 1 2

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Clone 48

Name Hypothetical protein (up-regulated in fully-open flower stage)

PCR and reverse Northern 1 (a) (b) 1 (a) (b) 1 (a) (b) (a) (b) (a) (a) (b) 1 (a) (b) 2 1 2 (b) 1 2 2 (a) (b) 2 (a) (b) 2

Housekeeping gene (elongation factor) 1 2

59

Unknown protein (down-regulated in fullyopen flower)

83

Hypothetical protein (equal expression)

(a) (b) 1 2

90

Hypothetical protein ( down-regulated in fullyopen flower stage )

96

Unknown protein (down-regulated in fullyopen flower)

(a) (b) 1 2

A28

Unknown protein (up-regulated in fully-open flower)

(a) (b) 1 2 1 2

A46

Unknown protein (a) (equal expression) (b)

(a) (b) 1 2 1 2

A54

Hypothetical protein (a) (equal expression) (b)

(a) (b)

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4.2.2 Cloning and Characterization of Selected Fragrance-related Transcripts

Molecular characterization was carried out on three putative fragrance-related transcripts that showed up-regulated expression in fully-open flowers of Vanda Mimi Palmer. The selected transcripts were putative 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (VMPCMEK), putative cytochrome P450 protein (VMPCyP450) and an unknown protein (VMPA28). In addition, a putative phenylacetaldehyde synthase (VMPPAAS) previously identified from the floral ESTs of Vanda Mimi Palmer by Miss Chan Wai Sun was also selected for molecular characterization. Putative fragrance-related cDNA transcripts were subjected to full-length cDNA isolation and gene expression analysis. Gene expression analysis was carried out in different tissues, at different developmental stages, and different time points in a 24-hour cycle.

4.2.2.1 Sequence and Expression Analyses of Putative Phenylacetaldehyde Synthase (VMPPAAS)

The full-length sequence of VMPPAAS cDNA is 1709 bp consisting of 1524bp open reading frame (ORF) flanked by a 92bp 5 untranslated region (UTR) and a 93bp 3UTR including a poly-A tail (Figure 15). The ORF encodes a protein of 508 amino acid residues (Figure 16). The predicted molecular weight of this protein is 56.1 kD with an isoelectric point (pI) of 7.1.

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3 48 93 1 138 16 183 31 228 46 273 61 318 76 363 91 408 106 453 121 498 136 543 151 588 166 633 181 678 196 723 211 768 226 813 241 858 256 903

GCG GGA TCG CGG CAA GGC AGA AGG CAA CCA AAA AAC AAA AAA AAA AAA AAC AAG CTT GTT GCC TTT CCC ATT CCC TAG GAA CTC CAG AAA >>>>>>>>>>>>>>> ATG GGC AGC CTT CCC ACC GAA CCA TTC CTG CCA CTA GAC CCA GAC M G S L P T E P F L P L D P D >>>>>>>>>>>>>>>>>>>>>>>>>>>>>
VMPPAAS ORF Forward primer

47 92 137 15 182 30 227 45 272 60 317 75 362 90 407 105 452 120 497 135 542 150 587 165 632 180 677 195 722 210 767 225 812 240 857 255 902 270 947

CGC TTC ACC AAA GAA TCC AAA GCC GTC GTC GAC TTC ATC GCC GAC R F T K E S K A V V D F I A D TAC TAC CGC CAA ATA GAA CTC TTC CCT GTT CGC AGC CAA GTA AAG Y Y R Q I E L F P V R S Q V K CCA GGC TAT CTC CAT GAC CGC ATT CCA AAC ACT CCC CCC ATC CTC P G Y L H D R I P N T P P I L TCC GAA CCC ATC ACC ACA ATC CTC CAC GAC ATT AAA ACA GAC ATC S E P I T T I L H D I K T D I TTT CCC GGA CTA ACC CAC TGG CAA AGC CCC AAT TTT TAC GGC TAC F P G L T H W Q S P N F Y G Y TAC CAA GCC AAT GCC AGC ACC CCC GGT TTC GCC GGA GAG ATG CTC Y Q A N A S T P G F A G E M L TGT TCC GGC CTC AAC GTC GTC GGC TTC AGC TGG ATC GCT TCC CCT C S G L N V V G F S W I A S P GCC GCC ACT GAA CTA GAA ACC ATC ATC ATG GAC TGG ATG GCC AAG A A T E L E T I I M D W M A K ATG CTC AAA CTT CCA TCA ACC TTC CTT TCC GGA CAC CTC GGC GGC M L K L P S T F L S G H L G G GGC GGT GGC GTA ATC CAC GGC AGC ACG TGC GAA GCG GTG CTC TGC G G G V I H G S T C E A V L C ACC CTC GCC GCT GCT AGA GAT AAC GCT TTG AGC AAG AGC GAC GGC T L A A A R D N A L S K S D G GAA GGG ATC ACG AAG CTG ACG GTA TAT GTC TCT GAT CAG ACA CAT E G I T K L T V Y V S D Q T H TTT ACG GTT CAG AAG GCG GCG AAG TTG GTT GGA ATC CCG ACG CGG F T V Q K A A K L V G I P T R AAC TTA CGG GTG ATA TCG ACT TCG AGG GAG ACA GGG TAT GCC TTG N L R V I S T S R E T G Y A L ACG GCG GAG ATT GTG AGG GCG GCG ATG GAT GCT GAT GTG GCG GCA T A E I V R A A M D A D V A A GGG ATG GTG CCG CTG TAT TTG TGT GGC ACG GTG GGG ACG ACG GCT G M V P L Y L C G T V G T T A GTG GGG GCG GTG GAC CCG ATA AGG GAG ATC GGG GAG GTT GCG AGG V G A V D P I R E I G E V A R GAG TTC GGG GTG TGG TTC CAC GTG GAC GCG GCG TAT GCG GGG AGC

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271 948 286 993 301 1038 316 1083 331 1128 346 1173 361 1218 376 1263 391 1308 406 1353 421 1398 436 1443 451 1488 466 1533 481 1578 496 1623 1668

E F G V W F H V D A A Y A G S GCT GGG ATT TGC CCT GAG TTC CGG CGG TTC TTT GAT GGA GTG GAG A G I C P E F R R F F D G V E ACG GCT GAT TCC TTT AGT TTG AAT CCG CAT AAA TGG CTG CTC GCA T A D S F S L N P H K W L L A AAC ATG GAC TGT TGT TGC CTT TGG GTA AGA TGT GCA ACG AAG CTC N M D C C C L W V R C A T K L GTA GAC TCG TTA TCG ACC AAG CCG GAG ATA TTG ACA AAC AGT GCT V D S L S T K P E I L T N S A AGC GAA GAT GGC AAA GTG ATT GAC TAC AAA GAT TGG CAG GTC GCA S E D G K V I D Y K D W Q V A CTG AGT CGT AGG TTT CGT GCA ATG AAG CTA TGG ATA GTC ATC AGA L S R R F R A M K L W I V I R CGA TTT GGA GTT GCT AAC CTG ATG GAG CAC ATC AGG AGC GAT GTG R F G V A N L M E H I R S D V GAG ATG GCC AAG CAT TTC GAG AGA CTT GTC GCC GAG GAT GAG AGG E M A K H F E R L V A E D E R TTT GAG GTG GTT GTA CCA AGA AGA TTC ACG CTC GTT TGT TTT AAA F E V V V P R R F T L V C F K TTG AGG TAT GTG GGA GAA GAT ATT GAT GAA GAG GAG GGG ACG AAA L R Y V G E D I D E E E G T K >>>>>>> TGT TGG GAG ATG AAT AAG AAG TTG CTC GAT TCG GTG AAC GAA AGT C W E M N K K L L D S V N E S >>>>>>>>>>>>>>>>>>>>>
VMPPAAS RT-PCR Forward Primer

285 992 300 1037 315 1082 330 1127 345 1172 360 1217 375 1262 390 1307 405 1352 420 1397 435 1442 450 1487 465 1532 480 1577 495 1622 508 1667

GGA CGA GCA TTC ATG ACC CAT GCG GTT GTT TGC GGG CAG TTT GTG G R A F M T H A V V C G Q F V CTG CGG TTT GCA CTT GGC GCC ACG TTG ACA GAG ATA CGA CAT GTG L R F A L G A T L T E I R H V GAG GAG ACA TGG AGG TTG GTT CAA GAG AAG GCA AGT GAG TTG TTG E E T W R L V Q E K A S E L L <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPPAAS RT-PCR Reverse Primer

ATG ATT ACG AAT GAG CTG GGT TGG AAA CTC AAA ACA CTC TGA GAT M I T N E L G W K L K T L * <<< <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPPAAS ORF reverse primer

AGC TCA CTT AAA ATA AAA AGA GAA ATA ATA GTT TCA AAT GAA ATA AAA TTA TAA AAA ATT TAA AAA TTT AAA GAA AAA AAA AAA AAA

1709

Figure 15: The Nucleotide and Deduced Amino Acid Sequence of VMPPAAS (Putative Phenylacetaldehyde Synthase). The open reading frame (ORF) of VMPPAAS starts at nucleotide 93. The asterisk (*) indicates stop codon. The putative polyadenylation signal is bold and underline. The location of gene specific primers used in real-time PCR analysis and ORF isolation are shown by arrow heads (>>>> and <<<<).

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3000bp 2000bp ~1550bp 1500bp 1000bp

500bp

Figure 16: PCR Product of the Open Reading Frame (ORF) of Putative Phenylacetaldehyde Synthase of Vanda Mimi Palmer (VMPPAAS). The ORF represents 508 amino acids. The PCR product was electrophoresed on 1% (w/v) agarose gel. Lane M: 100bp marker (Vivantis, Malaysia); lane 1 and 2: gap; lane 3: PCR product of ORF of VMPPAAS.

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BLASTX and BLASTP analysis showed that VMPPAAS has the closest similarity to putative tyrosine/dopa decarboxylase (AAK50420.1) and pyridoxal-dependent

decarboxylase conserved domain containing protein (ABB47543.1) of Oryza sativa from japonica-cultivar group, corresponding to 57% identity. The VMPPAAS also shows similarity to tyrosine decarboxylase of Aristolochia contorta (ABJ16446.1) and tyrosine/dopa decarboxylase of Papaver somniferum (AAC61844.1) corresponding to 55% and 54% identities, respectively. Besides that, VMPPAAS also shows similarity to fragrance-related amino acid sequences of well studied scented flowers including aromatic L-amino acid decarboxylase of Rosa hybrida (BAF64844.1) (56% identity), phenylacetaldehyde synthase of Rosa hybrida (ABB04522.1) (56% identity) and phenylacetaldehyde synthase of Petunia hybrida (ABB72475.1) (54% identity).

Localizome analysis using Localizome software (Lee et al., 2006) revealed a domain for pyridoxal-dependent decarboxylase in the amino acid sequence of VMPPAAS. In addition, the same software also predicted no signal peptide present in the amino acid sequence of VMPPAAS and in all of the closely related protein sequences. Prediction of motifs present in the deduced protein sequence of VMPPAAS and the closely related proteins by the Expasy tool showed a conserved motif for a pyridoxal phosphate attachment site (see Figure 17). Besides that, there are two N-myristoylation sites (GVxxGS and GAxxTE), three casein kinase II phosphorylation sites (TxxE, SxxE, and TxxE), two N-glycosylation sites (NAST and NESG), and a site for cAMP- and c-GMPdependent protein kinase phosphorylation (RRxT) in the VMPPAAS amino acid sequence (see Figure 17). There is another motif (VHVDAAY) shared by VMPPAAS

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NAST

TxxE

***

***

GVxxGS SxxE

**** **

VHVDAAY motif

Pyridoxal phosphate attachment site

TxxE

**

NESG

***

Figure 17: Alignment of VMPPAAS (Putative Phenylacetaldehyde Synthase) with Other Closely Related Protein Sequences from the GeneBank. The VMPPAAS amino acids sequence was aligned with putative tyrosine/dopa decarboxylase (AAK50420.1) of Oryza sativa, tyrosine/dopa decarboxylase of Papaver somniferum (AAC61844.1), aromatic L-amino acid decarboxylase of Rosa damascena (BAF64843.1), phenylacetaldehyde synthase of Rosa hybrida (ABB04522.1) and phenylacetaldehyde synthase of Petunia hybrida (ABB72475.1). The pyridoxal phosphate attachment site and HVDAAY motif are highlighted in box. Four stars (****) indicate the conserved motif for N-myristoylation sites (GVxxGS and GAxxTE) meanwhile two stars (**) indicate the conserved motif for casein kinase II phosphorylation sites (TxxE, SxxE and TxxE). The conserved motif for N-glycosylation sites (NAST and NESG) are marked with three stars (***).

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and other decarboxylases protein either from plants or bacteria (Connil et al., 2002) (see Figure 17). A phylogenetic tree (Figure 18) was constructed using MEGA version 4.0 (Tamura et al., 2007) to estimate the genetic relatedness between amino acid sequence of VMPPAAS with other plant decarboxylases. In the phylogenetic tree, VMPPAAS is clustered together with protein sequences from aromatic amino acid of Ricinnus communis, and supported by the bootstap value of 93 for the branch.

Relative expression study of VMPPAAS transcript in different tissues (Figure 19) of Vanda Mimi Palmer showed up-regulated expression in floral tissues including petal, sepal and lip but down-regulation in vegetative tissues including leaf, shoot, root and stalk. Among the floral tissues, petal has shown the highest expression of VMPPAAS transcript which is more than 20,000-fold expression compared to bud tissue as the calibrator. In the sepal, the expression of VMPPAAS transcript was also detected at high level (nearly to 15,000-fold expression) compared to the calibrator. Meanwhile in the lip, the VMPPAAS transcript was detected at 5000-fold higher expression compared to the calibrator.

Relative expression analysis at five different flower developmental stages shows that the VMPPAAS transcript was differentially expressed during the flower life cycle (Figure 20). In bud stages including young and mature buds, the expression of VMPPAAS transcript was down-regulated compared to half-open and fully-open flower stages. At 14-day fully-open flower, the expression of VMPPAAS transcript decreased drastically at very low level.

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VMPPAAS (Vanda Mimi Palmer) OsTyDC(Oryza sativa-japonica cultivar... RcAADC (Ricinus communis) PsTyDC(Papaver somniferum) RsAADC (Rosa hybrida) RhPAAS (Rosa hybrida) RdAADC (Rosa x damascena) PhPAAS (Petunia hybrida) CaTDC(Camptotheca acuminata) AtTyDC (Arabidopsis thaliana) ZmTyDC1 (Zea mays) OsTDC (Oryza sativa Japonica Group) ZmTyDC2 (Zea mays)

Figure 18: Phylogenetic Tree of VMPPAAS with Homologous Proteins. The phylogenetic tree is constructed by MEGA software version 4.0 using amino acid sequence of VMPPAAS with the homologous proteins available in GeneBank database (NCBI) including tyrosine decarboxylase of Oryza sativa (AAK50420.1), aromatic amino acid decarboxylase of Ricinus communis (EEF36965.1), tyrosine/DOPA decarboxylases Papaver somniferum (AAC61844.1), aromatic L-amino acid decarboxylase of Rosa hybrida (BAF64844.1), phenylacetaldehyde synthase of Rosa hybrida (ABB04522.1), aromatic L-amino acid decarboxylase of Rosa damascene (BAF64843.1), phenylacetaldehyde synthase of Petunia hybrida (ABB72475.1), tryptophan decarboxylases of Camptotheca acuminata (AAB39708.1), tyrosine decarboxylase of Arabidopsis thaliana (AAL69507.1), tyrosine/DOPA decarboxylase 1 of Zea mays (ACG29316.1), tryptophan decarboxylase of Oryza sativa (BAD35168.1) and tyrosine/dopa decarbxylase 2 of Zea mays (ACG46884.1).

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Relative Expression of VMPPAAS (fold change)

25000 20000 15000 10000 5000 0

bud

fullyopen flower

petal

sepal

lip

leaf

root

shoot

stalk

Tissues

Figure 19: Relative Expression Study of VMPPAAS Transcript in Different Tissues of Vanda Mimi Palmer. The floral tissues are bud, fully-open flower, petal, sepal and lip meanwhile the vegetative tissues are leaf, root, shoot and stalk. The quantitative expression level of VMPPAAS in each tissue was calculated relative to the calibrator which was the bud collected at 12.00 noon. Standard error between three replicates of relative gene expression in each tissue is indicated by error bar.
Relative Expression of VMPPAAS (fold change)

105000 90000 75000 60000 45000 30000 15000 0 young bud (green) mature bud (red) half-open flower fully-open flower 14-day old flower

Developmental Stages

Figure 20: Relative Expression Analysis on VMPPAAS Transcript at Different Developmental Stages During Flower Development. This study was carried out on five flower developmental stages of Vanda Mimi Palmer including young bud, mature bud, half-open flower, fully-open flower and 14-day old fully-open flower. Quantitative measurement of VMPPAAS expression in each flower developmental stage was expressed relative to the calibrator which was the young bud collected at 12.00 noon. Standard error between three replicates of relative gene expression at each developmental stage is indicated by error bar.

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Relative expression study of VMPPAAS transcript at different time points in a 24-hour cycle (12 hours in light, followed by 12 hours in dark) (Figure 21) shows a differential expression. From the analysis, the expression of VMPPAAS was higher at night (7.00pm to 7.00 am) compared to day time (7.00am to 7.00pm). From 8.00am until 6.00pm (in light) the expression level was lower compared to night. However, after 6.00pm the amount of VMPPAAS transcript increased more than three fold with the highest peak at 10.00pm.

4.2.2.2 Sequence and Expression Analyses of Putative 4-(cytidine 5'-diphospho)-2-Cmethyl-D-erythritol Kinase (VMPCMEK)

The full-length cDNA sequence of VMPCMEK is 1446bp, comprising 1200bp ORF, 51bp of 5 UTR and 195bp of 3UTR with a polyadenylation tail (poly-A tail) (Figure 22). The ORF encodes a protein of 400 amino acid residues (Figure 23) with a predicted molecular weight of 44.1 kD and an isoelectric point (pI) of 8.4.

BLASTX and BLASTP analysis (NCBI) shows the deduced amino acid sequence of VMPCMEK exhibited similarity to 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase from other plants (66-73% identity). It has the highest similarity to 4-(cytidine 5'diphospho)-2-C-methyl-D-erythritol kinase of Nicotiana benthamiana (ABO87658.1),

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Relative expression of VMPPAAS (fold change)

4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00

12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm

Time

Figure 21: Relative Expression Analysis of VMPPAAS at Different Time Points in a 24-hour Cycle. Quantitative expression of VMPPAAS at each time point was expressed relative to the calibrator which was the fully-open flower collected at 12.00 noon. Standard error between three replicates of relative gene expression at each time point is indicated by error bar.

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1 46

GGC TGC TGC AAA AAG CAT AAC TCC CTA CGC TTC TCA GCT TTT CTC >>>>>>>>>>>>>>>>>>>>>>> CTA ACA ATG GCC TCT TTC TCT AAT CAT CTG ATT TCA TCG TTC TCA M A S F S N H L I S S F S >>>>>>>>>>>>>>>>>
VMPCMEK ORF Forward Primer

45 90 13 135 28 180 43 225 58 270 73 315 88 360 103 405 118 450 133 495 148 540 163 585 178 630 193 675 208 720 223 765 238 810 253 855 268 900 283 945

91 14 136 29 181 44 226 59 271 74 316 89 361 104 406 119 451 134 496 149 541 164 586 179 631 194 676 209 721 224 766 239 811 254 856 269 901

TGC TCG AGA AGA AGT GCT TCA CTG CCC CGA AGA GGG AAT ACC CCC C S R R S A S L P R R G N T P TCG ATC TTT CGA CAT GGT CAT TAC TCT TTC AAA TCT TGG TCT GAG S I F R H G H Y S F K S W S E GTC AGC GGG AAC AAG TAT GGT AGA ATC CTC ATT TGT GCG GCA GAA V S G N K Y G R I L I C A A E ACT GGG AGG AGG CAA GTG GAG ATT GTT TAT GAT CCG GAG GAG AGG T G R R Q V E I V Y D P E E R TTT AGT GGA CTG GAG GGT GAA GTA GAT GAT AAC AAC AAG CTT TCT F S G L E G E V D D N N K L S AGG TTG ACC CTA TTC TCG CCG TGT AAG ATT AAT GTT TTC TTG AGG R L T L F S P C K I N V F L R ATA ACT GGA AAG AGG AAT GAT GGG TTT CAT GAT TTG GCC TCT TTG I T G K R N D G F H D L A S L TTT CAT GTA ATC AGT TTA GGA GAT ACG ATT AAA TTC TCC TTG TCA F H V I S L G D T I K F S L S CCA TTA AAG AGA AAG GAT CGC CTG TCA ACT AAT GTG CCG GGA GTT P L K R K D R L S T N V P G V CCA GTT GAT GAT AGA AAT TTG ATA ATC AGA GCT CTC AAT CTT TAC P V D D R N L I I R A L N L Y AGG AAG AAG ACA GGC ACA AAC AAT TTC TTC CAG ATT GAG CTT GAC R K K T G T N N F F Q I E L D AAA AAA GTT CCT ACT GGT GCT GGG CTT GGT GGT GGA AGT AGT AAC K K V P T G A G L G G G S S N GCA GCA ACT GCT TTA TGG GCT GCC AAC CAG TTC AGT CGT TCT CTT A A T A L W A A N Q F S R S L GTT ACT GAA AAA GAG CTT CAG GAT TGG TCA GGT GAA ATT GGT TCA V T E K E L Q D W S G E I G S GAT ATT CCT TTT TTT TTC TCT AAT GGG GCT GCA TAT TGT ACC GGT D I P F F F S N G A A Y C T G AGG GGA GAG GTT GTT AAA GAA CTT CCT TTT GCA TTG CCC AAG GAC R G E V V K E L P F A L P K D CTG CCA ATG GTT CTT ATA AAG CCC CAA GAA GCA TGT CCA ACC GCC L P M V L I K P Q E A C P T A GAA GTG TAC AAG CGA CTT CAT CTT GGT AAA ACT AGT TCA GTT GAC E V Y K R L H L G K T S S V D CCG TTG ACT CTG CTA GAA AAG ATA TCT CTA AAT GGA ATA TCT CAA

108

284 946 299 991 314 1036 329 1081 344 1126 359 1171 374 1216 389

298 990 313 1035 328 1080 343 1125 358 1170 373 1215 388 1260 400

GAT GTC TGC ATA AAT GAT CTT GAA CCC CCT GCA TTT GAT GTT TTG D V C I N D L E P P A F D V L CCA TCC TTG AAG AAG TTG AAG CAA CGT GTG CTA GCT GCA GGG CGT P S L K K L K Q R V L A A G R GGC CAG TAT AGT GCT GTT TTC ATG TCT GGA AGC GGA AGC ACC ATT G Q Y S A V F M S G S G S T I >>>>>>>>>>>>>>>>>>>>>>>>>>>
VMPCMEK RT-PCR Forward Primer

GTG GGA ATT GGT TCA CCA GAC CCA CCT CAA CTT GTT TAT GAT GAG V G I G S P D P P Q L V Y D E GAT GAA TAC AAT GAT GTT TTC ATA ACA GAG GCT TCC TTT CTC ACT D E Y N D V F I T E A S F L T CGG CAA CAG AAT CAG TGG TAC GCA GAG CCA ACT TCG TCC ACA GGG R Q Q N Q W Y A E P T S S T G TCT TTG AGC AGA GAA GAG CCG TCA CAA ACA GGA AAA TAA TTA CGA S L S R E E P S Q T G K * <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPCMEK ORF Reverse Primer

<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPCMEK RT-PCR Reverse Primer

1261 1306 1351 1396 1441

TAA TTT TTT TAC ATT CTA GAC CTT CTA ATT TTA ATT TTT CTC ACA TAA AAT CAT ATT GTA TTA CTG TAC TTA TTG TTC ATG CAA GAA AGA TCG ATC AAG CTA TCT TTC ATG AAT GAG CAA AAT ATG CAA TTT TAA AAG GCA CAT TTA CAT GCT TAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA 1446

1305 1350 1395 1440

Figure 22: The Nucleotide and Deduced Amino Acid Sequences of VMPCMEK (putative 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase). The open reading frame (ORF) of VMPCMEK starts at nucleotide 52. The asterisk (*) indicates stop codon. The putative polyadenylation signal in the nucleotide sequence is bold and underline. The location of gene specific primers used in real-time PCR analysis and ORF isolation are shown by arrow heads (>>>> and <<<<).

109

1200bp 1000bp 500bp

~1200bp

Figure 23: PCR Product of the Open Reading Frame (ORF) of Putative 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol Kinase of Vanda Mimi Palmer (VMPCMEK). The PCR product was electrophoresed on 1% (w/v) agarose gel. Lane M: 100bp marker (Vivantis); Lane 1: PCR product of ORF of VMPCMEK.

110

corresponding to 73% identity. The closest similarity was followed by 4-(cytidine 5'diphospho)-2-C-methyl-D-erythritol kinase of Solanum lycopersicum (AAF87717.1) and Catharanthus roseus (ABI35992.1), corresponding to 72% identity. Besides that, the VMPCMEK sequence also showed similarity to two 4-(cytidine 5'-diphospho)-2-Cmethyl-D-erythritol kinase proteins of Ginkgo biloba (AAZ80385.1 and AAZ80384.1), corresponding to 70% and 69% identity, respectively.

VMPCMEK and all of the closely related proteins were predicted to have no signal peptide. Prediction of motifs by Expasy tool revealed three conserved N-myristoylation sites (GAxxCT, GQxxAV and GSxxTI) shared by VMPCMEK and other closely related proteins (Figure 24). The software also predicted the presence of another conserved site in VMPCMEK and the closely related protein sequences which is cAMP- and cGMPdependent protein kinase phosphorylation site (RKxT). Besides that, a conserved motif for ATP-binding site for the functional activity of 4-(cytidine 5'-diphospho)-2-C-methylD- erythritol kinase (Kim et al., 2008) was also detected in the sequence of VMPCMEK and its closely related proteins (Figure 24).

A phylogenetic tree (Figure 25) was constructed using MEGA software version 4.0 (Tamura et al., 2007) to estimate the genetic relatedness between amino acid sequence of VMPCMEK with its closely related proteins. From the phylogenetic tree, VMPCMEK amino acid sequence is not clustered together with methyl-D- erythritol kinase proteins from 4-(cytidine 5'-diphospho)-2-Clycopersicum, Nicotiana

Solanum

benthamiana, and Cantharanthus roseus.

111

RKxT

**

GAxxCT

****

GQxxAV GSxxTI

**** ****

Figure 24: Alignment of VMPCMEK with Other Closely Related Protein Sequences Downloaded from the GeneBank Database. The VMPCMEK is aligned with HbCMEK (4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase) of Hevea brasiliensis (BAF98293.1), SlCMEK (4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase) of Solanum lycopersicum (AAF87717.1), CrCMEK (4-(cytidine 5'-diphospho)-2C-methyl-D-erythritol kinase) of Catharanthus roseus (ABI35992.1), ZmCMEK (4(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase) of Zea mays (ACG34338.1) and NbCMEK (4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase) of Nicotiana benthamiana (ABO87658.1). Residues 189 through 204 (highlited in box) are the conserved ATP-binding site for the functional activity of 4-(cytidine 5'-diphospho)-2-Cmethyl-D-erythritol kinase (Kim et al., 2008). There are three putative conserved motifs for N-myristoylation site shared by VMPCMEK with other closely related plant proteins (GAxxCT, GQxxAV and GSxxTI). The motifs are marked with four stars (****). Besides that, there is another conserved motifs for cAMP- and cGMP-dependent protein kinase phosphorylation site (RKxT) marked with two stars (**).

112

VMPCMEK (Vanda Mimi Palmer) HbCMEK (Hevea brasiliensis) SrCMEK (Stevia rebaudiana) SlCMEK (Solanum lycopersicum) NbCMEK (Nicotiana benthamiana) CrCMEK (Catharanthus roseus) AtCMEK (Arabidopsis thaliana) ZmCMEK (Zea mays) GbCMEK1 (Ginkgo biloba 1) GbCMEK2 (Ginkgo biloba 2)

Figure 25: A Phylogenetic Tree of VMPCMEK with Homologous Proteins. The phylogenetic tree is constructed by MEGA software version 4.0 using amino acid sequence of VMPCMEK with the homologous proteins available in GeneBank database (NCBI) including other 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase from Hevea brasiliensis (BAF98293.1), Stevia rebaudiana (ABB88838.3), Solanum lycopersicum (AAF87717.1), Nicotiana benthamiana (ABO87658.1), Catharanthus roseus (ABI35992.1), Arabidopsis thaliana (AAG01340.1) Zea mays (ACG34338.1) and two proteins from Ginkgo biloba (AAZ80384.1) and (AAZ80385.1).

113

Relative expression analysis of VMPCMEK in different tissues (Figure 26) of Vanda Mimi Palmer showed up-regulated expressions in floral tissues in contrast to vegetative tissues. Among the floral tissues, petal and sepal showed the highest expression of VMPCMEK transcript which was approximately two folds higher than the bud (calibrator). In the lip, the expression of VMPCMEK was detected lower than the bud. Relative expression analysis of VMPCMEK at different floral developmental stages showed that VMPCMEK was differentially expressed (Figure 27). The expression level of VMPCMEK increased gradually from young bud to half-open flower (the highest level), followed by a decrease in fully-open flower stage. Relative expression study of VMPCMEK at different time points in a 24-hour cycle (12 hours in light and followed by 12 hours in dark) (Figure 28) showed a differential expression pattern. From the analysis, expression of VMPCMEK increased gradually from 12.00am to 8.00am, followed by a gradual decrease until 2.00pm (the lowest peak). After 2.00pm, the expression increased again gradually until 10.00pm.

4.2.2.3 Sequence and Expression Analyses of Putative Cytochrome P450 Protein (VMPCyP450)

VMPCyP450 has a full-length cDNA transcript of 1785bp comprising 1614bp of ORF, 18bp of 5-UTR and 153bp of 3-UTR with a poly-A tail (Figure 29). The VMPCyP450 sequence encodes for 538 amino acid residues (Figure 30) with a predicted molecular weight of 62.1 kD and an isoelectric point (pI) of 8. A homologous sequence

114

Relative Expression of VMPCMEK (fold change)

2.50 2.00 1.50 1.00 0.50 0.00

bud

fullyopen flower

petal

sepal

lip

leaf

root

shoot

stalk

Tissues

Figure 26: Relative Expression Study of VMPCMEK Transcript in Different Tissues Including Floral and Vegetative Tissues. The floral tissues are bud, fully-open flower, petal, sepal and lip meanwhile the vegetative tissues used in the expression study are leaf, root, shoot and stalk. The quantitative expression level of VMPCMEK in each tissue was calculated relative to the calibrator which was the bud collected at 12.00 noon. Standard error between three replicates of relative gene expression in each tissue is indicated by error bar.

Relative Expression of VMPCMEK (fold change)

3.00 2.50 2.00 1.50 1.00 0.50 0.00 young bud (green) mature bud (red) half-open flower fully-open flower 14-day old flower

Developmental Stages

Figure 27: Relative Expression Analysis of VMPCMEK Transcript at Different Developmental Stages During Flower Development. This study was carried out on five different flower developmental stages of Vanda Mimi Palmer including young bud, mature bud, half-open flower, fully-open flower and 14-day old fully-open flower. Quantitative measurement of VMPCMEK expression in each flower developmental stage was expressed relative to the calibrator which was the young bud collected at 12.00 noon. Standard error between three replicates of relative gene expression at each developmental stage is indicated by error bar.

115

3.00

Relative expression of VMPCMEK (fold expression)

2.50 2.00 1.50 1.00 0.50 0.00 12am 2am 4am 6am 8am 10am 12pm 2pm Time 4pm 6pm 8pm 10pm

Figure 28: Relative Expression Analysis of VMPCMEK at Different Time Points in a 24-hour Cycle. Quantitative expression of VMPCMEK at each time point was expressed relative to the calibrator which was the fully-open flower collected at 12.00 noon. Standard error between three replicates of relative gene expression at each time point is indicated by error bar.

116

ATA CTG CTG CTG CCA CTA ATG TCT TCT TCC TCA AGC TCC TCA CTT M S S S S S S S L >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
VMPCyP450 ORF Forward Primer

45 9 90 24 135 39 180 54 225 69 270 84 315 99 360 114 405 129 450 144 495 159 540 174 585 189 630 204 675 219 720 234 765 249 810 264 855 279 900 294 945 309

46 10 91 25 136 40 181 55 226 70 271 85 316 100 361 115 406 130 451 145 496 160 541 175 586 190 631 205 676 220 721 235 766 250 811 265 856 280 901 295

CTG CCA TAC AAA CTC ATT GCT TTC AGC GCA ATC TTC CTC ATC AGT L P Y K L I A F S A I F L I S TGG ATC TTC CTT CAT AGA TGG GCA CAG AGA AAC CGC AGA GGT CCG W I F L H R W A Q R N R R G P AAG ACA TGG CCG CTC ATC GGA GCC GCC ATT GAA CTG CTC AAC AAC K T W P L I G A A I E L L N N TAT GAA CGC ATG CAT GAT TGG ATT ACA GAT TAC TTG TCT GAA TGG Y E R M H D W I T D Y L S E W AGG ACT GTT ACT GTC CCC TTG CCC TTC ACT TCA TAC ACT TAT ACT R T V T V P L P F T S Y T Y T GCA GAC CCT GCA AAT GTG GAG CAT ATT CTG AAG ACC AAC TTC AAC A D P A N V E H I L K T N F N AAC TAT CCC AAG GGA GAG CTA TTT AGA TCA TAT ATG GAG GTA TTG N Y P K G E L F R S Y M E V L CTG GGA GAT GGG ATA TTT AAC GCA GAT GGA GAG CTG TGG AGG AAG L G D G I F N A D G E L W R K CAG AGG AAG ACT GCA AGC TTT GAG TTT GCT TCA AAG AAC TTG AGG Q R K T A S F E F A S K N L R GAA CTG AGC ACC GTT GTG TTT AGA GAG TAT GCT TTG AAA CTA TCT E L S T V V F R E Y A L K L S GAC ATA TTA TGC CAA GCC TCT TGC AAA GAT CAT CAT CAA GCT GTA D I L C Q A S C K D H H Q A V GAT ATT CAG GAT TTA TTC ATG AGG ATG ACA ATG GAC TCC ATA TGC D I Q D L F M R M T M D S I C AAG CTT GGT TTT GGA GTG GAG ATA GGG ACA CTA TCT CCC CAA CTC K L G F G V E I G T L S P Q L CCT GAT AAC AGC TTT GCT CGA GCT TTC GAC ACC GCG AAC GCG ACC P D N S F A R A F D T A N A T GTC ACG CGT CGA TTC TTC GAT CCC TTG TGG AGG TTG AAG AGG TTT V T R R F F D P L W R L K R F CTT TGT GTG GGA TCA GAG GCT GCC CTC AAC CAA AAT ATC AGA ATT L C V G S E A A L N Q N I R I GTT AAT GAC TTC ACC TCT AAT GTT ATA CGT ACA AGA AAG GCT GAG V N D F T S N V I R T R K A E ATC ATG AGA GCT AAA CAA AAC GGG CAT CAT GAT GAG ACA AAG CAA I M R A K Q N G H H D E T K Q GAC ATA CTA TCA AGG TTC ATC GAG CTC GCC AAC ACC GAC AAA GAG D I L S R F I E L A N T D K E AGT GAT TTC AGC ACG GAA AAA GGT TTA AGA GAT GTG GTG CTA AAC S D F S T E K G L R D V V L N

117

946 310 991 325 1036 340 1081 355 1126 370 1171 385 1216 400 1261 415 1306 430 1351 445 1396 460 1441 475 1486 490 1531 505 1576 520 1621 535 1666 1711 1756

TTT GTT ATT GCA GGG AGG GAC ACT ACT GCT GCA ACG CTC TCA TGG F V I A G R D T T A A T L S W >>>>>>>>>>>>>>>>>> TTT ATA TAC ATA TTA GTC ACA CAA CCT CAG GTG GCA CAG AAA CTC F I Y I L V T Q P Q V A Q K L >>>>>> TAT ATA GAG ATG AAA GAG TTT GAG GAG ATC AGA GCT GAA GAA GAA Y I E M K E F E E I R A E E E

990 324 1035 339 1080 354 1125 369 1170 384 1215 399 1260 414 1305 429 1350 444 1395 459 1440 474 1485 489 1530 504 1575 519 1620 534 1665 538 1710 1755 1785

VMPCyP450 RT-PCR Forward Primer

AAT ATA AAT TTG GAT TTA TGT AAT TTG GAA GAT ATG GAT TCA TTC N I N L D L C N L E D M D S F AGA AAC AGA TTA TCA GAT TTT TCG AGG CTT TTG GAT TAT GAT TCA R N R L S D F S R L L D Y D S <<<<<<<<<<<<<<<<<<<<<<<<<<
VMPCyP450 RT-PCR Reverse Primer

TTA GCA AGG CTG CAA TAT CTG CAT GCA TGC ATT ACA GAG ACC CTG L A R L Q Y L H A C I T E T L AGG CTG TTT CCT CCT GTT CCT CAG GAC GCG AAA GGC ATT TTG AAG R L F P P V P Q D A K G I L K GAT GAT GTT CTC CCT GAC GGA ACA AAA CTG AGA GCC GGG GAA ATG D D V L P D G T K L R A G E M GTG CTA TAC GTC CCC TAT TCA ATG GGA AGA ATG GAG TAC ATT TGG V L Y V P Y S M G R M E Y I W GGC ATC GAC GCA TCA GAA TTT CGC CCC GAA AGA TGG CTA AAT AAC G I D A S E F R P E R W L N N GAC AAT AAT TCC GTC CAA AAT AAC GTC TCT CCA TTC AAG TTC ACG D N N S V Q N N V S P F K F T GCG TTT CAG GCT GGT CCC AGA ATG TGC TTG GGG AAG GAC TCC GCT A F Q A G P R M C L G K D S A TAT Y CAA Q ATG M AGA R TAA CTG L TTC F ATA I AGA R GTT CAG Q AGA R GTA V GGA G ATT ATG AAG ATG ACA GCA GCG TTA CTC TGC AGG TTC TTT M K M T A A L L C R F F CTT GCT CCT CAT CAT CCT CCT GTT AAG TAT AGG ATG L A P H H P P V K Y R M CTT TCC ATG GCG CAT GGC CTG CAT GTG CTC GTT TGT L S M A H G L H V L V C TCA TGA TTT TTG ATG CAT GGA TCT ATG TTT TAT TAT S * GCG TCT GTT TGT TGT CCA TAA GTG AAG TGC AGA CAA <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPCyP450 ORF Reverse Primer

CAT CAT TAT TAT ATG TCA TGC CCC CTA AAT GTT TAC TTC CGA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA

Figure 29: The Nucleotide and Deduced Amino Acid Sequence of VMPCyP450 (Putative Cytochrome P450 Protein). The open reading frame (ORF) of VMPCyP450 starts at nucleotide 19. The asterisk (*) indicates stop codon. The putative polyadenylation signal is bold and underline. The location of gene specific primers used in real-time PCR analysis and ORF isolation are shown by arrow heads (>>>> and <<<<).

118

1500bp

~1600bp

500bp

Figure 30: PCR Product of Open Reading Frame (ORF) of VMPCyP450 of Vanda Mimi Palmer. The ORF represents 538 amino acids. The PCR product was electrophoresed on 1% (w/v) agarose gel. Lane M: 100bp marker (Vivantis, Malaysia); Lane 1: ORF of clone VMPCyP450.

119

analysis by BLASTX and BLASTP (NCBI) shows that VMPCyP450 exhibited similarity to sequences that encode cytochrome P450 proteins from other plants (39-67% identity). From the BLASTX and BLASTP analyses, VMPCyP450 has the closest similarity to putative cytochrome P450 protein of Oryza sativa (ABF 94184.1), and cytochrome P450 protein of Populus trichocarpa (EEE89622.1), corresponding to 66% identity. Besides that, VMPCyP450 also showed similarity to cytochrome P450 monooxygenase of Petunia hybrida (AAZ39646.1) (45% identity) and cytochrome P450 monooxygenase from other plants.

Localizome analysis predicts that VMPCyP450 protein does not have any signal peptide. Most of other plant cytochrome P450 proteins were predicted by the Localizome software to have signal peptide except cytochrome P450 protein of Arabidopsis thaliana, cytochrome P450 protein of Populus trichocarpa and cytochrome P450 monooxygenase of Petunia hybrida. VMPCyP450 and all the closely related proteins were predicted to have a transmembrane domain by the Localizome software. The deduced VMPCyP450 protein was predicted to have 11 amino acids at the N-terminal (non-cytosolic), 21 amino acids located in the membrane and 506 amino acids in cytosol. Expasy programme prediction shows that three N -myristoylation sites (GxxxAD, GVxxGT and GsxxAL) were shared by VMPCyP450 and its closely related proteins (Figure 31). Besides that, there was another conserved site for tyrosine kinase phosphorylation (see Figure 31). A phylogenetic tree (Figure 32) was constructed using the MEGA version 4.0 (Tamura et al., 2007) to estimate the genetic relationship of VMPCyP450 with other cytochrome

120

GIxxAD

****

GVxxGT

****

GSxxAL

****

RxxExxxY

Figure 31: Alignment of VMPCyP450 with Other Closely Related Protein Sequences Downloaded from GeneBank Database NCBI. The VMPCyP450 was aligned with cytochrome P450 of Ricinus communis (EEF39957.1) putative cytochrome P450 of Arabidopsis thaliana (AAG60111.1), putative cytochrome P450 protein of Oryza sativa from Japonica Cultivar (AAL84318.1), cytochrome P450 of Zea mays (ACG35470.1) and cytochrome P450 monooxygenase of Petunia hybrida (AAZ39646.1). There are three N-myristoylation sites (GxxxAD, GVxxGT and GsxxAL) shared by VMPCyP450 with other closely related proteins which are marked with four stars (****). There is another putative conserved region for tyrosine kinase phosphorylation site (RxxExxxY) marked with five delta symbol ().

121

VMPCyP450 (Vanda Mimi Palmer) PhP450 (Petunia x hybrida) MtP450 (Medicago truncatula) GmP450 (Glycine max) RcP450 (Ricinus communis) PtP450 (Populus trichocarpa) AtP450 (Arabidopsis thaliana) CaP450 (Capsicum annuum) OsP450 (Oryza sativa-japonica cultiva... ZmP450 (Zea mays)

Figure 32: A Phylogenetic Tree of VMPCyP450 and Homologous Proteins. The phylogenetic tree is constructed by MEGA software version 4.0 using amino acid sequence of VMPCyP450 with the homologous proteins available in GeneBank database (NCBI) including cytochrome P450 of Ricinus communis (EEF39957.1), cytochrome P450 of Populus trichocarpa (EEE89622.1) cytochrome P450 of Arabidopsis thaliana (AAG60111.1), cytochrome P450 of Capsicum annuum (ACD10924.1), cytochrome P450 protein of Oryza sativa from Japonica Cultivar (AAL84318.1), cytochrome P450 of Zea mays (ACG35470.1), cytochrome P450 monooxygenase of Petunia hybrida (AAZ39646.1), cytochrome P450 monooxygenase of Medicago tranculata (ABC59094.1) and cytochrome P450 monooxygenase of Glycine max (Soy bean)(ABC68403.1).

122

P450 proteins. The phylogenetic tree shows that VMPCyP450 amino acid sequence was clustered together with cytochrome P450 monooxygenase of Medicago truncatula, Glycine max and Petunia hybrida and supported by the bootstrap value of 99 for the branch.

Relative expression analysis in different tissues (Figure 33) of Vanda Mimi Palmer including floral and vegetative tissues by real-time RT-PCR shows that the VMPCyP450 had up-regulated expression in floral tissues especially in the lip. The other floral tissues including petal and sepal showed lower expressions of VMPCyP450 compared to the lip. For vegetative tissues such as leaf, root, shoot, and stalk, the expression of VMPCyP450 was detected at a very low level. Expression analysis of VMPCyP450 transcript at different flower developmental stages (Figure 34) shows a developmentally regulated pattern. Meanwhile, the expression analysis of VMPCyP450 at different time points in a 24-hour cycle (Figure 35) also shows a differential expression pattern.

4.2.2.4 Sequence and Expression Analyses of Unknown protein (VMPA28)

The full-length sequence of VMPA28 cDNA transcript is 972bp containing 591bp ORF flanked with 148bp 5UTR and 233bp 3UTR including a poly-A tail (Figure 36). The ORF encodes a protein of 197 amino acid residues (Figure 37). Expasy tool (Prolite) has predicted 22.32kD as the molecular mass of VMPA28 protein with an isoelectric point of 9.06. The Expasy tool also predicts the presence of a N-glycosylation site (NTSN), two

123

Relative Expression of VMPCyP450 (fold change)

6.00 5.00 4.00 3.00 2.00 1.00 0.00

bud

fullyopen flower

petal

sepal

lip

leaf

root

shoot

stalk

Tissues

Figure 33: Relative Expression Analysis of VMPCyP450 in Different Tissues Including Floral and Vegetative Tissues. The floral tissues are bud, fully-open flower, petal, sepal and lip meanwhile the vegetative tissues used in the expression study are leaf, root, shoot and stalk. The quantitative expression level of VMPCyP450 in each tissue was calculated relative to the calibrator which was the bud collected at 12.00 noon. Standard error between three replicates of relative gene expression in each tissue is indicated by error bar.

Relative Expression of VMPCyP450 (fold change)

4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 young bud (green) mature bud (red) half-open flower fully-open flower 14-day old flower

Developmental Stages

Figure 34: Relative Expression Study of VMPCyP450 at Different Flowering Developmental Stages. This study was carried out on five flower developmental stages of Vanda Mimi Palmer including young bud, mature bud, half-open flower, fully-open flower and 14-day old fully-open flower. Quantitative measurement of VMPCyP450 expression in each flower developmental stage was expressed relative to the calibrator which was the young bud collected at 12.00 noon. Standard error between three replicates of relative gene expression at each developmental stage is indicated by error bar.

124

Relative Expression of VMPCyP450 (fold expression)

7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 12am 2am 4am 6am 8am 10am 12pm 2pm Time 4pm 6pm 8pm 10pm

Figure 35: Relative Expression Study of VMPCyP450 at Different Time Points in a 24-hour Cycle. Quantitative expression of VMPCyP450 at each time point was expressed relative to the calibrator which was the fully-open flower collected at 12.00 noon. Standard error between three replicates of relative gene expression at each time point is indicated by error bar.

125

2 47 92 137 182 12 227 27 272 42 317 57 362 72 407 87 452 102 497 117 542 132 587 147 632 162 677 177 722 192 767 812

AAA AAC GTC GTT GTG TCT CGG GGT CGT TGG GGA GAA TTT CTT AAT AAC AGT CGG AAA AAA GGT TCC CTA ATG ATA AGC GGG ACA GTT AGC GCA ACT AAA TTA ATG TGA GAT TAG TTC AAT TCT TAG GCA CCC CAG >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
VMPA28 ORF forward Primer

46 91 136 181 11 226 26 271 41 316 56 361 71 406 86 451 101 496 116 541 131 586 146 631 161 676 176 721 191 766 197 811 856

ATT TTA CAT TTT ATG TTT TCC GGT TCG TTT TTT TTG GGA AAT TTT M F S G S F F L G N F TTA GTG AAT AAC AAT TTC ACC CAG GAA AAC AGT TAT GAC CAT GAT L V N N N F T Q E N S Y D H D TAC GCC AAG CTT GCA TGC CTG CAG GTT GAC TTT AGA GGG GAT CCA Y A K L A C L Q V D F R G D P AAT TTT AAA TAT CCC GCA ACC TGT CCC ACC TTT TCT CCC CTT TTC N F K Y P A T C P T F S P L F GTC TCT TTT TCC CTG TTT CTC TTT CTC ATC CGA TTG ATG GAT CTT V S F S L F L F L I R L M D L AGG GCG GCG ATA GTC GCC GCC GCC GGC GAG CGT TGG ACG GAG GAG R A A I V A A A G E R W T E E CGG CAC TCC CGC TTC CTC AAC TCG ATC GAA AGT ACT TTC GTC CAT R H S R F L N S I E S T F V H CAA Q GCG A GAG E GCT A CTC CGC CGA TGC L R R C ATC GCC GGT AAA I A G K GAT AGG AGG CCT D R R P TGT ACA CGG AAA C T R K >>>>>>>>>>>>>> CGA TCA CTG CGG CGA TAT GAT GCG TCG CTA GAC CAG GTG GTG CCG R S L R R Y D A S L D Q V V P >>>>>>>>>>>
VMPA28 RT-PCR Forward Primer

ATG M GCG A TCT S GCC A

CTC L AGG R GCG A ATT I

GGC G CTC L AAG K ACT T

ATC I GAC D AGT S GCG A

CAT H CGT R TCT S GGC G

CCC P CGT R CAG Q GCC A

GAC D GTT V ATG M AAC N

GGC G CCC P CGA R ACC T

GAT D GAT D TCG S TCT S

AAC N TGC C CCG P AAT N

GAG TTC AAA AAT AAG AAC GTC GGC GAG GAT GCA TCC AAG CGA AAG E F K N K N V G E D A S K R K TTT GAA GAC GCA GCA CAT TAA CAA TGA GAA CAA TAT TGG AGT CTA F E D A A H * AAG CTG CAG CTG TCT TCA TCT CTT GCT GTC TAC AAG CAA TGT TTG <<<<<<<<<<<<<<<<<<<<<<<<<<<
VMPA28 ORF Reverse Primer

VMPA28 RT-PCR Reverse Primer

ACT TGC ATG TTG AAA GGA ATG TAG AAA TGT GTT CTT ATA TAT TAT <<<<<<<<<<<<<<

857 902 947

<<<<<<<<<<<<<< GTT TTA GAG GAA ACG TAT TTA TGA TAA TTT TTG CTT GTT AAA GTT GGT TTT TGC CAT TTC AAC ATG GTT TCA TTC TGT GAA CAT TTT AGA TCA AAA AAA AAA AAA AAA AAA AAA AAA 973

901 946

Figure 36: The Nucleotide and Deduced Amino Acid Sequences of VMPA28. The open reading frame (ORF) of VMPA28 starts at nucleotide 149. The asterisk (*) indicates stop codon. The putative adenylation signal is bold and underline in the nucleotide sequence. The location of gene specific primers used in real-time PCR analysis and ORF isolation are shown by arrow heads (>>>> and <<<<).

126

1000bp ~ 700bp 500bp

Figure 37: PCR Product of Open Reading Frame (ORF) of an Unknown Protein of Vanda Mimi Palmer (VMPA28). The PCR product was electrophoresed on 1% (w/v) agarose gel. Lane M: 100bp marker (Vivantis); Lane 2: ORF of clone VMPA28.

127

protein kinase C phosphorylation sites (SxK, SxR) and a N-myristoylation site (GAxxSN). BLASTX and BLASTP analyses (NCBI) show the deduced amino acid of VMPPA28 had no significant similarity to any known sequence in the GeneBank database. Localizome analysis shows there was no signal peptide in the deduced protein sequence of VMPA28.

Expression analysis of VMPA28 was carried out in different tissues, at different flower developmental stages and also at different time points in a 24-hour cycle by real-time RTPCR. Analysis of VMPA28 transcript in different tissues (Figure 38) shows a slight upregulated expression in floral tissues compared to vegetative tissues. For analysis of VMPA28 transcript at different flower developmental stages (Figure 39), the expression of VMPA28 was developmentally-regulated where the transcripts level increased gradually from the bud to the fully-open flower stage, followed by a gradual decrease until the end of flower life-time. The transcript was also found to be differentially expressed at different time points in a 24-hour cycle (Figure 40).

128

Relative Expression of VMPA28 (fold change)

1.60 1.20 0.80 0.40 0.00

bud

fullyopen flower

petal

sepal

lip

leaf

root

shoot

stalk

Tissues

Figure 38: Relative Expression Study of VMPA28 Transcript in Different Tissues Including Floral and Vegetative Tissues. The floral tissues are bud, fully-open flower, petal, sepal and lip meanwhile the vegetative tissues used in the expression study are leaf, root, shoot and stalk. The quantitative expression level of VMPA28 in each tissue was calculated relative to the calibrator which was the bud collected at 12.00 noon. Standard error between three replicates of relative gene expression in each tissue is indicated by error bar.

Relative Expression of VMPA28 (fold change)

4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 young bud (green) mature bud (red) half-open flower fully-open flower 14-day old flower

Developmental Stages

Figure 39: Relative Expression Study of VMPA28 at Different Flower Developmental Stages. The study was carried out in five flower developmental stages of Vanda Mimi Palmer including young bud, mature bud, half-open flower, fully-open flower and 14-day old fully-open flower. Quantitative measurement of VMPA28 expression in each flower developmental stage was expressed relative to the calibrator which was the young bud collected at 12.00 noon. Standard error between three replicates of relative gene expression at each developmental stage is indicated by error bar.

129

Relative Expression of VMPA28 (fold change)

5.00 4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 12am 2am 4am 6am 8am 10am 12pm Time 2pm 4pm 6pm 8pm 10pm

Figure 40: Relative Expression Study of VMPA28 at Different Time Points in a 24hour Cycle. Quantitative expression of VMPPAAS at each time point was expressed relative to the calibrator which was the fully-open flower collected at 12.00 noon. Standard error between three replicates of relative gene expression at each time point is indicated by error bar.

130

CHAPTER 5 DISCUSSIONS

5.1 Biochemical Analysis of the Scent of Vanda Mimi Palmer

Biochemical analysis of the scent of Vanda Mimi Palmer by gas chromatography-mass spectrometry (GC-MS) showed a fluctuating pattern of volatile emission by fully-open flower of Vanda Mimi Palmer in a 24-hour cycle with the highest level detected during the daytime (in light) but none at night (in dark). This emission pattern was observed in most roses and snapdragon (Anthirrhinum majus) whereby the highest level of scent emission was reported to occur during daytime (Helsper et al., 1998; Picone et al., 2004; Dudareva et al., 2003). Meanwhile, for some other plants such as in Petunia hybrida and Stephanotis floribunda, the highest level of scent emission occurs at night (Verdonk et al., 2003; Pott et al., 2002). The pattern of scent emission of Vanda Mimi Palmer in a 24hour cycle might be controlled by some factors such as photoperiod and circardian clock like in other scented plants (Lu et. al, 2002). Besides that, flowers of Vanda Mimi Palmer also showed a developmentally regulated pattern of scent emission whereby no volatile compound was detected in the bud stage. Emission of volatile compounds was detected from the half-open flower stage and increased to the maximum level in the fully-open flower stage. The same pattern of scent emission was previously reported in other scented flowers including Clarkia breweri and Anthirrhinum majus (Pichersky et al., 1994; Nagegowda et al., 2008).

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Based on the GC-MS analysis of the scent of Vanda Mimi Palmer, volatile compounds emitted by the flowers are derived from the terpenoid, benzenoid and phenylpropanoid groups. Thus, there are possibly two main pathways involved in the fragrance biosynthesis of Vanda Mimi Palmer which are terpenoid as well as benzenoid and phenylpropanoid pathways. Hsiao et al. (2006) reported that the volatile compounds in Phalaenopsis bellina (a scented orchid from Taiwan) are derived from the three pathways: terpenoid, lipoxygenase, as well as benzenoid and phenylpropanoid pathways (Hsiao et al., 2006). These three fragrance biosynthetic pathways were also reported in other well studied scented flowers like Rosa hybrida (Guterman et al., 2002), Clarkia breweri (Pichersky et al., 1995), Petunia hybrida (Boatright et al., 2004) and Anthirrhinum majus (Nagegowda et al., 2008).

The GC-MS analysis of the scent of Vanda Mimi Palmer showed that there are four volatile compounds (linalool, ocimene, linalool oxide and nerolidol) which might have been derived from the terpenoid pathway. Linalool, ocimene and linalool oxide are classified as monoterpenes (Croteau and Karp, 1991; Knudsen and Gershenzon, 2006) while nerolidol is classified as a sesquiterpene (Knudsen and Gershenzon, 2006; Nagegowda et al., 2008). Monoterpenes and sesquiterpenes are the common compounds in the scent of scented orchids including Phalaenopsis bellina (Hsiao et al., 2006), Dendrobium beckleri and Phalaenopsis violacea (Kaiser, 1993). The terpenoid compounds emitted by Vanda Mimi Palmers flower were also detected in the scent of other scented flowers including Anthirrhinum majus (Linalool, ocimene and nerolidol) (Dudareva et al., 2003; Nagegowda et al., 2008), and Clarkia breweri (Linalool)

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(Pichersky et al., 1994). Thus, the terpenoid pathway in Vanda Mimi Palmer involved in the biosynthesis of linalool, ocimene and nerolidol compounds might be closely related to the terpenoid pathway in other well studied scented flowers. The terpenoid pathway for the floral scent biosynthesis in those well studied scented flowers could be used for the study of the terpenoid pathway in Vanda Mimi Palmer.

Besides terpenoids, there were four other compounds in the scent of Vanda Mimi Palmer which might be derived from the benzenoid and phenylpropanoid pathway such as methylbenzoate, benzyl acetate, phenylethanol and phenylethyl acetate. The same benzenoid and phenylpropanoid compounds were reported to be present in the scent of other scented orchids like Dendrobium trigonopus, Dendrochilum cobbianum and Vanda tessellata (Kaiser, 1993), and in other scented flowers like Rosa hybrida (Shalit et al., 2003), Petunia hybrida (Verdonk et al., 2003), Clarkia breweri (Raguso and Pichersky, 1995; Dudareva et al., 1998) and Anthirhinum majus (Dudareva et al., 2000). In Phalaenopsis bellina, some other benzenoid and phenylpropanoid compounds such as 3phenyl-2-propen-1-ol, 3-methylphenyl butanoic ester, and 2-methylphenyl butanoic ester were detected in its floral scent (Hsiao et al., 2006). Benzyl acetate which was detected in the scent of Vanda Mimi Palmer, was also reported to be present in the scent of Rosa hybrida (Shalit et al., 2003) and Clarkia breweri (Raguso and Pichersky, 1995; Dudareva et al., 1998). Another detected compound, methylbenzoate was also reported in many scented flowers like Petunia hybrida (Verdonk et al., 2003) and Anthirhinum majus (Dudareva et al., 2000). Besides that, phenylethanol, a compound which is also present in the scent of Vanda Mimi Palmer, was identified in the scent of Rosa hybrida (Shalit et

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al., 2003), Petunia hybrida (Verdonk et al., 2003), Clarkia breweri (Raguso and Pichersky, 1995; Dudareva et al., 1998), and Rosa hybrida (Shalit et al., 2003). Thus, the benzenoid and phenylpropanoid pathways in Vanda Mimi Palmer which could possibly be involved in the biosynthesis of methylbenzoate, benzyl acetate, phenylethyl acetate and phenylethanol compounds might be closely related to the benzenoid and phenylpropanoid pathways in well studied scented flowers especially like Petunia hybrida, Clarkia breweri and Rosa hybrida.

Solid phase Micro-extraction (SPME) method used in this study is one of the advanced methods utilised to capture volatile compounds emitted by scented flowers. In this study, a modified funnel was used to collect and accumulate the volatiles emitted by Vanda Mimi Palmer flowers in a special trap before being captured by the SPME. It is possible that there might be some other compounds in the scent of Vanda Mimi Palmer emitted in traces amount that could not be detected in this study using the above method (capturing was just for 15 minutes). Trace compounds could be detected and identified using a combination of modern headspace attached directly to the GC-MS where the volatiles emitted by the scented flowers are accumulated in a special chamber for 2-3 hours and then concentrated with a special pump prior injection into the GC-MS port. Unfortunately, this approach was not employed in this study due to inavailability of the system in Universiti Putra Malaysia. There is another method used in the floral scent studies in rose by Hendel-Rahmanim et al. (2007) and Farhi et al. (2010) on Rosa hybrida whereby a few grams of the rose petals were soaked in hexane for a few hours. The debris was removed and the supernatant containing hexane with the extracted compounds

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was filtered and then concentrated with nitrogen before being injected into the GC-MS injector port. In this study, the above mentioned method was applied to extract the essential oil from Vanda Mimi Palmer using hexane and a large amount of flowers (100grams). Most of the compounds detected at high levels in the scent of Vanda Mimi Palmer were also detected in the essential oil of Vanda Mimi Palmer. However, there were some compounds including ocimene and nerolidol which were not found in the essential oil. One possible reason for non-detectability of ocimene is because the compounds might not be stored in the cell long enough prior to its release into the air. As for nerolidol, this compound could be synthesized at very low amount compared to other compounds such as linalool, methylbenzoate, benzyl acetate and phenylethyl acetate in the floral tissues of Vanda Mimi Palmer. Besides that, a lot of other compounds which are not emitted by fully-open flower of Vanda Mimi Palmer were also detected in the essential oil (see Appendix D, subsection (c) and (d)). This could be due to the nature of hexane itself as a powerful solvent to extract out non-polar compounds from the plant samples.

In Vanda Mimi Palmer, the emitted scent is dominated by ocimene and linalool compounds in the morning (8.00am-12.00 noon). At this time, other compounds were detected at very low levels especially the compounds derived from the benzenoid and phenylpropanoid pathways. However, the emission levels of benzenoid and phenylpropanoid compounds especially benzyl acetate and phenylethyl acetate increased drastically after 12.00 noon, higher than the levels of the terpenoid compounds (linalool and ocimene). Thus, the percentage of each compound of the scent of Vanda Mimi

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Palmer is different at different time points during daytime. This might be the special characteristic of the scent of Vanda Mimi Palmer to attract different pollinators at different time points during daytime. Unfortunately no comparison can be made with other well studied scented flowers such as Petunia hybrida, Clarkia breweri, Antirrhinum majus and Rosa hybrida as their percentage of each compound in the scent at different time points and their functions to attract specific pollinators at different time points were not investigated.

Comparison of the volatiles emitted by Vanda Mimi Palmer (scented orchid) and its parents, Vanda Tan Chay Yan (non-scented orchid) and Vanda tessellata (scented orchid) in Table 6 (section 4.1.1) shows ocimene was the only compound detected in the scent of Vanda Mimi Palmer and both of its parents. Ocimene is a monoterpene derived from the terpenoid pathway (Dudareva et al., 2003). An ocimene synthase from Vanda Mimi Palmer is postulated to be involved in the final step of terpenoid biosynthetic pathway. This enzyme could be involved in catalyzing the formation of ocimene from geranyl diphosphate, a precursor for monoterpenoid biosynthesis. The transcript of this enzyme has yet to be identified in Vanda Mimi Palmer. For another monoterpene compound which is linalool, a linalool synthase transcript has been identified in the floral ESTs of Vanda Mimi Palmer (Teh Siow Ling, Master student, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, pers. comm. on 18th September 2008). This enzyme might be involved in catalyzing the formation of linalool from geranyl diphosphate. The linalool synthase gene might be derived from Vanda tessellata since

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linalool was also detected in the scent of Vanda tessellata previously by Kaiser (Kaiser, 1993) using GC-MS.

Besides that, the formation of nerolidol (a sesquiterpene) was postulated to be catalyzed by sesquiterpene synthase (Chan, 2009). The transcript of sesquiterpene synthase was identified from the floral ESTs of Vanda Mimi Palmer. This sesquiterpene gene in Vanda Mimi Palmer could be contributed by the gene pool from Vanda Tan Chay Yan since coupane, a sesquiterpene was identified to be emitted by fully-open flower of Vanda Tan Chay Yan (see Figure 10, in section 4.1.1). In contrast, no sesquiterpene was detected in the scent of Vanda tessellata (another parent of Vanda Mimi Palmer) as reported by Kaiser (Kaiser, 1993). The combination of GC and MS used by Kaiser in his work (Kaiser, 1993) at that time was sensitive enough to detect the presence of sesquiterpenes since some sequiterpenes were detected in other scented orchids including Aerangis confuse (germacrane D and nerolidol), Dendrobium virgineum (nerolidol), Laelia anceps (nerolidol), Oncidium longipes (nerolidol), and Polystachya fallax (caryophyllene and farnesene) (Kaiser, 1993). In Vanda tessellata, sesquiterpene synthase might not be expressed if it is present as a recessive allele of the gene. While in Vanda Tan Chay Yan, seisquiterpene synthase gene might be represented by a dominant allele. The hybrid of these orchids could bring heterozygous dominant characteristic of the sesquiterpene synthase. Interestingly, there were another two sesquiterpenes identified in the essential oil of of Vanda Mimi Palmer (germacrene and copaene) (see Table 6) besides nerolidol that was identified in the scent of Vanda Mimi Palmer.

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Volatiles comparison between Vanda Mimi Palmer and its parents (see Table 4, in section 4.1.1) shows that two benzenoids (benzyl acetate and methylbenzoate) were detected in the scent of both Vanda Mimi Palmer and Vanda tessellata (Kaiser, 1993). These compounds could be derived from the benzenoid pathway. A number of benzenoids identified in the scent of Vanda tessellata (Kaiser, 1993) were not detected in the scent of Vanda Mimi Palmer such as benzaldehyde, benzyl alcohol, cinnamyl alcohol, methyl cinnamate and methyl salicylate. Similarly, some compounds were only detected in the scent of Vanda Mimi Palmer including phenylethanol and phenylethyl acetate. Based on the differences, it was postulated that some modifications might have occurred in the benzenoid and phenylpropanoid pathways of Vanda Mimi Palmer compared to its parent Vanda tessellata which showed emission of benzenoids and phenylpropanoids (Kaiser, 1993). Benzaldehyde, benzyl alcohol and cinnamyl alcohol which were detected in the scent of Vanda tessellata as final products might be intermediates or precursors for methylbenzoate, benzylacetate and phenylethyl acetate biosynthesis in Vanda Mimi Palmer. In addition, phenylethanol compound was identified in the essential oil of both Vanda Mimi Palmer (scented orchid) and Vanda Tan Chay Yan (non-scented orchid). The phenylethanol might be used directly or acts as an intermediate for other phenylpropanoids involved fragrance and non-fragrance metabolism. In addition, the benzenoid and phenylpropanoid pathway has been involved in the biosynthesis of other non-fragrance compounds including flavanoids and high molecular weight

phenylpropanoids (Lacombe et al., 1997; Shirley, 2001; Boerjan et al., 2003; Takashi et al., 2007; Derikvand et al., 2008). Interestingly, in Vanda Mimi Palmers essential oil, the phenylethanol compound was detected at high level, 11.18% of the total essential oil. In

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Vanda Mimi Palmer, the phenylethanol emission was only detected at trace level during the highest peak, while the other phenylpropanoid which is phenylethyl acetate was detected to be emitted during day time at very high level (see Figure 7). This suggested that phenylethanol might be the main precursor for phenylethyl acetate in Vanda Mimi Palmer.

Most of the compounds in the scent of Vanda Mimi Palmer were also detected in its essential oil including linalool, methylbenzoate, benzyl acetate, phenylethanol and phenylethyl acetate. The compounds might be stored in special oil glands or trichomes for few hours before they are released into the air as volatiles since a lot of oil glands or trichomes were detected on the surface of the petal and sepal of fully-open flowers of Vanda Mimi Palmer (Janna Ong Abdullah, unpublished data). Besides that, two intermediates of the scents compound such as benzyl alcohol and benzyl benzoate were detected in the essential oil of Vanda Mimi Palmer (see Table 6, in section 4.1.1). The intermediate compounds might be the precursors for the production of benzyl acetate and phenylethyl acetate which were detected in the scent of Vanda Mimi Palmer. In addition, none of the fragrance compounds in the scent of Vanda Mimi Palmer was detected in the essential oil of Vanda Tan Chay Yan except for phenylethanol. This phenylethanol might be involved in non-fragrance activities since the compound was not detected in the volatiles of fully-open flowers of Vanda Tan Chay Yan. In the essential oils of Vanda Mimi Palmer and Vanda Tan Chay Yan, a lot of non-fragrant compounds were identified (see Appendix D, subsection (c) and (d)). This might be due to the nature of the hexane extraction method itself which could extract out most of the non-polar compounds from

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the sample. Alternatively, pure essential oil could be isolated by a technique called hydro-distillation. However, the hydro-distillation method has its limitation since it requires a large amount of flowers.

Based on biochemical analysis on volatiles of Vanda Mimi Palmer and comparison with its parents scent compounds, it seems that the fragrant characteristic of Vanda Mimi Palmer might be contributed by a pool of genes from both parents. In other scented flower especially roses, the scent produced by Rosa chinensis (Chinese rose), an ancestor of modern roses (Rougetel, 1988) is different from the scent of modern rose cultivars (Wu et al., 2004). A lot of compounds in the scent of the Chinese rose are not found in modern roses cultivars and some of them do not have any fragrance because breeding was only focused on the beautiful colour and shape of the flower instead of the fragrance itself (Yomogida, 1992; Zuker et al., 1998; Wu et al., 2004). For example, 1,3,5trimethoxybenzene compound (TMB) synthesized from ploroglucinol, was identified in the scent of Rosa chinensis but was not detected in the scent most of modern rose varieties but a related compound which is 3,5-dihyroxytoulene synthesized from orcinol was detected in many modern roses. Biochemical modifications could have occured in the fragrance biosynthetic pathway of roses by interaction of genes and enzymes derived from parents of rose hybrids (Flament et al., 1993; Lavid et al., 2002; Wu et al., 2004).

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5.2 Molecular Studies of Vanda Mimi Palmer

5.2.1 Isolation of Putative Fragrance-related cDNAs

Thirteen putative fragrance-related cDNA clones were isolated from the floral cDNA library of Vanda Mimi Palmer and preliminarily characterized by reverse-Northern analysis with two different probes representing mRNAs of fully-open flower and bud stage separately. The aim was to select cDNAs with up-regulated expression in fullyopen flower stage (high fragrance emission) compared to bud stage (no fragrance emission) of Vanda Mimi Palmer which might be potential fragrance-related cDNAs.

From the verification of the putative fragrance-related cDNAs with up-regulated expression in fully-open flower compared to bud of Vanda Mimi Palmer in section 4.2.1.4, VMPCMEK (putative 4-(cytidine 5-diphospho)-2-C-methyl-d-erythritol kinase) was selected for further characterization because it showed higher expression in fullyopen flower of Vanda Mimi Palmer compared to the bud. This could be due to the involvement of VMPCMEK transcripts in the early steps of the terpenoid pathway (see Figure 41) for the biosynthesis of monoterpenes and sesquiterpenes such as linalool, ocimene and nerolidol as reported in section 4.1.1.

Besides VMPCMEK, VMPCyP450 was also selected for further characterization because of its high expression in the fully-open flower of Vanda Mimi Palmer compared to the bud. In the bud, this VMPCyP450 might be involved in other metabolism especially for

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PLASTID
Pyruvate + glyceraldehyde 3-phosphate
VMPDXS

CYTOSOL

1-Deoxy-D- 5-phosphate (DXP)

VMPDXR

2-C-Methyl-D-erythritol 4-phosphate (MEP) 4-(Cytidine 5-diphospho)-2-CMethyl-D-erythritol (CDP-ME) 2 Acetyl-CoA

VMPCMEK

Acetoacetyl-CoA
VMPAACT

4-(Cytidine 5-diphospho)-2-CMethyl-D-erythritol 2-phosphate (CDP-ME)

3S-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)
VMPHMGR

3R-Mevalonic acid (MVA) 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate (cMEPP) Mevalonic acid 5-phosphate 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP)
VMPHDS

Mevalonate diphosphate

Dimethylallyl diphosphate (DMAPP)

VMPLIS

Isopentenyl diphosphate (IPP)

Isopentenyl diphosphate (IPP)

VMPFPPS

Dimethylallyl diphosphate (DMAPP)

Geranyl diphosphate (GPP)

Farnesyl diphosphate (FPP) Linalool

VMPCyP450

Ocimene

VMPSQS

Nerolidol Linalool oxide

Figure 41: Elucidation of Terpenoid Pathway in Vanda Mimi Palmer. The pathway is elucidated based on the terpenoid pathway of Anthirrhinum Majus (Nagegowda et al., 2008). VMPCMEK and VMPCyP450 which was identified and isolated in this study are shown in circles. Other putative fragrance related cDNAs that were isolated from floral ESTs of Vanda Mimi Palmer are shown in boxes (Janna Ong Abdullah, Associate Professor, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, pers. comm. on 26th October 2009).

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the biosynthesis of non-fragrance compounds since cytochrome P450 has been reported to be involved in metabolism in the lipoxygenase pathway and also phenolic metabolism (Ehlting et al., 2006). Another transcript selected for further characterization is an unknown protein designated as VMPA28. The verification result shows the expression of VMPA28 transcript was slightly higher in the fully-open flower compared to the bud, suggesting this transcript might be involved in other metabolisms in the plant system which has yet to be investigated.

5.2.2 Molecular Characterization of Putative Fragrance-related Transcripts

5.2.2.1 Sequence and Expression Analysis of Putative Phenylacetaldehyde Synthase (VMPPAAS)

The BLASTX and BLASTP analyses (NCBI) show the deduced amino acid sequence of VMPPAAS is 49-63% similar to other plant decarboxylases such as phenylacetaldehyde synthases, tryptophan decarboxylases, tyrosine decarboxylases and aromatic amino acid decarboxylases. GC-MS analysis of the scent of Vanda Mimi Palmer shows the presence of phenylethyl acetate, which could be synthesized via the benzenoid and phenylpropanoid pathways. Phenylacetaldehyde synthase from Petunia hybrida catalyzed the decarboxylation of phenylalanine to phenylacetaldehyde (Kaminaga et al., 2006), which might be the potential precursor for the production of phenylpropanoids including phenylethanol and phenylethyl acetate in Vanda Mimi Palmer. Previously in floral scent studies, two phenylacetaldehyde synthases have been identified from Petunia hybrida (PhPAAS) and Rosa hybrida (RhPAAS). The PhPAAS and RhPAAS share ~50-60%

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identity with other plant decarboxylases including tyrosine decarboxylases, tryptophan decarboxylases and aromatic amino acid decarboxylases (Kaminaga et al., 2006). Motif prediction by Expasy tool showed a pyridoxal phosphate attachment site in the deduced amino acid sequence of VMPPAAS and other plant decarboxylases (see figure 17). Besides that, a conserved VHVDAAY motif has been identified in the deduced amino acid sequence of VMPPAAS as reported in other decarboxylase proteins from plants and bacteria by Connil et al., 2002 (see figure 17). Thus, VMPPAAS could be classified as aromatic amino acid decarboxylase which might be involved in decarboxylation of aromatic amino acids in Vanda Mimi Palmer especially for phenylalanine. The involvement of VMPPAAS in decarboxylation of phenylalanine to phenylacetaldehyde can only be confirmed by enzymatic assay (see Figure 42). Phylogenetic analysis of VMPPAAS with other plant decarboxylases shows VMPPAAS is not clustered together in the same group as PhPAAS and RhPAAS since Rosa hybrida and Petunia hybrida come from different family and genera compared to Vanda Mimi Palmer. Localization analysis by Localizome software shows no transit peptide in VMPPAAS amino acid sequence, suggesting this protein might be localized in cytosol.

Relative

expression

analysis

in

different

tissues

shows

that

the

putative

phenylacetaldehye synthase (VMPPAAS) was up-regulated in floral tissues compared to vegetative tissues. Among the floral tissues of Vanda Mimi Palmer, petal showed the highest expression of VMPPAAS and followed by sepal. In other scented flowers, petals have been identified as the main source of scent emission and biosynthesis. Most of the

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Figure 42: Elucidation of Benzenoid and Phenylpropanoid Pathways of Vanda Mimi Palmer. This elucidation is based on benzenoid and phenylpropanoid metabolism of Petunia hybrida (Boatright et al., 2004; Pichersky and Dudareva, 2007).

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well studied fragrance-related transcripts have shown higher expression levels in petal compared with the other floral parts including in Petunia hybrida (Verdonk et al., 2003), Clarkia (Onagraceae) (Pichersky et al., 1994), and Rosa hybrida (Shalit et al., 2003). In sepal, the high level of VMPPAAS expression (slightly lower than petal) might be due to the similar structure and function of sepal and petal in orchid. This is supported by earlier histological work on Vanda Mimi Palmer that showed the presence of many oil glands (trichomes) in petals and sepals (Janna Ong Abdullah, unpublished data) suggesting potential sites for fragrance biosynthesis and accumulation.

Relative expression analysis of putative VMPPAAS at different developmental stages shows a developmentally regulated pattern. This expression study complements the result of GC-MS analysis on the scent of Vanda Mimi Palmer at different developmental stages (in section 4.1.1) whereby the emission of phenylethyl acetate in Vanda Mimi Palmer is developmentally regulated. In bud stages including young and mature buds, the expression of VMPPAAS showed a down-regulated expression compared to half-open and fully-open flower stages. In addition, from the GC-MS analysis, no volatile compound was detected in the bud stage. In other scented flowers including Clarkia breweri, Anthirrhinum majus, Rosa hybrida and Petunia hybrida (Pichersky et al., 1994; Nagegowda et al., 2008), no emission of fragrance-related compounds were detected at the bud stage. Thus, during early stages of flower development, the floral tissues of Vanda Mimi Palmer might not be ready for fragrance biosynthesis. In half-open flower stage, the expression of VMPPAAS increased drastically and reached the highest level at fully-open flower stage. At 14-day old fully-open flower, the VMPAAS expression level

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decreased drastically compared to the early fully-open flower stage. The developmentally regulated pattern of fragrance biosynthesis in Vanda Mimi Palmer is also similar to other fragrance-related transcripts of S-adenosyl-L-methionine: benzoic acid carboxyl methyl transferase (BAMT) from Anthirrinum majus (Dudareva et al., 2003), geranyl diphosphate synthase from Phalaenopsis bellina (Hsiao et al., 2008), and linalool synthase from Antirrhinum majus (Nagegowda et al., 2008).

Relative expression analysis of VMPPAAS in the fully-open flower of Vanda Mimi Palmer in a 24-hour cycle shows a differential expression whereby the expression is upregulated at night time and down-regulated during day time. The expression pattern of the VMPPAAS in a 24-hour cycle is opposite compared to the emission of phenylethyl acetate in a 24-hour cycle whereby the emission of phenylethylacetate compounds from the fully-open flower was detected at high levels during day time and not detected at night (see section 4.1.1). This might be due to VMPPAAS being not directly involved in catalyzing the formation of the end product (phenylethyl acetate) but involved in the formation of the main precursor for the end product. In addition, the precursor might be stored in cells for few hours before being used for subsequent reaction and released as volatile (phenylethyl acetate) during day time. The gene that encodes the rate-limiting enzyme for the formation of phenylethyl acetate (not identified yet) might show high expression during day time because phenylethyl acetate emission was detected high during day time (see section 4.1.1). In other scented flowers such as Petunia hybrida and Stephanotis floribunda, the expression of fragrance-related genes and emission of volatiles were very high level during night time and very low level during the day (Pott et

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al., 2002; Boatright et al., 2004). Meanwhile, in Anthirrhinum majus, both fragrancerelated genes expression (mycrene synthase and ocimene synthase) and volatiles emission were detected at high level during the night (Dudareva et al., 2003). In other scented flowers including Petunia hybrida (Boatright et al., 2004), Anthirrhinum majus (Dudareva et al., 2003; Nagegowda et al., 2008) and Stephanotis floribunda (Pott et al., 2002), the volatiles emission pattern and expression profile of fragrance-related genes which are involved in the formation of end product are usually similar.

5.2.2.2 Sequence and Expression Analysis of Putative 4-(cytidine 5'-diphospho)-2-Cmethyl-D-erythritol Kinase (VMPCMEK)

The deduced amino acid sequence of VMPCMEK shows 66-72% identity to 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase from other plants including Hevae brasiliensis, Solanum lycopersicum, Catharanthus roseus and Ginkgo biloba. Besides that, VMPCMEK shared the conserved ATP-binding site for functional activity of 4(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (Kim et al., 2008). In addition, there are few other putative conserved motifs shared by VMPCMEK and other plant 4(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase as predicted by the Expasy tool. The 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase has been reported to be involved in phosphorylation of 4-(cytidine 5-diphospho)-2-C-methyl-D-erythritol into its phosphorylated form, 4-(cytidine 5-diphospho)-2-C-methyl-D-erythritol 2-phosphate in the terpenoid pathway (Kim et al., 2008) (see Figure 41 in section 5.2.1). The subsequent reactions in the terpenoid pathway might produce the main precursors including

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dimethylallyl diphosphate (DMAPP) and Isopentenyl diphosphate (IPP) (see Figure 41 in section 5.2.1) for biosynthesis of monoterpenes and sesquiterpenes (Ogura and Koyama, 1998; Poulter and Rilling, 1981; McGarvey and Creteau, 1995). The Localizome software predicted the presence of signal peptide in VMPCMEK amino acid sequence. Thus, VMPCMEK might be localized in plastid since it is predicted to be involved in the early step of terpenoid pathway, the methyl erythritol phosphate (MEP) pathway which has been reported to occur in plastid (Lichtenthaler, 1999; Rohmer, 1999).

Relative expression analysis of putative 4-(cytidine 5'-diphospho)-2-C-methyl-Derythritol kinase (VMPCMEK) in different tissues showed up-regulated expression in the tested floral tissues compared to vegetative tissues. VMPCMEK is predicted to be involved in the earlier path of terpenoid pathway which might contribute to the biosynthesis of volatile terpenoids including linalool, ocimene and nerolidol, as detected in the floral scent of Vanda Mimi Palmer by GC-MS analysis (see section 4.1.1). In the bud, no fragrance compounds had been detected by GC-MS but the VMPCMEK expression level was relatively high in bud compared to vegetative tissues. This might be due to the involvement of terpenoid pathway for synthesis of other non-volatile terpenoids including carotenoids, gibberelin and sterol which might be used either in primary or secondary metabolisms (Bremly, 1997).

Analysis of VMPCMEK expression at different flower developmental stages shows a developmentally regulated pattern in accordance to volatile emission pattern (in section 4.1.1), suggesting the terpenoids emission is developmentally regulated. Terpenoid

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pathway was also reported to be important for gibberellin biosynthesis whereby gibberellins were reported to play an important role in plant growth and development (Bremley, 1997). VMPCMEK might be involved in the early step of the terpenoid pathway which contributes for bud development. Thus, during bud stage of Vanda Mimi Palmer, VMPCMEK might play an important role for flower development while at open flower stage (once the flower open until end of the flower life-time), VMPCMEK might contribute for fragrance biosynthesis.

VMPCMEK shows a differential expression in a 24-hour cycle but the expression pattern is totally different compared to VMPPAAS. This could be due to VMPCMEK and VMPPAAS might be involved in different pathways in fragrance biosynthesis of Vanda Mimi Palmer. Besides that, the expression of VMPCMEK and emission pattern of terpenoids including linalool and ocimene (see Figure 6 in section 4.1.1) are also different. At 2pm, the expression of VMPCMEK is at the lowest level while the emission level of linalool and ocimene compound are detected at the highest level. The expression of VMPCMEK increased gradually from 12.00am until 8.00am, and then decreased gradually until the lowest peak at 2.00pm while the emission of terpenoids including linalool and ocimene (see Figure 6 in section 4.1.1) were detected at very low levels as early as 6.00am and increased gradually until the highest peak at 2.00pm. This might be due to the fragrance compounds or their intermediates might not be directly emitted after being synthesized and the compounds might be stored in trichomes for a few hours before being released into the air as scent. The precursors of the final compounds might be accumulated in cytosol or plastid before being used for the final product biosynthesis.

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Besides that, the volatiles emission might not be controlled at gene expression level, but influenced by environment factors like heat and light. This is supported by the fact that the emission of volatiles from the flower of Vanda Mimi Palmer were detected at very high level in the early afternoon, a period when highest temperature usually occur in Malaysia.

5.2.2.3 Sequence and Expression Analysis of Putative Cytochrome P450 Protein (VMPCyP450)

BLASTX and BLASTP analysis (NCBI) shows the deduced amino acid of VMPCyP450 exhibited 39-67% similarity to other plant cytochrome p450 proteins including p450 protein from Oryza sativa, Populus trichocarpa, Ricinus communis, Capsicum annum and also cytochrome P450 monooxygenase of Petunia hybrida, a well studied scented flower. Cytochrome p450 proteins have been reported to be involved in modification of monoterpene compounds into another compound such as oxidation of linalool to linalool oxide (Hsiao et al., 2006; Dudareva et. al., 2004; Dudareva and Pichersky, 2000). Thus, VMPCyP450 is postulated to be involved in fragrance biosynthetic pathway of Vanda Mimi Palmer since GC-MS analysis of the scent of Vanda Mimi Palmer detected traces of linalool oxide. Besides that, cytochrome P450s have been reported to be involved in other fragrance biosynthetic pathways including benzenoid, phenylpropanoid and lipoxygenase pathways (Ehlting et al., 2006). Thus, in Vanda Mimi Palmer, VMPCyP450 might be involved in fragrance biosynthesis eventhough the exact function of the protein in fragrance biosynthetic pathway of Vanda Mimi Palmer is still far from understood. Phylogenetic analysis of VMPCyP450 with closely related proteins from other plants

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shows VMPCyP450 is clustered together with cytochrome P450 monooxygenase of Petunia hybrida (a well studied scented flower). Thus, VMPCyP450 might be involved in fragrance biosynthesis like Petunia hybrida eventhough the cytochrome P450 monooxygenase is not well studied in Petunia hybrida. Localization analysis by Localizome shows no signal peptide in the VMPCyP450 amino acid sequence. The same analysis shows the presence of a transmembrane domain in the VMPCyP450 amino acid sequence. Thus, VMPCyP450 could possibly be located in the plastid membrane like cyctochrome C which is involved in photosynthesis. So, VMPCyP450 might be involved indirectly in the terpenoid pathway of Vanda Mimi Palmer since monoterpene biosynthesis in plant mostly occurs in plastid (Lichtenthaler, 1999; Rohmer, 1999).

Relative expression analysis of putative cytochrome P450 protein (VMPCyP450) in different tissues showed up-regulated expression in floral tissues compared to vegetative tissues. Among the floral tissues, the lip shows the highest expression, more than two times higher than petal and sepal. This contrasting result compared to other putative fragrance-related transcripts (VMPPAAS and VMPCMEK) in Vanda Mimi Palmer could be due to the involvement of VMPCyP450 in other metabolisms besides fragrance biosynthesis. As reported by Seidenfaden and Wood (1992), lip of orchid, which is a modified petal, has a complicated structure. Transformation of a putative P450 gene into Phalaenopsis flowers (orchidecae genera) showed a possibility for colour modification of flowers compared to wild type where the anthocyanins level showed an increased (Su and Shu, 2003). Thus, in Vanda Mimi Palmer, VMPCyP450 might also be involved in the bright colour formation especially in the lip. In petal and sepal, VMPCyP450 might be

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involved indirectly in fragrance biosynthesis as cytochrome P450 proteins were reported to be involved indirectly in fragrance biosynthesis including terpenoid, benzenoid and phenylpropanoid, as well as lipoxygenase pathways as in other plants (Ehlting et al., 2006). In Vanda Mimi Palmer, VMPCyP450 might be involved in terpenoids as well as benzenoid and phenylpropanoid pathways because volatile fragrance compounds from both classes were detected in the scent of Vanda Mimi Palmer (see section 4.1.1).

Real-time PCR result on VMPCyP450 expression at different developmental stages shows a developmentally regulated pattern like other putative fragrance-related transcripts (VMPPAAS and VMPCMEK). This result is expected since the fragrance compounds emission detected by GC-MS (see section 4.1.1) is developmentally regulated eventhough the specific location of VMPCyP450 in the fragrance biosynthetic pathway is still not well understood. Expression analysis of VMPCyP450 at different time points in a 24-hour cycle shows a differential expression throughout the day. The pattern is totally different compared to other putative fragrance-related transcripts (VMPPAAS and VMPCMEK). This could be because VMPCyP450 is not only involved in fragrance biosynthesis but also other metabolic functions in Vanda Mimi Palmer.

5.2.2.4 Sequence and Expression Analysis of Unknown Protein (VMPA28)

Analysis of the deduced amino acid sequence of an unknown protein transcript (VMPA28) of Vanda Mimi Palmer shows no significant hit to any known proteins in the Genebank database. Thus, sequence analysis with closely related proteins including clustal W alignment and phylogenetic analysis cannot be carried out. Localization

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analysis by Localizome software shows VMPA28 does not have any signal peptide in its amino acid sequence. Thus, VMPA28 might be localized in the cytosol. Motif search by Expasy tool shows the presence of some sites including a N-glycosylation site (NTSN), two protein kinase C phosphorylation sites (SxK, SxR) and a N-myristoylation site (GAxxSN). The function of the VMPA28 can only be confirmed by functional study of this protein.

Real-time PCR analysis on the expression of the VMPA28 transcript in different tissues showed a slightly higher expression in floral tissues compared to vegetative tissues. This could be due to the involvement of VMPA28 in both fragrance and non-fragrance metabolisms eventhough the function of VMPA28 protein is still far from understood. Expression analysis of VMPA28 transcript at different flowering developmental stages shows a developmentally regulated pattern like other putative fragrance related transcripts (VMPPAAS, VMPCMEK and VMPCyP450). The result is in accordance to the GC-MS result on the scent of Vanda Mimi Palmer whereby the scent emission is developmentally regulated. Meanwhile, expression analysis of VMPA28 transcript in a 24-hour cycle showed a differential expression which is totally different compared to other putative fragrance-related transcripts selected (VMPPAAS, VMPCMEK and VMPCyP450) for molecular characterization. Thus, the involvement of VMPA28 in fragrance metabolism in Vanda Mimi Palmer could be different to these other putative fragrance-related transcripts selected for molecular characterization.

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CHAPTER 6 CONCLUSION

The objectives of this study have been successfully achieved by biochemical analysis of the scent of Vanda Mimi Palmer as well as isolation and molecular characterization of putative fragrance-related transcripts that might be involved in the fragrance biosynthetic pathways of this orchid hybrid. Two fragrance biosynthetic pathways have been elucidated to play a role in biosynthesis of the fragrance which are terpenoid and also benzenoid and phenylpropanoid pathways. In the terpenoid pathway, a putative fragrance-related transcript, putative 4-(cytidine 5-diphospho)-2-C-methyl-d-erythritol kinase (VMPCMEK) which was identified by differential screening of floral cDNA was postulated to be involved in the early step of this pathway. The VMPCMEK transcript might play an important role in the biosynthesis of monoterpenes and sesquiterpene including linalool, ocimene, and nerolidol. Meanwhile, a putative phenylacetaldehyde synthase (VMPPAAS) isolated from ESTs of Vanda Mimi Palmer might be involved in the early path of benzenoid and phenylpropanoid pathway, possibly involved in the biosynthesis of phenylethanol and phenylethyl acetate compounds which were detected in the floral scent of this orchid. Besides that, there were two putative fragrance-related genes encoding putative cytochrome P450 protein (VMPCyP450) and an unknown protein (VMPA28) were also isolated and characterized at molecular level. These genes might contribute to the fragrance of Vanda Mimi Palmer indirectly.

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Combination of GC-MS analysis of the scent of Vanda Mimi Palmer and expression analysis of putative fragrance-related transcripts at different developmental stages have shown that the floral scent biosynthesis and emission in Vanda Mimi Palmer is developmentally and rhythmically regulated. The knowledge from this work is important for preliminary understanding of fragrance characteristics and its biosynthetic pathway in Vanda Mimi Palmer. In order to gain a deeper insight on the fragrance biosynthesis in Vanda Mimi Palmer, future work should focus on isolation of full-length cDNA of ratelimiting enzymes that are responsible for the biosynthesis of fragrance compounds. The fragrance-related cDNA should be cloned into bacterial system and confirm their involvement in fragrance biosynthetic pathway by enzymatic assays. Besides that, future work should also focus on transformation of the fragrance-related cDNA into other plant system including non-fragrance orchids and other flowers to increase their commercial values.

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165

APPENDIX A GENERAL SOLUTIONS

TAE buffer 242 g Tris base 57.1 ml glacial acetic acid 100 ml 0.5 M EDTA pH 8

LB broth (1 L) 10 g tryptone 10 g NaCl 5 g yeast extract Adjust to pH 7.0 (For LB agar, 20g of bacteriological agar must be added)

SM buffer 5.8 g NaCl 2 g MgSO4.7H2O 50 ml 1 M Tris-HCl pH 7.5 5 ml 2% (w/v) gelatin

TE buffer 10 mM Tris-HCl pH 8 1 mM EDTA

NZY agar (1 L) 5 g NaCl 2 g MgSO4.7H2O 5 g yeast extract 10 g NZ amine 15 g agar Adjust to pH 7.5

Pre-hybridization buffer (100 ml) 10 ml 50 X Denhardts solution 25 ml 20 X SSC 2.115 ml 1 M NaH2PO4 2.885 ml 1 M Na2HPO4 0.5 g SDS

NZY top agar (1 L) 5 g NaCl 2 g MgSO4.7H2O 5 g yeast extract 10 g NZ amine 7 g agar Adjust to pH 7.5

20 X SSC buffer 3 M NaCl 0.3 M sodium citrate

166

APPENDIX B

Cymbidium mosaic Virus Coat Protein Transcript Sequence

1 AATAAAGTAGCTATAGGCCCCTGGCGAGGGTTAAGTTACCACAATAATTTGAAATAATCA 61 TGGGAGAGCCCACTCCAACTCCAGCTGCCACTTACTCCGCTGCCGACCCCACTTCTGCAC >>>>>>>>>>>>>>>>>>>> Forward Primer (CMV-F)

5-AGAGCCCACTCCAACTCCAG-3

121 CCAAGTTGGCCGACCTGGCTGCCATTAAGTACTCACCTGTCACCTCCTCCATCGCCACCC 181 CCGAAGAAATCAAGGCCATAACCCAATTGTGGGTCAACAACCTTGGCCTCCCCGCCGACA 241 CAGTGGGTACCGCGGCCATTGACCTGGCCCGCGCCTACGCTGACGTCGGGGCGTCCAAGA 301 GTGCTACCCTGCTCGGTTTCTGCCCTACGAAACCTGATGTCCGTCGTGCCGCTCTTGCCG 361 CGCAGATCTTCGTGGCCAACGTCACCCCCCGCCAGTTTTGCGCTTACTACGCAAAAGTGG 421 TGTGGAATCTGATGCTGGCCACTAACGATCCGCCCGCCAACTGGGCCAAGGCTGGTTTCC 481 AGGAGGATACCCGGTTTGCTGCCTTTGACTTCTTCGACGCCGTCGATTCCACTGCCGCGC 541 TGGAGCCTGCCGAATGGCAGCGCCGCCCTACTGACCGTGAACGTGCTGCGCACTCGATCG 601 GGAAGTACGGCGCCCTTGCCCGTCAGCGTATCCAAAACGGCAACCTCATCACCAACATTG 661 CCGAGGTCACCAAGGGCCATCTTGGCTCCACCAACAGTCTCTATGCTCTGCCTGCACCCC <<<<<<<<<<<<<<<<<<<<< Reverse primer (CMV-R)
5-GCAGGCAGAGCATAGAGACTG-3

721 CTACTGAATAACGCCAAACTTAATAAGGCGTGTGGTTTTCTAAAGTTTGTTTCCACTACT 781 GGCGTAATATATTTAGCCAGATAAATAAAAAAAAAAAAAAAAA

167

APPENDIX C

Equations Used in Expression Analysis of the Putative Fragrance-related Transcripts

Equation A (Standard curve) y = mx + c where y = CT values, m = slope of the standard curve, x = log input of template amount, c = y-intercept of the standard curve line. Equation B (PCR amplification efficiency) E = (10 -1/slope 1) x 100 where E = PCR amplification efficiency, slope = m as mentioned in Equation A Equation C (Relative quantity) Q = E(lowest CT sample CT) where Q = Relative sample quantity, E = PCR efficiency Equation D (Normalization expression) Normalized expression level of target n = Q target n/ NFtarget n where Qtarget n = relative sample quantity of target n, NFtarget n = normalization factor of target n

Equation E (Normalization Factor) NF = (Q sample (ref 1) * Q sample (ref 2) * . * Q sample (ref n))1/n Where NF = normalization factor, Q sample (ref 1) = relative quantity of reference gene 1 (endogenous control), Q sample (ref 2) = relative quantity of reference gene 2 (endogenous control), n = number of endogenous control genes used Equation F (Scaled Normalized Expression)

Rescaled normalized expression target = normalized expression leveltarget n normalized expression levelcalibrator

168

APPENDIX D GC-MS Analysis on Vanda Mimi Palmer and Vanda Tan Chay Yan

a) Data for GC-MS analysis of the scent of Vanda Mimi Palmer

169

170

171

172

173

174

175

176

177

178

179

180

b) Data for GC-MS analysis of the volatiles emitted by Vanda Tan Chay Yan

181

182

183

184

c) Data for GC-MS analysis of the essential oil of Vanda Mimi Palmer

185

186

d) Data for GC-MS analysis of the essential oil of Vanda Tan Chay Yan

187

188

APPENDIX E Contigs and Singletons

No
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Clone name
1 29 17 47 11 40 A44 57 60 72 82 2 3 4 5 10 24 25 27 30 31 33 34 35 36 38 42 44 45 48 51 52 59 61

cDNA transcript
putative zinc finger (C2H2 type) family protein putative zinc finger (C2H2 type) family protein putative ATP binding / alanine-tRNA ligase putative ATP binding / alanine-tRNA ligase putative phospholipid transfer protein putative phospholipid transfer protein putative phospholipid transfer protein putative S18.A ribosomal protein putative S18.A ribosomal protein putative enoyl-ACP-reductase putative enoyl-ACP-reductase putative elongation factor putative 2,3-biphosphoglycerate independent phosphatase putative DNA ligase putative cycloartenol synthase putative coated vesicle membrane like protein putative vacuolar sorting protein putative zinc finger (C3HC4-type RING finger) family putative protein kinase putative ATP-dependent protease putative esterase/ lipase/ thioesterase family protein hypothetical protein putative LITAF-domain-containing protein putative kinesin no significant hit protein putative pleiotropic drug resistance like protein putative flowering time control protein isoform putative glycosyl hydrolase family protein putative Leucine aminopeptidase hypothetical protein putative cytochrome P450 monooxygenase Putative photosystem II core complex protein no significant hit protein putative abscisic stress ripening protein

Contig/ singletone
Contig 1 Contig 1 Contig 2 Contig 2 Contig 3 Contig 3 Contig 3 Contig 4 Contig 4 Contig 5 Contig 5 Singleton 1 Singleton 2 Singleton 3 Singleton 4 Singleton 5 Singleton 6 Singleton 7 Singleton 8 Singleton 9 Singleton 10 Singleton 11 Singleton 12 Singleton 13 Singleton 14 Singleton 15 Singleton 16 Singleton 17 Singleton 18 Singleton 19 Singleton 20 Singleton 21 Singleton 22 Singleton 24

189

No
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

Clone name
62 63 66 68 69 71

cDNA transcript
putative alpha tubulin putative syntaxin-like protein putative KH domain-containing protein / zinc finger protein-like putative RNA binding protein putative GRF protein putative 4-diphosphocytidyl-2-C-methyl-Derythritol kinase putative dihydrolipoamide S-acetyltransferase precursor putative Ubiquinol-cytochrome c reductase complex ubiquinone-binding protein putative Aci-reductonedioxygenase putative thioredoxin-dependent peroxidase putative mitochondrial dicarboxylate carrier protein putative 6-phosphogluconolactonase family protein putative calmodulin-binding protein hypothetical protein putative jasmonate ZIM-domain protein putative translation elongation factor 1 alpha hypothetical protein putative MADS box AP3-like protein putative 40S ribosomal protein putative 60S ribosomal protein no significant hit protein no significant hit protein putative GRF-interacting factor putative chaperon/ heat shock protein no significant hit protein hypothetical protein putative pectate-lyase like protein beta-ketoacyl-CoA synthase

Contig/ singletone
Singleton 25 Singleton 26 Singleton 27 Singleton 28 Singleton 29 Singleton 30 Singleton 31 Singleton 32 Singleton 33 Singleton 34 Singleton 35 Singleton 36 Singleton 37 Singleton 38 Singleton 39 Singleton 40 Singleton 41 Singleton 42 Singleton 43 Singleton 44 Singleton 45 Singleton 46 Singleton 47 Singleton 48 Singleton 49 Singleton 50 Singleton 51 Singleton 52

73 74 75 76 77 78 81 83 85 86 90 91 94 95 96 A28 A38 A40 A46 A54 A58 A66

190

APPENDIX F BLASTX Results Sequencing results of 62 clones up-regulated expressed in fully-open flower of Vanda Mimi Palmer after elimination of Cymbidium mosaic Virus transcripts

No

Clone name

cDNA transcript

Classification

Score

E.value
5.00e-39

putative zinc finger (C2H2 type) family protein putative elongation factor putative 2,3biphosphoglycerate independent phosphatase

protein with binding function

164

protein synthesis

211

3.00e-53 3.00e-77

Metabolism

291

putative DNA ligase

cell cycle and DNA processing biogenesis of cellular component

432

1.00e-119 2.00e-77

5 5

putative cycloartenol synthase

291

10

putative coated vesicle membrane like protein putative phospholipid transfer protein putative ATP binding / alanine-tRNA ligase putative vascoular sorting protein putative zinc finger (C3HC4-type RING finger) family putative protein kinase

cellular transport

325

2.00e-87 5.00e-44 2.00e-76 2.00e-124 3.00e-67

11

cellular transport

180

17

Transcription

289

24

cellular transport

448

10

25

protein with binding function

258

11

27

cell cycle and DNA processing protein with binding function

161

2.00e-38 1.00e-30

12

29

putative zinc finger (C2H2 type) family protein

135

191

No

Clone name

cDNA transcript

Classification

Score

E.value
1.00e-108 1.00e-89

13

30

putative ATP-dependent protease putative esterase/ lipase/ thioesterase family protein hypothetical protein

Energy

396

14

VMPEST

Metabolism

333

15 16

VMPA33 34

Unknown Metabolism

106 191

1.00e-32 3.00e-47 3.00e-32

17

35

putative LITAF-domaincontaining protein putative kinesin

cell cycle and DNA processing Unknown Cell rescue, defence and virulence

141

18 19

VMPA36 38

no significant hit protein putative pleiotropic drug resistance like protein

128

3.00e-28

20

40

putative phospholipid transfer protein putative flowering time control protein isoform

cellular transport

180

6.00e-44 5.00e-20

21

42

Development

102

22

44

putative glycosyl hydrolase family protein

Metabolism

241

1.00e-62

23

45

putative Leucine aminopeptidase putative ATP binding / alanine-tRNA ligase hypothetical protein putative cytochrome P450 monooxygenase Putative photosystem II core complex protein

protein synthesis

234

4.00e-60 2.00e-76 3.00e-26 3.00e-36 3.00e-23

24

47

Transcription

289

25 26

VMPA48 VMPCyP450

Unknown Metabolism

121 154

27

52

Energy

111

28

57

putative S18.A ribosomal protein

protein synthesis

254

2.00e-66

192

No

Clone name

cDNA transcript

Classification

Score

E.value

29

VMP59

no-significant hit protein

unknown

30

60

putative S18.A ribosomal protein putative abscisic stress ripening protein

protein synthesis

254

2.00e-66 1.00e-56

31

61

221 Cell rescue, defence and virulence cell cycle and DNA processing cell cycle and DNA processing protein with binding function 90.1

32

62

putative alpha tubulin

4.00e-17 5.00e-45 1.00e-24

33

63

putative syntaxin-like protein

184

34

66 putative KH domaincontaining protein / zinc finger protein-like

115

35

68

putative RNA binding protein putative GRF protein putative 4diphosphocytidyl-2-Cmethyl-D-erythritol kinase putative enoyl-ACPreductase putative dihydrolipoamide S-acetyltransferase precursor

protein with binding function Development Metabolism

214

8.00e-54 2.00e-123 7.00e-41

36 37

69 VMPCMEK

445 169

38

72

Metabolism

154

6.00e-38 1.00e-51

39

73

Metabolism

204

40

74

putative Ubiquinolcytochrome c reductase complex ubiquinonebinding protein

Energy

123

4.00e-27

41

75

putative Acireductonedioxygenase

Metabolism

294

5.00e-78

193

No

Clone name

cDNA transcript

Classification

Score

E.value
1.00e-36

42

76

putative thioredoxindependent peroxidase putative mitochondrial dicarboxylate carrier protein

Energy

155

43 77

cellular transport

138

1.00e-31

44

78

putative 6phosphogluconolactonas e family protein putative calmodulinbinding protein putative enoyl-[acylcarrier protein] reductase

Metabolism

102

1.00e-20

45

81

protein with binding function Metabolism

94.4

3.00e-18 4.00e-115

46

82

417

47 48

VMP83 85

hypothetical protein putative jasmonate ZIMdomain protein

Unknown Cell rescue, defence and virulence

255 105

2.00e-66 3.00e-21

49

86

putative translation elongation factor 1 alpha

protein synthesis

259

8.00e-68

50 51

VMP90 91

hypothetical protein putative MADS box AP3-like protein putative 40S ribosomal protein

Unknown Development

82.4 183

2.00e-14 3.00e-45 2.00e-62

52

94

protein synthesis

241

53

95

putative 60S ribosomal protein

protein synthesis

176

1.00e-42

54

VMP96

no significant hit protein

Unknown

194

No

Clone name

cDNA transcript

Classification

Score

E.value

55

VMPA28

no significant hit protein

Unknown

56

A38

putative GRF-interacting factor putative chaperon/ heat shock protein

Development

117

5.00e-25 2.00e-38

57

A40

Cell rescue, defence and virulence

161

58

A44

putative phospholipid transfer protein no significant hit protein hypothetical protein putative pectate-lyase like protein

cellular transport

180

4.00e-44

59

VMPA46

Unknown 3.00e-28 2.00e-50

60 61

VMPA54 A58

Unknown biogenesis of cellular component

127 201

62

A66

beta-ketoacyl-CoA synthase

Metabolism

191

3.00e-47

Note: The no significant hit proteins are the sequences which not homologous to any sequence in NCBI database for the cut off e-value of 1e-05 and score more than 80. The clones selected for verification by RT-PCR are highlighted and the abbreviations is only apply for the selected clones for the verification.

195

APPENDIX G Expressed Sequence-Tags (ESTs) of Vanda Mimi Palmer

>1-1-T3 hypothetical protein
TTGGAGATTCTCGCTTCGCCGCACCCACAGCCGCGGCAGCATCTGCGGCGGTGCAGACCCGGATACGGTGAAATAAAA GGTAGAATTTTGGTCAGAGGGCGGTAAGGAGAGGGAGCGGAAGGGGAGGAAGTGGAAGATGACGGG AAAAGCGAAG CCGAAGAAGCACACGGCGAAGGAAATAGCAGCGAAGGTTGATGCAGCGACGACGAATCGCGGCGGGGGTAAGGCTGG GCTCGCGGACCGATCGGGACTGGATAAGGGAGGGCATGCTAAATTCGAGTGCCCTCACTGCAGGATAACCGCGCCTGA TATGAAGTCGATGCAGATCCATCATGAAGCCCGACACCCTAAGATTCCTTTCGAAGAATCGAAGCTCACCGATCTTCAT GCCTCCAACGTTGGCGATTCGTCCAAGCCCCGCCCAGGGGTTCGGGGAAGCTTCAAAAAATAGCTTCAGCTTTGCTATT CCGAATAGCACTGTGAGCTATTCAATAACCTTTGCTTTCCTTCTGTCATATTTAGCTTCAACTTTTACGATGCCCTAATGT ATTTTTATGATATTTGCTCGACTGCATCTTGTCATCGCTGCTACAAGTAAGAACATGTGAGATTCCTTGTGTCATGGTTA TCGGAGGACTTGAATTTGAACTATTTGTAGTAGTTTTCTTAAACGGTTGCTTGCTTTAAGGTAATGCGCTTCCCATAGCC GATTA

>2-2-T3 elongation factor

CACCCTGGTCAGATTGGCAACGGCTACGCTCCCGTCCTGGACTGCCACACCTCTCACATTGCCGTCAAGTTTGCCGAAA TCCTGACCAAGATCGACAGGCGGTCTGGCAAGGAGCTCGAGAAGGAGCCCAAGTTCCTGAAGAATGGCGATGCCGGTT TCGTGAAGATGATTCCCACGAAGCCGATGGTGGTCGAGACCTTCTCAGAGTATCCTCCGCTGGGCCGTTTCGCCGTGAG GGACATGAGGCAAACAGTGGCTGTTGGAGTCATCAAGAGCGTCGAGAAGAAGGATCCCAGTGGAGCCAAGGTTACCA AATCTGCTGCCAAGAAGAAATAAAGCTGAATGGATAAACTCTTTGAGTCTTGTTTATGTTAAGTTATCTTTGTGTTGGTC TAATCCGGTGCAGCTTTCCAGTATCAGATTCCAGATCCGGTGCTTGATATGCTGTGGCGGTGAGGTTTTGGTGTCTTTTA AGCTTTGTTTTATGCGACTGTTTTAATTTGTTTGAGTTGAGAAGACTGCGTTTTCATTTTAACAGCTCTTTTGATTATGTT TCTTGCACTGCTTGTTAAATTTATATTTATACATTTTTTAGGGTCTATATTATAAATTTATTTTTGTCTT

>3-3-T3 2,3 phosphoglycerate independent phosphatase

CTTTAACGTTCAACCTAAAATGAAGGCACTTGAAATTGCCGAGAAGGCAAGGGATGCTATTCTCAGTGGCAATTTTGAC CAGGTACGAGTTAATATACCAAACGGTGACATGGTAGGCCACACTGGTGATATTGGGGCTACTACTGTGGCTTGTGAG GCTGCAGACGTAGCTGTAAAGATGATTCTTGATGCCATTGAGCAAGTGGGTGGCATCTATGTTGTCACTGCAGATCATG GCAATGCTGAGGACATGGTGAAAAGGGGTAAATCTGGGCAGCCACTTTTTGACAAAGAAGGGAAAGTTCAAATTCTCA CCTCTCACACCCTTCAACCGGTTCCTATCGCCATCGGCGGCCCCGGCTTGAGTCCAGGTGTTCGTTTCCGGAGTGATGTG CCTGATGGTGGCCTTGCCAATGTTGCTGCTACTGTGCTGAACTTACATGGTTTTGAAGCTCCCAGTGACTATGAAACCAC CCTAATTGAAGTAGTTGAGAACTAAATATACTGTGTAATTTTCTATTTATAATATGCTAATGTGATTTTTATATTAGTTA AGCTCATTGAAAATCTTAAAATAAGTTTACTCCATTGAACTGGAGAATGTTGTCAGGTTTATGTTGTCACAGTTGAGGG GGCTGTGGGCAAAGTTTTGGGTTGTTTCACTATAGTTTATATTTGTGTGGAATCTCTCCTCACAATGACTTTCTTGCTTTG AATATTTACGCCACTTTCGAGGGATGTTGTTTTTGCTTAAAGTTTCATATGTTGTATTATTGGATAGTTGGTTATGGCAA AGACGATGATTGCTT

>4-4-T3 DNA ligase

TTTTGATATTCTCTACATTAATGGAAGGCCTCTCCTCCATGAGCAACTCAAAGTTCGCAAGGAGGAACTTTACAAATGT TTTGTGGAGATACCCGGAGAATTTAGTTTTGCTACTGCAATAACATCTAATGATCTTGAAGAAATACAGAAGTTCTTAG AAAACGCTGTCAGCTCCAGTTGTGAAGGGTTGATTATCAAAACTTTGGACAAAGATGCTACATATGCGCCTTCAAAACG GTCAAACAACTGGTTAAAATTGAAAAAGGATTATATGGACAGTGTAGGAGACACTTTAGATCTGGTGCCCATTGCTGCC TTCCATGGCCGAGGAAAACGCACAGGTGTTTATGGTTCTTTTCTTCTTGCTTGCTATGATGAACAAAATGATGAATACC AAAGCATATGTAACATTGGTACTGGATTTTCTGAATTGCAGCTTGAAGAGAGGTCCAAAAGTCTTGGAAGTAAAGTTAT TCCAAAACCAAAACCATACTACAGAGTAGCTGATTCAATGAATCCTGATGTCTGGTTCGAACCTGCAGAGGTTTGGGTA GTAAAGGCAGCGGATTTGAGCATTAGTCCTGTTCATCGTGCTGCTTCTGGCATTGTGGATCCAAATAAGGGTATTTCTCT GCGATTTCCTCGTCTACAACATGTCCGAGATGACAAAAACCCAGAACAAGCCACAACGTCTGAGCAGGTTGCTGAAAT GTATCGTGCACAAAAGATCAATCATATGAACAATCAAGAGGAAGAGGATGACGAATGACGTGGTTTGCTGCTTGCAAT CAAGTCTATCATTTCTTTGAAGTTGGCAGTGTAGTTTTCCTGGTAGAGTCTAATGCATTTACTTGATAAACTTCTTATTCT ACTTTCTTGTGCTAGTACTTTGCCTATTAAATGTAAACNTGAATCTATGCTATGACGTTTCGTTTC

>5-5-T3 cycloartenol synthase/ squelene cyclase

ATTTCATTGAGAAGATACAGAAACCAGATGGTTCATGGTATGGTTCTTGGGCTGTTTGCTTCACTTATGGAATATGGTTT GGAGTGAAAGGACTAATTGCAGCTGGAAAATCATATCAAAATAGTCCTTCTATCCAAAAAGCATGTAATTTCCTGTTAT CTAAACAACTAGCTTCTGGTGGTTGGGGGGAGAGTTATCTGTCTTGTCAACATAAGGTGTACACAAATCTTGAAGGAGG TCGCACTCATGCAGTAAACACAGCATGGGCCATGTTGGCTCTAATAGATGCTGGACAGGCTGAAAGAGATCCAAAGCC ATTGAATCGAGCAGCAAAAACCTTGATTAACATGCAGCTGGAGAGCGGCGAATTCCCTCAACAGGAAATTATGGGAGT

196

TTTCAACAGAAACTGCATGATCAGCTATTCAGCATATCGCAACATCTTCCCAATCTGGGCGCTCGGAGAATACCGCACT CGGGTTTTGAAGGCAAGCAATTAACATAACTCCATGTCTGAGATAAACAGCTCCTCGAACCTCATGCTTTTGTCTCTTGC AAAAGCATTCTTGCTTGGAAAACAATAATGTGATATTTTAATGGAGGAAAATATATTAAAGATTCCATTCT

>6-10-T3coated vesicle membrane like protein

CTCAGCGGTGGGCCTTTTTTCATGTTTGCAATCGGCTGTTGGGATTAGGTTTGTGATAGACAGGGAAGAGTGCTTCTCCC ATGAAGTTCCATATGAAGGAGACACTGTTCATGTTTCGTTTGTTGTGATTAAGTCTGAGACACCCTGGCATTACGGAAA TGAGGGCGTGGATCTCGTGGTGAAAGATCCATCTGGCAATCAAATTCATGATTTTCGTGATAAGATAAGCGATAAATTT GAATTTATAGTTCGCAAGAAAGGGCTTCATCGCTTTTGCTTCACCAACAAATCTCCATATCATGAAACAATAGATTTTG ATGTTCAAGTCGCCCACTTCACATACTTTGAAGAACATGCGAAGGATGAGCATTTATCTCCTCTCCTTGAACAAATCAA CAAGTTAGAAGACGCTCTTTACAATATTCAATTTGAGCAGCATTGGCTGGAGGCTCAGACCGAACGTCAAGCAATTGTA AATGAAGGGATGAGCAGAAGGGCAATACACAAGGCACTTTTTGAATCTGCAGCACTGATTGGAGTCATTGTGCTACAA GTGTATCTCCTCCGTCGCCTTTTTGAGCGAAAGCTTGGAACCTCTAGGGTTTAGCCAACTCAGGAGCCTGCAACTTCCAT CACATGTTTCTTCTTTCTTCCAAACCTCTGACTTCATTCGTAACAAAATGAGCTTGTTATACAGGGAATCTTCATTCAAC ACTGGCTTGTCTTTTTGGCTTGTTCCAGTGCTTCCCTGATCTAATAACCAGGTAATTATCAAATTATTCAGTTTGTTGCTT AGTAAATGAGAATATATCTGTTTGTGGCAGC

>7-11-T3 phospholipid transfer protein

GTTATCACTCATTGTCAATGGCTCGCTCCACCGCTTTTGCAGTCGTATGCATAGTGTCCTTCCTCCTTGTTTCCGGCGTTT TCCGCGAGGCGAGCGGGGCCATCAGTTGCGGTCTGGTGGCCTCATCCGTGTCATCGTGTATCAACTATATTCGAGGCGT TGGCTCGCTCTCACCAGCATGTTGCAGTGGGGTGAAGAGACTCAACTCGCTGGCGCGCACAACCCCTGACCGTCAGGC GGCATGTTCTTGCCTCAAGAGTTTCGCCAACCGTATCCCTAATCTGATCCCTTCCCGTGCTGCTGGACTTCCTAGCAGCT GCGGAGTCAGCGTTCCATATCCCATCAGTACCTCCACCGACTGCTCCAAGGTGCACTGAGCTTCTTGGACGGGCTACAA ATTACTGTTTTCGTATGATCTACAGAATAAAGTGGTCTCTGGGTCTAGTGAGGGTTGTTAATATCTTGTTGTTTGTGCGT TTCTGTCTTATTTTAATTTATGTTCCTGTATTGTTATAAGGCTACACCCTTGGATGTGGTGAGTTTAATATTCCTGCT

>8-17-T3 ATP binding/ tRNA ligase

CGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGGCACGAGGGCGACATCCTCCGGCACCTCGTTTGC AATGGTACAAGTACCAGCTCGCAGGTCCTGAACGACGAGGCTGACGACGACTATGGCGAAGCAGAGTCCAACGAAGC TTGCCTACTTCGAGGGCATGTACGCGCTTCAGTCCACTGCCACCTTCCTCTCATTCGAGCAGGTAGATGGCCGATTGGC GATGATCCTTGATTCAACCATCTTCCACCCTCAGGGTGGTGGGCAGCCAGCTGACAAAGGGGTCATTTGCGGTTCAGGC TTCAGGTTCGTGGTGGAGGATGTCCGGTCCAACGATGGGGTGGTCTTTCATTTTGGATACATTGATGACTCTCAAGCTA ACTGTGAATCCAACCTCAAAGAGAGACAAGAAGTTACCTTACAAGTTGATGCACAACGACGTGATCTCAATTCCAGGA TTCATTCTGCTGGCCATTTATTGGATATTTGTATGCACAATGTAGGCTTATCTCACCTAAAGCCTGGAAAGGGTTATCAT TTCCCTGACGGGCCATTTGTTGAATATATTGGAGTAATCTCCCCAGATCAACTGCAGATCAAACAAAAAGAGCTTGAAG AAGAAGCAAATTCATTAATCAGTATTGGTGGAAAGGTTTCTGCTTCTGTTTTACCTTATGATGAAGCTGCCAGGTGGTG CAATGGTGATCTCCCTAGTTACATTTTGAAGGGCAGCACCCCAAGAATTGTGAGGCTAGGTAACAATGCCGGATGCCCC TGTGGAGGGACTCACGTTGCTGATATTTCAGACATAAAAAGCTTTAAGATTACCCAAATTCGATCAAAGAAAAGAATC ACAAAGATCAGCTACAGCATCAACCCATGAGCTCTGTATTTCTCTGCAACATTGTTGATCTATTGCGATGATTAGTGAG CTCCTATCATACCTGAATGTATCACCTAATTTTATTATGTTTTTGTATGATTAATCACAGTTGTTATTATTGGTCTATAGA AATGTGAACTTTGCCT >9-24-T3

Vascoular sorting protein

GGAACATTCTGCCTAGGCTGTACCTTCTTTGCACGGTCAGATCTATTTATATAAAATCAAAAGAGGCTCCTGCAAAGGA TATCCTTAAGGATATTGTGGAAATGTGCCGGGGTGTACAACACCCTGTCCGGGGTCTCTTTCTGAGGAATTACCTCTGTC AGATCAGTAGGGATAAGTTGCTTGATATTGGTTCAGACTATGAAGGGGATGGCGGCAGTGTTATCGATGCTGTTGAATT TGTGCTTCAGAACTTTACAGAGATGAATAAACTGTGGGTCCGAATGCATTATCAGGGACCAGTTTGGGAAAAGGAGAA ACGTGTAAAAGAGAGGAGTGAGCTTCGTGATCTTGTGGGAAAAAACCTCCATGTTCTTAGCCAAATAGATGGTGTTGAT CTTGGTATGTACAAAGAAAATGTGCTTCCTAGGGTATTAGAACAGGTGGTCAATTGCAAAGATGAACTAGCCCAACATT ATTTGATGGATTGTATAATCCAAGTGTTTCCAGATGAATATCACTTGCAAACTCTTGAATCATTATTAGGCGCATGTCCA CAACTTCAGCCCACTGTTGATGTTATGGCAGTCCTATCTCAGCTCATGGATAGATTATCCAATTATGCAGCCTCTAGTAC GGAGGTTTTACCAGAATTTTTGCAAGTTGAAGCTTTTGCCAAATTAAGCAGTTACATTGGAAAGGTCATTGATTCGCAG CCTGAAATGCCAATTTTTGGTGCCATCGGCTTATACGTCTCACTTCTTACATTTACTCTTCATGTCCATCCAGATCGTCTT GATTATGTGGATCAAGTTCTGGGAGAGTGTGTTAGAAAATATCTGGAAAATCGAGATTGAGGATAGCAAAGCATTAAA CAAATAGTTGCTCTTTAAGTGCTCCAATGGAAAAGTACAATGACATAGAATATTGTTTTAAAGCTGCTANTATCCAGTG TCATGAGCACCTTGNTATGCNNAAGAAATCATGCAGTGNNTATCAGAGCATATGAGATAACCGCATATC

197

>10-25-T3 zinc finger (c3Hc4-type ring finger) family

CTCCCTTATGTTGACCACCAAAAGGCAAAGACTATAAAGAATGATTTTAATTTGCATAAAGACATGATTCGGTTGGAAG TGGATGTCCAGAACCCGGAGGAGTATCTGGTTTCCTTTGTCTATGATGCTGTCGTGGACGGGAGGTAATCCGCTTGCGT CACCATATACTACTTTGCTAAAGAAGGAGCCAACTGTAGTTTTTCCTCAACCGAAGAAGACATCTATAAACCCAGGAAT TATCTTTTTGAGAAGGGAACGGCCAAAAATTTCTGTCAACCATCTGGGCATGGCATTGATTTAGGCTTCTTTGAGTTAG AGAGTCTTTCAAAGCCAGTAAATGGAGAAGTCTTCCCACTAGTTATTTATGCCAAGGCATGCGGGCCAACTGATGGAG ATGGCCACTCTTCTCAATCTTCTGCCACTCATGTGCAGATTACATTAGCCGTCATTGAGAAGAATAGTAAAGGAGAGTT TGAAGCTAAAGTTATAAAGCAAATCCTATGGATTGCCGGCGAACGCTACGAGCTGCAAGAAATCTTTGGCCTGGAAAA TTCTTCAACTGAACAGATTGATGATGGTGATGATGATATTGGTAAGGAGTGCGTAATCTGCTTGACAGAACCTAGGAGC ATTGCAGTCTTTCCCTGCAGGCATTTGTGTATGTGTAGTGATTGTGCAAAATCTTTGAGGGTCAAATCAAATAAGTGTCC CATTTGTCGTCAACCTGTTGAGAAACTAATGGAAATCAATGTAAATTTGAGGAGCTCTGATAGGTAAGATCGGCATCAG TCTTCATGAAATTTGACAGAGGT

>11-27-T3 putative protein kinase

ATATTTGTTTGGCTGATTTTGCTAAGAGATGAGTTACGGCAGAGATGATCAAAATGATGGAATAGAGAAAAGGAAGTT TATGAATTCTGAACCAAAAACAGCACCAGATTCTGTCCATCCTGGCAGGCCGTCCTTTGACTGCAACTATACTATTCCA GTTGAGAACTTGGAGATTGATAAAAAGCTTTTGATTGATCCGAAGAATCTTTATATAGGATCCAAGATTGGTGAAGGTG CTCATGGAAAAGTTTATGAGGGAAAATTTAGGGAACAAGTTTTAGCACTCAAAATTATCAATAGTGGCAACACGAATG AGGAGAAAATGACAATTCAGGCTCGTTTCATTCGAGAAGTCAATATGATGTCTAGAGTGAAACATGAGAATCTTGTTA AGTTCATTGGAGCTTGCAAGCACCCTATCATGGTTATTGCTACAGAGTTGCTACCTGGGATGTCCTT

>12-29-T3zinc finger (C2H2 type) family protein

GGAAGAAGACGGGAAAAGCGAAGCCGAAGAAGCACACGGCGAAGGAAATAGCAGCGAAGGTTGATGCAGCGACGAC GAATCGCGGCGGGGGTAAGGCTGGGCTCGCGGACCGATCGGGACTGGATAAGGGAGGGCATGCTAAATTCGAGTGCC CTCACTGCAGGATAACCGCGCCTGATATGAAGTCGATGCAGATCCATCATGAAGCCCGACACCCTAAGATTCCTTTCGA AGAATCGAAGCTCACCGATCTTCATGCCTCCAACGTTGGCGATTCGTCCAAGCCCCGCCCAGGGGTTCGGGGAAGCTTC AAAAAATAGCTTCAGCTTTGCTATTCCGAATAGCACTGTGAGCTATTCAATAACCTTTGCTTTCCTTCTGTCATATTTAG CTTCAACTTTTACGATGCCCTAATGTATTTTTATGATATTTGCTCGACTGCATCTTGTCATCGCTGCTACAAGTAAGAAC ATGTGAGATTCCTTGTGTCATGGTTATCGGAGGACTTGAATTTGAACTATTTGTAGTAGTTTTCTTAAACGGTTGCTTGC TTTAAGGT

>13-30-T3 ATP dependent protease

GGATTTGGTCGATTAGGGATGATTTGCTTGTGCCGTCTTCTCCCTACTTCCCTGTGGAGGCTCAGGGCGGACAAGGACC ACCGCCAATGGTGCAGGAGCGGTTTCAGAGCGTTATTAGCCAGCTATTCCAGCATAGAATTATACGCTGTGGTGGTGCG GTCGATGATGACATGGCAAATATAATTGTAGCGCAGCTTCTTTATCTCGATGCAGTGGATCCTACGAAGGACATCGTCA TGTATGTGAACTCTCCTGGAGGATCAGTGACAGCTGGTATGGCTATATTTGATACGATGAGGCATATTCGTCCTGATGT TTCCACTGTATGTGTTGGATTAGCAGCCAGTATGGGTGCTTTCATACTAAGTTCTGGCACCAAGGGAAAACGCTACAGC CTACCAAACTCCAGAATCATGATTCATCAGCCCCTTGGCGGCGCCCAAGGAGGACAAACTGATATAGACATTCAGGCA AATGAGATGCTTCATCACAAGGCCAATTTGAATGGATATCTAGCTTACCACACGGGCCAGAGTTGGGAGAAAATAAAC CAGGATACTGATCGAGACTTCTTCATGAGCGCCAAAGAGGCAAAAGATTATGGCTTAATTGATGGAGTAATAATGAAT CCACTCAAAGCTCTTCAGCCTCTCTCAGCTTGAGATGATGAAGAAGAAGAATCAGATGAGTTCTACGACCATGATCAGA GAAATCAGAGCTTAAGGATTGCAGTAATCTGCTCTGCTGCGATACTAAAACTTCCGACCGTTGAGAGCACTCATTGCCT GATATAGTCAGTATTATTCGTCTTATTGTGCCTATCTAGAGTTAGAGTTAGCTCACAATGTTGCTTGAATATTTATCAAT TAAATGAATATCAGAAAATTGCGGTTTT

>14-31-T3 esterase/lipase/thioesterase family protein

GTAAGGCGTTTCCGCGAAGAGGTCAGTTCACAGTTGGAATTCTGCGTCTCTATCGACGATGTCGTCTCCTGGAGTGTCC GAGCAAAGGGTTGAAATTTTAAACAACTATGGAGAGAAACTTGTTGGTGTGCTGCATTTAGCAGGTTCCAAGAATCTG GTGATCTTATGCCATGGATTTCGCTCCACAAAGGATGAGAAACTGTTACTTAACCTCATTGCTGCACTAACGAAAGAAG GTGTGAGCGCCTTTCGCTTTGATTTTGCTGGAAATGGGGAAAGTGAAGGTGAATTTCAATATGGAAACTATCGCAAGGA AGCTGACGACTTACGAGCTGTGGTGCTATATTTCTTAGAGCAAAATTTTAAAATTTGTGCTATTACTGGGCATAGTAAA GGAGGAAATGTGGTGCTTCTTTATGCATCTACGCATAATGATGTCCCTCTCATCATTAATCTTTCTGGCCGTTTTGCATT GGAAAGAGGAATTGAAGGACGCCTGGGGAAAGAATTCATGCAAAGAATAAAGAAAGATGGCTTTATTGATGTCAAGG ATAAAACAGGAAAATTTGAATACCGGGTGACAGAAGAAAGCTTGATGGATCGTCTAACCACAGACATGCGTGCAGCAT GCCATGCCATTGATAAGAAATGCAGGGTTTTGACAATCCATGGTTCAGCAGATGAAATTGTACCTTCAGAAGATGCCTT TGAGTTCGACAAAGTCATACCCAACCACAAGCTTCATATCATTGAAGGTGCTGACCATTGCTATACTGCATGCCAAGCA GAGCTGGCTTCTCTTGTTCTAGACTTCATAAAATCTGATCAGGTCGTCGATGCTACCACAGCACAAGTGATGTAAGAGT TTTTCAAGCTCATTGTTGTTACTTTATCTTTCAATTTGTCACGCCATGGTCATTACTGTCTAGCTTGATGTTTCATTTTCTA TATGTAATG

198

>15-33-T3 Hypothetical protein

ATGAAACCCTAATTTTTGAGCTTTTGGATTTCAAAAGCGAGATTGAAGACAATGCCAGTGCTGTTTGGTTTCTACGTGA CATTGCTCGTGAACAGGATGCAGAAGATAGCATGGTCCAAGAACACTCAGGAACAATAGAAGTTGCTGGCTTACAGTA TAGAGATGCTCCTGTAATATATCAAACAGCAGTTGGTCAAATGACCATTTCCAAGGAAAGCCAAGGAATGGAGGGACA CAAAAATGGGGGGGGGTTTAAGGGGAAATATTTCGCGTCCCAGGATTTTGCGGTGAGGGGGGCGCGTGATCACTGCAT ATGAGCAAATTTTGGACAATTCCTTAAGTGAGAGTAGAAGCATTGAGGAAGCTGATCCAACAGTTTCTGGCAGCCTATC TGCTGCAGAAGTCTTTAAACTATCAGCTGCAAGCTTTAAGGTACATGACTGGAATCTCTTTGGTGCTGGGGCTTGACTA ATTATTTAATATGATTTTAAGAATTTTTGGTTATGAAAAACATTTTCATATTATTTTAATATGATTTTAAGATTTTATTTG ACTTGTATTTTTATTTTTTAAAACTGGTGCTTAATCTGTAATTCTGTTCAACTATAATTATTTTTACAGGCGATGAAGCAA CTGATGATGGGAATTT

>16-34-T3 LITAF domain containing protein

GTGAAACGGAAGAACTGATGGCAGCGAAGGGTTCAGGCGACGAACCTGCTCTTGGGGTGCCCTACCACTATGCATCCG GCGGTGCCGGGGTTTCCGGATATGCCCAAGGTCCGAATCCACAGCCTGCATTCTACGTGGCGCAGCACCCGCATCAAG CTGGGTTGATCCCGCCGAATGCGGTTTTCGGAGATCCAAAGGGCATCCCGCTCCAGCAGACGATGTTCCGGGATACCCC CGCACCTTTCGAGTGCGTTTACTGTGGATCATCCAGTCTTACCACCATCAGATCCAAACCAAGTCTAGCTGCTCTTGTTG GTTGCATGATGCCATTTATGCTGGGAGTATGTTTTCTTTGCCCTTCCATGGACTGCCTCTGGCACAAATATCACTATTGC CCAAATTGTAAAGAGAAGGTTGCCTACTTTGAAAAATCAGACCCTTGTATTGTGGTGGATCCAACTAATTGGACCGAGC CCAGCTATGCCATCCCCAGGTTGGTTTGAACTCTCTTTGAAGTTTTTGCGGATATATATATATATATATATATATATATA TATATATATATACGGTTTTTGCTTTTGGATTTGTAAATAATGAAGTCCTCTTTGT

>17-35-T3 putative kinesin

AGGAAGAGNCCTTAATAGCAGCTCATAGAAAAGAAATTGAAAATACAATGGAGATAGTTCGGGAGGAAATGAATTTA TTGGCGGAGGTGGACCGACCAGGAAGCCAAATTGACACGTATGTATCGCAGCTCAGCTTCCTGCTGTCGCGGAAGGCT GCCGGGCTGGTCGGCCTACAAGCTCGCCTGGCCAGATTTCAGCACCGGCTGAAAGAGCACGAGATTCTCAGTCGCAAG AAGGCTTTGAGGTAATAAATGATCTGCGTGCTTTTCTTTCTTTTTAATGTATCTTCTACCAGCAGAATGTCCCCTTCCCTC TCTCTGCTTTTTCTTGCTGATTGTTATTGCCACAGATTTCTCATATTCTCATTCTTGAAGTTATACAATAGCGGCAATGCC GTGTGATTTACCTTTATGTGTCGTGTTTGGCCTTCTTTTATTGTTGGGAGTGAGGGTGGGTTGGTCTTTCGGAATGAAAG AAGAAGAATGTATGGAAGCCATTGTTGATAATAGTATAGTATTTATTT

>18-36-T3 hypothetical protein

ANNAGCCNTGTGCTTTTATTTCTGGGGCCTGCTTTTGTCTATCTTGTGCCTGAGGACAATGTCTGGGAGGTAGTCTTACA GGTTCTGGTGGCCTTAGTGTGTGTCGTTGGCGGATCCGCCTCCTTCGCTGCTTCAAATTTCTTGTCAAACTTGCAGAGAT CCAATTGAGTTTTGGTAGTCTTGTGCTTCTGACGTGGTCATTTTGGTGCAAATGTTAGCTTGTTACTTGCTCGGATTCCTT CTGAACTCTATGTATTAGGCAATAACAAGCAAGTCAGCTTCTGGGGAGAACTATGCATGGACAGTAGGTATTTGCTAAA TTCTTTTTAAGATTCAGGTAATATATATATATATATATATATATATATATATATATATATATAGATATGTATGTATGTATT TATGTATGTATGTATGGAACTGTACCTTCGGAGTATCATACAATAATTAAGCTCTAAAAATTGAATGCAATAATTTTCA GCT

>19-38-T3 pleiotropic drug resistance like protein

GTNNAAATTCCACGGCCGAGGATGCCTATATGGTGGAGATGGTATTTCTGGGCATGCCCAATTGCGTGGACACTTTATG GATTAGTCGCTTCGCAGTTTGGTGATATAGAAGACGAGCTCGAATCTGGTGAGATTGTGTCTCAATTTGTAAAAAGTTA CCTTGGATTCAGACATGACTTCTTGGGCGTGGTTGCGGCTGTAATCGTTGGGATTCCAGCACTCTTTGCCTTCATGTTTG GATTTTCAATTAAAGCTCTAAACTTCCAAAGAAGATGAAGGAATTGATGAAGTCCTAAAAGATGCCCTGGTGCTAGGA AGTTTAACATATTCATTTTGTTAATATTCTTCCACAAGAAGAATGAAAAGTTCTCAGAGGGGGTAATAATGAAGTGAAG GATATAATCTTTCCTGACCTATATATGAACAAGAAAATATGCTCATGAATATGTTCCTCGATGTACTTAATTTGGGCATC AGTTCAAATGTAGAGAAGTTAGGATGGATGTTGAGAGGTCTGATTTATCTTTGTCAACTATTCAAATGTAATAGTGCAA ATATAAAACTTTACTTGGTGGATATATAATGGGGAGATATATTAACACT

>20-40-T3 phospholipid transfer protein

GTAGAGNGTTATCACTCATTGTCAATGGCTCGCTCCACCGCTTTTGCAGTCGTATGCATAGTGTCCTTCCTCCTTGTTTCC GGCGTTTTCCGCGAGGCGAGCGGGGCCATCAGTTGCGGTCTGGTGGCCTCATCCGTGTCATCGTGTATCAACTATATTC GAGGCGTTGGCTCGCTCTCACCAGCATGTTGCAGTGGGGTGAAGAGACTCAACTCGCTGGCGCGCACAACCCCTGACC GTCAGGCGGCATGTTCTTGCCTCAAGAGTTTCGCCAACCGTATCCCTAATCTGATCCCTTCCCGTGCTGCTGGACTTCCT AGCAGCTGCGGAGTCAGCGTTCCATATCCCATCAGTACCTCCACCGACTGCTCCAAGGTGCACTGAGCTTCTTGGACGG GCTACAAATTACTGTTTTCGTATGATCTACAGAATAAAGTGGTCTCTGGGTCTAGTGAGGGTTGTTAATATCTTGTTGTT TGTGCGTTTCTGTCTTATTTTAATTTATGTTCCTGTATTGTTATAAGGCTACACCCTTGGATGTGGTGAGTTTAATATTCC TGCTAT

199

>21-42-T3 flowering time control protein isoform

TATAATCTCCTTTGCTAGACCTGCAAACAGCAGAATACGATGGAGAGGCATCGCTTCGGTGACGGTGGAAAGTACTCG AGAATGCCGTCACGGTGGCCATCAGATACCCAACCAAACCAGCACCAACGTTACCCTAGAGGCGGTGGAATAGCCGGC GGCGGCGAAGGCTTTGGTGGCCGATACCACCCTTACCGTGGCCAGCCTGATTATTCTTCCGCCGGCGGTCAGGGTTTCC GCGATGGTCATGGGGACTTGGGTAGTCATCAGATGCCGATGGGTGGTCAGAAGCGAGGGTTTGCGGGCAGAGGAGGTT CTCCAGACTTTGGTGAGGGAAACAAGTTTGCCAAACTGTTCATTGGGTCGGTTCCAAAGACTGCAACTGAAGAAGATAT TCGTCCTTTATTTGAGGAACATGGTGATGTTGTTGAAGTGGTTTTAATTAAGGATTGGCGAACAGGTCACCAACAAGGT AGATTCAGATGTTTTCTCATGTTCGTGATACATTGAGGTTTTCTTTAACAGGTCCTTTGGGGTTTGGGGAGCAATTTATT TATAAGTCCTCTCAGATAATGTTTTTATCTGATTAGAGTGGTTGTATTCTGTTGCAACCAGCCCATCATTATGAGGTCAG GGTGGTATTGAGTTTCTCAAAGAAAAAGGGAAACTTGACATTGTCGTTTCTATATTTACAGCAGCCAAAATGTTGATGA ACTGTTTACACGTGTTGATGCTTATTGATTAAAAGCTTGGTATGCAATTAAGGCATTTTTGAACTGCTTATAGCAGTGGA AGGGAATGATGTCTTTTGAGCCTTTCTTCTCTAAAAGATCTATGGCAGTTGTTCTTATAAGCTTAGTATTTATGTGCAAC GGTGATGAGAACTATACACATATTTTTACTTAATAAAGCTTTGCTCTTCTTGCATTTTATGCAAATACT

>22-44-T3 glycosyl hydrolase family protein 43

GAAGCACCGGCCGTGTTCAAACATAAAGGAACATACTATATGATAACTTCAGGGTGCACAGGGTGGGCGCCGAACAGG GCAATGGCGCATGCGGCGGAGGCGATGATGGGGTTATGGGAGACGATTGGTAACCCTTGTGTCGGCGGGAACAAAATA TTCGAACTGACCACTTTCTTTTCACAGAGCACTTTCATAGTTCCGATTTCGGGGCTGCCGGGGGTGTTCATTTTCATGGC AGATCGGTGGAATCCGTCGGAGCTGAGAGATTCCAGGTATGTTTGGCTGCCTCTGACAGTAGGTGGAGTGGTTGATGA ACCATTAGAATACAATTTTGGGTTTCCAGTTTGGTCTAAGGTCTCCATTTTCTGGCATAGGAGATGGAGACTTCCGAATT GGTGAACTGCTTAACATTGAGTGTACAATATTTTGTAGATTGTTTTACATATGGTGTATAGTGTAATAAATGTTTTTGTA GATTTT

>23-45-T3 Leucine aminopeptidase

GTAAANNNGGTGTCGAAAAGATTGTTGATTTGGCAACGCTAACTGGTGCTTGCATTGTTGCCCTTGGACCCAGCATTGC AGGAATCTTCACTCCCAACGATGAACTAGCAAAAGAGATCACTGCTGCTTCCGTGTTAACCGGTGAGAAATTTTGGAGG TTGCCTTTGGAGGAGAGTTATTGGGAGTCAATGAAGTCGAGTGTTGCTGATATGGCTAATAGTGGAGGCCGTCATGGCG GCGCTATCTTAGCTGCACTTTTCCTCAAACAGTTTGTGGATGAAAAGGTCCAGTGGGTGCATATAGATGTAGCTGGTCC AGTTTGGAATGATAAGAAGCGCGTAGCCACAGGGTTTGGTGTATCAACTTTGGTTGAATGGGTTCTCAAGAATTCTTCT TAAATCAAATACTGAGATGCAGCAAATGGAATAGAACAAACTTTTTTCAGGTTCAGCTCATGAAAGCATTAGGAAACA TTCTCAAATTTACTTCAAATAAAAGAACTTTGATCTGATTTTCTGTTGTCTGATATTTTATTAGTCTGCTATGTATTAGTG TTGTCTGTTGTCATAATTTTTATGATTTGTAGCTCTGAGTGGCAGTGTAAACTAGCTGAATTTTGAATGAATTTGAAGTG TTGTGAGGCTTTTGGCTTGTTAAATAAACAGTCAAAACTTTGTTGTGTTTGACTTGTTTATTGATGCAGTTTGCAAACAT GAAAATTTATTTCAAAGCTTTATAAAAATTTTAAAGTC

>24-47-T3 ATP binding / alanine-tRNA ligase

GCGANNCCTCCGGCACCTCGTTTGCAATGGTACAAGTACCAGCTCGCAGGTCCTGAACGACGAGGCTGACGACGACTA TGGCGAAGCAGAGTCCAACGAAGCTTGCCTACTTCGAGGGCATGTACGCGCTTCAGTCCACTGCCACCTTCCTCTCATT CGAGCAGGTAGATGGCCGATTGGCGATGATCCTTGATTCAACCATCTTCCACCCTCAGGGTGGTGGGCAGCCAGCTGAC AAAGGGGTCATTTGCGGTTCAGGCTTCAGGTTCGTGGTGGAGGATGTCCGGTCCAACGATGGGGTGGTCTTTCATTTTG GATACATTGATGACTCTCAAGCTAACTGTGAATCCAACCTCAAAGAGAGACAAGAAGTTACCTTACAAGTTGATGCAC AACGACGTGATCTCAATTCCAGGATTCATTCTGCTGGCCATTTATTGGATATTTGTATGCACAATGTAGGCTTATCTCAC CTAAAGCCTGGAAAGGGTTATCATTTCCCTGACGGGCCATTTGTTGAATATATTGGAGTAATCTCCCCAGATCAACTGC AGATCAAACAAAAAGAGCTTGAAGAAGAAGCAAATTCATTAATCAGTATTGGTGGAAAGGTTTCTGCTTCTGTTTTACC TTATGATGAAGCTGCCAGGTGGTGCAATGGTGATCTCCCTAGTTACATTTTGAAGGGCAGCACCCCAAGAATTGTGAGG CTAGGTAACAATGCCGGATGCCCCTGTGGAGGGACTCACGTTGCTGATATTTCAGACATAAAAAGCTTTAAGATTACCC AAATTCGATCAAAGAAAAGAATCACAAAGATCAGCTACAGCATCAACCCATGAGCTCTGTATTTCTCTGCAACATTGTT GATCTATTGCGATGATTAGTGAGCTCCTATCATACCTGAATGTATCACCTAATTTTATTATGTTTTTGTAATGATTAATC ACAGTTGTTATTATGGGTCTATAGGAAAATGTGAAACTTTGCCT

>25-48-T3 hypothetical protein

NNNAGATTGAGGTGTAGTGGAATTTGCTCGGATGAACACCCTATCAGAACTGAGCTTGTGAGTTTTGCTTCCCTCTTTG CCCCATTGCGGCCATCAGTGAAGATAAATCCTCAAGCAGCTACCAGATTCATTGAGCATTCTTTGCCTGATTTGGCACC AGACCAGAGGAAGAGTCTCCATAATATCAGTACAGGCAAGGGTGACCGGACTCCTTTCATGACAAACAGAGCGAAGA AGAAAACGAAATACCAATCTTTTGAGCAGCAGTCTGCGCGTGCTGCTGCGCAGGAGTTTCTTGAGAAGGCAGCAAGGG AGCTCTTTGGCTCAAACGATTCGGACGTGAAGGGTCCATTGCAGAACCTTAAATCAGATGATGAAGATGATGAATAAA TGCTTTGATTTATTTTTTATTTTTTTATTTATTTCTTTTACTTCAGATAAATTGTTCTCTTTTCTTCGTGAAGTATTTGGTAA ATTATGTTTGATAAAAAACACTTTTTTTTTCTAAGAACCTTAATTTTATGGTAGT

200

>26-51-T3 cytochrome P450-like protein

CTGAGATCATGAGAGCTAAACAAAACGGGCATCATGATGAGACAAAGCAAGACATACTATCAAGGTTCATCGAGCTCG CCAACACCGACAAAGAGAGTGATTTCAGCACGGAAAAAGGTTTAAGAGATGTGGTGCTAAACTTTGTTATTGCAGGGA GGGACACTACTGCTGCAACGCTCTCATGGTTTATATACATATTAGTCACACAACCTCAGGTGGCACAGAAACTCTATAT AGAGATGAAAGAGTTTGAGGAGATCAGAGCTGAAGAAGAAAATATAAATTTGGATTTATGTAATTTGGAAGATATGGA TTCATTCAGAAACAGATTATCAGATTTTTCGAGGCTTTTGGATTATGATTCATTAGCAAGGCTGCAATATCTGCATGCAT GCATTACAGAGACCCTGAGGCTGTTTCCTCCTGTTCCTCAGGTTGGGTTCAAAAAAATAAAAGGGAAGATTTACATAGA TTTACATATATTTATGTTAAATATATAAATATTT

>27-52-T3 putative photosystem II core complex proteins

CCAACCACCCTCTCTTCCATCCCTGACCTCCTCTGCCATCGCCGCTGCGGTATTCTCCTCCCTAAGCTCAGCGGACGCTG CCCTTGCAGTGCAACAGATCGCCGACATTGCCGATGGTGACAGCCGGGGCATTGCTCTCCTAATTCCCATAGTTCCGGC CATCGGTTGGGTTCTTTACAACATTCTCCAGCCGGCGTTGAACCAGATCAACAGGATGAGGAGTGTGAAAGGGCTTGCC ATTGGGTTGGGGCTCGGACTGGGCCTGAACTCAGCAACCAGTGCATCAGCTGGAGAAGTGGCGGTGATTGCTGAGGCG GCTTCTAGGGATAATAGGGGCCTACTGTTGCTAGTTCCAGTGGGTGTGGCGATTGCTTGGGTGCTGTTCAATATACTTCA GCCAGCGTTAAACCAGATCAATAGGATGAGGGAAAGGTAAGGTACGAACGAAGTAGAGTTTGAGCCTTGCCGTTTGTG ATTGAGTTCTTGGATGATTATAGCTTCCTTTGTATTCATGGTCATTATATTTTATCGAGGCACATTGTAGTATGCCATGAT TATTAGATATAATTCTTTTGCTGC

>28-57-T3 S18.A ribosomal protein

AAACCCTAGCATTCGGAAAATTTTTGAAGCAGAGAAAAAATGTCTCTAATTGCGAACGAGGATTTCCAGCACATTCTTC GTGTGCTGAACACGAACGTGGATGGGAAGAAGAAGATCATGTTTGCTCTCACCTCCATCAAGGGTATTGGTCGCCGTTT TGCTAACATCGTCTGCAAGAAGGCCGATGTCGACATGAACAAGAGAGCTGGTGAACTCTCTGCTGCTGAACTTGAGAA TCTCATGACTGTAGTTGCAAATCCACGCCAATTCAAAATCCCAGATTGGTTTTTAAACAGAAAGAAGGATTACAAGGAT GGTCGGTACTCACAAGTGGTTTCAAATGCATTGGACATGAAATTGAGAGATGATCTCGAAAGATTGAAGAAGATCAGA AATCATCGGGGCCTGCGTCATTATTGGGGGCTTCGTGTTCGTGGGCAGCACACAAAGACTACTGGGAGGAGAGGAAAG ACTGTCGGCGTGTCGAAGAAGCGTTGATCCTCCACCTCATTCCTGCTTTGCTTCTCTACTGCATTGTTATGTTGTCTCTCC ATTGGAGGTTGAAACTGAAATTTTGGGCGACTT

>29-59-T3 unnamed protein product

NNNNAGNGCTCGAGAACGATGCTTCCACCTCCACCCAATAGCATGAGAACGATGCCTCCACCTCCGCCGAAATTCCAG TCGGATCATAACTTGAGAATAATGCCTCCACCTCCGAAGTTTCGATCCGTTCTTAATGGTAATTCAGAAATGAGAGCAG CAGCTGTTCACAGAGATTCAGAGAAAGCTAGTTTAGAACGGGTTCCTGATACCCTATTGAAGCTGGTTGAGTATGGCGA GGAAGACGAAGAGGAAGATGACATGGTTGGCTCAGCCGAAGAATCCTGTCGAAGTGATATAATGCAAAGCACGAGCT CAAAACCTTTTTGGGCTGTATAATAAGTAGTCTTTTGAAATTTTAGTTGGCATGTCAACATGTTTAATCTCCTTCCATTG GCTCTCAGGCCTTCTCTGATTGATGATGAGCTTTGAAGCTATGGCTCTGTTCTATTTTTCGATTTGGATGATAAATTTAGT GAGAAAATTTGATTTAT

>30-60-T3 S18.A ribosomal protein

CGGCGGCGGGGAGTGGAAACCCTAGCATTCGGAAAATTTTTGAAGCAGAGAAAAAATGTCTCTAATTGCGAACGAGGA TTTCCAGCACATTCTTCGTGTGCTGAACACGAACGTGGATGGGAAGAAGAAGATCATGTTTGCTCTCACCTCCATCAAG GGTATTGGTCGCCGTTTTGCTAACATCGTCTGCAAGAAGGCCGATGTCGACATGAACAAGAGAGCTGGTGAACTCTCTG CTGCTGAACTTGAGAATCTCATGACTGTAGTTGCAAATCCACGCCAATTCAAAATCCCAGATTGGTTTTTAAACAGAAA GAAGGATTACAAGGATGGTCGGTACTCACAAGTGGTTTCAAATGCATTGGACATGAAATTGAGAGATGATCTCGAAAG ATTGAAGAAGATCAGAAATCATCGGGGCCTGCGTCATTATTGGGGGCTTCGTGTTCGTGGGCAGCACACAAAGACTAC TGGGAGGAGAGGAAAGACTGTCGGCGTGTCGAAGAAGCGTTGATCCTCCACCTCATTCCTGCTTTGCTTCTCTACTGCA TTGTTATGTTGTCTCTCCATTGGAGGTTGAAACTGAAATTTTGGGCGACTT

>31-61-T3 abscisic stress ripening protein

GCTCGGTGCCATAGCCGCTGGTGCTTTTGCACTGCATGAGAAGCACAAGGCAGAGAAAGACCCTGAGCACGCCCATAA GCACAAGATAGAAGAGGAAATTGCAGCAGCAGCTGCAGTTGGTGCCGGTGGTTATGCCTTCCATGAGCATCACGAGAA GAAAGAAGCCAAGGAAGAGGAGAAAAAGCACCATCACCACCACTTTTAAAGCTTTCAACTATATCAAGACATCCATTA CTATGTGTTTGTAATTTATATATATATATATATATATATATATATTTTTTGGG

>32-62-T3 alpha tubulin

CTCCCGCATTGACCATAAATTTGATCTCATGTATGCGAAGCGTGCTTTTGTGCATTGGTATGTTGGAGAAGGAATGGAA GAAGGTGAGTTTTCCGAGGCTCGGGAGGATCTTGCAGCCCTCGAGAAAGACTACGAGGAAGTCGGTGCTGAGGGTGCT GAAGATGAAGGGGAAGATCCGGATGACTATTGAGTTAGTGGGGATTCATTGAGAGTTTGGGTGTGGTTCCAGTCTTGTT GATTTGTTTTGTTGTACTCGTGCTAGATATGCTTTCATATTGGCATATATTTCAAACCTTTGTGGTGGTGTTCTTCCATGC GTGATTTTCCTTTTTGGATTTTTAAAAGTTTACTGGGTGTTAAATGGAATTGCTTAGATT

201

>33-63-T3 syntaxin-like protein

GTANCGGCCTGATGAAGAGGTAGAACTTTGTCTTTTTTTAATTTCTGAATGATTTGAATGTTTTTAAGATCCATAGTTTC TACCATAATTGTTGGCTCTGCAGACTATCGACCAGCTGATAGAGACAGGAAATAGCGAGCAAATTTTCAAGAAGGCAA TACAAGAGCAAGGACGTGGCCAGATAGTGGACACCGTTGCAGAGATCCAGGAGCGTCATGATGCCATTAAAGATTTAG AAAGGAAGCTTCTTGAGCTGCAGCAGGTATTCTTCGACATGGCGATATTGGTCGAAGCACAAGGTGACTTGCTCGATAA CATTGAATCCCAGGTTTCAAGTGCGGTTGACCACGTTCAATCTGGAACAACAGCTCTGCAGACGGCGAAGAGACTGCA AAAGAATTCTCGTAAGTGGATGTGCATAGCCATTATAATTCTTCTAATTATTGTCATAGTGATCGTTGTAGCTGTCATTA AGCCTTGGAGCAAATAGTTTCCTTACAACAGACAGCTGAAGGAACTTCAGGATCGATCGATGGATCAATCAATCAATC AGGTGATCAACTGTTTTGTATGTCATATATTTAATATTATTATTTATTTTCGCCAACTCTTTCTGCATCTGTTGATATTTTT GTCGTATACGCTGTCGAGAGCAGCAATTTTTTTTTAATGAATTGGATACTGTTAGCGAAAAATAAATTTGTTATTATTAT TATTATTTTTAT

>34-66-T3 KH domain-containing protein / zinc finger protein-like

AGNGANCTGCCGTTTAACTGGAGCTAGACTGTTCATACGTGAGCATGAGAGTGATCCAAATCTTAGGAACATTGAGCT GGAAGGAACATTTGATCAGATCAAGCAGGCGACTGGGTTGGTCAGGGAACTGATTGTCAACATCAGTACTAGTGCTGC TCCTGTGCCCGTGAAGGCAGCTGGTGGTCTTGGGGCAGGAGGTGGTGGTGGTAGTGGTGGTCCTGGGAGCAATTTTAA GACAAAGATGTGTGACAATTTCACCAAGGGAAGCTGCACCTTTGGAGAGAGGTGCCATTTTGCTCATGGGGCGGCTGA GCTGCGCAAGGCAACTGCTTGAAATGTTTTAACCACAACTTTCTAAGGTCATTTGTAGCAAGCGCCGGTTCGTTCGTTTT GTAGGTGGACTCGGAACTGTTTATGAACTAGTTGATTTCTTT

>35-68-T3 putative RNA binding protein

GTTAGGATTGCAGATTCGAGCGAGCAGATTCGGGCATTGACGGAAATGAATGGTGTTTATTGTTCTTCTAGACCTATGC GGGTTGGTCCAGCAGCTAGTAAGAGGTCTGCCGATGCACAACAGCATGACTCTGCAAAAGCTTCATTCCAAAGCTCAC AGGGAAACCAATCAGAAAGTGACCCAAACAACACTACAATTTTTGTCGGTGGCCTGGATCCTAATGCTACGGAGGATA TGCTGCGGCAGGTTTTTAGCCCATATGGGGAACTGGCTCATGTGAAGATACCTGTTGGAAAGCGTTGTGGCTTTGTCCA GTTTGTTAGGAGGGCTTGTTCAGAGGAGGCTCTACTGATGCTTCAGGGAACTCAGCTTGGCGGTCAAAAGATGCGGCTT TCATGGGGTCGTAGTCCTTCAAGCAAGCAGTCTCAGCAGCAGGAAACTAATCAGTGGAATGGAAACTACTATGGTTAT GGCCAAAGCTATGAAGGATATGGGTATGCTCAAGCTCCTCAGGATCCGAACATGTATTCATATGGAACTTATCCAGGAT ACGCAAATTATCAGCAACCACAGTGAGCTTTACTCGGATTGTGGCCTTTAACTTGCAAATGGTAGCAGTTTGCAAGCGG TATCTTCAACTTCACCAGAAGACGGTCTATAGTTATGTCTGCAGGGTATTTTAACATGGATATAATTGGTTGTGCTTTCT TCATCTTCAGAGGCTGCGTTAGTTATGCTTTTTAAGCTTCTCTTAGCTATGATTCCTGAGTGTGTTGTCTTAACGCTTGTT ACCTTTTCTCCTGAGTTTCAATGGTTAAAGAGTATCAATTTAACTGAGTACAAGTTATGATTTATGGATTATTAATGAAT CAATCTGGATCAATTCTAGCTTTGCTGA

>36-69-T3 GF14 protein

AAAAGAGANCGCGATCGTTGAGACGAAGATCTAGAAAAGGTTACTGGATCATTATAAAGCAGAAGTAAAGATGTCGTC TTCTGAGTCTTCTCGTGAGGATAATGTTTATATGGCAAAGCTTGCAGAGCAAGCTGAGCGATATGAGGAAATGGTTGAG TTCATGGAGAAGGTGGCCAAGACGGGAAGTGTGAATGAGTTGTCTGTGGAGGAGCGCAACCTCCTCTCTGTGGCCTAT AAAAATGTTATCGGAGCTAGACGGGCCTCGTGGAGGATAATATCCTCCATTGAGCAGAAAGAGGAAGGCCGTGGCAAT GAGGATCGTGTGACCATTATCAAGGAATATCGTGGAAAAATTGAGACCGAGCTCAGCAAGATCTGTGATGGAATCTTG AAGCTGCTTGATTCTCATCTGATTCCCTCTGCTTCTGCTGCTGAATCAAAGGTTTTCTACCTAAAGATGAAGGGTGATTA CCACAGGTATCTTGCTGAGTTTAAAACTGGAGCAGAGAGGAAGGAGGCTGCTGAGAGCACACTACTGGCGTACAAATC TGCTCAGGATATTGCATTGGCGGAACTGGCCCCTACTCATCCAATAAGACTGGGGTTGGCGCTGAATTTCTCAGTTTTCT ATTATGAAATCCTTAACTCTCCAGATCGTGCCTGCAATCTTGCAAAACAGGCCTTTGATGAGGCCATCTCTGAATTGGA CACCCTTGGCGAAGAGTCCTACAAGGATAGCACATTGATCATGCAACTTCTCCGAGACAACTTGACTCTATGGACTTCT GACATAACGGAGGATGCTGGGGATGAGATCAAAGAATCCTCAAAACATGAATCATGAGGAGCTGGACCTCATTTTCAT TTCATTTTAAGTAGATATTATTGCTTTGAAAGACTTTGTTGTGTGCGTGAACTTCTTACTTTTAACTAATGAAACAACAG CCCTGTATATGGCGACTGGATGTGAAGATGGCGTTTTTTAATGGTTATATTAAGCTTGTGGTGTGCATCATTATGCTATT TAAGGGCTTTCGAATTACTTTTTAGTAGATGTCAGCTTGCTACTGTTT

>37-71-T3 isopentenyl monophosphate kinase

GTNAAGCGACTTCATCTTGGTATAACTAGTTCAGTTGACCCGTTGACTCTGCTAGAAAAGATCTCTCTAAATGGAATAT CTCAAGATGTCTGCATAAATGATCTTGAACCCCCTGCATTTGATGTTTTGCCATCCTTGAAGAAGTTGAAGCAACGTGT GCTAGCTGCAGGGCGTGGCCAGTATAGTGCTGTTTTCATGTCTGGAAGCGGAAGCACCATTGTGGGAATTGGTTCACCA GACCCACCTCAACTTGTTTATGATGAGGATGAATACAATGATGTTTTCATATCAGAGGCTTCCTTTCTCACTCGGCAAGA GAATCAGTGGTACGCAGAGCCAACTTCGTCCACAGGGTCTTTGAGCAGAGAAGAGCCGTCACAAACAGGAAAATAATT ACGATAATTTTTTTACATTCTAGACCTTCTAATTTTAATTTTTCTCACATAAAATCATATTGTATTACTGTACTTATTGTT CATGCAAGAAAGATCGATCAAGCTATCTTTCATGAATGAGCAAAATATGCAATTTTAAAAGGCACATTTACATGCTT

202

>38-72-T3 enoyl-[acyl-carrier-protein] reductase (NADH)/ oxidoreductase

AAGTGATGAGTCCCGACGGCTGTCGGGGGGGCTCTTTTCCAGATTGCTATCCGANCAAAGAGAGTGATGCAGTGTTTGA TAGTCCTGAGGATGTCCCTGATGAAATTAAGACGAACAAGCGTTATTCAGGTTCTTTATACTGGACTGTGAAGGAAGTT GCTGAATGCGTAAAGGATGACTTTGGAAGTATAAATATTCTTGTGCATTCCCTTGCTAACCGGCCAGAGGTGACCTAGC CTCTATTGGAGACTTCTGAACAGGGGTATCTTGCAACTTTATCTGTTTTCTTCTACTCCTTTAAATCTCTACTCGAGGGG GGCCGTCCTATAATAAATCCATGGGTAGCTAACATATCTCTAACTTACATTGCTTCTGAGAGAATCTTTCCAGTATATGT TGTAGCATGATTTCGGCAAAAGCTGCACTTGAGAGTGATACTCAATTACTACCTTTTAGAATCA

>39-73-T3 dihydrolipoamide S-acetyltransferase precursor

AAAAGACTCAGTTAATGACATTGTCATAAAAGCTGTAGCACTAGCGTTGAGGAATGTTCCTGAAGCAAATGCT TATTGG AGTGATGAGAAGGGTGAGGCCATCATATCCAGCTCCATTGACATATCAATTGCGGTGGCTACAGAGAAGGGCTTAATG ACACCAATTGTAAGGAACGCAGACGAGAAGACATTATCAACTATATCCTCAGAGGTTAAAGAATTGGCTGAGAAGGCA CGTAATGGAAAACTTAAACCTGAGCAGTTTCAAGGTGGGACTTTTAGCATATCAAATCTGGGAATGTTCCCTGTAGACC ACTTTTGCGCGATTATAAATCCTCCACAGGCATGCATTCTTGCTGTTGGTCGAGGTTATCAAGTGGTTGAGCCTGTCAGT GGCAGCGGTGGAATCGAGAAGCCTGGAATGGTGACAAAGATGAGCTT

>40-74-T3 Ubiquinol-cytochrome c reductase complex ubiquinone-binding protein QP-C

GAGGAAAGCAGAGCGAGAGGGAGAGATGGGGAAGACGCCGGTGAGGATGAAGGCAGTGGTTTATTCCCTATCGCCAT TCCAGCAAAAGGTTATGCCGGGATTGTGGAAAGATCTTCCGACCAAGATTCACCACAAAATCTCTGATAATTGGCTAAG CACTGTTCTTCTCCTAGGCCCGCTCATCGGAACCTACTCGTATGTTCAGCATTACAAGGAGAAGGAGAAGCTCGCGCAC AGGTATTGAATCTGAAGTCTTGCAGAAGATGCTGAATATGGATTGCAGTATCTGAAGATTTTTTCCTGCATAAATACTG AGACTTTTTTTGAAGTAAATTAGATTAATAACTGCATTTTGGGCACTGGTGGGAGTATTTGTTTAATTATTTGAGTAATA ATTATCGACATAATTGAATGTGATAGATCTTCCCAGCGTTGGTGTTTTATTTTTTGGGTCATTTACATGCATACAATACA ATTCTT

>41-75-T3 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 2 (Aci-reductonedioxygenase 2)

NTGAGCACCGCACGCCCAAGCAGTTTGTTTCTATTGACAAACTTGCAGAACTTGGTGTTCTCAGCTGGCGACTGAATGC TAATGACTATGAAAATGATGAAGCGCTAAAAAAGATTCGTAAGGCGAGGGGCTACTCTTATATGGACATAGTTGAAGT TTGCCCAGAGAAGCTGCCAAATTATGAAGCCAAGATCAAGAGCTTCTTTGAGGAACATTTGCACACCGATGAAGAAAT TCGTTATTGTCTTGAAGGAAGCGGTTATTTTGATGTGAGGGATGAAAATGACCAATGGATTCGTGTAGCTGTGAAGAAA GGGGCAATGATTGTATTGCCTGCTGGAATTTACCATCGATTTACTCTAGACACAAATGACTACATAAAGGCTTTACGAC TTTTTGTCGGTGAGCCTGTGTGGACTCCACACAACCGACCCAATGATCATCTCCCTGCCAGAAAGGAGTACCTTGATTT GTTGGGGAAGAAGTCAGCTGCTGGTGGCCATCAAGCCATTGAGGCTCACTGAGGTCATTGGATAAGTACATCTCTTAAC ACCAGTCTTAGCTGCCGACTGCGATTCCTGCTTTAATCTTATATAATCTCATTTCATGAGTGGTTTTTCACGTGTGGGTTT GATGTGATGCCAGATGTTTCTGTTGTCTTATAGAATAAAATCGGTTGCTTTATGCCATGCCAAAAGCGTGAACATGTGG GATTTGTTTATCTTCTGTTCCTGCTGCATGCTGGCTACTTGTATGACGCTTTATGTTAAATAAGTTTGATGAGTTTGCC

>42-76-T3 thioredoxin-dependent peroxidase

GCGTGGACGAGATCCTTCTAATTAGCGTCAATGANCCATTTGTGTTGAAGGAGTGGGCGAAGAGTTACACGGACAACA AGCACGTGAAGTTCCTCGCCGATGGCTCAGCTAACTACACGAAGGCTCTTGGCCTGGAGCTGGATCTGAGCGAGAAGG GGCTCGGGCTGCGTTCTCGCAGGTATGCGATCCTGGTGGACGACCTCAAGGTTAAGGTGGCTAACATTGAGGAGGGTG GAGATTTCACCATCTCTGGTGCTGATGAGATCCTCAAGTCTCTGTGATACGCCTTCTGCTGTTTGTTGCCTCTTTTTGAGA CGCGCTCGTTTCACGTTTCTCTTTGAATAAAAGCGCTTCTTAGCTCTTCCTGTTTTGTTTTGTGTGAAAGAGAACACAGTT CTATAAAGTTCAAGTGTTTATTTGGTTTACTCTTTAAGGTTTACCTGCAAGAAGTTTGTATTGTTGTTTGAAGTTGGTAA AGATCTTAATTTTATGCC

>43-77-T3 putative mitochondrial dicarboxylate carrier protein

GANTCCGCCGCCGGATTAGTGGCAGCGGTGGCGTCGAATCCGGTGGATGTGGTGAAGACGAGGGTGATGAATATGAAG GTTGAGAAGGGGGCACCGCCGCCGTATGCCGGAGCGCTGGACTGTGCGTTGAAGACGGTGAGGGCGGAGGGACCTAT GGCGCTTTATAAAGGGTTTATTCCTACTGTTTCTCGTCAGGGGCCCTTCACCGTCGTTCTCTTCGTCACGCTGGAGCAGG TGCGCGCGCTTCTGAAAGATTTCTAGGTTCATCTCTATGTGAGATTTGTTGGATTGATTTGTTATGATGCCGAAAATGTA TTGCCGCTCTGGGGGCTATGAGCAGCAGAGCGGCTAGTGTTTGTTCTTCAGAAGAATCAATTAAAATCTGAGTTTTTCT ATTATT

>44-78-T3 6-phosphogluconolactonase family protein

NAANANACCCGAGTCACCGCCAGAAAGGATAACCTTTTCGATTCCAGTGATCAACTCAGCATCAAACGTCGCCATTTTG GCTACGGGTGATGACAAGGCCATGGCTCTGCAGTTCGTTGTCGATCACAGCTCTAGTTTTGATGCCTTTTCGCTGCCCGC AAGGTTGGTGAACCCGACCGAAGGGAAGCTGTTGTGGTTCGTGGACAAAGCGGCAGCCTCGTTCATCGACTCTGCCGA TGAAAGCGAGCATTTTGAAATTTAGCAGCTATGTATGTGTTCTGTATGTAGATGAGTACAGTGGGTTTGATTAGTTACTT CTTTTGTTTAGGCCCTCTTTGAGATATCTGAAGGAAGCTGTGGTTAAGATGTGAGGGTAAAGCAGTAGATGAGGAAGTG GATTAATAAGCATAGTGCAACATTTTGGATGGAGGGCAGTAATCTGTGAGAGACATTGCTTTTATTTTAATTAGTGAAA GAAAATTCTCAAGAGTTCTGAATTTTC

203

>45-81-T3 calmodulin-binding protein

TTGAAGAGGAAGACGGCGCTGCCGTACAAGCAGAGTTTTTTAAACTGGCTCAGATTCGCTCCGGCGTTGCGTAGTTTTA GTTCTTCGGGGGCCGGCGATAATCATTCACCCTATCGTGGGAAAACCATGGCAGGGTCGAAGTCAATGCCTCGCTTCAG AGAAGGGAATTAATTGAGCAAAATTGAATGAATTTGGTCAAGAAAGTTAGTTAGATTTTTTTTTCTTTTTTTTTTATTTG CTATGATAATTGATGAGTTTTAAAGTAGTTAAATTCATTTTAAGATGAGCATAATATAAATTTTAAATGATTTATCATGA TTATGCTTATCTTAAAATAAATATAATAATTTTAAACCTATCTTCATCAAAACATTCTGACTATTTTTTCTTCATCTAATC AGCTATGCTTGTTGTACATAAAGGTGAGTGTGTTGTGTCTAAGTGTGTAAATTTCTACTTTGAAATGAGAAAGGATGTC T

>46-82-T3 enoyl-[acyl-carrier protein] reductase

AGATTCTTGTTGGAACATGGGTGCCTGCATTGAACATATTTGAGACTAGCTTAAGGCGTGGAAAGTTTGATGAGTCCCG ACGGCTGTCAAATGGCTCTCTTTTCGAGATTGCTAAGGTTTATCCCCTTGATGCAGTGTTTGATAGTCCTGAGGATGTCC CTGATGAAATTAAGACAAACAAGCGTTATTCAGGTTCTTCAAATTGGACTGTGAAGGAAGTTGCTGAATGCGTAAAGG ATGATTTTGGAAGTATAGATATTCTTGTGCATTCCCTTGCTAACGGGCCGGAGGTGACCAAGCCTCTATTGGAGACTTC CAGAAAGGGGTATCTTGCAGCTTTATCTGCTTCCAGCTACTCCTTTATATCTCTACTCCAGCATTTCCTTCCTATAATAA ATCCAGGGGGAGCTACGATATCTCTAACTTACATTGCTTCTGAGAGAATCATTCCAGGATATGGTGGAGGCATGAGTTC GGCAAAAGCTGCACTTGAGAGTGATACTAAAGTACTAGCATTTGAAGCAGGAAGGAAAAAGCAAGTTAGAGTAAATG CAATCTCTGCTGGTCCGCTGGGAAGCCGTGCTGCAAAAGCCATTGGTTTCATTGAGAAGATGATAGAGTATTCACATGC TAATGCGCCATTACAGAAAGAACTGCTAGCAGATGAAGTTGGTAATGCTGCGGCTTTCTTGGTGTCCCCATTAGCTTCT GCTATCACTGGCTCCGTAGTTTATGTCGACAATGGATTAAATACAATGGGTTTGGCAATTGACAGCCCATCTCTCTCAGT CGAGTGATAAAGCAGAAAATCTGCATAATGATTGGTTTTTTAATAACAAGTATCCCCTTCTGTTTCAATCTCCTGCGGCA TTAATGAATAATAATTGAGTTGGTTTCTTTCCTAGTTTCATTCGAGGTCCAGTGGTTTTATACATGTAGCTATGTGATGA TATGATGATTCCTTTCAGTGNGTACAGTGAAAATACATTTTGATG

>47-83-T3 hypothetical protein OsI_021037

GNAANAATTCAGGAAGAAGGTCCTTATGATGATCTTGATGAGGAAAAGGGCCTCTCTCGTCGTTGCCAGCTTGTGCTCG CATTCCTTGCATTTTTTTTACTGTTTACAACATTTTGCCTTATTATCTGGGGAGCGAGCAGACAGTACAAAGCAGATGTG GTGGTGAAGAGCTTGTCAATGGATGATTTTTATTCGGGAGAGGGTTCGGATAGCACTGGTGTTCCGACCAAGTTGATCA CGGTGAACTGTTCTCTGAAGATCAATGTGTATAATCCAGCATTCACATTTGGTATTCATGTCACTTCTAGTCCCATTAAT CTCAAGTACTTGGATATTGTCATTGCTACTGGTCAAATAGAGAACTACTACCAGCCCAGAAAAAGCCATAGAACCATGT CTTTAATACTAAGGGGAGACAAGGTTCCTCTATATGGAGCTGGTGCTGCCTTACCTCTATCCAATAGTGGTGGTTCTGTT CCATTAACTTTGGAATTTGATTTGGTTACCCGAGCTAATGTGGTGGGAAAGCTGGTTAGGGTGAAGCATCAGAAGCATG CCTCATGCCAAATTGTAGTTGATTCTAGCAAGAACAAGGCCATAAAGTTGTCAAAAAATGCTTGTACCTATGACTAAGT CCCTTTATCTTCATGTTTTACTAGCAGATAATCTGCTCATGATGAGGTGAAAGAAGGAAGAGTAGTTGCATGGTCTCCA TTTGTCTATGTTTTTTTTATGTTCATTTTTGTATATTGTTGCAATAAGACAATTTGAAGATGAGTTTTGACTTGAATTTCC TGTACTGTAGAACTTATACTTTGTTGGTAAGTGGAAGAAGGCATTTCTCTTGT

>48-104013_85_T3 jasmonate ZIM-domain protein 1

AAGAAATCGATCGATCGAGGCGGAGGATTCAACTAGTCTTTGGGATTTTCACACTCAACGATAGAGGGTATATGGCAA AGAGGCAGGAGAAGTCGAACTTCTCCATCACCTGCGGCCTCTTGAGCCAGTACATCAAGGAAAAGGGCAGCCTTGCTG ATCTAGGGCTTCTCGATTCTGCTCGATTAGGCAAACATGAGGCTTATCGGCCGCTGACAACCAGGAGCTTGCTCTCAGG AGTAGGTTTCTCCATCAATGACCCTAAAGACACCAAATCCATGGAGCTTTTTCACAAGAGTATTGGTTTCCTTCCGGCC

>49-86-T3 translation elongation factor 1 alpha

GNNAAGACCTGAAACGTGGTTTTGTGGCTTCTAGTTCAAAGGATGATCCTGCTAGGGAAGCCGCCAATTTCACTTCTCA GGTCATCATCATGAACCACCCTGGCCAGATTGGCAATGGTTATGCTCCGGTCCTTGACTGCCACACTTCCCACATTGCC GTCAAATTTGCAGAGATCTTGACCAAGATAGACAGGCGGTCCGGCAAGGAGCTCGAGAAGGAGCCAAAATTTCTCAAG AATGGAGATGCAGGTTTCGTGAAGATGATTCCCACCAAGCCTATGGTGGTTGAGACGTTCTCCGAGTATCCGCCGCTCG GAAGGTTTGCTGTGAGGGACATGAGGCAGACGGTGGCGGTCGGGGTCATCAAGAGCGTGGAGAAGAAGGATCCGAGT GGAGCGAAGGTGACCAAATCGGCTGCCAAGAAGAAATGAATTGTGCGTTGTTGTTTGAATAAGGAGGAGCGGGAATCC ATCGAGTTGGTTTCTGGTGTTTGGATGCAGAACTGGGTGCTTGACAGACGGTGGCACTGCTCGCTTCAGTTATCTTTTTA GTTGTTGTCTGTGTTGTTTGTTTTTCTTGTGTTGAAGGCTGTTGTACTGCTTTTCTATGGTTGTATTATTTATCGCTGCTTA TTATGATTGTACCGTTGTTGTTGTATGAGTTTGAATTTGATGTTATTTGAGTTTTTGTGTTCT

>50-90-T3 hypothetical protein

NGCGATCAGGTTCCAAGACTTGATTTTTATTTTCCTATGGAAGTTCACAAAGATAGCAATGAACGTTTATCCATACCCA CATTTGGCGAGTGGGATGGGAAGGTGGGTCTACCGGACTACTCGGTTGATTTTACTAAGATTCGAGAAAATCGGCGGC AGAACAAGAGTCGGGTGAGCTTGGGGAATGAGGATGAGCTCCTACATCGTACTGGTGTAAATAGTGACCAGAATGTTG ACGCTCTCAAGAAGACACTCCCACTTCATCAGAAGCATGTTAACTCCACAGTGGAAAGGAAGAAAAGCAGCAGATATT TCAACTGTTGTCGTTGTTTGGGGGCCTGAAACTGTGGCTTCATCCACACATTATGTTGCACGTGGAGGTGAGGGCCTTTG AAGCTCTTCAGCCATCCTTTTCAAATTTCTTTCACCTCCGTTTTGTCGCTCAATCAAATGTTATGGTGATTGCAAACTACT

204

TGGATTAGGATTTTTGTTATGAAGAAATGCAGGATTGGAGATGTTTCTTGGTGTCGTTCTATAAAGATGTAATGTTTGGA TTGCTTGTTAGATTCACATATTTTTATTATTATTCGATCGCATTGTTGTAGATAGATTTTATATTTTATGTACATGAAATT TTATCTTATTTATGATTAGTAATTTCTTATTCTAATCC

>51-91-T3 MADS box AP3-like protein 17

NCNCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCCTTGAGCAAACTTTGGAAGAGTCTCTGAGAATTGT TAGGCATAGAAAGTATCATGTGATCGCCACACAAACTGACACTTACAAGAAAAAGCTTAAAAGCACAAGGGAAACAT ACCGCGCTCTAATACATGAACTGGATATGAAAGAGGAGAATCCGAACTACGGTTTTAATGTTGAGAACCACAGCAGAA TTTATGAAAACTCAATTCCAATGGTGAATGACTGCCCTCAGATGTTTTCCTTTAGGGTTCATCCAAATCAGCCTAATCTG CTCGGTTTGGGTTATGAATCACACGATCTTAGCCTTGCATGATCAGCAGTACTATTATAAAAGTTTATGATTTTATTGCA TTTTTATTATATGTTTGAAACTTTAGAATTATGAGATGGGGGATCTAGTC

>52-94-T3 40S ribosomal protein S15a

TTTGAANGATGCTCTCAAGAGCATGTACAATGCTGAGAAGCGTGGGAAAAGACAAGTTAAAATCAGACCATCTTCCAA AGTTATCATCAAGTTCCTCCTCGTCATGCAGAAGCATGGCTATATCGGTGAGTTCGAGTATGTCGATGATCATAGAGCG GGCAAGATTGTGGTGGAATTGAATGGGCGTTTGAACAAGTGTGGTGTCATTAGCCCAAGGTTTGATGTTGGTGTTAAGG ATATTGAGCCATGGACTGCAAGATTGCTCCCTTCGCGTCAGTTTGGGTATATTGTGCTGACAACATCAGCAGGCATCAT GGATCATGAGGAAGCTAGGAGGAAGAATGTAGGAGGCAAGGTTCTTGGATTCTTTTACTAGTTAAAAAATGTTTTGAA TGAATTTTGAAGATTGTTGTCTTTTAAGTGCATTTATTTGATGTTAGCCTGCATTTCTCCTTAACTTTGAATGGTGTAAGG CTACTTGTTATCTTTGGATCTTGAGGCATATTTCCCATTTGTGTTTGCGTATTTTTTTGCTAAGGTTACCGTTGAAAATAT TGCAGAAAATTTTATTTT

>53-104016_95_T3 60S ribosomal protein L14 (RPL14A)

CCCTTCATAGCTTCCAGCAGATCGCAGCAGCAGTCATGCCTTTCCAGAGGTACGTGGAGATCGGGAGGGTGGCACTCGT CAACTATGGGAAGGAGTATGGGAGACTTGTTGTTATCGTCGATGTCATCGATCAGAATCGAGCCTTAGTTGATGCTCCT GACATGGTTCGAGGACAAATGAACTTCAAGAGGCTGTCGCTGACTGATATCAAGATTGACATCCCCCGAATTCCCAAG AAGAGCAAACTGATCGAGGCCATGCAAGCAGCTGATGTGAAGAATAGATGGGAGAAGAGCTCTTGGGGGAGGAAGCT GATTGTGCAAAAGAAGAGAGCTGAACTGAATGATTTTGATAGATTTAAGGTCATGTTGGCTAAAATTAAGAGAGGAGA TTCCATCCGACGTGAGCTTTCAAAGCTAAAGAAGAGTGCTTCAAAGTGAGGCTTTTGAAGAATTAATGGCCGATTCTGT GTCCATTATTGCAGAATTTAAGTTTGAGAGTCAACCGTTAGAACAAAATGTAGTTTTGGTCGCTGTTATGAACTTAAAA GTTTGATGTTTTGATATTTGGATTTTTTTAAAAAAATTTGTAACTGTTTGCTTTATTATTTGAATGAATTTAAGAATTGGA TTATTATTAATTATTCCACAGTAGAAGGAGTAATTTGTGAAACTGATTTCTGTTAATGTTTGATTGCAGATTCTACAAAC GCAATCTTCAAAATGTATTTTTGATTGTTCAATATAGATTTTAGAAATGTCTCAATTTGTTT

>54-96-T3 hypothetical protein OsJ_006705

CTTGATTTTGCTCAGAGATCTATGCCTGTTATTGGATATAGCGCTCATGCAGGTAACAGCGGGCCAATGGCTTCTGAGT CAGTGCCTGGTTAAAATGTCCATTTCTAAGTCGCCGAATAATGCACCTCCATAATCAATCATGAAAGGACCCCCTGGGC TAGTTTATGTATGAATGCAAAGCCTTTATCACCATCATTGTCTGCACCATCATCGTCAACATGGGAAGGAGGTTAGCTTT TGTTGAAGTTTAGGCGAAATTGCTCATGCCTTAACGAGCACATATCAGTTTTCGCCTTCTCTCATCTCTGAATTTGGTCT CTTCTAACATGTAAACTTTAGAAACAGTGGGGAGTAGCCGGGAGGTTTCTTCTTCCGGGGAGCTTTATTTTGTTCGCTCT GCATGGGAACTCCAGTAAATTTATTAGTGTCCTGGACTATACAGGTTTGATCCTGGAGATCGATCGTTTCTGCAGTTTTA AAACTTGAAACTATCAAAAAGGTACTTTTATGCGTGGGCTTGGTGGTGTAATTATTCTGTTAGAATCTATATTTAGAGG CTTC

>55-28 hypothetical protein

AAATATCCCGCAACCTGTCCCACCTTTTCTCCCCTTTTCGTCTCTTTTTCCCTGTTTCTCTTTCTCATCCGATTGATGGATC TTAGGGCGGCGATAGTCGCCGCCGCCGGCGAGCGTTGGACGGAGGAGCGGCACTCCCGCTTCCTCAACTCGATCGAAA GTACTTTCGTCCATCAAATGCTCGGCATCCATCCCGACGGCGATAACCTCCGCCGATGCGCGGCGAGGCTCGACCGTCG TGTTCCCGATTGCATCGCCGGTAAAGAGTCTGCGAAGAGTTCTCAGATGCGATCGCCGGATAGGAGGCCTGCTGCCATT ACTGCGGGCGCCAACACCTCTAATTGTACACGGAAACGATCACTGCGGCGATATGATGCGTCGCTAGACCAGGTGGTG CCGGAGTTCAAAAATAAGAACGTCGGCGAGGATGCATCC

>56-38 GRF interacting factor

CTCCTCACTTGCCCGCCGCCATTCTTGTCGATTACCCACTGTTCCCTCAAAGATCCCCTTTATTCGCTTAACCCACCACC AAAGCTGAATTTCAGAGTCCAGGACTCAAAAAGCCGAAATCTTTCCGTGTAGAAAATACGACAAGCCAACACAGGAAT GCAGCAGCCCTCGCATCCGATGTCCCAAATTTCTCCGGGCAACATTACCACAGAGCAGATTCAGAAGTATTTGGATGAA AACAAGCAGCTGATTTTGGCAATTTTGGATAATCAGAACTTGGGCAAACTTGCTGAATGTGCTCAGTACCAAGCCCAGC TCCAAAAGAATCTGCTTTATCTCGCCGCCATAGCTGATGCACAGCCTCCAACTCCTTCAGTCCGTCCTCAGGTTTTCTCT

205

GCACAATTTTCAAGCTTATCCTCATATATCTGGTTGTGCATAGAAGTAGAAATTTGTTAATGCTGACAGTTTGAGGAGC GAGCTCATGGAACGAAGACTATTCAAAATTTGGGTTTGTTTATGGCTCGGTGACTATCATGCTTACTTGAAACAACTTT GGTTTACCTCGGTATAGATCAAATAATTTTGCTTGAGATGAAATCTAAATAAAGGGTTGGTTTGTTCTATATATAATGA AGTCTTTTACCTAGCACACTATTAGATATGGTTGTGAAGAGCTTCTTATAATAAAAAATTTTGGGAAAGC

>57-40 chaperon heatshock protein

GCAAGCTGAAGATGGATGCCAGAGTTTTCAGATTGGAGAACCCCCTCTTCTACGCACTGCAAAATTTGGTGGACCTGCC GGAAGAAATGGAGAAAGCCTTCAACGCTCCGACGAAGAAATATGTACGCGACGCGCGGGCGATTGCTTCCACGCCGGC AGACGTGATTGAGAAGCCGACGGCCTACGAGTTCGTCCTAGATATGCCGGGAGTGAAATCTGGCGATATAAAGGTCCA GGTAGAGGAGGGTAACGTGCTCGCCATTACAGGGGAGCGGCGCCGGGAGGACGAAGACAAGGAAGGCGGCGTAAGTT ACCTTCGATTGGAACGACGGGTAGGGCAATTTCTGCGCAAGTTCTCTCTCCCGGATGACTCCAATCTCGAAGCGATAAC CGCTGTCAGCCGTGATGGTGTTCTGACAGTTACCGTGCAGAAGCTGCCGCCGCCCGAGCCTAAGAAACCCAAGACAAT CGAGGTCAAGATTGCGTGAATTGATCTGTTCGTTTTGAATTTTTAGGGTTTGCTTTACTGTCATTATGAAGTATGTTGAG GTTGTGTCTAAGGATTGGATCCTTGTGATCTGCTTTGTTTCCAATCGCTTTTTGATATATCTATAATACTATCAGTAAAG ATTTTGTGGCTTTGGG

>58-44 phospholipid transfer protein

CTCGCTCCACCGCTTGTGTTGCAGTCGTATGCATAGTGTCCTTCCTCCTTGTTTCCGGCGTTTTCCGCGAGGCGAGCGGG ACCGTCACTTGCGGCCAGGTGGCCTCAAACGTGGCACAGTGCATCGGCTATATTCGAGGCGCTGGCTCGCTCACACCAG CATGTTGCAGTGGGGTGAGGAAACTCAACGGGCTGGCGAGCACCACCCCTGACCGTCAGACGACATGTTCTTGCCTCA AGACTCTCGCCGGCAGTATCAAGGATCTGAACCGTGGCCTTGCTGCTGGACTTCCTGGAAAGTGCGGCGTCAACGTTCC ATATCCCATCAGCACCTCCACCGACTGCTCCAAGGTGCGCTGAGCTCCAAGGACGGGCTACTCATCGAGCACTGCTTTG GTTCGATGATCTGCAGAATAAAGTGGAGACTGGATCTATTGAAGCTATTTTATGTATTGTTGTTTGTGGGTTCTGTCTTT CGATTTTAGTTTATTGTTTTGCAAGAGGGTGCACCCTTGGTTGTGGTGTGCTGGATATTGTTGCTGTAATATTATCACTTT TTCGACTAATATGAGTTGCAGCATATTATAATT

>59-46 hypothetical protein

GGAGAAGCCTAAGGAGCCGGAGAAGGAGAAGCCTAAACAGCCTGAGCCGCCTCAAGGGGGCCAGCCGGTCTGGCCGC CCGGTCCACACATGCCATGCTGCTGCTGGAGACCATGCTATGAGCAGTACTACGGCGGCTGTCGGTGCTGCTCCTGTGG GATGTTCTACGGGTGGACGGGGCCGCCATTGCCACCGGCTATGGGCACTCCGTCAACATCCACGTGTCAGTTCTTCTGC GAGGAGGATCCTTCTACATGTACCATTATGTAATAATATAGTCCCTTTCGTTCAAATTATGAGCCATTAATAGAGATGA TAAATAAAGCTGTGCTCCTCTCTATAGTGCATTGAGTTATAATAACTCAAGAGATATATGTCAGTTTTATAATATTTTGT CTTCTTTATTTGAATATGTAATTTTGTTTTCATCATGGTGAAAGATATATGTCAACTTTGCAATATTTTGTTTTCTTT

>60-54 hypothetical protein

CTGAGAAGCAGTCCTCGTACACGTACTGGGTGAGGGAGACGAAGGACGACGCGGCGCCGCTGCAGGTGCCTCGGAAG CTCACCCCGGAGGACGTTTCTCAGCAATCCCAGCCTAACTTGTTGGGATCCGTATGGAATCAGGCCGGAACAtGGGAGG AAAAAAATCTAAATTCATGGGCAAATAGTAGAATTAAGGAGCTTCTCAGTTCATTGTCCTTGGAGTTTTCTAATGGAAA AGCAGCGGTTTATGAAGTTACCAAATGTTCAGGGGATGCATTTTTGATCACGGTGCGGAAC

>61-58 pectate lyase protein

GCGATTATGATGATGGTTTGATTGATATCaCaCgAGAGAGCACTGACaTCaCTATCTCgAGATGCCACTTTGCAATGCATG ATAAAACAATGCTTATTGGGGCTGATAGCAGCCATATTACTGATAGATGTATCCGGGTGACAATACATCACTGTTTTTT TGATGGAACAAGACAGAGACACCCTCGTGTTAGATTTGGGAGAGTTCACCTCTACAATAATTATACAAGAAATTGGGG TATATATGCAGTATGCGCTAGTGTGGAAGCaCAGGTTCTCTCTCAGTGCAATATATATGAAGCCGGAGAG

>62-66 hypothetical protein

CCCACCCCTTCCCTATCTTCCATGATAATCAACCACTACAAGCTTAGGGGAAACATCATCAGCTACAACCTTGGTGGTA TGGGATGTAGTGCTGGACTCATCTCCATAGATCTTGCAAATCGTCTTCTTCAAGTTCATCCCAACTCCTATGCTTTGGTT ATCAGCATGGAGAACATTACTCTCAATTGGTACTTTGGAAATACCCGTTCCATGCTCGTTTCGAATTGCTTGTTTCGAAT GGGCGGCGCAGCTATTCTCCTATCCAACAGACGGTCAGATCGCCGTCGATCCAAGTATCAGTTAGTCCATACGGTTCGC ACGCATAAGGGCGCCGACGAAAAATGTTTTGCTTGTGTGACTCAACAAGA

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APPENDIX H Real-time RT-PCR Data 1) PCR Efficiency for Putative Fragrance-related cDNAs and Housekeeping Genes

(A)

(B)

(C)

(D)

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(E)

(F)

(G)

Notes: PCR efficiency of putative fragrance-related cDNA and housekeeping gene cDNA transcripts for real-time RT-PCR analysis. (A) putative actin (B) putative cyclophylin (C) putative alpha tubulin (D) VMPPAAS (E) VMPCMEK (F) VMPCyP50 and VMPA28

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2) Expression Analyses of Putative Fragrance-related cDNA Transcripts and Reference Genes Transcripts

a) Expression Analyses in Different Tissues

VMPPAAS Tissues bud fully-open flower petal sepal lip leaf root shoot stalk VMPCMEK Tissues bud fully-open flower petal sepal lip leaf root shoot stalk VMPCyP450 Tissues bud fully-open flower petal sepal lip leaf root shoot stalk

Calibrator x

Relative Quantity 1 8333 20256 15879 4439 0.5 0.3 0.3 0.4

Standard Error 0.238 168.76 888.56 487.98 567.47 0.007 0.0065 0.0054 0.076

Calibrator x

Relative Quantity 1 1.63 1.763 1.81 0.609 0.009 0.015 0.012 0.012

Standard Error 0.16757 0.20777 0.2645 0.1436 0.02515 0.00095 0.0042 0.00155 0.00121

Calibrator x

Relative Quantity 1 3.094 1.12 1.912 5.497 0.001 0.003 0.001 0.002

Standard Error 0.17897 0.27044 0.17177 0.18797 0.30796 0.00008 0.00047 0.00005 0.00016

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VMPA28 Tissues bud fully-open flower petal sepal lip leaf root shoot stalk

Calibrator x

Relative Quantity 1 1.17 1.266 1.352 1.054 0.586 0.62 0.482 0.644

Standard Error 0.10546 0.09434 0.10744 0.04426 0.06094 0.06464 0.04724 0.04496 0.06778

b) Expression Analyses at Different Flower Developmental Stages

VMPPAAS Stages young bud (green) mature bud (red) half-open flower fully-open flower 14-days old fully-open flower VMPCMEK Stages young bud (green) mature bud (red) half-open flower fully-open flower 14-days old fully-open flower VMPCyP450 Stages young bud (green) mature bud (red) half-open flower fully-open flower 14-days old fully-open flower VMPA28 Stages young bud (green) mature bud (red) half-open flower fully-open flower 14-days old fully-open flower Calibrator x Relative Quantity 1 1.98 2.5 3.5 2.8 Standard Error 0.25742 0.24924 0.34808 0.26529 0.28955

Calibrator x

Relative Quantity 1 8.711 49277.86 83509.328 7617.29

Standard Error 0.045 0.578 4691.799 6075.784 1163.816

Calibrator x

Relative Quantity 1 1.508 2.273 1.822 0.123

Standard Error 0.10003 0.21186 0.22069 0.23132 0.10549

Calibrator x

Relative Quantity 1 2.734 3.344 3.041 2.094

Standard Error 0.10579 0.27015 0.49873 0.22863 0.29847

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c) Expression Analyses at Different Time Points in a 24-hour Cycle

VMPPAAS Time 12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm VMPCMEK Time 12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm VMPCyP450 Time 12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm

Calibrator x

Relative Quantity 1 0.601 0.474 0.328 0.023 0.012 0.011 0.001 0.001 0.154 1.404 3.361

Standard Error 0.09914 0.01281 0.03276 0.13436 0.05484 0.00981 0.0071 0.00021 0.00021 0.04827 0.01737 0.29116

Calibrator x

Relative Quantity 1 1.87 1.954 2.437 2.613 2.179 1.424 0.857 1.13 1.15 1.342 1.778

Standard Error 0.18511 0.22595 0.27654 0.30942 0.34981 0.33652 0.25509 0.09788 0.12365 0.17864 0.18711 0.25471

Calibrator x

Relative Quantity 1 0.158 0.347 0.654 0.813 0.819 0.445 1.56 2.378 3.808 4.586 6.367

Standard Error 0.18057 0.02676 0.06629 0.06927 0.10608 0.09146 0.07637 0.16879 0.11831 0.23655 0.33069 0.10896

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VMPA28 Time 12am 2am 4am 6am 8am 10am 12pm 2pm 4pm 6pm 8pm 10pm

Calibrator x

Relative Quantity 1 1.562 2.013 2.196 2.981 1.376 0.635 1.879 4.188 2.18 0 0

Standard Error 0.2131 0.17269 0.2717 0.27402 0.39576 0.02264 0.18099 0.2764 0.2486 0.2475 0 0

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3) Melting Curve analysis (a) VMPPAAS

(b) VMPCMEK

(c) VMPCyP450

(d) VMPA28

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(e) Actin (Reference gene)

(f) Cyclophilin (Reference gene)

(g) Tubulin (Reference gene)

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BIODATA OF THE AUTHOR

Mohd Hairul Ab. Rahim was born on 18 September 1985 in Telok Mas, Melaka. He went to SK Telok Mas primary school from 1992-1997. He continued his secondary education in SMK (A) Sultan Muhammad, Melaka from 1998-2000, and then SM(A)P Kajang from 2001-2002 to obtain his Sijil Pelajaran Malaysia (SPM). His pre-university education was completed in Malacca Matriculation College. In July 2004, he entered Universiti Putra Malaysia to begin his undergraduate study and completed his Bachelor of Science degree in Biotechnology with a CGPA of 3.372. He continued his Master degree in the field of Plant Biotechnology at Universiti Putra Malaysia in 2007.

He was active in extracurricular activities throughout his undergraduate studies. He was a member of the Students Representative Council of Universiti Putra Malaysia for 2005/2006 session and also an Exco of BioMix Society, an undergaduate society of Faculty of Biotechnology and Biomolecular Sciences UPM for 2004/2005 session. Besides that, he was involved in many students programs including Sambutan Hari Assyura Universiti Putra Malaysia 2006 organized by the Students Representative Council of Universiti Putra Malaysia (The Director of the programme), commitee members of Faculty Night 2005 and 2006 organized by BioMIX Society and a facilitator for Minggu Perkasa Putra 2005 (orientation week for incoming new students of Universiti Putra Malaysia). During his undergraduate studies, he was selected to present a poster presentation at 18th Intervarsity Biochemistry Seminar for his final year project. He received the Public Service Department (JPA) scholarship for his undergraduate studies.

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His interest on molecular biology and plant biotechnology started in 2006 during his practical training at Molecular Biology Laboratory, Makmal Biaklon, Felda Agricultural Services under the supervision of Dr. Sharifah Shahrul Rabiah Syed Alwee. During his postgraduate studies, he had presented in some conferences and seminars such as Asia Pacific Conference on Plant Tissue Culture and Agrobiotechnology (APaCPA) 2007 (poster presentation), 5th Malaysian International conference on essential oils, fragrance and flavour Materials (MICEOFF5) (oral presentation), 18th Scientific Meeting of the Malaysian Society for Molecular Biology and Biotechnology (MSMBB) (Poster presentation) and 2009 Plant Biotechnology Postgraduate Symposium (Oral

presentation). He won the first prize for best oral presenter for the Molecular Biology, Agriculture and Physiology category in the 2009 Plant Biotechnology Postgraduate Symposium. Besides that, he was also a recipient for a research grant from Malaysia Toray Science Foundation (MTSF) 2008 for the screening of fragrance-related cDNAs from Vanda Mimi Palmer. During his postgraduate studies, he was financially supported by the Graduate Research Fellowship (GRF), Universiti Putra Malaysia.

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LIST OF PUBLICATIONS

Terpenoid, Benzenoid and Phenylpropanoid Compounds in the Floral Scent of Vanda Mimi Palmer. Mohd-Hairul, A.R., Namasivayam, P., Cheng Lian G.E. and Abdullah, J.O. (2010). Journal of Plant Biology 53: 358-366.

Putative Phenylacetaldehyde Synthase Transcript of Vanda Mimi Palmer: Sequence and Expression Analysis. Mohd-Hairul, A.R., Chan, W.S., Namasivayam, P., Cheng Lian G.E. and Abdullah, J.O. (2010) International Journal of Botany. Published online on September 2010. http://scialert.net/abstract/?doi=ijb.0000.20116.20116#

Screening, Isolation and Molecular Studies of Putative Fragrance-related Transcripts of Vanda Mimi Palmer. Mohd-Hairul, A.R., Namasivayam, P., Abdullah, J.O. and Cheng Lian G.E. (2010). Status: Submitted

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