(ACC) Oxidase Gene in Cattleya Spp

SPECIAL PROBLEM

TITLE Isolation and molecular characterization of 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene in Cattleya spp.

BY Miss Chanthra Timclub

PROGRAM IN AGRICULTURAL BIOTECHNOLOGY FACULTY OF AGRICULTURE KAMPHAENGSAEN

KASETSART UNIVERSITY 2004 Bachelor’s Degree Special Problem Program in Agriculture Biotechnology TITLE Isolation and molecular characterization of 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene in Cattleya spp.

BY Miss Chanthra Timclub

This special problem has been accepted

Date……...March 2005 (Mrs. Parichart Burns, Ph. D.)

Isolation and molecular characterization of 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene in Cattleya spp.

Miss Chanthra Timclub

Abstract

The plant hormone ethylene is known to stimulate a wide variety of plant response to flower senescence and fruit ripening. A gene in ethylene synthetic pathway, 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene, was identified from flower of Cattleya spp. Lucky Strike variety. In this study, reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods were used to amplify the ACC oxidase sequences. The cDNA of ACC oxidase (ACO) was cloned and characterized. The cDNA from Cattleya-ACOs, open reading frame (ORF) and 3’untranslated region (3’UTR), were 1,081 nucleotides in length. The sequence was further analysed by comparing with other ACC oxidase genes in GenBank Database. The results show that major difference between Cattleya spp. located at 3’UTR while the amino acid sequence alignment indicated high percent identity at 96.7%. In addition, the amino acid sequence of Cattleya-ACC oxidase was compared with 11 ACC oxidase genes of other plants from Genbank database. Ascorbate binding domains, ACC binding domain, ferrous ion binding domain, bicarbonate binding domain, leucine zipper and basic zipper domain were found in all ACOs.

Keyword : Cattleya spp., Cattleya, Ethylene, ACC oxidase genes, senescence. Degree : Bachelor of Science, Program in Agricultural Biotechnology, Faculty of Agriculture Kamphaeng saen, Kasetsart University Advisor : Parichart Burns, Ph.D Year : 2005 Page : 54 การหาลาดํ ับเบส และวิเคราะหยีน 1-aminocyclopropane-1-carboxylate (ACC) oxidase ในกลวยไมสก ลแคทลุ ียา

นางสาวจันทรา ทิมคลับ

บทคัดยอ

เอทิลีนเปนฮอรโมนพชทื าหนํ ากระตนการเสุ ื่อมสภาพของดอกไมและการสุกของผลไม ใน การทดลองครงนั้ ี้ไดศึกษา ACC oxidase ซึ่งเปนยีนที่เกยวขี่ องในกระบวนการสราง เอทธีลนี โดย ใชหลักการ Reverse Transcription Polymerase Chain Reaction (RT-PCR) และ Rapid Amplification of cDNA Ends (RACE) ในการ Amplify ลําดับนิวคลโอไทดี ของยีน ACC oxidase (ACO)ในกลวยไมสกุลแคทลียาพนธั ุ Lucky Strike จากการโคลนยีน และหาลาดํ ับนิวคลีโอไทด นํามาวิเคราะห พบวาล ําดบนั ิวคลีโอไทดในบร ิเวณ 3’untranslated region (3’UTR) มีความ แตกตางท่เหี นได็ อยางชัดเจน เมื่อนาไปเปรํ ียบเทียบกบลั ําดับนวคลิ ีไทดของแคทลียาพนธั ุ Bicolor และแคทลียาพันธ ุ Intermedia ซึ่งมีรายงานใน GenBank แตเมื่อเปรยบเที ียบลําดบกรดอะมั ิโนใน สวน Open Reading Frame (ORF) พบวาม ีความเหมือนถึง 96.7% นอกจากนยี้ ังพบ Ascorbate binding domain, ACC binding domain, ferrous ion binding domain, bicarbonate binding domain, leucine zipper and basic zipper domain เมื่อนํามาเปรียบเทียบกับยนี ACC oxidase ของพืชชนิดอื่นๆที่มีรายงานไวในฐานขอมลู Genbank

คําสําคัญ : Cattleya spp., แคทลียา, เอทธีลนี , ยีน ACC oxidase, การเหี่ยว. ปญหาพิเศษ : ปริญญาตรี สาขาวิชาเทคโนโลยีชวภาพทางการเกษตรี คณะเกษตร กาแพงแสนํ มหาวิทยาลยเกษตรศาสตรั  อาจารยท ี่ปรึกษา : ดร.ปาริชาติ เบิรนส ป : 2548 จํานวนหนา : 54 ACKNOWLEDGEMENTS

I am highly thankful to my supervisor, Mrs. Parichart Burns, Ph.D., for her kindness, generous help, suggestion and criticism of my special problem. I would like to thank the members of my supervisor committee, Ms. Orawan Kumdee, Ms. Suwanna Bandee, Ms. Saifon Cheuabunmee and Ms. Yada Mukjang, for their helpful advices and criticisms of the manuscript. Thanks are also expressed to the Plant Genetic Engineering Unit, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom province for making available research facilities. I would like to express special appreciation and gratitude to all of my friends for their help and fellowship during the period of my study. Finally, I gratefully and respectively thank to my family for their understanding, consolation and encouragement. Chanthra Timclum March, 2005

TABLE OF CONTENTS Page TABLE OF CONTENTS…………………………………………………………………………….i LISTS OF TABLE…………………………………………………………………………………...ii LISTS OF FIGURES……………………………………………………………………………….iii INTRODUCTION…………………………………………………………………………………..1 LITERATURE REVIEW Cattleya……………………………………………………………………………………2 Ethylene…………………………………………………………………………………...6 Senescence……………………………………………………………………………….9 MATERIALS AND METHODS Plant Material……………………………………………………………………………14 Plant Sample…………………………………………………………………………….14 Cloning, Sequencing and Characterization of Cattleya-ACO……………………..14 Identification of functional domains in ACO putative proteins…………………….22 RESULTS AND DISCUSSIONS Cloning, Sequencing and Characterization of Cattleya-ACO..……………………24 Identification of functional domains in ACO putative proteins…………………….37 CONCLUSION……………………………………………………………………………...... 41 LITERATURE CITED……………………………………………………………………………..42 APPENDIX………………………………………………………………………………………...46

LISTS OF TABLE

Table Page

1. The nucleotie sequence alignment indicated percent Identity between Cattleya spp. Variety……………………………………………....33 2. The amino acid sequence alignment indicated Percent Identity between Cattleya spp. Variety……………………………………………….34

Appendix Table

1. Abbreviations of amino acids………………………………………………...53 2. The genetic code………………………………………………………………54

iii

LISTS OF FIGURE

Figure Page

1. The ethylene biosynthetic pathway of higher plants………………………..7 2. Schematic illustration of morphological changes in mesophyll cells……11 3. Hypothetical scheme for the action of ethylene in inducing flower senescence…………………………………………………………….12 4. Flow diagram outlining the strategy used for the cloning, sequencing and characterization of Cattleya-ACO gene……………………………...... 14 5. Flow diagram outlining the strategy used for the identification of functional domains in Cattleya-ACO putative proteins…………………….22 6. Diagramatic representation of the Cattleya-ACO cDNA………………..…23 7. Ethidium bromide stained 1% agarose gel of Cattleya flower total RNA ……………………………………………………..25 8. Ethidium bromide stained 1% agarose gel of open reading frame Cattleya- ACO genes……………………………………...... 26 9. Identification the recombinant clones by amplifying the recombinant plasmids with M13F and M13R primers………………...27 10. Ethidium bromide stained 1% agarose gel of 3’untranslated region of Cattleya- ACO genes ACO genes……………..28 11. The colony hybridization analysis of clone 3’UTR Cattleya-ACO……..…29 12. Identification the recombinant clone by amplifying the recombinant plasmids with with 3’ end Cattleya-ACO specific primer and VFO2.……………………………………………………30 13. Nucleotide and amino acid sequence of Cattleya-ACO …………………31 14. The sequence comparison of Cattleya spp ………………………..……...32 15. Phylogenetic tree of ACO nucleotide sequences………………………….35 16. Phylogenetic tree of ACO amino acid sequences…………………………36

LISTS OF FIGURE (cont’d)

Figure Page

17. Alignment of ACC binding domain and ascorbate binding domain in ACO sequences……………………………………………………………….38 18. Alignment of basic zipper domain and leucine zipper domain in ACO sequences………………………………………………….39 19. Alignment of Fe2+ binding domains and bicarbonate binding domain in ACO sequences………………………………………..40

Appendix Figure

1. Disection of Cattleya flower…………………………………………………..47 2. pDrive Cloning Vector……………………………………………………….48

INTRODUCTION

Cattleya Orchid (Cattleya spp.), is native to Central and South America. With its beauty, large flower, fragrance and colorful flowers, it is widely known as Queen of the orchid (Buranachonnabud, 1991; Papp, 2004). Although well adapted and cultivated in various areas around the world including Thailand, Cattleya flower has short vast life. This limits its market and popularity. There is little known about its senescence and the role of ethylene in the process. Therefore, a gene in ethylene synthetic pathway, 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene is identified and characterized. This study will provide a basic knowledge of Cattleya senescence.

Objective 1. Isolate ACC oxidase (ACO) gene from Cattleya spp. flower 2. Characterize ACC oxidase (ACO) gene from Cattleya spp. flower

Place and Duration All experiment was done at Plant Genetic Engineering Unit, Kasetsart University Kamphaengsaen Campus, Nakhorn Pathom. The experiment began in November, 2003 and completed in January, 2005.

LITERATURE REVIEW

Cattleya

Cattleyas were among the first tropical orchids to come into cultivation. Their culture is often used as the basis for comparison with other types of orchids, being highly prized for their large blooms. They have well-adaptation to their habitat organs include thickened stems for food storage called pseudo-bulbs, roots that cling to the substrate to hold the plant in place, and thick, leathery leaves that transpire little water (Rittershausen, 1999; Ombrello, no date).

1. Origin

Most of the Cattleyas have been developed from plants native of the America’s, especially Central and South America. The species range through tropical across the Panama Isthmus as far north as the rainforests of Mexico, but the best are found in the steamy Brazilian rainforests. The epiphytic or air plants grow on the trunks and stout branches of the forest trees, in their wild state existing for hundreds of years, growing into huge clump several metres across (Rittershausen, 1999). The flower was introduced to England, when in 1818; William Cattleya imported some tropical plants from Brazil. In 1821, the genus was named Cattleya by the plant taxonomist, John Lindley (Bechtel et. al, 1992; Papp, 2004). While the number of Cattleya species is comparatively small, they are so closely related to other genera in the alliance that much interbreeding has taken place, resulting in many intergeneric hybrids. The first Cattleya cross, Cattleya hybrida, appeared in 1857. It was the first tropical hybrid to be recognized by the Royal Horticultural Society, who awarded it a first class certificate. During the first half of the twentieth century cattleyas were in great demand as cut flowers and today they are cultivated by a growing band of devotees. Due to their size and irregular flowering habits, they have not 3 entered the pot-plant trade in large number, but will be found in specialist nurseries (Rittershausen, 1999). The Cattleya has ventured a long way since it was first seen and the beginning of hybridization to produce some of the most flamboyant, largest, best scented, and colorful flowers amongst the Cattleya family. Eventually it became the “florists orchid” or more commonly known as the “Corsage orchid” (Papp, 2004). Cattleyas have been the most hybridized, and has resulted in a treasure of beautifully colored flowers available in almost any color except blue and black; there are no blue or black orchids of any kind. The intermarriages between Cattleya and other genera such as Laelia, Brassavola, Sorphronitis and many more has produced plants far removed from the original Cattleya species of decades ago (Papp, 2004).

2. Classification

Cattleya is classified Kingdom Plantae, Division: Magnoliophyta, Class: Liliopsida, SubClass: Liliidae, Order: Asparagales, Family: Orchidaceae, Subfamily: Epidendroideae, Tribe: Epidendreae, Subtrip: Laeliinae and Genus: Cattleya (Bechtel et. al, 1992; http://en.wikipedia.org/wiki/Cattleya). There are approximately 53 species of Cattleya from Mexico to South America (see Appendix). The genus is divided into two groups; bifoliate and monofoliate cattleyas. The first group, such as Cattleya trianae, is found in Mexico and Brazil while the latter one, such as Cattleya bicolor, is found in Brazil, Columbia, Panama, Peru and Venezuela. Bifoliate Cattleya contains two broad leaves growing from each pseudobulb. Monofoliate Cattleya, on the other hand, contains only one, narrower and more erect leaf originating from each pseudobulb. The typical flower has three rather narrow petals: two are fringed; the third is the conspicuous lip with a fringed margin and various markings and specks. At the base, the fringed margins are folded into a tube. Each flower stalk originates from a pseudobulb. (http://en.wikipedia.org/wiki/Cattleya) 4

3. Botanical

The Cattleya is either epiphytic or lithophytic. The stems are thickened called pseudobulb and have one or two leaves at apex of each. The Leaves are usually thick and coriaceous or fleshy. The Inflorescence occurs at terminal (one flower) or racemose as peduncle usually subtended by a large spathaceous sheath. The Sepals are free or more or less equal and fleshy. The Petals are mostly much broader than sepals and less fleshy. The Lip is sessile, erect, free or rarely to column-base and enfold column. The Column is usually long, wingless, semiterete, arcuate as anther terminal, and has somewhat compressed to four pollinia. The Capsule is ellipsoidal (Bechtel et. al, 1992). Disection of Cattleya flower (Appendix figure1.)

4. The culture

The Cattleyas are epiphytes and called “tree dwellers” as they inhabit the branches of trees and sometimes barren rocks. Their nutrition is derived from the atmosphere or from decaying organic matter that accumulates on branches or in crotches between limbs so they are not parasites of trees. Cattleyas thrive in this nutrient poor and freely draining medium (Ombrello, no date). Cattleya orchids are slow-growing, taking 5-7 years or more to flower from seed since most produce relatively few and large flowers at maturity (Ombrello, no date). There are long-lived perennials and will usually flower annually. Commercial growers maintain plants for 8-10 years before replacing them (http://en.wikipedia.org/wiki/ Cattleya). The Culture is relatively straightforward for orchids, and they are considered by many to be the archetypical epiphytic "orchid" in that they require very well-drained media, frequent wet/dry cycles, good air circulation, moderate light and temperature, judicious watering and an occasional dose of fertilizer (Ombrello, no date; http://en. wikipedia.org/wiki/Cattleya).

In nature, Cattleyas start off life as tiny seeds within a seedpod or capsule on the mother plant. When the seedpod matures and splits open, Cattleya seeds are released and dispersed by the wind. The germination is occurred when seeds fall on a suitable medium and contact with a microscopic fungus. The fungus converts complex starches to simple sugars that seeds can use for energy. Cataleyas could be growth with artifical mediums that supply the necessary nutrients in a small flask. In recently, Cattleyas are propagated by tissue culture technique. Tissue from growing point of single shoot can yield hundreds of identical plants. Conventional methods, asexual propagation, would take many years to accomplish and will probably lead to an almost unimaginable variety of types in the future (Ombrello, no date).

Ethylene

1. Introduction

Ethylene (C2H4) is a simple organic molecule gas with biological activity. This molecule gas, regulates various plant physiological processes, the growth and development of plants initial germination to senescence, including seed germination, root hair development, root nodulation, flower and leaf senescence, responsiveness to stress and pathogen attacks, abscission and fruit ripening (Johnson and Ecker, 1998). The ethylene molecule isn’t direct in effect to plant physiological processes but it result from action together of ethylene, receptor protein and some metal ions to stimulate gene expression into other responsive aspect many physiological process (Brady and Speirs, 1991). In general, plant tissues produce few amounts ethylene. The production of ethylene is induced by internal signal during development and in response to environmental stimulant from biotic stress, such as pathogen attack or insect attack and abiotic stress, such as wounding, flooding, drought, chilling injury, auxin and auxin inhibitor treatment (Theologis, 1992). These factors activate to ethylene production in normal state of plant.

2. Ethylene Biosynthetic Pathway

The pathway for ethylene biosynthesis was elucidated by Yang and Hoffman (1984). The precursor of ethylene is methionine, which plants produce itself. Methionine is converted to S-adenosyl-L-methionine (SAM or AdoMet) by the enzyme methionine-s- adenosyl transferase with adenosine triphosphate (ATP). Then SAM is converted to 1- aminocyclopropane-1-carboxylic acid (ACC) and 5-methylthioadenosine (MTA) is recycled through the pathway by converting to methionine (Figure 1.). ACC is converted to ethylene, CO2 and HCN by ethylene forming enzyme (EFE) or ACC oxidase. In oxygen 2+ state use cofactor, Fe ion and ascorbic acid (John, 1997). The CO2 and HCN are converted to β-cyanoalanine for protection to toxic substance, HCN, accumulator. In 7 addition, ACC is also converted to 1-(malonylamino) cyclopropane-1-carboxylic acid (MACC) (Kende, 1993).

Figure 1. The ethylene biosynthetic pathway of higher plants. Source : Arshad et al., 2002

3. Ethylene forming enzyme (EFE) or ACC oxidase

The last step in ethylene pathway is the conversion of ACC to ethylene. Since this step requires O2, the enzyme is also called ACC oxidase which is a bisubstrate enzyme.

It requires both O2 and ACC as substrates (Abeles et al, 1992; Hooykass et al., 1999; Arshad and Frakenberger, 2002). In addition, ascorbic acid, bicarbonate, ferrous ion and dioxygen were required for activity of this enzyme. The ACC oxidase is classified as a nonheme Fe (II) enzyme (Rocklin et al., 1999). The production of ethylene in vivo was inhibited by several types of chemicals including analogs of ACC, uncouplers of oxidative phosphorylation, free-radical scavengers, metal chelators sulfhydryl reagents and heavy metal ions action on the conversion of ACC to ethylene (Yang and Hoffman, 1984). The metal ions, Co2+, Cu2+ and Zn2+, are more effective in inhibiting ACC oxidase activity. They might replace Fe2+ and forming inactive enzyme-metal complexes (Arshad and Frakenberger, 2002).

4. Regulation of Ethylene Production

In ethylene synthesis pathway, ACC oxidase and ACC synthase, are highly regulated. Two systems of ethylene regulation in higher plants, system I and system II, have been proposed. In system I, ethylene is an auto-inhibitor controlling the basal levels of ethylene production in non-ripening climacteric fruits and vegetative tissues of both climacteric and non-climacteric fruits. In system II, ethylene acts as auto-stimulator. It operates during ripening of climacteric fruits and petal senescence. The mechanisms of regulation in system II require the induction of ACC synthase and ACC oxidase (Yang and Hoffman, 1984; Nakatsuka et al., 1998; Alexander and Grierson, 2002).

Senescence

1. Introduction

Senescence is a pervasive developmental process operating at many stages and levels during the life cycle of an organism. It has an important function in cell differentiation, not only in the most obvious example of xylem differentiation, but also in other cases such as the development of leaf lobing patterns and the breakdown of specialized cells in the embryo and the female gametophyte. Its function at the organ level is illustrated by senescence of leaves, flower parts, and fruits. Perhaps, the most remarkable senescence is the post reproductive senescence of the whole organism (Noodén, 1988). Alteration of plastid structure and function in senescence are often reversible and it is argued that such changes represent a process of transdifferentiation or metaplasia rather than deterioration. It may be that the irreversible senescence of many flowers and some leaves represents the loss of ancestral plasticity during evolution. Reversibility serves to distinguish senescence fundamentally from programmed cell death (PCD), as does the fact that viability is essential for the initiation and progress of cell senescence. Senescence, particularly its timing and location, requires new gene transcription, but the syndrome is also subject to significant post-transcriptional and post-translational regulation. The reversibility of senescence must relate to the plastic, facultative nature of underlying molecular controls. Senescence appears to be cell-autonomous, though definitive evidence is required to substantiate this. The vacuole plays at least three key roles in the development of senescing cells : it defends the cell against biotic and abiotic damage, thus preserving viability, it accumulates metabolites with other function, such as animal attractants, and it terminates senescence by becoming autolytic and facilitating true cell death (Thomas et al., 2003).

2. Flower senescence

Flowering, which is one of the most dramatic and spectacular events in plant development, is often associated with senescence and death. The process is sudden and comprehensive in monocarpic plants, such as Agave Americana and Phyllostachys edulis, where the entire plant dies after flowering or fruiting. In polycarpic plants, such as Dianthus superbus and Cattleya spp., senescence and death are restricted to some parts of the flower itself-mainly the corolla and stamens, which normally abscise or wither soon after flowering. The flower is a more complex organ and the interrelationship between the various flower parts may determine their rate of senescence. Since senescence of the whole flower is so complex, the present discussion will deal primarily with processes occurring during senescence of the corolla (Leshem et al., 1986). The first change in cells during senescence (Matile and Winkenbach, 1971) was observed in the vacuole membrane-the tonoplast showing invagination, that is, formation of enclosed vesicle containing cytoplasmic components. This may indicate autophagic activity of the vacuole and the loss of compartmentation, which in young cells maintains separation of the cytoplasm and organelles from vacuoles containing hydrolytic enzymes. The breakdown of compartmentation is expressed by increased hydrolytic activity of the cellular macromolecules-mainly proteins and nucleic acids. The autolysis of the cell components leads to complete disintegration and death (Leshem et al., 1986) These changes are schematically illustrated in Figure 2.

Figure 2. Schematic illustration of morphological changes in mesophyll cells of senescing corollas of morning glory. (A) Autophagical (digestive) activity of the vacuole. Invagination of the tonoplast results in incorporation of cytoplasmic material into the vacuole. (B) Shrinkage of the vacuole, dilution of the cytoplasm and swelling of the cytoplasmic membrane systems. (C) Autolysis of the cell components is initiated by the breakdown of the tonoplast. Source : Leshem et al. (1986)

3. Ethylene action and flower senescence

A hypothetical model for the action of ethylene in flower senescence is shown in Figure 3. This scheme suggests a membrane-based binding site that is activated or repressed by a “sensitivity factor”. The ethylene molecule binds to a site where the inhibitors of ethylene action, Ag+ and 2, 5-norbornadiene (NBD), can also bind. When the binding site is sensitized and ethylene binds to it, a second message is generated which interacts with the 5’ (promoter) region of genes involved in ethylene-regulated senescence, inducing transcription of the genes, and synthesis of the proteins encoded by these genes. (Reid and Wu, 1991)

Figure 3. Hypothetical scheme for the action of ethylene in inducing flower senescence. Source : Reid and Wu (1991)

4. Ethylene in the control of flower senescence

Ethylene, a group of plant hormones, influences the rate of ageing of various flowers and has been implicated to participate in the regulation of flower senescence. The symptoms of ethylene effects vary in various flowers. It can cause in-rolling of petals, as in carnation or morning glory, loss of turgor, as in petunia and some orchids, change in pigmentation or it can induce abscission of flower buds or corollas, as in snapdragon, geranium and sweet pea (Leshem, 1986). Petal and other floral organs are derived from leave and share common biochemical processes during senescence. Both leaves and flowers exhibit a combination of mobilizatin, wilting, and abscission during senescence. One difference between these organs is the ability of pollination a similar rapid deterioration and abscission of the leaf (Abeles et al., 1992). Not all flowers use an increase in ethylene production as the signal indicating the end of their functional life. In some cases, externally supplied ethylene does not induce floral senescence. Orchids exhibited a range of responses to applied ethylene. Vanda spp. is the most sensitive. It showed accelerated senescence after a day of 0.3 µl /liter ethylene. Cattleya spp., Cymbidium spp., and Paphiopedilum spp., are intermediately sensitive. These plants showed a response to ethylene within 3 to 7 days after ethylene treatment. Orchids that almost insensitive to externally applied ethylene are Dendrobium spp., and Oncidium spp., (Abeles et al., 1992).

MATERIAL AND METHODS

1. Plant Material

Cattleya spp. Lucky Strike variety was selected for this experiment. Cattleya was grown in Net house at Greenhouse Complex of Central Laboratory,Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom.

2. Plant Sample

The Cattleya flowers were collected at first day of blooming. The flowers were washed with distilled water followed by 70% ethanol and being air-dried. Flowers were separated in to 4 parts (sepal, petal, labellum and staminal column), cut into small pieces, frozen with liquid nitrogen and kept at -80oC.

3. Cloning, Sequencing and Characterization of Cattleya-ACO

Total RNA extraction

RT-PCR

Cloning

Sequencing

Sequences analysis

Figure 4. Flow diagram outlining the strategy used for the cloning, sequencing and characterization of Cattleya-ACO gene. 15

3.1 Total RNA Extraction

a. Method 1

Total RNA was extracted from Cattleya flower by Chang et al. (1993) Tissue was ground in liquid nitrogen to fine powder. Five grams of each powder was thawed with 15 ml of warmed RNA extraction buffer and then 300 µl of 2-mercaptoethnol was added before shaken at 65 oC for 10 min. The cellular debris was pelleted by centrifugation at 12,000 rpm at 4oC for 10 min and extracted twice with equal volume of cloroform:isoamyl alcohol (24:1). The RNA in the aqueous phase was precipitated by the addition of 0.25 volume of 10 M LiCl and shaked before being incubated at 4oC overnight. The RNA was collected by centrifugated at 12,000 rpm for 20 min, resuspended the pellet in 500 µl of SSTE buffer and transferred the content into sterile eppendorf tube. The content was added with an equal volume of chloroform:isoamyl alcohol (24:1) and mixed by vortexing. Following centrifugation at 12,000 rpm at RT for 5 min, the aqueous phase was transferred to a sterile eppendorf tube. An Equal volume of isopropanol was added to the supernatant and kept at -20oC for 2 hr. The RNA was precipitated by centrifugation at 4oC for 20 min, dried and resuspended in 50 µl of DEPC-treated water. The RNA was kept at -80oC. The concentration and purity of total RNA were determined using spectrophotometer (UltroSpec® 500/1100) at 260 and 280 nm.

b. Method 2

Total RNA was extracted from Cattleya flower by RNA extraction Mini kit according to the manufacturer (Qiagen). The weighed sample was immediately placed in liquid nitrogen and thoroughly grinded with a mortar and pestle. The 100 mg of tissue powder was thawed with 450 µl RLT Buffer and vigorously vortex. The lysate was directly piped onto a QIA shredder spin column (lilac) and centrifuged for 2 min at 14,000 rpm. The supernatant of the flow-through fraction was carefully transferred to a new microcentrifuge tube. This supernatant was added 0.5 volume of 100% ethanol and 16 transferred to an RNeasy mini column (pink) and centrifuged for 15 sec at 10,000 rpm. The flow-through was discarded and the RNeasy column was added 700 µl RW1 Buffer and centrifuged for 15 sec at 10,000 rpm. Then the RNeasy column was added 500 µl of RPE Buffer and centrifuged for 15 sec at 10,000 rpm. The RNeasy column was added another 500 µl of RPE Buffer, centrifuged for 2 min at 10,000 rpm and discarded the flow-through. The RNeasy silica-gel membrane was allowed to dry by centrifuged 14,000 rpm. for 1 min. The RNeasy column was transferred to a new 1.5 ml collection tube and 30-50 µl of RNase-free water was directly piped onto the RNeasy silica-gel membrane and stood at room temperature for 1 min. The mixture was centrifuged for 3 min at 14,000 rpm and total RNA was eluted. The RNA was resuspended in RNase-free water and stored at –80oC. The concentration and purity of Total RNA were determined using spectrophotometer (UltroSpec® 500/1100) at 260 and 280 nm.

3.2 Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Five hundred nanograms of total RNA from flower tissue were added to reverse transcription reaction containing 30 pmoles of ACO degenerate primer VFO2 (TCG TCT AGA GYT CYT TNG CYT GRA AYT T), 1 µl of formamide and 1µl of 10 mM dNTPs . The reaction was heated at 65oC for 5 min and quenched on -80oC 100% ethanol. Then the mixed of 4 µl of 5X First-Strand Buffer (25 mM Tris-HCL (pH 8.3), 375 mM KCl, 15 mM TM MgCl2), 2 µl of 0.1 M DTT and 1 µl of RNaseOUT Recombinant RNase Inhibitor (40 units/µl) was added and incubated at 42oC for 2 min. The mixture was added with 1 µl of SuperScriptTM III RT (200 units/µl) and incubated at 42oC for 50 min. Then the reaction was inactivated by heating at 70oC for 15 min. The first strand cDNA was used as template for PCR amplification. The open reading frame (ORF) of ACO reaction was comprised of 5 µl, 1µl and 1:10 µl of cDNA from first-stand reaction, 5 µl of 10X PCR buffer (10 mM Tris (pH 8.8), 50 mM KCl, 1.5 mM MgCl2 and 0.1% triton X-100), 1 µl of 10 mM of dNTPs, 30 pmol each of degenerate forward primer VFO1 (GTG AAT TCG CNT GYG ARA AYT GGG GHT T) and degenerate reward primer VFO2, 0.5 µl of Taq DNA polymerase (5 units/µl) and adjusted to a final volume of 50 µl with dH2O.The cycle of 17

PCR reaction was as followed; 1 cycle at 94oC for 3 min. Then 30 cycle at 94oC for 30 esc, 50oC for 1 min and 72oC for 1 min. and a final step at 72oC for 10 min.

3.3 Determination the 3’end of Cattleya-ACO

The 3’end of ACO genes was determined using RT-PCR with oligo-dT primer and 3’ end Cattleya-ACO specific primer (ATC GGA GAT CAG CTT GAG). Five hundred nanograms of total RNA from flower tissue were added to reverse transcription reaction containing 30 pmole of oligo-dT primer and 1 µl of formamide and 1µl of 10 mM dNTPs. The reaction was heated at 65oC for 5 min and quenched on -80oC absolute ethanol. The reverse transcription mixture was added 4 µl of 5X First-Strand Buffer (25 mM Tris-HCL TM (pH 8.3), 375 mM KCl, 15 mM MgCl2), 2 µl of 0.1 M DTT and 1 µl of RNaseOUT Recombinant RNase Inhibitor (40 units/µl) and incubated at 42oC for 2 min. The reaction was incubated at 42oC for 50 min. after adding 1 µl of SuperScriptTM III RT (200 units/µl). Then the reaction was inactivated by heating at 70oC for 15 min. The first strand cDNA was used as template for PCR amplification. The 3’end of ACO reaction was comprised of 5 µl and 1 µl of cDNA from first-stand reaction, plus 5 µl of 10X PCR buffer (10 mM

Tris (pH 8.8), 50 mM KCl, 1.5 mM MgCl2 and 0.1% triton X-100), 1 µl of 10 mM of dNTPs, 30 pmol each of oligo-dT and ACO specific primer 3’ end ,0.5 µl of Taq DNA polymerase

(5 units/µl) and adjusted to a final volume of 50 µl with dH2O.The cycle of PCR reaction was as followed; one cycle at 94oC for 3 min.,then 35 cycle at 94oC for 30 sec, 52oC for 30 sec and 72oC for 1.30 min and a final step at 72oC for 10 min.

3.4 Purification of PCR product

PCR reactions was adjusted to 100 µl with distilled water and 2.5 volume of 100% ethanol, 0.1 volume of 3 M sodium acetate and 1 µl of glycogen (20 mg/ml) were added. The mixture was incubated at –80oC for 15 min. The DNA was precipitated by centrifugation at 10000 rpm for 10 min. The pellet was washed with 70% ethanol to 18 air-dry at room temperature. The pellet was suspended in 10 µl of distilled water and stored at 4 oC.

3.5 Ligation of DNA Fragments

PCR products were ligated into pDrive vector using Qiagen®PCR Cloning kit essentially as described by the manufacturer. The reactions were performed in the 0.5 ml microcentrifuge tube. The reaction mixture contained 0.5 µl of pDrive Cloning vector (50ng/µl), 5 µl of 2X ligation Master Mix and 4.5 µl of purified PCR product in the total volume of 10 microliters. The reaction was incubated overnight at 16 oC and heated at 65oC for 10 min to inactivate the reaction. The ligation product was used directly in transformation reaction or stored at -20oC until use.

3.6 Transformation of Plasmid DNA

a. Preparation Competent cells Escherichia coli DH5α cells were streaked on Luria Bertani agar (LBA: 1% bacto- tryptone, 0.5% bacto-yeast extract, 1% NaCl and 1.5%bacto-agar) and incubate at 37oC overnight. A single colony was transferred into 10 ml of Luria Bertani (LB: 1% bacto- tryptone, 0.5% bacto-yeast extract, 1% NaCl) and incubated at 37 oC for 16 hr. One microliter from 10 ml overnight cuture was incubated in 250 ml of LB culture at 37oC to an OD600 of 0.2-0.4. The culture was transferred to a centrifuge tube and incubated on ice for 15 min. The cells were collected by centrifugation at 3,500 rpm for 15 min at 4oC. The supernatant was removed and the pellet cells were treated with 33 ml of RF1 solution (100mM KCl, 50 mM MnCl2.4H2O, 50mM CH3COOK,10 mM CaCl2, 15% Glycerol, pH5.8) and place on ice for 15 min. The cell were pelleted by centrifugation at 3,500 rpm for 15 min at 4oC and resuspended in 4 ml of RF2 solution (10 mM 3-(N-Morpholino)

Propane Sulfonic Acid, 10 mM KCl, 75 mM CaCl2.2H2O, 15% Glycerol, pH 6.8). The 100 µl aliquot of cell suspension was quickly transferred to each cool sterile eppendorf tube into liquid nitrogen and stored at -80oC. 19

b. Transformation

Four microliter of ligation mix were added to 100 µl aliquots of competent cells and the mixture was incubated on ice for 30 min. Cells were then heat shocked at 42oC for 90 sec and placed on ice for 2 min. The cells were resuspended in 800 µl of LB and incubated in a incubator at 37oC for 60 min. The culture was centrifuged at 8,000 rpm. for 1 min and the pellet cells were resuspended in 200 µl of LB. The suspenstion cell were plated on LB agar containing 29 µg/ml isopropyl β-D-thiogalactopyranoside (IPTG), 0.06% 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and ampicillin (50 µg/ml) and incubated at 37oC for 16 hr. Recombinant clones were identified by blue/white selection and used to incubate overnight LB cultures containing ampicillin (50 µg/ml) or using for colony hybridization.

3.7 Colony Hybridization

a. Preparing Colony

White colonies were selected to two new LBA, master plate and replica plate, and incubated at 37oC overnight. Master plate was retained for reference set of colonies, and replica plate was pre-cooled for 30 min at 4oC. The Nylon membrane disc was placed onto the surface of pre-cooled plate for 2 min. The membrane disc with the colonies side facing up was removed to new empty plate. Then denaturation solution I (0.5 M NaOH, 1.5 M NaCl) was added, placed for 15 min and removed on a dry sheet of Whatman 3M paper. The membrane disc was removed to new plate containing Neutralization solution (1.5 M NaCl, 1.0 M Tris-HCl) and placed for 15 min and removed on a dry sheet of Whatman 3M paper before removing to a new plate containing 2X SSC (0.3M NaCl, 3mM Sodium citrate, pH 7.0), placed for 10 min and allowed to dry on a sheet of Whatman 3M paper. While the membrane disc is drying, the positive control (Cattleya ACO plasmid) was spotted on new membrane size 3x3 cm. Then the membranes were baked at 80oC for 30 min. 20

b. Hybridization a DIG-labeled DNA Probe to the Colony Lifts

The membrane was prehybridized in 15 ml of Standard hybridization buffer (5X SSC, 0.1% N-lauroylsarcosine, 0.02% SDS, 1% blocking reagent) at 60oC for at least 60 min., following by hybridization with ACC oxidase probes, were denatured by boiling for 10 min. and quenched on ice for 5 min. before used. The hybridization was carried out overnight at 60oC. The membrane were washed with 2X washing solution (2X SSC, 0.1% SDS, pH 7.0) at RT for 15 min, and twice with 0.5X washing solution (0.5X SSC, 0.1% SDS, pH 7.0) at 65oC for 15 min, respectively before the detection.

c. Detection Probe-Target Hybrids with Chemiluminescence

The membrane was agitated in 10 ml of 1X blocking solution for 30 min and incubated in 10 µl of antibody solution (Anti-Digoxigenin-AP was diluted in 1X blocking solution 1:10,000) for 30 min. The membrane was washed twice with washing buffer for 15 min at RT and equilibrated with Detection buffer for 5 min. following by incubation in chemiluminescent substrate CDP-StarTM (Roche) diluted in detection buffer 1:200 for 15 min at room temperature. The membrane was placed in a plastic bag and sealed. Then the membrane was exposed to the X-ray film for detection of the chemiluminescent signal. Colonies were identified by comparison the signal with positive control and the positive colonies were grown incubated overnight at 37oC in 3 ml of LB containing ampicillin (50 µg/ml).

3.8 Small Scale Isolation of Plasmid DNA, Mini-Prep

Plasmid DNA was extracted by a modification method of Sambrook et al.(1989). A recombinant clone was grown overnight at 37oC with vigorous shaking in 3 ml of LB containing 50 µg/ml of ampicillin. Cell suspension with volume of 1.5 ml was centrifuged for 30 sec at 12,500 rpm. The pellet was resuspended in 100 µl of chilled solution I (50 mM glucose, 10 mM EDTA and 25 mM tris-HCl, pH 8.0) and mixed vigorously. Two 21 hundred microliters of freshly prepared solution II (0.2 M NaOH, 1% SDS) was added and mixed gently by inverting 2-3 times. Then the solution was neutralized by addition of 150 µl of ice-cold solution III (3M potassium acetate, 5M glacial acetic acid). The contents was extracted with equal volume of chloroform and centrifuged at 12,000 rpm for 10 min at 4oC. The supernatant was transferred to a new tube and precipitated with 2 volume of ice-cold 100% ethanol, incubated at -20oC for 10 min and centrifuged at 12,000 rpm for 10 min at 4oC. The pellet was washed with 70% ethanol, allowed to dry at room temperature. The pellet was resuspended in 20 µl of distilled water containing 10 µg/ml RNase A and incubated at RT for 30 min. The Plasmids DNA were identified with specific ACC oxidase primer for the PCR amplification.

3.9 Nucleotide Sequences of Cattleya-ACO cDNA was determined using DNA sequence (Applied Biosystem)

The Clones were identified and was grown overnight at 37oC with vigorous shaking in 3 ml of LB containing ampicillin (50 µg/ml). After The Clones were used for determined nucleotide sequences by using a DNA automates sequencer.

3.10 Sequence analysis

a. Nucleotides sequence analysis

The programs EditSeq was used to determined ORFs and codon usage. Genbank nucleotides databases were search for sequences having homology with ACO sequences using BLASTN program (NCBI). Comparisons of all nucleotide sequences were done using the ClustalW (EBI) and MegAlign program (Lasergene).

b. Amino acid sequences analysis

The program EditSeq was used to change nucleotides to amino acid sequences. Genbank amino acid databases were search for sequences having homology with ACO sequences using BLASTN program (NCBI). Comparisons of all amino acid sequences were done using the ClustalW (EBI) and MegAlign program (Lasergene).

4. Identification of functional domains in ACO putative proteins

Amino acid sequence of Amino acid sequence of isolated Cattleya ACC oxidase ACC oxidase from (Lucky strike variety) Genbank database

Alignment

Searching for functional domains

Figure 5. Flow diagram outlining the strategy for identification of functional domains in Cattleya-ACC oxidase putative proteins.

The functional domains of ACC oxidase have been reported including ACC binding domain (Dilley et al., 2001), ferrous ion binding domain (Rocklin et al., 1999), bicarbonate binding domain (Dilley et al., 2001), ascorbate binding domain (Lukacin et al., 1999), leucine zipper and basic zipper domain (Liu et al., 1999). The amino acid sequences of isolated Cattleya-ACO (Lucky strike variety) were compared with ACOs amino acid sequences of other plant from Genbank database using MegAlign program.

Figure 6. Strategy for cloning of Cattleya- ACO. (A) Open Reading Frame, (B) 3’ Untranslated region (UTR) and (C) identified Cattleya-ACO size 1081 bp.

Result and Discussion

1. Cloning, Sequencing and Characterization of a Cattleya-ACO

A Cattleya-ACO was cloned and sequenced from flower Cattleya orchid Lucky Strike variety using RT-PCR and 3’RACE techniques. Total RNA was purified from Cattleya flower (Figure 7). The presence of genomic DNA was later removed by DNase treatment. The RT-PCR products using ACO degenerate primers were shown in Figure 8. The major band that had expected size of 800 bp. was cloned into pDrive vector (Appendix Figure 2). The recombinant clones were identified by amplifying the recombinant plasmids with M13F and M13R primers (Figure 9). The 3’UTR was amplified using RT-PCR with oligo-dT and 3’end Cattleya-ACO specific primer (Figure 10). The recombinant clones were screened using colony hybridization with DIG labeling Cattleya ACO probe (Figure 11). The plasmid from selected clone was amplified with VFO2 and 3’end Cattleya-ACO specific primer to confirm the authenticity (Figure 12). The Cattleya- ACO cDNA sequence, missing at 5’end, was 1,081 bp. in length (Figure 13) contain an open reading frame which encoded 299 amino acid and 179 nucleotide of 3’ UTR region were. The comparison between Cattleya spp. was done at both nucleotide and amino acid sequences. The nucleotide sequence (ORF and 3’UTR) alignment between Cattleya spp. Lucky Strike variety and Cattleya bicolor indicated that the identity was 89.4% while the identity between Cattleya spp. Lucky Strike variety and Cattleya intermedia was 93.2%. The 3’UTR nucleotide sequence alignment indicated that Cattleya Bicolor and Cattleya spp. Lucky Strike variety was at 88.6% identity. The 3’UTR nucleotide sequence alignment indicated that Cattleya Intermedia and Cattleya spp. Lucky Strike variety was at 85% identity (Table 1.). The significantly difference between Cattleya spp. located at 3’UTR and deletion 8 bp of nucleotide at 3’UTR (TTAATTAG) was observed (Figure 14.). While the amino acid sequence alignment indicated high percent identity between Cattleya spp. at amino acid level were 96.7% (Table 2.). The phylogenetic tree of ACO nucleotide and amino acid sequences from Cattleya spp. Lucky Strike variety between other plants indicated that ACO gene of Cattleya spp. Lucky Strike variety has the closer 25 relationship with Cattleya Bicolor and Cattleya Intermedia, respectively. Plant ACO genes were distinguished into two clusters of monocot and dicot plants. (Figure 15. and Figure 16.).

Figure 7. Ethidium bromide stained 1% agarose gel of Cattleya flower total RNA following an electrophoresis at 60 V for 1 hr.

Figure 8. Ethidium bromide stained 1% agarose gel of open reading frame Cattleya-ACO genes following an electrophoresis at 60 Volt for 1 hr. Lane 1; PCR product size 800 bp.

1000 bp 1000 bp

Figure 9. Identification the recombinant clones by amplifying the recombinant plasmids with M13F and M13R primers. Products sizes are 1000 bp.

Figure 10. Ethidium bromide stained 1% agarose gel of 3’untranslated region Cattleya- ACO genes following an electrophoresis at 60 Volt for 1 hr. RT-PCR was carried out using 500 ng of total RNA sample with 3’end Cattleya-ACO specific primer and oligo-dT. Lane 1-2; PCR product size 426 bp.

Selected colony

Control

Figure 11. Colony hybridization analysis of clone 3’UTR Cattleya-ACOs. Using ORF Cattleya-ACOs as probes. The positive clones were shown in circular. .

500 bp 250 bp 229 bp

Figure 12. Identification the recombinant clones by amplifying the recombinant plasmids with with 3’ end Cattleya-ACO specific primer and VFO2. Products size is 229 bp.

1 GTGAATTCGC TTGCGAGAAC TGGGGATTCT TCGAGCTACT GAACCACGGT ATCTCCACAG E F A C E N W G F F E L L N H G I S T 61 AGCTGATGAA CCGAGTCGAA GCCGTGAACA AAGAAAATTA CCGGCGATTC CGCGAGCAGC E L M N R V E A V N K E N Y R R F R E Q 121 GGTTCAAAGA ATTCGCCGCC AAAACCCTTG ATTCCGGCGA GAAGGTCGAC GGCGATAATC R F K E F A A K T L D S G E K V D G D N 181 TGGACTGGGA AAGCACCTTC TTCCTCCGCC ATCTTCCGAA CTCCAACATC TCCCAAATCC L D W E S T F F L R H L P N S N I S Q I 241 CGGATCTGGA CGAGGACTGC CGGAGCACGA TGAAGGAGTT CGCCCGAGAG CTGGAGAAAC P D L D E D C R S T M K E F A R E L E K 301 TGGCGGAGTG GTTGCTGGAT CTGTTGTGCG AGGATTTGGG GCTTGAGAAA GGGTATTTGA L A E W L L D L L C E D L G L E K G Y L 361 AGAGGGTTTT TTGCGGGGGA TCGGATGGAT TGCCGACGTT CGGGACGAAG GTTAGCAATT K R V F C G G S D G L P T F G T K V S N 421 ATCCGCCGTG TCCGAAGCCG GAATTGATAA AGGGGCTGAG AGCTCATACG GATGCGGGAG Y P P C P K P E L I K G L R A H T D A G 481 GGATTATTCT ACTGTTTCAG GACGATAAGG TCAGCGGGCT TCAGCTGCTC AAGGATGGCC G I I L L F Q D D K V S G L Q L L K D G 541 AGTGGATTGA TGTTCCCCCG CTGCGCCATT CCATTGTTGT CAATATCGGA GATCAGCTTG Q W I D V P P L R H S I V V N I G D Q L 601 AGGTGATAAC AAATGGAAAA TACAAGAGTG TGATGCACAG GGTGGTCGCC CAAACCGACG E V I T N G K Y K S V M H R V V A Q T D 661 GCAACCGCAT GTCCATCGCC TCGTTCTACA ACCCCGGCAG CGACGCCGTC ATCTCCCCGG G N R M S I A S F Y N P G S D A V I S P 721 CGCCGGAGCT GGTGGAGAAA GAGGCGGAGG AGGAGAAGAA GGAAACAACT TATCCAAAAT A P E L V E K E A E E E K K E T T Y P K 781 TTGTGTTTCA GGACTACATG AATCTGTATA TTCGCCAGAA ATTTGAGGCT AAGCAGCCGA F V F Q D Y M N L Y I R Q K F E A K Q P 841 GGTTTGAGGC TATGAAGACC ATGGAAACTG TTTCCGGCTC ACAGCTCATT CCTACTGCTT R F E A M K T M E T V S G S Q L I P T A 901 AATTAATTAA TTAATTAGTA GAACATTTTA GTTCATATGG TTTAATTTAT GTGTTGCTGT . 961 TTGTTAGGGT CTAGTGGTTT GATTTGAACA ATTATAATGT GTATTTATCT TGTGTATGTT

1021TGTACCCTAT ATTTATCTAT ATATTATCTT TGGATGGTAA GGTATTAAAAAAAAAAAAAAA

Figure 13. Nucleotide sequence of Cattleya ACC oxidase cDNA and the deduced amino acid sequence. The translation start site is underlined. The asterisk denotes the stop codon.

Figure 14. The sequence comparison of Cattleya spp. Major difference located at 3’UTR and deletion 8 nucleotide are TTAATTAG (respectived by box).

(A) open reading frame and 3’untranslated region

Bicolor Intermedia Lucky Bicolor ***** 95.2 89.4 Intermedia ***** ***** 93.2 Lucky ***** ***** *****

(B) 3’untranslated region

Bicolor Intermedia Lucky Bicolor ***** 83.2 88.6 Intermedia ***** ***** 85 Lucky ***** ***** *****

Table1. The nucleotie sequence alignment indicated percent Identity between Cattleya spp. using MegAlign program (Lasergene). (A) In open reading frame and 3’untranslated region part, (B) In 3’untranslated region part.

Bicolor Intermedia Lucky Bicolor ***** 97.7 96.7 Intermedia ***** ***** 96.7 Lucky ***** ***** *****

Table 2. The amino acid sequence alignment indicated Percent Identity between Cattleya spp. using MegAlign program (Lasergene).

Figure 15. Phylogenetic tree of ACO nucleotide sequences from Cattleya spp. Lucky Strike variety and other plants. Cluster analysis was done using the MegAlign program (Lasergene). Cattleya intermedia accession : AY598794, Cattleya bicolor accession : AY598793, Laelia anceps accession : AY598795, Phalaenopsis sp. “True Lady” accession : AF004662, Dendrobium crumenatum accession : AF038840, x Doritaenopsis sp. accession : L07912, Oryza sativa accession : AK058296, Phyllostachys edulis accession : AB044747, Carica papaya accession : L76283, Carica papaya ripening- induduced ACO accession : AY077461, Musa acuminate accession : X95599

Figure 16. Phylogenetic tree of ACO amino acid sequences from Cattleya Lucky Strike variety and other plants. Cluster analysis was done using the MegAlign program (Lasergene). Cattleya intermedia accession:AY598794, Cattleya bicolor accession : AY598793, Laelia anceps accession : AY598795, Phalaenopsis sp. “True Lady” accession : AF004662, Dendrobium crumenatum accession : AF038840, x Doritaenopsis sp. accession : L07912, Oryza sativa accession : AK058296, Phyllostachys edulis accession : AB044747, Carica papaya accession : L76283, Carica papaya ripening- induduced ACO accession : AY077461, Musa acuminate accession : X9559

2. Identification of Functional Domains in ACO Putative Proteins

ACC oxidase is a member of the non-heme iron and ascorbate dependent oxidase family. The functional domains presented in other plant ACC oxidases and members of the non-heme iron and ascorbate dependent oxidase family including ACC binding domain, ascorbate binding domain, ferrous binding domain, bicarbonate binding domain, leucine zipper and basic zipper domains were determined. ACC is a substrate of all ACOs. Tyrosine, Argenine and Serine which found to be in ACC binding domain through mutagenesis (Dilley et al., 2001) also presented in Cattleya-ACO Lucky Strike variety (Figure 17A). Ascorbate is a cofactor for ACC oxidase activity. Ascorbate binding domain (Arg-X-Ser) is conserved in all ACOs and found in Cattleya-ACO Lucky Strike variety when X is represented by methionine (Figure 17B). Basic zipper (bZIP) domain facilitates oligomerization (Liu et al., 1999). The bZIP domain (consensus sequence, E..L..L..N..L) is found in all ACOs and present in Cattleya-ACO Lucky Strike variety (Figure 18A). Leucine zipper domain is facilitates oligomerization (Liu et al., 1999). The 4 haptad repeats of leucine in Leucine zipper domain are found in all ACOs. The 4 haptad repeats of leucine were also found in Cattleya-ACO Lucky Strike variety (Figure 18B). Ferrus ion is a cofactor for ACC oxidase activity. Fe2+ binding domain is conserved in all ACOs with consensus sequences of HXD/E…H (Rocklin et al., 1999). This conserved region is found at position histidine, threonine, aspatic acid and histidine in Cattleya-ACO Lucky Strike variety when X is represented by threonine (T) and aspartic acid (D) is found in all ACOs (Figure 19.). Bicarbonate is an active form of carbondioxide that activates ACC oxidase activity. Bicarbonate binding domain found through mutagenesis experiment located at argenine 175 (Dilley et al., 2001) is conserved in all ACOs also presented in Cattleya-ACO Lucky Strike variety (Figure 19).

(A) ACC binding domain

(B) Ascorbate binding domain

Figure 17. Alignment of amino acid sequences in ACOs. (A) Conserved Thr, Arg and Ser in ACC binding domain are respectived by box. (B) Consensus sequence of RXS (R = arginine, X = methionine, S = serine) in ascorbate binding domain are respectived by box.

(A) Basic zipper (bZIP) domain

(B) Leucine zipper domain

Figure 18. Alignment of amino acid sequence in ACOs. (A) Consensus sequence of Glu…Leu…Leu…Asn…Leu (E…L…L…N…L) in basic zipper (bZIP) domain are respectived by box. (B) The 4 haptad repeats of leucine in leucine zipper domain are respectived by box.

Figure 19. Alignment of amino acid sequences in ACOs. Consensus sequence of His-X-Asp/Glu…His (HXD/E…H) in Fe2+ binding domains are respectived by box. The X is represented by threonine (T) and aspartic acid (D) is found in all ACOs. The Arg in the bicarbonate binding domain is respectived by box.

CONCLUSIONS

In this study, the cloning, sequencing and characterizing of ACC oxidase gene in Cattleya spp. variety Lucky Strike flower was investigated. The results could be summarized as followed: 1. Partial sequence of Cattleya Lucky Strike ACO was identified (1081 bp) and characterize. 2. A major difference between Cattleya spp. was located at 3’UTR resulting in shift of stop condon. 3. Amino acid sequence comparison indicated a close relationship between ACOs from three Cattleya spp. 4. Functional domains of ACOs were also found in Cattleya ACOs.

This knowledge could be applied to other varieties of Cattleya or other orchid flowers for ACO identification. It also indicated that there might be certain differences in the ethylene production/regulation due to a distinct variation of the gene sequences at 3’UTR.

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Appendix

Appendix figure1. Disection of Cattleya flower. Source : http://www.nihongo.com/matsuura/Cattleya/Kakubu.htm.

Appendix Figure 2. pDrive Cloning Vector Source : http://www1.qiagen.com/literature/images/pDrive_cloning_vector

53 species of Cattleya from Mexico to South America

- Cattleya aclandiae : Lady Ackland's Cattleya (Brazil) - Cattleya amethystoglossa : Amethyst-lipped Cattleya (Brazil) - Cattleya araguaiensis : Araguaia Cattleya (Brazil) - Cattleya aurantiaca : Orange Cattleya (Mexico to C. America). - Cattleya bicolor : Bicolored Cattleya (SE. Brazil) - Cattleya bicolor subsp. bicolor (Brazil). Pseudobulb epiphyte - Cattleya bicolor subsp. canastrensis (Brazil) . Pseudobulb epiphyte - Cattleya bicolor subsp. minasgairensis (Brazil). Pseudobulb epiphyte - Cattleya boissieri Colombia. - Cattleya bowringiana : Bowring's Cattleya (Mexico to Honduras). - Cattleya × brasiliensis (= C. bicolor × C. harrisoniana) (Brazil) . - Cattleya × brymeriana (= C. violacea × C. wallisii) (N. Brazil). - Cattleya candida (Colombia). - Cattleya × colnagiana (Brazil). - Cattleya × dayana (= C. forbesii × C. guttata) (Brazil). - Cattleya × dolosa (= C. loddigesii × C. walkeriana): Dolose Cattleya, Crafty Cattleya, Deceitful Cattleya (Brazil). - Cattleya dormaniana : Dorman's Cattleya (Brazil) - Cattleya dowiana : Queen of the Cattleyas, Dow's Cattleya (Costa Rica to Colombia). - Cattleya dowiana var. aurea : Golden-yellow Cattleya (S. Panama to Colombia). Pseudobulb epiphyte - Cattleya dowiana var. dowiana (Costa Rica). Pseudobulb epiphyte - Cattleya × dukeana (C. bicolor × C. guttata) (SE. Brazil). - Cattleya dupontii (Brazil). - Cattleya × duveenii ( = C. guttata × C. harrisoniana) (SE. Brazil). - Cattleya elegantissima (Venezuela). - Cattleya elongata : Cattleya with the Elongated Stalk (Brazil) - Cattleya forbesii : Forbes' Cattleya (Brazil) 50

- Cattleya gaskelliana : Gaskell's Cattleya (Colombia to Trinidad). - Cattleya × gransabanensis (= C. jenmanii × C. lawrenceana) (Venezuela). - Cattleya granulosa : Granulose Cattleya (Brazil) - Cattleya × guatemalensis (= C. aurantiaca × C. skinneri.) : Guatemalan Cattleya (SE. Mexico to C. America). National flower of Guatemala - Cattleya guttata : Spotted Cattleya (Brazil). - Cattleya × hardyana ( = C. dowiana var.aurea × C. warscewiczii): Hardy's Cattleya (Colombia). - Cattleya harrisoniana : Harrison's Cattleya (SE. Brazil). - Cattleya herbacea (NE. Argentina). - Cattleya × hybrida (= C. guttata × C. loddigesii) (SE. Brazil). - Cattleya × imperator ( = C. granulata × C. labiata) (NE. Brazil). - Cattleya intermedia : Intermediate Cattleya (SE. & S. Brazil, Paraguay, Uruguay). - Cattleya × intricata (=. C. intermedia × C. leopoldii) (S. Brazil). - Cattleya iricolor : Rainbow-colored Cattleya (Ecuador to Peru). - Cattleya × isabella (.= C. forbesii × C. intermedia) (SE. Brazil). - Cattleya × itatiayae (SE. Brazil). - Cattleya jenmanii : Jenman's Cattleya (Venezuela to Guyana). - Cattleya × joaquiniana ( = C. bicolor × C. walkeriana) (Brazil) . - Cattleya × kautskyi (= C. harrisoniana × C.) (SE. Brazil). - Cattleya kerrii : Kerr's Cattleya (Brazil). - Cattleya labiata : Crimson Cattleya, Ruby-lipped Cattleya (Brazil) - Cattleya lawrenceana : Sir Trevor Lawrence's Cattleya (Venezuela, Guyana, N. Brazil). - Cattleya loddigesii : Loddiges' Cattleya (SE. Brazil to NE. Argentina). - Cattleya loddigesii subsp. loddigesii (SE. Brazil to NE. Argentina). Pseudobulb epiphyte - Cattleya loddigesii subsp. purpurea (Brazil). Pseudobulb epiphyte - Cattleya × lucieniana ( = C. forbesii × C. granulosa) (SE. Brazil). - Cattleya lueddemanniana : Lueddemann's Cattleya (N. Venezuela). 51

- Cattleya luteola : Pale-yellow Cattleya (N. Brazil, Ecuador to Bolivia). - Cattleya maxima : Greatest Cattleya, Christmas Flower (Venezuela to Peru). - Cattleya × measuresii ( = C. aclandiae × C. walkeriana) (E. Brazil). - Cattleya mendelii : Mendel's Cattleya (NE. Colombia). - Cattleya × mesquitae ( = C. nobilior × C. walkeriana) (Brazil). - Cattleya × mixta ( = C. guttata × C. schofieldiana) (Brazil). - Cattleya × moduloi (C. schofieldiana × C. warneri) (Brazil). - Cattleya mooreana : Moore's Cattleya (Peru). - Cattleya mossiae : Easter Orchid, Mrs. Moss' Cattleya (N. Venezuela) - Cattleya motae (Brazil). - Cattleya nobilior : Noble Cattleya (WC. Brazil to Bolivia). - Cattleya patinii : Patin's Cattleya (Costa Rica to Venezuela, Trinidad). - Cattleya × patrocinii (= C. guttata × C. warneriana): Patrocinio's Cattleya (SE. Brazil). - Cattleya percivaliana : Christmas orchid, Percival's Cattleya (Colombia to W. Venezuela). - Cattleya × picturata ( = C. guttata × C. intermedia) (SE. Brazil). - Cattleya porphyroglossa : Purple-lipped Cattleya (Brazil). - Cattleya × resplendens ( = C. granulosa × C. schilleriana) (NE. Brazil) - Cattleya rex : King of the Cattleyas (Colombia to N. Peru). - Cattleya schilleriana : Consul Schiller's Cattleya (Brazil). - Cattleya schofieldiana : Schofield's Cattleya (Brazil) - Cattleya schroderae : Easter Orchid, Baroness Schroder's Cattleya (NE. Colombia). - Cattleya × scita (= C. intermedia × C. tigrina) (S. Brazil). - Cattleya skinneri : Flower of San Sebastian, Skinner's Cattleya (SE. Mexico to C. America). - Cattleya storeyi (Windward Is.) (Barbados.) - Cattleya × tenuata (= C. elongata × C. tenuis) (Brazil) . - Cattleya tenuis : Slender-stemmed Cattleya (NE. Brazil). - Cattleya tigrina (SE. & S. Brazil). 52

- Cattleya trianae : Dr. Triana's Cattleya (Colombia). - Cattleya × undulata ( = C. elongata × C. schilleriana) (Brazil). - Cattleya velutina : Velvety Cattleya (Brazil) - Cattleya × venosa (= C. forbesii × C. harrisoniana) (Brazil). - Cattleya × victoria-regina ( C. guttata × C. labiata) (NE. Brazil). - Cattleya violacea : Superba of the Orinoco, Violet Cattleya (S. Trop. America). - Cattleya walkeriana : Walker's Cattleya (WC. & SE. Brazil). - Cattleya wallisii (N. Brazil). - Cattleya warneri : Warner's Cattleya (E. Brazil). - Cattleya warscewiczii : Warscewicz's Cattleya (Colombia). - Cattleya whitei (Brazil) - Cattleya × wilsoniana ( = C. bicolor × C. intermedia). (Brazil).

Source : http://en.wikipedia.org/wiki/Cattleya

Appendix Table 1. Abbreviations of amino acids

Amino acid Three-letter abbreviation One-letter symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Appendix Table 2. The genetic code

First position Third position (5’end) Second position (3’end) U C A G Phe Ser Tyr Cys U U Phe Ser Tyr Cys C Leu Ser Stop Stop A Leu Ser Stop Trp G Leu Pro His Arg U C Leu Pro His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg G Ile Thr Asn Ser U A Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr Lys Arg G Val Ala Asp Gly U G Val Ala Asp Gly C Val Ala Glu Gly A Val Ala Glu Gly G