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Studies of wax ester production and biochemical characterization of jojoba-type wax synthase in Arabidopsis… Liang, Wei-Wan Scott 2011

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STUDIES OF WAX ESTER PRODUCTION AND BIOCHEMICAL CHARACTERIZATION OF JOJOBA-TYPE WAX SYNTHASE IN Arabidopsis thaliana    by Wei-Wan Scott Liang B.Sc., Carleton University, 2007 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies (Botany) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2011  © Wei-Wan Scott Liang, 2011 	
   ii	
   Abstract  Wax esters are components of surface lipids, serving as surface protectants for both terrestrial plants and animals. Wax esters are also specialized energy storage reserves for the organisms living in extreme environments, such as marine animals in Arctic and Antarctic oceans, the jojoba plant in the desert, and Acinetobacter species in the soil. Wax esters are also important substrates for industrial applications, such as the production of biodiesel, lubricants, cosmetics and polishes. Our current sources and production methods of wax esters from living organisms are not sufficient to meet market demands, so alternative sources including engineered oil crops that can generate sufficient amounts of wax esters are being sought. For my MSc project, I 1) investigated the feasibility of producing high levels of wax esters in the seed of Arabidopsis thaliana; 2) attempted to biochemically characterize new wax ester synthases that share amino acid similarity to the jojoba wax ester synthase; and 3) studied the promoter activity of the wax ester synthase encoded by the At5g55330 gene in Arabidopsis thaliana by a GUS assay. The first objective has been achieved, and the jojoba-type wax esters accumulated when the jojoba wax ester biosynthetic pathway was introduced in the seeds of Arabidopsis. I found that the enzyme encoded by the At5g55330 gene has wax ester synthase activity, but I was unable to characterize its substrate specificity. The third objective is still in progress, but my preliminary results to date indicate that the At5g55330 gene is transcribed in specific tissues of flowers, leaves, stem, and siliques. 	
   iii	
   Table of contents Abstract…...……………………………………………………………………………………...ii Table of contents...……...……………………………………………………………………….iii List of tables……..……………………………………………………………………………….v List of figures……..……………………………………………………………………………..vi 1	
   Introduction ......................................................................................................................................1	
   1.1	
   Wax esters.................................................................................................................................................1	
   1.2	
   The distribution of wax esters and their function in living organisms........................................1	
   1.2.1	
   Land plants ...........................................................................................................................................................1	
   1.2.2	
   Land animals .......................................................................................................................................................2	
   1.2.3	
   Marine animals ...................................................................................................................................................3	
   1.2.4	
   Bacteria..................................................................................................................................................................5	
   1.2.5	
   Summary of the distributions and functions of wax esters in all living organisms .....................5	
   1.3	
   Wax ester biosynthesis ...........................................................................................................................6	
   1.4	
   Studies of wax ester synthases (WS) ...................................................................................................7	
   1.4.1	
   Mammalian-type WS........................................................................................................................................7	
   1.4.2	
   Bacterial-type WS..............................................................................................................................................9	
   1.4.3	
   Jojoba-type WS................................................................................................................................................13	
   1.5	
   The current industrial production of wax esters and the possible improvements by biotechnology .................................................................................................................................................. 16	
   1.5.1	
   Applications of wax esters ...........................................................................................................................16	
   1.5.2	
   Bottlenecks in wax ester production.........................................................................................................16	
   1.5.3	
   Overcoming the production bottlenecks by engineering wax ester biosynthetic pathways in transgenic oil crops........................................................................................................................................................17	
   1.6	
   MSc project objectives ........................................................................................................................ 17	
   2	
   Methods and materials ................................................................................................................ 19	
   2.1	
   Preparation of DNA for molecular cloning .................................................................................... 19	
   2.1.1	
   Isolation of genomic DNA from Arabidopsis thaliana leaves ........................................................19	
   2.1.2	
   Preparation of cDNA from Arabidopsis thaliana siliques ................................................................19	
   2.2	
   Generation of bacterial, yeast, and plant expression constructs ............................................... 19	
   2.2.1	
   Design of bacterial, yeast, and plant expression constructs ..............................................................19	
   2.2.2	
   Preparation of E. coli competent cells, transformation, and routine growth ...............................21	
   2.2.3	
   Amplification of DNA fragments by polymerase chain reaction (PCR)......................................22	
   2.2.4	
   Plasmid purification, DNA gel purification, and PCR product purification ...............................26	
   2.3	
   Heterologous protein expression in Escherichia coli .................................................................... 26	
   2.3.1	
   Bacterial growth condition and protein extraction...............................................................................26	
   2.3.2	
   Heterologous protein expression assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblotting and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB).. ...........................................................................................................................................................................27	
   2.4	
   Heterologous protein expression in Saccharomyces cerevisae .................................................... 28	
   2.4.1	
   Yeast growth conditions, introduction of the expression plasmid, and induction of the heterologously expressed proteins............................................................................................................................28	
   2.4.2	
   Yeast lipid analysis.........................................................................................................................................29	
   2.5	
   Analysis of transgenic plants ............................................................................................................. 29	
   2.5.1	
   Transformation of Agrobacterium tumefaciens and Arabidopsis thaliana and the screening of the transgenic plants.................................................................................................................................................29	
   2.5.2	
   Plant growth conditions ................................................................................................................................30	
   2.5.3	
   Transgenic seed wax ester analysis...........................................................................................................31	
   	
   iv	
   2.5.4	
   Identification and quantification of the jojoba-type wax ester produced by transgenic seeds…...............................................................................................................................................................................31	
   2.5.5	
   Gene expression analysis by the β-glucuronidase (GUS) gene reporter system .......................32	
   3	
   Jojoba-type wax ester production in transgenic Arabidopsis thaliana seeds expressing the jojoba KCS, FAR, and WS......................................................................................................... 33	
   3.1	
   Introduction .......................................................................................................................................... 33	
   3.2	
   Results and discussion......................................................................................................................... 33	
   3.2.1	
   A. thaliana transformation and identification of transgenic seeds .................................................33	
   3.2.2	
   TLC analysis of T2 transgenic seeds.........................................................................................................35	
   3.2.3	
   GC-FID quantification ..................................................................................................................................36	
   3.2.4	
   Identification of unsaturated wax esters by GC-MS ...........................................................................37	
   3.2.5	
   Determination of isomer composition of unsaturated wax esters by GC-MS ............................41	
   4	
   Enzymatic activity of the Arabidopsis thaliana jojoba-type WS homologues .................. 43	
   4.1	
   Introduction .......................................................................................................................................... 43	
   4.2	
   Results and discussion......................................................................................................................... 44	
   4.2.1	
   Bacterial expression .......................................................................................................................................44	
   4.2.2	
   S. cerevisiae expression ................................................................................................................................48	
   5	
   Arabidopsis thaliana WS7 expression patterns ....................................................................... 55	
   5.1	
   Introduction .......................................................................................................................................... 55	
   5.2	
   Results and discussion......................................................................................................................... 55	
   6	
   Conclusions and future directions............................................................................................. 58	
   References..…...………………………………………………………………………………………………………..60	
   Appendix…………………………………………………………………………………………………………………71 	
   v	
   List of tables Table	
  1:	
  Summary	
  table	
  of	
  steady	
  state	
  transcripts	
  of	
  the	
  jojoba-­‐type	
  WS	
  homologues	
  detected	
  by	
  RT-­‐PCR	
  in	
  different	
  organs	
  of	
  Arabidopsis	
  thaliana	
  (Klypina	
  and	
  Hanson,	
  2008).. ...............................................................................................................................................................15	
   Table 2: Temperature program for cloning PCR of the jojoba WS, Arabidopsis thaliana jojoba- type WS homologues, and the WS7 promoter. ....................................................................................23	
   Table 3: The primers used to amplify the jojoba WS and Arabidopsis thaliana jojoba-type WS homologues coding sequences for the construction of fusion MBP protein in bacterial expression pMAL-c2x constructs (New England BioScience). .....................................................24	
   Table 4: The primers used to amplify the jojoba WS and Arabidopsis thaliana jojoba-type WS homologues coding sequences for heterologous protein expression in yeast using yeast expression vector pESC-URA (Stratagene)..........................................................................................25	
   Table 5: Different wax esters and the signature ions used for isomer composition determination..............................................................................................................................................................................41	
   Table 6: Summary table of isomer compositions of the observed long chain unsaturated wax esters in the transgenic line FWS3-UB4 ................................................................................................42	
   	
   vi	
   List of figures Figure 1: General chemical structure of wax esters. .......................................................................................1	
  Figure	
  2:	
  The pMAL-c2x bacterial expression constructs........................................................................20	
   Figure 3: The pESC-URA yeast expression constructs..............................................................................21	
   Figure 4: Plant expression construct pCAMBIA1381-WS7P:GUS. ......................................................21	
   Figure 5: The plant wax ester expression constructs pBinGlyRed-FWS1 (FWS1) and pBinGlyRed-FWS3 (FWS3) generated by Dr. Edgar Cahoon (University of Nebraska – Lincoln).............................................................................................................................................................34	
   Figure 6: DsRed-positive transgenic seeds of A. thaliana carrying pBinGlyRed empty vector (V), pBinGlyRed-FWS1 (FWS1) or pBinGlyRed-FWS3 (FWS3)........................................................35	
   Figure 7: TLC analysis of wax ester accumulation in the seeds of Columbia wild type (ColWt), transgenic Arabidopsis containing pBinGlyRed empty vector (VB1, VB4, and VB12), pBinGlyRed-FWS1 (FWS1-B12, B13, B20, B26, B27, and S3), and pBinGlyRed-FWS3 (FWS3-UB4, UB5, UB6, UB7, UB10, UB14, US1).........................................................................36	
   Figure 8: The GC-FID analysis of wax ester accumulation in 100 seeds of Columbia wild type (ColWt), transgenic Arabidopsis containing pBinGlyRed (VB1, VB4, and VB12), pBinGlyRed-FWS1 (FWS1-B12, B13, B20, B26, B27, and S3), and pBinGlyRed-FWS3 (FWS3-UB4, UB5, UB6, UB7, UB10, UB14, US1).........................................................................37	
   Figure 9: Mass spectrum of the standard 9-hexadecenoic, 9-hexadecenyl ester from NISTO2 (a) and the presumed C42:2 wax ester from the transgenic T2 line FWS3-UB4 (b). ....................39	
   Figure 10: Mass spectrum of the standard palmitic acid, 9-hexadecenyl ester (a), 9-hexadecenoic acid, hexadecanyl ester from NISTO2 (b), and the presumed C42:1 wax ester from the transgenic T2 line FWS3-UB4 (c). ...........................................................................................................40	
   Figure 11: Western blot of the soluble protein extract from BL21(DE3)codon+ cells containing the pMALc2x or pMALc2x-Jojoba WS  expression constructs grown at 37 and 16 OC. .....45	
   Figure 12: Optimization of protein expression: Western blot of the soluble protein extract from BL21(DE3)codon+ cells containing the pMALc2x or pMALc2x-jojobaWS expression construct grown at 16OC..............................................................................................................................46	
   Figure 13: Chemical reaction catalyzed by wax ester synthase...............................................................47	
   Figure 14: Biochemical colorimetric assay using DTNB as a reagent on the soluble protein extract from the bacteria transformed with pMALc2x or pMALc2x-jojobaWS expression construct.. .........................................................................................................................................................48	
   Figure 15: Neutral lipid analysis of mutant yeast strain H1246 and the H1246 strain transformed with pESC-URA, pESC-URA-WS2, WS4, WS5, WS7, WS12, and jojoba WS expression vectors.. .............................................................................................................................................................50	
   	
   vii	
   Figure 16: GC-FID analysis on the wax ester fraction extracted from the TLC. Yeast strain H1246 expressing different enzymes produces different wax ester profiles even when fed with the same fatty acid and primary alcohol. .....................................................................................51	
   Figure 17: Neutral lipid accumulation in mutant yeast strain H1246 expressing WS4, WS7 and WS12 after feeding with palmitic (C16:0) fatty acid and primary alcohols of various chain lengths. ..............................................................................................................................................................52	
   Figure 18: Neutral lipid accumulation in mutant yeast strain H1246 expressing WS4, WS7 and WS12 after feeding with fatty acids of various chain lengths and hexadecanol (C16:0). .....53	
   Figure 19: GC-FID analysis of the wax ester fractions of three different transgenic yeast experiment samples extracted from the TLC.. .....................................................................................53	
   Figure 20: Organ and tissue-specific expression patterns of WS7 detected in WS7promoter:GUS transgenic lines.. .............................................................................................................................................56	
   	
   1	
   1 Introduction 1.1 Wax esters Wax esters are a group of organic compounds that are composed of long chain fatty acids and long chain alcohols. These two components are linked around a carbonyl center (Figure 1), forming a dimeric compound.  Figure 1: General chemical structure of wax esters. These dimeric compounds consist of fatty acyl- groups (on the left side of the carbonyl structure) and an alcohol- group (on the right side of the carbonyl group)  The variation in the numbers of the carbons, double bonds, or side chains in either component provides this group of chemicals with a wide range of chemical and physical properties (Carlsson et al, 2006; Patel et al, 2001). For example, an addition of one carbon atom increases the melting temperature of the chemical by 1~2OC, and the presence of one double bond decreases the melting temperature by ~30OC (Patel et al, 2001). Mixing wax esters of different physiochemical properties with hydrocarbon-based oil gives a wide range of lubricating properties, which are useful for industry (Carlsson et al, 2006).  1.2 The distribution of wax esters and their function in living organisms 1.2.1 Land plants In land plants, thin waxy structures called cuticles, cover the surfaces of aerial tissues and reduce the rate of non-stomatal water loss (Riederer and Schreiber, 2001). The cuticles are composed of 	
   2	
   cutin matrices, which are insoluble in organic solvents, and cuticular waxes, in which wax esters are found. Wax esters are not the major component of cuticular waxes in most plant species, but they are abundant cuticular wax components in carnauba (Copernicia cerifera, 40% of total cuticular wax) and candelilla (Euphorbia cerifera and E. antisyphilitica, 23.5% of total cuticular wax; Gniwotta et al, 2005; Kolattukudy, 1970; Wolfmeier et al, 2000).  Both carnauba and candelilla are currently major plant sources for the production of wax esters (Jetter and Kunst, 2008; Wolfmeier et al, 2000). Another major plant source for wax ester production is the North American desert shrub jojoba (Simmondsia chinensis). In contrast to carnauba and candelilla, the wax esters of jojoba are found in the seeds and used as energy storage. The wax esters are stored in the oil bodies of the cotyledons, the same organelles where other oil crops, such as Brassica napus, store triacylglycerols (Muller et al, 1975). The composition of jojoba wax esters is similar to sperm whale oil, and its high content in the seed (40-60% by mass) makes it a good replacement for sperm whale wax esters as a commercial source (Yermanos, 1975).  1.2.2 Land animals Wax esters can be found in sebum, a mixture of skin lipids, secreted by sebaceous glands associated with hair follicles in humans (Nicolaides and Kellum, 1965; Nicolaides, 1965; Stewart et al, 1986; Yamamoto et al, 1987). The human sebum is composed of wax monoesters and has limited importance. Furthermore, the activity of the sebaceous gland decreases dramatically as we age (Jacobsen et al, 1985). Sebaceous wax esters from other mammals, such as rat skin or sheep wool wax, are more complicated containing diesters with α-hydroxy fatty 	
   3	
   acids (Nicolaides, 1965). Their function is to coat the fur with a hydrophobic layer to protect the animal against over-wetting and to provide insulation (Zouboulis et al, 2008). Additional specialized sebaceous glands producing wax esters in other tissues or species include the meibomian glands in the eyes, the preputial glands in mice, and the uropygial glands in birds. The uropygial glands located at the root of the feathers in birds have both waterproof and anti- parasitic functions (Moyer et al, 2003). The wax esters produced by the human meibomian glands at the edge of the eyes are similar to those produced by sebaceous glands, and are believed to protect eyes from blepharitis (Dougherty et al, 1991). The preputial glands are bilateral glands that flank the reproductive tract of male mice and produce 48% of the total lipid as wax esters (Sansone and Hamilton, 1969). The surfaces of most insects are covered by thin waxy cuticles, which protect the insects from water loss. Some of the species, including grasshoppers Melanoplus packardii and Melanoplus sanguinipes contain wax esters with secondary alcohols (Blomquist et al, 1972). The scale insects and bees can accumulate a large amount of wax esters that are commercially important (Tulloch, 1970). Scale insects are plant parasites that pierce the plant surface tissues with their mouthparts and drain the fluid. They produce wax ester mixtures to protect their eggs and their younglings from other predators and hot weather. Bees, on the other hand, secret wax from the gland on the inner side of ventral shield, chew the wax, and mold hexagonal cells of their hives. 1.2.3 Marine animals Wax esters are not found in marine plants, but only found in marine animals (Sargent et al, 1977; Sargent et al, 1981). There are 30 known marine animal species that contain wax esters, including sperm whales (Physeter catodon), dolphins (Globicephala melaena), copepods (Calanus helgolandicus, Calanoides acutus, Metria gerlachei, and Euchaeta antarctica), polar 	
   4	
   krills (Euphausiia crystallorophias and Thysanoessa inermis), and lantern fish (Symbolophorus evermanni, Stenobrachius leucopsarus) (Lee et al, 2006; Nevenzel, 1970; Sargent et al, 1977). The major marine zooplankton, Arctic and Antarctic krill and copepods, accumulate wax esters in their oil sacs before the winter to cope with food shortage (Lee et al, 2006). When the spring arrives, the animals use the stored wax esters as an energy source to develop reproductive organs, and transfer wax esters into eggs or convert them into triacylglycerols before the transfer (Sargent et al, 1977). The diet of copepods and krill are dinoflagellate algae and diatoms, both of which do not contain wax esters. These findings indicate that they synthesize wax esters de novo (Lee et al, 1971; Benson and Lee, 1972). Copepods can accumulate up to 97% of total lipid as wax esters, as krill accumulate 50-65% of the total lipid as wax esters (Lee et al, 2006). In addition to the energy storage function, these two crustaceans also use wax esters as a buoyancy agent. Buoyancy agent requires being at a constant state in the body, instead of fluctuating due to metabolism, and have lower density to help the body float, and wax esters in marine animals have both theses features. The correlation between the wax ester content and the marine vertical moving behavior has also been observed in other marine vertebrates (Nevenzel, 1970) Marine fish that prey on copepods and krill have non-specific lipases that break wax esters into fatty acids and alcohols, both of which are later incorporated into predators’ metabolisms. Some fish, such as lantern fish, also have the ability to produce their own wax esters, shown by the biochemical assay on their muscle and liver tissue (Nevenzel, 1970). There has not been biochemical data to date to show that the tissues of sperm whales can generate wax esters on their own. However, as an analogy to the lantern fish, sperm whales might have their own enzyme to break down wax esters in their diet and produce their own wax esters in their blubber tissue (Challinor et al, 1969; Hansen and Cheah, 1969).  Sperm whales feed 500-1000 meters below the sea level, and the wax esters in their blubbers help them surface for air exchange. 	
   5	
   1.2.4 Bacteria The accumulation of wax esters is not common in bacteria, but the genus Acinetobacter, gram- negative soil bacteria, are known to accumulate wax esters (Fixter et al, 1986). Wax esters are produced at a faster rate when there is high carbon to nitrogen ratio in the growth medium, and they are metabolized when the bacteria are transferred into a medium with a low carbon to nitrogen ratio in several different strains of Acinetobacter calcoaceticus (Fixter et al, 1986). This showed the bacteria converted the excess carbon source in the medium into wax esters as energy storage, and broke down wax esters when the carbon or energy source in the medium is low (Fixter et al, 1986). Wax esters are stored in a membranous structure analogous to the oil bodies in higher plants (Stöveken et al, 2005).  1.2.5 Summary of the distributions and functions of wax esters in all living organisms Wax esters have specialized adaptive roles in different organisms. They are used as energy storage alternatives for species living in extreme areas. For example, jojoba survives in the arid desert; sperm whale and some crustaceans live in the deep polar ocean where there are dramatic changes in food sources between different seasons. For the sperm whale and fishes that migrate over great distances vertically in the sea, the wax esters are also used as a buoyancy agent. On the surface of the land plants and animals, wax esters are used as a hydrophobic barrier. In terrestrial plants and insects, they are found in the cuticle, which prevents excessive water loss. In mammals or birds, they are found on the surface tissues and keep the fur or feathers dry and parasite-free. Wax esters are also found in specialized tissues, such as meibomian glands and preputial glands, where they serve as lubricating and antibacterial agents. 	
   6	
   1.3 Wax ester biosynthesis Wax ester biosynthesis involves three key enzymes: a β-ketoacyl-Coenzyme A (CoA) synthase (KCS) found in a fatty acid elongation (FAE) complex, a fatty acyl reductase (FAR), and a wax ester synthase (WS).  The fatty acid elongation complex carries out elongation cycles adding two carbons at a time, and contains four enzymes: KCS, enoyl-CoA reductase (ECR), β- hydroxyacyl-CoA dehydratase (HCD), and β-ketoacyl-CoA reductase (KCR). The only component of the elongation complex that is substrate specific is the KCS, determining the carbon chain lengths and amounts of the pool of fatty acyl-CoAs, which was shown by the ectopic expression in yeast and plant tissues (Millar and Kunst, 1997; Trenkemp et al, 2004). Some of the fatty acyl-CoAs are utilized by FAR to generate a pool of primary alcohols. Some of the acyl-CoAs and primary alcohols from each pool are then esterified by wax ester synthases to make wax esters. In summary, the biosynthesis of a wax ester involves three steps: elongation, reduction and esterification. The wax ester biosynthesis pathway is well established in the jojoba seed cotyledon tissue (Ohlrogge et al, 1978; Pollard et al, 1979; Wu et al, 1981; discussed in jojoba-type wax ester synthase section), and the genes encoding the jojoba KCS, FAR, and WS have also been identified (Lardizabal et al, 2000; Lassner et al, 1996; Metz et al, 2000). Forward and reverse genetic approaches have also identified the genes involved in cuticular wax ester biosynthesis in Arabidopsis thaliana epidermis (Fiebig et al, 2000; Li et al, 2008; Millar et al, 1999; Rowland et al, 2006). In addition to these two plant tissues, wax ester biosynthesis has also been studied in bacteria, mammals, and deep-sea fish. The muscle and liver tissue of the fish have been shown to have wax ester synthase activities (Nevenzel, 1970). In both bacteria (Acinetobacter calcoaceticus) and mammals (human and mice), the FAR and WS genes have been characterized 	
   7	
   (Cheng and Russell, 2004a; Cheng and Russell, 2004b; Kalscheuer and Steinbüchel, 2003; Reiser and Somerville, 1997). I am interested in the last step of the wax ester biosynthesis, so I will focus on the studies related to the WS. Based on the species where the enzyme was first characterized, they are termed the jojoba-type WS (Lardizabal et al, 2000), the bacterial-type WS (Kalscheuer and Steinbüchel, 2003; Kalscheuer et al, 2004; Stöveken et al, 2005), and the mammalian-type WS (Cheng and Russell, 2004b; Turkish et al, 2005; Yen et al, 2005). In the following sections, I will describe the research on different types of WS enzymes. 1.4 Studies of wax ester synthases (WS) 1.4.1 Mammalian-type WS Preputial glands are specialized sebaceous glands found in some mammals, including mice, that produce pheromones enriched with wax esters. Therefore, they provide a good model to study the wax ester biosynthesis in a mammalian system. A murine wax ester synthase cDNA was identified by expression cloning from a cDNA library of preputial gland (Cheng and Russell, 2004b). This enzyme is highly expressed in preputial gland, eyelid, thymus, spleen and skin, where there is measurable wax ester accumulated. This wax synthase has activity toward saturated fatty acids and alcohols with 10~18 carbons, polyunsaturated fatty acids and alcohols with 16~20 carbons, and isoprenoid alcohol, all of which are constituents of sebum (Cheng and Russell, 2004b). There are two human wax ester synthases, acyl-CoA wax alcohol acyltransferase 1 and 2 (AWAT1 and AWAT2) (Turkish et al, 2005), both of which belong to acyl-CoA: diacylglycerol acyltransferase 2 (DGAT2) family and share high amino acid similarities with the murine WS. AWAT1 and AWAT2 are the only genes in the DGAT2 family that are expressed in human skin, 	
   8	
   and in situ hybridization expression analysis showed that AWAT2 is expressed in undifferentiated peripheral sebocytes, while AWAT1 is expressed in more mature sebocytes that are about to rupture (Turkish et al, 2005; Yen et al, 2005). The substrate specificity assays demonstrated AWAT2 preference toward C16:1 and C18:1 acyl- CoA, and C16:0, C16:1, and C18: 1 alcohol, while AWAT1 has maximal activity with C18:0 acyl- CoA and C16:1 alcohol (Turkish et al, 2005). The detailed biochemical studies showed that AWAT2 also has acyl-CoA: monoacylglycerol acyltransferase (MGAT) and acyl-CoA: retinal acyltransferase (ARAT) activities (Yen et al, 2005). The difference in the substrate specificities and tissue expression of AWAT1 and AWAT2 suggest modifications of wax esters during sebocyte development. Both AWAT1 and AWAT2 genes are two of the three DGAT2 family members located on the human X chromosome, and the third one, designated hDC3 (human DGAT2 candidate 3), has yet to be studied (Turkish et al, 2005). The remaining 4 DGAT2 family members are DGAT2, MGAT1, MGAT2, and MGAT3. DGAT2 enzyme transfers long chain acyl-CoAs to diacylglycerol and is highly expressed in the liver and white adipose tissue, suggesting that it functions in mammalian triacylglycerol metabolism (Cases et al, 2001). MGAT1, 2, and 3, transfer long chain acyl-CoAs to monoacylglycerol and are expressed in the digestive tissues involved in dietary fat absorption (Cheng et al, 2003; Yen et al, 2002; Yen and Farese, 2003). MGAT1, 2, and 3 provide an alternative pathway to glycerol phosphate pathway (Kennedy pathway) to generate diacylglycerol, a major substrate for triacylglycerol biosynthesis. All of these three enzymes also have weak DGAT activities (Cheng et al, 2003; Yen et al, 2002; Yen and Farese, 2003). 	
   9	
   In addition to the wax ester synthase activities observed in some of the DGAT2 family members, DGAT1, an unrelated enzyme that transfers acyl-CoA to diacylglycerol, also has WS activity in mammals (Cases et al, 1998; Yen et al, 2005). DGAT1 belongs to acyl-CoA: cholesterol transferase (ACAT) family. The other two members of ACAT family are ACAT1 and ACAT2. ACAT1 is widely expressed, with the highest expression in macrophages, steroid genic tissues, and sebaceous glands, and is involved in forming cytoplasmic oil droplets with cholesterol esters. ACAT2 is only expressed in liver and small intestine, and is involved in lipoprotein assembly (Turkish and Sturley, 2009). In summary, wax ester biosynthesis can be considered as a branch of neutral lipid biosynthesis in mammalian tissues, which also includes the biosynthesis of triacylglycerol, cholesterol ester, and retinol ester. The studies of neutral lipid biosynthesis have helped understand the mechanisms that relate the lipid homeostasis to human disease, such as diabetes and obesity (Turkish and Sturley, 2007). There are 10 genes to date in two unrelated gene families that contribute to the production of different neutral lipid classes. The lipotoxicity caused by the accumulation of the free fatty acids or alcohols (primary alcohols, cholesterol, and diacylglycerol) due to mutations in one of the neutral lipid synthesis genes leads to lethality or developmental defects in certain tissues, such as skin or eyes (Turkish and Sturley, 2007; Turkish and Sturley, 2009). The existence of different classes of neutral lipids and the overlapping roles of different biosynthetic enzymes are the product of the evolutionary process to help organisms maintain lipid homeostasis. 1.4.2 Bacterial-type WS The bacterial genus Acinetobacter can accumulate both wax esters and triacylglycerols as their energy storage compounds. A mutant of Acinetobacter calcoaceticus, which was deficient in 	
   10	
   wax ester accumulation and contained trace amounts of triacylglycerols, was isolated from a transposon-mutagenized population (Kalscheuer and Steinbüchel, 2003). Using positional cloning, the gene responsible for the phenotype was found, and a deletion mutant was made to confirm the wax ester and triacylglycerol deficiency phenotype (Kalscheuer and Steinbüchel, 2003). The enzyme encoded by this gene was termed wax synthase/diacylglycerol acyltransferase (WS/DGAT) since it has both enzyme activities (Kalscheuer and Steinbüchel, 2003). This enzyme does not share any homology with DGAT2, ACAT, or the jojoba WS. The homologues of WS/DGAT are reported in various bacteria, and are especially abundant in Mycobacterium tuberculosis, leading to an interest to study the relationship between the invasiveness of the bacteria and the accumulation of the neutral lipid (Kalscheuer, 2010; Wältermann, et al, 2007). The heterologous expression of the WS/DGAT in bacteria and yeast led to the purification of the protein and detailed biochemical analyses of the substrate specificities of the enzyme (Kalscheuer et al, 2004; Kim et al, 2009; Stöveken et al, 2005; Uthoff et al, 2005).  The WS activity of this bacterial acyltransferase is maximal with C16 alcohol and C14-18 acyl-CoA (Stöveken et al, 2005). The enzyme can also use the substrates diacylglycerol, monoacylglycerol, secondary alcohol, cyclic alcohol, phenol alcohol, glycidols, and sterol (Kim et al, 2009; Stöveken et al, 2005). Site- specific mutagenesis showed that the two histidines of the conserved HHXXXDG motif of WS/DGAT genes are essential for the wax ester synthase activities (Stöveken et al, 2009). The second histidine of this motif is proposed to deprotonate the oxygen of the free hydroxy group of any alcohol, and the deprotonated oxygen reacts with acyl-CoA and form an ester group with acyl-chain, releasing a free CoA-SH (Stöveken et al, 2009). 	
   11	
   The substrate specificities of WS/DGAT using alkanethiols were also explored with substrate containing sulfur atom (Uthoff et al, 2005). When the oxygens of the free hydroxy groups of the primary alcohols are replaced with sulfur atoms, the chemicals are called alkanethiols. Alkanethiols share similar structural characteristics with primary alcohols, and the free thio groups share the similar chemistry to free hydroxy groups. Due to the broad range of the substrate specificities of WS/DGAT and the similarities between alkanethiols and primary alcohols, WS/DGAT was tested with the following substrate of 1-hexadecanthiol, 1,8- octanedithiol, and 1-S-monopalmitoyloctanedithiol, and shown to have the activities to make thioesters  (Uthoff et al, 2005). An antibody raised against Acinetobacter calcoaceticus purified WS/DGAT was used to study the cellular localization using transmission electron microscopy (TEM). The protein was shown to localize on the cytoplasmic side of the plasma membrane and the lipid bodies, and sparsely in the cytoplasm (Stöveken et al, 2005). With the use of fluorescence labeling, confocal microscopy, TEM, artificial bacterial membranes, quartz crystal microbalance, and scanning force microscopy (SFM), a novel mechanism of lipid body formation was discovered (Wältermann et al, 2005). Different from eukaryotic systems where the synthesis of the lipid was proposed to occur in between two leaflets of the ER membrane, the synthesis of the storage lipids in Acinetobacter occurs at the plasma membrane. The bacterial WS/DGAT is soluble in the cytoplasm and becomes active when it docks on the plasma membrane. It generates small lipid droplets of wax esters on the cytoplasmic side, and the oleaginous layers of phospholipids form to cover these small lipid droplets. Lipid droplets eventually form as the small lipid droplets conglomerate, and then bud off from the plasma membrane (Wältermann and Steinbüchel, 2005). This type of oil droplet formation can also be found in the recombinant Escherichia coli (E. coli) (Kalscheuer et al, 2006). 	
   12	
   The biotechnological application of the WS/DGAT gene was also explored (Kalscheuer et al, 2006; Kim et al, 2009; Steen et al, 2010; Stöveken and Steinbüchel; 2008). When the WS/DGAT gene is heterologously expressed with the jojoba FAR, a class of wax esters similar to the jojoba- type wax esters was produced (Kalscheuer et al, 2006). In addition, the recombinant bacterium also produced fatty acid methyl esters (FAME) and fatty acid ethyl esters (FAEE), which are possible substitutes for biodiesel. Currently, the industrial synthesis of wax esters, FAMEs, and FAEEs is either accomplished by chemical esterification or a lipase-based system using plant or animal lipids, both of which use organic solvents and have limited substrate sources. The application of WS/DGAT in a bacterial system for the production of wax esters, FAMEs and FAEEs in vivo is therefore a viable alternative. Recently, a recombinant bacterium expressing several foreign sugar and lipid metabolic enzymes was created, and the bacterium was shown to break down complex sugars and use them as a carbon source for making wax esters, biodiesel (FAMEs and FAEEs), and alcohols (Steen et al, 2010). There are 11 genes in Arabidopsis thaliana found to share nucleotide similarities to bacterial WS/DGATs (Kalscheuer and Steinbüchel, 2003). An epidermal microarray showed that 4 of these 11 homologues are highly expressed in the epidermis, where cuticular wax is synthesized (Suh et al, 2005). An Arabidopsis thaliana T-DNA insertional mutant in the At5g37300 gene encoding one of the 4 epidermally-expressed WS/DGAT candidates had a dramatic stem wax ester reduction (Li et al, 2008). This gene was named WSD1, and its product was demonstrated to have only WS activity, but no DGAT activity. The promoter activity assays of WSD1 using β- glucuronidase (GUS) reporter gene showed that WSD1 was expressed in all cell types in the stem, but its functions in tissues other than the epidermis remain unknown (Li et al, 2008). In addition, a homologue of WS/DGAT was also characterized from Petunia hybrida, which only 	
   13	
   has wax synthase activity and is responsible for the accumulation of wax esters in the petal (King et al, 2007). In summary, Acinetobacter WS/DGAT was characterized as an enzyme capable of generating wax esters or triacylglycerols in bacteria. The WS/DGAT homologues can be found in many different bacterial species, with the genus Mycobacterium having multiple copies. The Acinetobacter WS/DGAT has a broad range of substrate specificities and its involvement in forming the bacterial lipid bodies has been studied in detail. The biotechnological applications of this enzyme have also been explored. 1.4.3 Jojoba-type WS Jojoba is the only known plant to store wax esters in its seed oil, and 40-60% of the total dry seed weight is comprised of wax esters, making it a good commercial source of these compounds (Yermanos, 1975). The storage compartment of jojoba wax esters is the oil body, like the triacylglycerols in oil crops (Muller et al, 1975). During germination of the jojoba seed, the wax ester content decreases as the carbohydrate increases. Simultaneously the activity of wax ester lipase, which breaks down wax esters into fatty acids and primary alcohols, increases (Huang et al, 1978; Moreau and Huang, 1977). A biochemical assay on cellular fractions showed that the activity of the wax ester lipase was found in the wax ester body fraction, suggesting that it breaks down the wax ester into a fatty acid and an alcohol. Both the fatty acid and the alcohol later enter glyoxysomes where they are modified and activated for β-oxidation (Moreau and Huang, 1977). The appearance of the wax ester lipase only after germination suggests that it requires activation, or that it is inhibited before the onset of germination (Huang et al, 1978). The wax esters are synthesized in the cotyledon tissue from glucose (Ohlrogge et al, 1977). Glucose is converted into acetyl-CoA, which is used in the plastid for the de novo fatty acid 	
   14	
   biosynthesis, including palmitoyl (C16:0)-ACP (acyl carrier protein) and steroyl (C18:0)-ACP synthesis, and steroyl (C18:0)-ACP desaturation to generate oleoyl (C18:1)-ACP. Oleoyl-ACP is converted into oleic acid and subsequently into oleoyl-CoA, which is then transported to the ER for elongation to generate eicosenoyl (C20:1)-CoA and erucyl (C22:1)-CoA (Ohlrogge et al, 1978). These two very long chain acyl-CoAs are reduced to their corresponding primary alcohols, and the pools of primary alcohols and acyl-CoAs are esterified to generate wax esters (Pollard et al, 1979). The wax ester synthase activity was detected in the wax ester body fraction (Pollard et al, 1979; Wu et al, 1981). The gene encoding WS activity in the wax ester body fraction was characterized after the method developed for the purification of the jojoba KCS was modified to enrich for the WS activity, and used to purify the jojoba WS enzyme (Lardizabal et al, 2000; Lassner et al, 1996). The partial sequence of this protein was obtained and used to isolate the jojoba WS cDNA. The identity of the WS cDNA was confirmed by the detection of WS activity and increased wax ester accumulation when it was co-expressed with the jojoba FAR and the Lunaria annua KCS in Arabidopsis seed (Lardizabal et al, 2000). The enzyme is predicted to have 9 transmembrane domains, and its cellular localization is on the membrane of the ER and the wax ester bodies (Lardizabal et al, 2000; Pollard et al, 1979; Wu, 1981). The substrate preference of the jojoba WS enzyme is toward very long chain unsaturated acyl-CoAs and primary alcohols, corresponding to the wax ester composition of the jojoba seed (Lardizabal et al, 2000). The Arabidopsis thaliana has 12 jojoba-type WS homologues, and WS4, encoded by At3g51970, has been shown to be involved in phytosterol ester formation (Chen el al, 2007). WS4 is expressed in all organs along with WS12, while the rest of the family members are expressed in flowers and siliques except WS1, WS2, and WS5 (Table 1; Klypina and Hanson, 2008). These 12 Arabidopsis homologues and jojoba WS belong to a membrane bound O-acyltransferase 	
   15	
   (MBOAT) superfamily, which includes the human ACAT and the yeast ARE families. The DGAT1, belonging to the ACAT family (discussed in mammalian-type WS section), has both WS and DGAT activities (Case et al, 1998; Hofmann, 2000; Yen et al, 2005). The enzymes ARE1 and ARE2, homologues of human ACAT, are sterol ester biosynthetic enzymes in yeast Saccharomyces cerevisiae with weak DGAT activities (Sandager et al, 2002). Thus, similar to the other members of the MBOAT superfamily, the 12 Arabidopsis WS homologues might have more than one biochemical function. For example, I have shown that WS4 has both wax ester and sterol ester-forming activities. Table	
  1:	
  Summary	
  table	
  of	
  steady	
  state	
  transcripts	
  of	
  the	
  jojoba-­type	
  WS	
  homologues	
  detected	
  by	
  RT-­PCR	
  in	
   different	
  organs	
  of	
  Arabidopsis	
  thaliana	
  (Klypina	
  and	
  Hanson,	
  2008).	
  The	
  gene	
  that	
  is	
  expressed	
  is	
  marked	
  as	
  “+”,	
   and	
  the	
  gene	
  that	
  is	
  not	
  expressed	
  is	
  marked	
  as	
  “-­”.	
  	
   Gene Loci Rosette leaf Cauline leaf Stem Flower Silique WS1 At1g34490 - - - - - WS2	
   At1g34500 - - - - - WS3	
   At1g34520 - - - + + WS4	
   At3g51970 + + + + + WS5	
   At5g51420 - - - - - WS6	
   At5g55320 - - - + + WS7	
   At5g55330	
   - - + + + WS8	
   At5g55340	
   + - + + + WS9	
   At5g55350	
   - - + + + WS10	
   At5g55360	
   - - + + + WS11	
   At5g55370	
   - - + + + WS12	
   At5g55380	
   + + + + +  In summary, the metabolism and the biosynthesis of the wax esters have been well established in the jojoba seed embryo. The genes that are responsible for the biosynthesis have been characterized, such as the jojoba KCS, FAR, and WS. The jojoba WS has many homologues in other species, but their true function has yet to be determined. 	
   16	
   1.5 The current industrial production of wax esters and the possible improvements by biotechnology 1.5.1 Applications of wax esters Wax esters are a very diverse group of compounds based on their chemical structures. They are highly valued as outstanding lubricants due to their high oxidation stability and resistance to hydrolysis. Mixing wax esters of different physiochemical characteristics with hydrocarbon- based oils results in lubricants with a wide range of unique properties, such as viscosity, compressibility, stability, pouring point, melting point, cloud point, flash point, and acid number. (Carlsson et al, 2006). In addition, wax esters also have the properties of surface protection, adhesive, impregnation, and energy releasing, and are used as furniture and shoe polishes, dental treatment products, cosmetics, candles, and fuels (Carlsson et al, 2006; Jetter and Kunst, 2000; Wolfmeier et al, 2000). 1.5.2 Bottlenecks in wax ester production Currently, the major source of wax esters is fossil deposits, such as petroleum oil and brown coal deposits (Jetter and Kunst, 2008; Wolfmeier et al, 2000). The production of wax esters from fossil deposits involves solvent extraction, distillation, refinery, filtration, and chemical derivatization. Theses processes generate toxic and polluting byproducts, which are harmful to humans and the environment (Wolfmeier et al, 2000). In addition, the availability of fossil deposits is limited. Another source for wax ester production are living organisms. The main plant wax ester sources are jojoba (Simmondsia chinensis), carnauba (Copernicia cerifera), candillela (Euphorbia cerifera and E. antisyphilitica), retamo (Bulnesia retama), ouricury (Syagros coronata), and sugarcane (Saccharum officinarum), and the main animal sources include bee’s wax and sheep wool wax (lanolin) (Jetter and Kunst, 2008; Wolfmeier et al, 2000). Due to the limited 	
   17	
   production of wax esters from living organisms, they are mostly used for expensive products, such as pharmaceuticals, cosmetics, and food additives (Jetter and Kunst, 2008). Besides the low production, the diversity of the wax esters is also low because all the living organisms produce only wax esters with specific acyl- and alcohol- chain lengths (Jetter and Kunst, 2008). 1.5.3 Overcoming the production bottlenecks by engineering wax ester biosynthetic pathways in transgenic oil crops In an effort to increase wax ester production, we are interested in engineering the biosynthetic pathways for wax ester production in the seeds of the oil crops. To achieve this, we need to: 1) understand the wax ester biosynthetic pathway; 2) introduce the pathway into oil crops; 3) co- express unique modification enzymes to generate the wax esters with novel acyl- and alcohol- groups (Jetter and Kunst, 2008). The wax ester biosynthetic pathway in several plant species is well understood, and the introduction of the pathway into Arabidopsis thaliana seeds has been shown to be feasible (Lardizabal et al, 2000; Ohlrogge et al, 1978; Pollard et al, 1979; Wu et al, 1981). In order to increase the chemical diversity of the wax esters, I am interested in identifying wax ester synthases with novel specificities that can produce wax esters that are currently not available from the natural sources. 1.6 MSc project objectives As part of the Industrial Crops producing added value Oil for Novel chemicals (ICON) initiative, my MSc project has three main goals: 1) To introduce the jojoba wax ester biosynthetic pathway (jojoba KCS, FAR, and WS) into Arabidopsis thaliana seeds to determine the levels and the identities of wax esters that can be achieved in transgenic seeds; 2) To determine substrate specificities of a family of jojoba-type WS homologues from Arabidopsis thaliana in an attempt to identify those with novel substrate specificities; 3) To study the expression pattern of the 	
   18	
   jojoba-type WS homologue WS7 encoded by the At5g55330 gene with the highest WS activity in Arabidopsis thaliana. 	
   19	
    2 Methods and materials 2.1 Preparation of DNA for molecular cloning 2.1.1 Isolation of genomic DNA from Arabidopsis thaliana leaves To obtain genomic DNA from Arabidopsis thaliana, 10 young rosette leaves were ground in liquid nitrogen to fine power, and 700 µL of extraction buffer containing 2% of centrimonium bromide (CTAB), 1.4 M NaCl, 100 mM Tris-HCl pH 8.0, 20 mM EDTA, and 1% β- mercaptoethanol were added. The mixture was heated at 65 OC and chilled on ice. The mixture of chloroform:isoamyl alcohol (24:1) was added to precipitate the proteins. Isopropanol was used to precipitate the DNA and 75% ethanol was used to wash residual salts. The final DNA was dissolved in 100 µL TE buffer containing 10 mM Tri-HCl pH 8.0 and 1mM EDTA. 2.1.2 Preparation of cDNA from Arabidopsis thaliana siliques Total RNA was isolated from Arabidopsis thaliana siliques where 9 of 12 jojoba-type WS homologues are expressed, using Trizol Reagent (Invitrogen). The concentration and the purity of the RNA were determined by measuring the absorbance values at 260 nm and 280 nm. First stand cDNA was synthesized using 1 µg of total RNA, 500ng of oligo(dT)18, and SuperScript II reverse transcriptase following manufacturers instruction (Invitrogen). 2.2 Generation of bacterial, yeast, and plant expression constructs 2.2.1 Design of bacterial, yeast, and plant expression constructs The bacterial expression vector used was pMAL-c2x (New England Biolabs). The coding sequences of the jojoba WS and Arabidopsis thaliana jojoba-type WS homologues (WS2, WS4, WS5, WS7, and WS12) were directionally cloned into BamHI site and PstI site. The restriction 	
   20	
   enzymes BamHI, PstI, and BglII (Invitrogen) were used following the manufacturers instructions to cut and create the sticky ends on the vector and the amplicons. The T4 ligase (Invitrogen) was used to ligate the vector with the DNA fragments. All the introduced gene fragments were in phase with the malE gene, which encodes maltose-binding protein (MBP), to create fusion WS proteins with MBP at the N-terminus (Figure 2). The identities of all finished expression vectors were confirmed by diagnostic digestion and sequencing using primers provided by the manufacturer of the vector (New England Biolabs). 	
   Figure	
   2:	
   The pMAL-c2x bacterial expression constructs. The coding sequences of jojoba WS and the Arabidopsis thaliana jojoba-type WS homologues were cloned into BamHI and PstI sites of the vector.	
    The yeast expression vector pESC-URA (Stratagene) was used to create yeast expression constructs. Jojoba WS, and Arabidopsis homologues WS1, WS2, WS5, WS6, WS7, WS8, WS9, WS11, and WS12 were cloned into the BamHI and NheI sites. WS4 and WS10 were cloned into the XbaI and KpnI sites. The coding sequences of all the genes were downstream of yeast GAL1 promoter. His-tags containing a 6-histidine sequence were fused to the C-terminus of all proteins 	
   21	
   (Figure 3). The identities of all the constructs were confirmed in the same way as bacterial expression vectors.  Figure 3: The pESC-URA yeast expression constructs. The coding sequences of jojoba WS and Arabidopsis thaliana jojoba-type WS homologues were cloned behind the yeast GAL1 promoters.  The plant expression vector pCAMBIA1381 (Cambia Enabling Innovation) was used to design a β-glucuronidase (GUS) expression construct. The 1119 base pairs immediately upstream of the WS7 translation start site was used as the promoter since the WS7 gene does not contain any introns. The WS7 promoter (WS7P) was cloned into the BamHI and PstI sites (Figure 4).  The identity of the expression construct was confirmed by diagnostic digestion tests and sequencing.  Figure 4: Plant expression construct pCAMBIA1381-WS7P:GUS.  2.2.2 Preparation of E. coli competent cells, transformation, and routine growth To prepare competent cells for transformation, E. coli strain DH5α was grown to lag phase in LB medium (EMD Bioscience). The harvested cell culture was treated with ice cold 0.1 M MgCl2 and 0.1 M CaCl2 and resuspended in 0.1 M CaCl2 and 15% glycerol. The cells were 	
   22	
   frozen in liquid nitrogen and stored in -80OC until use. The cells were thawed on ice for the transformation. 1~10 ng of the plasmid or 10 µL of the ligation product was added to the cell suspension, mixed gently, and left on ice for 30 minutes. The cells were heat-shocked at 42 OC for 30 seconds and left on ice for 2 minutes. LB liquid medium was added and the cell suspension was incubated at 37 OC for 1 hour with vigorous shaking. The cell suspension was plated on LB medium containing agar (14 g/L) with the selected antibiotics (ampicilin 50µg/mL or kanamycin 50µg/mL) and allowed to grow for 12~16 hours. The colonies were then inoculated in LB liquid medium with selected antibiotics and grown at 37 OC with vigorous shaking for plasmid preparation to confirm its identity. 2.2.3 Amplification of DNA fragments by polymerase chain reaction (PCR) All the cloning PCR amplifications were performed with Finnzymes Phusion high fidelity DNA polymerase (New England Biolabs). The DNA template used to amplify the jojoba WS coding sequence was the pBinGlyRed-FWS3 plasmid (donated by Dr. Edgar Cahoon, University of Nebraska- Lincoln), and the coding sequences of the jojoba-type WS homologues were amplified using the silique cDNAs as templates. 1119 base pairs directly upstream of the WS7 translation start codon were amplified from the leaf genomic DNA. The PCR reaction (50 µL) contained Phusion HF buffer, 200 µM dNTP, 0.5 µM of the forward and reverse primers, 10 ng DNA template, and 0.5 µL of Phusion HF DNA polymerase. The PCR program used to amplify the jojoba WS, the Arabidopsis thaliana jojoba-type WS homologues, and the WS7 promoter region are shown in Tables 2. 	
   23	
    Table 2: Temperature program for cloning PCR of the jojoba WS, Arabidopsis thaliana jojoba-type WS homologues, and the WS7 promoter. Annealing temperature X OC was recommended by Finnzyme Tm calculator for each different PCR reaction (https://www.finnzymes.fi/tm_determination.html) Jojoba WS, jojoba-type WS homologues WS7 promoter Cycle step Temperature Time Temperature Time Cycles Initial denaturation 98 OC 30 seconds 98 OC 60 seconds Denaturation Annealing Extension 98 OC X OC 72 OC 10 seconds 25 seconds 30 seconds/kb 98 OC 64.5 OC 15 seconds 90 seconds/kb 35 Final Extension 72 OC 4 OC 10 minute forever 72 OC 4 OC 10 minutes forever   The BamHI and PstI restriction enzyme sites were introduced to the 5’ and 3’ ends of the amplicons of WS2, WS5, WS7, WS10, WS12, and Jojoba WS for bacterial expression constructs. The amplicon of WS 4 has BglII on 5’ end instead (Table 3), the sticky end after the digestion is compatible with BamHI. 	
   24	
   Table 3: The primers used to amplify the jojoba WS and Arabidopsis thaliana jojoba-type WS homologues coding sequences for the construction of fusion MBP protein in bacterial expression pMAL-c2x constructs (New England BioScience). The underlined and bold nucleotides indicate the restriction site Gene Forward Primer (5’3’) Reverse Primer (5’3’) WS2 GGG GGA TCC ATG GAG GAA GAA CTC AAG AAT TTC AGA CTG CAG CTA GAA AAT GGA AAA CTT GCG C WS4 GGG AGA TCT ATG GCG AGT TTC ATC AAG GC AGA CTG CAG TTA AAA AAG ATA TGC GGT CAG TTT CC WS5 GGG GGA TCC ATG GAG GAA GAA CTC AAG AAC TTG AGA CTG CAG TCA CCA GCC AAA AGT AAA CAA C WS7 GGG GGA TCC ATG GAG GAA GAA CTC AAG TTA TTC AGA CTG CAG TTA AAG ACT CGT AAA CAA CCC TAA G WS10 GGG GGA TCC ATG GAA GAA GAA CTC AAG AAC TTC AGA CTG CAG TTA ATC ACC CAA AAG TAA CAG AAG WS12 GGG GGA TCC ATG GAA GAA AAG TTT AGA AAC TTA ATC G AGA CTG CAG TCA TGA AGA AGT GAA TAA CTT GGC Jojoba WS GGG GGA TCC ATG GAG GTG GAG AAG GAG C AGA CTG CAG TCA CCA CCC CAA CAA ACC  To make yeast expression construct, the BamHI and NheI restriction enzyme sites were introduced into the amplicons of WS1, WS2, WS5, WS6, WS7, WS8, WS9, WS11, WS12, and jojoba WS. The XbaI and KpnI sites were introduced into the amplicon of WS4 and WS10 (Table 4). 	
   25	
   Table 4: The primers used to amplify the jojoba WS and Arabidopsis thaliana jojoba-type WS homologues coding sequences for heterologous protein expression in yeast using yeast expression vector pESC-URA (Stratagene). The underlined and bold nucleotides indicate the restriction site. A polyhistidine tag (underlined) was added to the C- terminus of each construct. Gene Forward Primer (5’3’) Reverse Primer (5’3’) WS1 TTT AAA GGA TCC ATG GAG GAA GAA CTC AAG AGC TTT AAA GCT AGC TCA ATG ATG ATG ATG ATG ATG CGA AAC AGT GTA ATT GAA AAC CTT GCG WS2 TTT AAA GGA TCC ATG GAG GAA GAA CTC AAG AAT TTC ATC TTT AAA GCT AGC TCA ATG ATG ATG ATG ATG ATG GAA AAT GGA AAA CTT GCG CTT CAC G WS4 TTT AAA AGA TCT ATG GCG AGT TTC ATC AAG GCA TGG TTT AAA GGT ACC TCA ATG ATG ATG ATG ATG ATG AAA AAG ATA TGC GGT CAG TTT CCT CCT G WS5 TTT AAA GGA TCC ATG GAG GAA GAA CTC AAG AAC TTG ATC TTT AAA GCT AGC TCA ATG ATG ATG ATG ATG ATG CCA GCC AAA AGT AAA CAA CTT GAG C WS6 TTT AAA GGA TCC ATG GAG GAA GAA ATC AAG AGC TTG TTT AAA GCT AGC TCA ATG ATG ATG ATG ATG ATG CTG GGG AAA ATA AAA TAA CTT GC WS7 TTT AAA GGA TCC ATG GAG GAA GAA CTC AAG TTA TTC ATC TTT AAA GCT AGC TCA ATG ATG ATG ATG ATG ATG AAG ACT CGT AAA CAA CCC TAA GAG G WS8 TTT AAA GGA TCC ATG GAT GAA GAA CTC AAG AAC TTG ATC TTT AAA GCT AGC TCA ATG ATG ATG ATG ATG ATG TCG CTT AAT GAA CTC AAC GAG C WS9 TTT AAA GGA TCC ATG GAG GAA GAA CTC ATG AGC TTT AAA GCT AGC TCA ATG ATG ATG ATG ATG ATG CGA TTT TAG CGA TTG GTT TCT ACA  AAGG WS10 TTT AAA AGA TCT ATG GAA GAA GAA CTC AAG AAC TTC ATC TTT AAA GGT ACC TCA ATG ATG ATG ATG ATG ATG ATC ACC CAA AAG TAA CAG AAG AAA C WS11 TTT AAA GGA TCC ATG GAA GAA GAG TTG AGA AAC C TTT AAA GCT AGC TCA ATG ATG ATG ATG ATG ATG CAT TAA AAT ACA GAC AAC GTG CC WS12 TTT AAA GGA TCC ATG GAG GTG GAG AAG GAG CTA AAG TTT AAA GCT AGC TCA ATG ATG ATG ATG ATG ATG CCA CCC CAA CAA ACC CAT CAA TTT C Jojoba WS TTT AAA GGA TCC ATG GAA GAA AAG TTT AGA AAC TTA ATC G TTT AAA GCT AGC TCA ATG ATG ATG ATG ATG ATG TGA AGA AGT GAA TAA CTT GGC TAG G  The primers used to amplify WS7 promoter are AAA AAT TTT GGA TCC GTT ATT TTA TTT ACG TTG TTG GGG ATA TTT TAT AAC GAG TG as forward primer and AAA AAT TTT CTG CAG GGT TAC TGG TTA AGT GAT AAT AAA CCA AGA AAC ATA GG as reverse primer. 	
   26	
   2.2.4 Plasmid purification, DNA gel purification, and PCR product purification To extract plasmids from bacteria, 3~5 mL of cell culture were harvested and resuspended in the 250 µL suspension buffer (50 mM Tris-HCl, 10 mM EDTA, pH 8.0, and 50 µg/mL RNase A). The 250 µL lysis buffer (0.2 M NaOH and 1% SDS) was used to break the cells, 350 µL neutralization buffer (4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2) was added to neutralize the solution and precipitate the protein. The supernatant of the mixture was then added to the EconoSpin prepacked silica membrane spin column (Epoch Life Science). After the spin at 10000 rpm, the wash buffer (20 mM NaCl, 2 mM Tris-HCl, pH 7.5, 80% ethanol) was added to wash the residual salts. The elution buffer (10 mM Tris-HCl, pH 8.5) or sterile distilled water was used to elute the plasmid. To extract DNA fragment from agarose gel after PCR reaction or digestion, the gel containing the fragment was sliced and dissolved in 3 gel volumes of gel solubilization buffer (5.5 M guanidine thiocyanate, 20 mM Tris-HCl, pH 6.6). 1 gel volume of isopropanol was added to increase the efficiency. The mixture was loaded to the Econospin column and washed with the wash buffer, and the product was eluted with the elution buffer. The PCR product was also cleaned up using 5 reaction volumes of absorption buffer (5.5 M guanidine hydrochloride, 20 mM Tris-HCl, pH 6.6) and following the same washing and eluting procedure. 2.3 Heterologous protein expression in Escherichia coli 2.3.1 Bacterial growth condition and protein extraction The E. coli bacterial strain used for the protein expression was BL21-CodonPlus (Stratagene). The made expression construct pMAL-c2x with jojoba WS or jojoba-type WS homologues were transformed into BL21-CodonPlus using the same transformation protocol as DH5α. The transformed BL21-CodonPlus transgenic bacteria were grown at 16 OC for fusion protein expression. The bacteria were grown to A600= 0.5, and isopropylthiogalactoside (IPTG, Sigma- 	
   27	
   Aldrich) was added to the medium to give a final concentration of 0.3 mM to induce the protein expression. The LB medium with sorbitol (660 mM), betaine (2.5 mM), sucrose (0.3 M) and glycerol (10 g/L) were used to increase the solubility of the fusion protein. The LB medium pH 5.6 and tryptose phosphate medium (20 g/L tryptose, 2 g/L glucose, 5 g/L sodium chloride, and 2.5 g/L disodium phosphate) were also used to optimize the fusion protein expression. The cell cultures were harvest 24~48 hours after the induction with IPTG. 2.3.2 Heterologous protein expression assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblotting and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) The harvested cells were suspended in cold extraction buffer (20 mM Tris-HCl pH=8, NaCl 200 mM, and EDTA 1mM). The cell mixtures were then sonicated for 2 minutes with 15-second interval on ice. The supernatant was then collected as soluble protein extract for both immunoblotting analysis and biochemical assay. Bio-Rad protein assay kit was used to determine the protein concentration of the soluble fraction. 10 µg of total soluble proteins of each sample were separated by SDS-PAGE on a 10% SDS-polyacylamide gel. After the separation, the protein was transferred to a nitrocellulose membrane (0.2 µm, Bio-Rad) using Trans-Blot SD semi-dry transfer cell following the manufacturer instructions (Bio-Rad). To detect the presence of fusion maltose binding protein (MBP), anti-MBP mouse monoclonal antibody (NEB) was used as primary antibody, and anti-mouse IgG (whole molecule)-alkaline phosphatase antibody produced in goat (Sigma-Aldrich) was used as secondary antibody. Nitro blue tetrazolium chloride (NBT)/ 5-bromo-4-chloro-3-indolyl phosphate toluidine (BCIT) was used to detect the binding of antibodies. The biochemical assay reaction contained 100 mM Tri-HCl pH 8.0, 100 µM DTNB, 50 µM palmitoyl-CoA, 50 µM hexadecanol, and 5 µg of soluble protein. The sample without 	
   28	
   hexadecanol was used as negative control. The free CoA released from the acyltransferase reaction binds to DTNB and released yellow colour with the wavelength at 412 nm (Srere, 1963). The absorbance at 412 nm was measured every 15 seconds for 3 minutes. 2.4 Heterologous protein expression in Saccharomyces cerevisae 2.4.1 Yeast growth conditions, introduction of the expression plasmid, and induction of the heterologously expressed proteins Heterologous protein expression in baker’s yeast (Saccharomyces cerevisae) was carried out in the mutant H1246 (w303a; MATα are1::HIS3 are2::LEU2 dga1::KanMX4 lro1::TRP1 ADE2) (Sandager et al, 2002). The yeast mutant was grown on selective dropout (SD) medium with 0.67% yeast nitrogen base without amino acids, 2% dextrose, amino acid dropout powder, and the selected amino acids (pH 5.8). The yeast expression construct pESC-URA containing the jojoba WS and jojoba WS homologues driven by the GAL1 promoter were transformed into the H1246 mutant according to Gietz and Woods (2004). To assay the biochemical activity of each WS enzyme, the amounts of wax esters produced by the transgenic mutant yeast after feeding with fatty acids and primary alcohols were measured. The different chain length fatty acids and alcohols (C16~C30) were dissolved in 100% hot ethanol and added to the medium to give a final concentration of 0.1% (v/v).  The yeast was grown in the SD medium with galactose instead of glucose. The yeast culture was grown at 30OC for 48 hours before harvesting. The biochemical screen assay was done by feeding the transgenic yeast with pESC-URA-WS1, WS2, WS4, WS5, WS6, WS7, WS8, WS9, WS10, WS11, WS12, and jojoba WS with C16:0 fatty acids and C16:0 primary alcohol. The substrate specificities assays were done by feeding the transgenic yeast with WS4, WS7, and WS12 with various carbon chain length of fatty acid and fatty alcohols (C16:0-30:0). 	
   29	
   2.4.2 Yeast lipid analysis After the yeast was harvested, the cells were freeze-dried and 25 mg of the lyophilized cells was extracted with methanol at 95 OC. Hexane and 0.9% NaCl were used to extract the neutral lipids. The lipid extracts were then run on a thin layer chromatography (TLC) plate with a solvent system of hexane-diethylether-acetic acid (90:7.5:1). Hexadecyl palmitate (C16:0-C16:0) and vegetable oil were used as reference standards. The wax ester fraction was scraped from the TLC plate and analyzed by gas chromatography (GC)- flame ionization detector (FID) (7890A; Agilent) and mass spectrometry (MS) (5973; Agilent). The injection program was splitless injection at 300 OC for GC-FID and on-column injection at 53 OC for GC-MS. The temperature program started at 50 OC for 2 minutes, and was raised at 40 OC/minute to 200 OC, held at 200 OC for 1 minute, then raised at 3 OC/minutes to 320 OC, and held at 320 OC for 15 minutes. The carrier gas used for GC-FID was hydrogen, and helium for GG-MS. The pressure program used was constant flow at 2 mL/minutes. The FID detector program was heating at 300 OC, with hydrogen flow at 30 mL/minute, compressed air at 350 mL/minute, and helium at 23 mL/minutes. The column used was HP-1 methyl siloxane (Agilent). 2.5 Analysis of transgenic plants 2.5.1 Transformation of Agrobacterium tumefaciens and Arabidopsis thaliana and the screening of the transgenic plants Competent Agrobacterium tumefaciens cells (A. tumefaciens, strain GV3101, pMP90) were harvested at lag phase, treated with ice cold 50 mM CaCl2 and then resuspended in 50 mM CaCl2 with 15% glycerol. The cells were frozen in liquid nitrogen and stored at -80 OC until use. Before transformation the cells were thawed on ice and 2 µg of the plasmid was added. The mixture was incubated at 37 OC for 5 minutes. LB medium was added and the mixture was incubated at 28~30 OC for 3 hours with vigorous shaking. The cell suspension was then plated and grown 	
   30	
   under the appropriate antibiotic selection (rifampicin 25 µg/mL, getamycin µg/mL, and kanamycin 50 µg/mL) at 30 OC for 2 nights. For the transformation of Arabidopsis thaliana, the plants were grown as a lawn at a density of approximately 100 plants/pot (12.5 cm in diameter) until they just started bolting (5 cm tall inflorescences). The A. tumefaciens was grown to O.D. 600 = 1.5 and harvested. The cells were washed with 5% sucrose and resuspended in 5% sucrose with 0.025% (v/v) Silwet L-77 (LEHLE SEEDS, Round Rock, Texas). The bacterial suspension was then used to spray the plants. The plants were covered in a plastic bag for 24 hours and returned to the standard growth conditions described below. The transgenic plants that carried pCAMBIA 1381 were selected by screening the seeds resistant to hygromycin (25 µg/mL). The transgenic seeds that carried pBinGlyRed, pBinGlyRed-FWS1, and pBinGlyRed-FWS3 were selected by their red glow when excited with green light due to the presence of the DsRed marker. The fluorescence stereomicroscope Leica MZ16 FA with the TXR filter setting or the green LED flashlight (INOVA) coupled with No.25 red filter (Vancouver Telescope Centre Ltd.) were used to identify the DsRed positive seeds. The selected DsRed positive seeds were propagated or analyzed for their seed wax ester content. 2.5.2 Plant growth conditions Seeds of wild type or transgenic Arabidopsis thaliana were sterilized with 20% (v/v) bleach containing 1 µL/mL of 20% Triton-X100. After the sterilization, the seeds were sown on the plates with Arabidopsis thaliana (AT) medium (Somerville and Ogren, 1982) solidified with agar (7g/L). The seeds were imbibed, germinated and grown on plates at 20 OC under continuous light (75 µEm-2s-1 photosynthetically active radiation (PAR)) for 10-14 days. The seedlings were then transferred to soil (Sunshine Mix 4, SunGro, Kelowna, BC), fertilized with the AT medium and grown at 20OC under continuous light (90-110 µEm-2s-1 PAR). 	
   31	
   2.5.3 Transgenic seed wax ester analysis 100 transgenic seeds were ground with pestle and mortar to a white fine powder and chloroform was used to extract the lipid of the seeds. The lipid extract was then concentrated under gentle nitrogen stream to 50 µL. The whole sample was loaded onto a TLC plate and the hexane- diethylether-acetic acid (90:7.5:1) solvent system was used to separate the wax esters and triacylglycerols. Hexadecyl palmitate (C16:0-C16:0) and vegetable oil are used as reference standards to determine the locations of wax ester and triacylglycerol fractions on the TLC plate. After the run, 5 µg of hexadecyl palmitate was added to the spot of wax ester fraction of the sample as an internal standard for quantitative analysis. The wax ester fraction was scraped from the TLC plate and extracted with chloroform for at least 2 hour. The sample was then concentrated under the nitrogen stream to 50 µL and analyzed by GC-FID. 2.5.4 Identification and quantification of the jojoba-type wax ester produced by transgenic seeds The mass spectrum library of the National Institute of Standards (NISTO2; in MSD ChemStation software) was used to identify the unsaturated wax esters. The most similar structures that can be used in the library as references are: 9-hexadecenoic acid, 9-hexadecenyl ester (C16:1-C16:1), palmitic acid, 9-hexadecenyl ester (C16:0-C16:1), and 9-hexadecenoic acid, hexadecanyl ester (C16:1-C16:0). The signature fragment ions provided by these three structures were used to identify the wax ester isomers from the transgenic seeds. To determine the percentage of the different wax ester isomer species within the same wax ester homologues, the signals of prominent fragmented signature ions were used to divide the total signals to give the percentages (explained in detail in Chapter 3). 	
   32	
   2.5.5 Gene expression analysis by the β-glucuronidase (GUS) gene reporter system Flowers, siliques, stem, and leaves of the transgenic plant containing WS7Promoter::GUS construct were removed and immersed in GUS solution containing 100 mM phosphate buffer pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.5% Triton X-100 and X- gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide, 1mg/mL) for 6 hours. The tissues were then cleared with 70% ethanol and examined under the dissecting microscope. 	
   33	
    3 Jojoba-type wax ester production in transgenic Arabidopsis thaliana seeds expressing the jojoba KCS, FAR, and WS 3.1 Introduction The jojoba seeds contain long chain di- or mono-unsaturated wax esters with chain lengths between C36 and C48, with the predominant molecular species being C40 (30% of total wax ester), C42 (50%), and C44 (10%).  The acyl-chains of the wax esters are mainly composed of C18:1 (10%), C20:1 (71%), and C22:1 (13%), while C20:1 (44%) and C22:1 (45%) moieties are the main alcohol species (Miwa, 1984). The genes that are involved in the biosynthesis of these chemicals were characterized from jojoba seed cotyledons where the wax esters are stored (Lassner et al, 1996; Metz et al, 2000; Lardizabal et al, 2000). The jojoba KCS is responsible for elongation of the fatty acyl-CoA to 20 and 22 carbons, generating C18:0, C20:0, C18:1, C20:1, and C22:1 fatty acyl- CoAs (Lassner et al, 1996). The jojoba FAR reduces the pool of acyl-CoA to the corresponding fatty alcohols (Metz et al, 2000). Finally, the jojoba WS ligates fatty acyl-CoA and primary alcohols to generate wax esters (Lardizabal et al, 2000). One of the goals of my MSc project is to introduce these three genes into Arabidopsis thaliana seeds and determine if the jojoba-type wax esters can be made, as well as the levels of the jojoba-type wax esters that can be generated in Arabidopsis thaliana seeds.  3.2 Results and discussion 3.2.1 A. thaliana transformation and identification of transgenic seeds The plant expression constructs containing the jojoba KCS, FAR and WS genes pBinGlyRed- FWS1 (FWS1) and pBinGlyRed-FWS3 (FWS3) (Figure 5) were a gift from Dr. Edgar Cahoon, 	
   34	
   (University of Nebraska - Lincoln). The difference between these two constructs is the reverse orientation of the jojoba KCS in the FWS1 in comparison with the FWS3. The empty vector termed pBinGlyred (V) was used as negative control.  Figure 5: The plant wax ester expression constructs pBinGlyRed-FWS1 (FWS1) and pBinGlyRed-FWS3 (FWS3) generated by Dr. Edgar Cahoon (University of Nebraska – Lincoln).  These constructs were introduced into A. thaliana by Agrobacteriaum-mediated transformation, and transgenic seeds carrying the DsRed selectable marker were identified  based on their red fluorescence (583 nm) when excited with green light (558 nm) (Figure 6). The DsRed positive seeds were hand-picked for the seed oil analysis. 	
   35	
    Figure 6: DsRed-positive transgenic seeds of A. thaliana carrying pBinGlyRed empty vector (V), pBinGlyRed-FWS1 (FWS1) or pBinGlyRed-FWS3 (FWS3). The picture was taken using fluorescence Stereomicroscope Leica MZ16 FA with the TXR filter  3.2.2 TLC analysis of T2 transgenic seeds To measure the wax ester level, the seed oil was extracted from the DsRed-positive seeds (100 seeds; T2 generation; Figure 6), and TLC was used to separate different classes of seed oil (Figure 7). Most of the T2 transgenic lines showed increases in wax ester accumulation in comparison with the background levels detected in the negative controls, Columbia wild type and empty vector (V) lines. Comparing the DsRed signals from the seeds (Figure 6) to the wax ester accumulation on TLC (Figure 7), there was no direct correlation observed. The wax ester fraction was extracted from TLC and quantified by GC-FID. 	
   36	
    Figure 7: TLC analysis of wax ester accumulation in the seeds of Columbia wild type (ColWt), transgenic Arabidopsis containing pBinGlyRed empty vector (VB1, VB4, and VB12), pBinGlyRed-FWS1 (FWS1-B12, B13, B20, B26, B27, and S3), and pBinGlyRed-FWS3 (FWS3-UB4, UB5, UB6, UB7, UB10, UB14, US1). TLC silica gel 60 was used. The solvent system was hexane:ethyl ester:acetic acid= 90:7.5:1.  3.2.3 GC-FID quantification After the TLC data were recorded, 5µg of hexadecyl palmitate (C32:0) were added to the wax ester spot as internal standard for later quantification. The wax ester fractions were then extracted with chloroform and analyzed by GC-FID. All the wax ester peaks were quantified by comparison to the peak area of hexadecyl palmitate. Columbia wild type seeds and transgenic lines containing empty vectors (negative controls) contained low amounts of endogenous unsaturated and saturated wax esters with chain lengths of 36, 38, and 40 carbons (Figure 8). These compounds were confirmed by GC-MS analysis. These are likely the background levels seen on the TLC plate (Figure 7). Only one transgenic line, FWS1-B13, showed a significant increase of unsaturated wax esters with carbon chain lengths of 36 and 38. Most transgenic lines showed increases of unsaturated wax esters 40, 42, and 44 carbons long, except FWS1-B26 that contained only background levels of wax esters (Figure 8). Interestingly, in contrast to the pBinGlyRed-FWS1 seeds, all the transgenic seeds that received pBinGlyRed-FWS3 constructs contain unsaturated wax esters 46 and 48 carbons long. Our results also demonstrated that transgenic lines expressing the jojoba KCS, FAR, and WS produced more unsaturated than saturated wax esters. 	
   37	
    Figure 8: The GC-FID analysis of wax ester accumulation in 100 seeds of Columbia wild type (ColWt), transgenic Arabidopsis containing pBinGlyRed (VB1, VB4, and VB12), pBinGlyRed-FWS1 (FWS1-B12, B13, B20, B26, B27, and S3), and pBinGlyRed-FWS3 (FWS3-UB4, UB5, UB6, UB7, UB10, UB14, US1). C36:0 standard is the wax ester with 36 carbons and 0 double bonds.  3.2.4 Identification of unsaturated wax esters by GC-MS To identify the wax ester composition, the wax esters usually underwent transmethlyation and derivatization with trimethylsilyl compounds, and this method gave the information of acyl- and alcohol- chain of the total wax ester, but can not indicate which acyl or alcohol chain each wax esters were composed of (Miwa, 1984; Spencer et al, 1976). In our experiment, the wax ester pool of the transgenic seeds was directly analyzed by the GC-MS, and we know the detailed acyl and alcohol chain composition of each wax ester. To identify the unsaturated wax esters, the mass spectrum library of National Institute of Standards (NISTO2) installed in MSD ChemStation software was used. The closest structures 	
   38	
   that can be used in this library as references are: 9-hexadecenoic acid, 9-hexadecenyl ester (C16:1-C16:1), palmitic acid, 9-hexadecenyl ester (C16:0-C16:1), and 9-hexadecenoic acid, hexadecanyl ester (C16:1-C16:0). In this report, I used the suspected C42:2 and C42:1 wax esters from FWS3-UB4 transgenic line as examples to show how I confirmed that they are the unsaturated wax esters. The mass spectrum of the 9-hexadecenoic, 9-hexadecenyl ester (C16:1-C16:1) showed two signature ions, RCO+ and [R’-1]+, with the M/Z of 237 and 222, (Figure 9a). The suspected C42:2 wax esters from my samples showed the M/Z of 293 and 321, both of which are acyl- fragmented ions RCO+ from C20:1-C22:1 and C22:1-C20:1 wax esters (Figure 9b). In addition, the wax ester peaks also showed the M/Z of 306 and 278, representing the alkyl-fragmented ion [R’-1]+ from C20:1-C22:1 and C22:1-C20:1 wax esters (Figure 9b). The fragmented ions of RCO+ and [R’-1]+ and the approximate ratio of these two ions showed that this suspected wax ester was indeed a wax ester with at least two different isomers. Using the same principle, I confirmed the other unsaturated wax esters from my samples as C40, C44, C46, and C48 species with 2 unsaturations. 	
   39	
    Figure 9: Mass spectrum of the standard 9-hexadecenoic, 9-hexadecenyl ester from NISTO2 (a) and the presumed C42:2 wax ester from the transgenic T2 line FWS3-UB4 (b). R stands for the fatty acyl-chain, and R’ stands for the fatty alcohol chain  When the unsaturation occurs only in the alkyl moiety of wax esters, the fragmentation yields [R’-1]+, RCO2H2+, and RCO+ ions (Figure 10a). When the unsaturation occurs only in the acyl moiety of wax esters, the fragmentation yields [RCO-1]+ and RCO2H2+ ions (Figure 10b). The suspected C42:1 wax esters in my sample showed the expected fragmented ions and the similar ion ratio to the standard (Figure 10c), so I conclude that this is a monounsaturated wax ester. In a 	
   40	
   similar fashion, I identified all the wax esters from my samples C40, C42, C44, C46, and C48 species with 1 unsaturation.  Figure 10: Mass spectrum of the standard palmitic acid, 9-hexadecenyl ester (a), 9-hexadecenoic acid, hexadecanyl ester from NISTO2 (b), and the presumed C42:1 wax ester from the transgenic T2 line FWS3-UB4 (c). R stands for the fatty acyl-chain, and R’ stands for the fatty alcohol chain  	
   41	
   3.2.5 Determination of isomer composition of unsaturated wax esters by GC-MS To complete the compositional analyses of wax esters in transgenic lines, I needed to determine the percentage of different wax ester isomers within a certain wax ester homologue species. For example, C42:2 wax ester contains several different isomer species including C20:1-C22:1 and C22:1-C20:1, and I was interested to know the percentage of these two isomers within the C42:2 wax ester. The standard (Figures 9a, 10a, and 10b) showed that the wax esters with the unsaturations located on acyl or alkyl chain gave different predominant signature ions (Table 5). I used the signals of these signature ions to represent the amount of the particular wax ester isomer, and then divided them by the sum of the total signals detected in the particular wax ester homologue to get the composition percentage (Supplemental table 1).  For example, to determine the percentage of C20:1-C22:1 isomer in C42:2 wax ester homologue, I took 69288, the signal of RCO+ C20:1 acyl-chain, to represent the amount of C20:1-C22:1 species, and divided it by 120459, the sum of all the signals of RCO+ with different carbon chain length. Using this approach, I calculated that C42:2 wax ester is made up of 57.52% of C20:1-C22:1 wax ester. Table 5: Different wax esters and the signature ions used for isomer composition determination  Wax ester containing unsaturated fatty acid and unsaturated fatty alcohol Wax ester containing only unsaturated fatty acids Wax esters containing only unsaturated fatty alcohol Most abundant signature ion RCO+ [RCO-1]+ [R’-1]+  The isomer composition of the wax esters of interest, C40:2, C40:1, C42:2, C42:1, C44:2, C44:1, C46:2, C46:1, C48:2, and C48:1, from FWS-B12, FWS1-B13, FWS3-UB4, and FWS3-UB10 is shown in Table 6. The detail percentage of each wax ester isomer is in Supplemental table 1. My results are in agreement with the reported substrate specificity of the jojoba wax ester synthase, which shows preference for unsaturated fatty acids and alcohols with 20 and 22 carbon chain 	
   42	
   lengths (Lardizabal et al, 2000). The determined carbon chain length profiles also agreed with the jojoba wax ester profiles (Spencer et al, 1977). My results showed that the introduction of the enzymes involved in jojoba wax ester biosynthesis into the seeds of Arabidopsis thaliana lead to the accumulation of long chain unsaturated jojoba-type wax esters  (C40:2, C40:1, C42:2, C42:1, C44:2, and C44:1) in the seeds. Table 6: Summary table of isomer compositions of the observed long chain unsaturated wax esters in the transgenic line FWS3-UB4 	
   43	
    4 Enzymatic activity of the Arabidopsis thaliana jojoba-type WS homologues 4.1 Introduction The only tissue of A. thaliana for which the wax ester composition and content have only been reported is the stem epidermis. These wax esters present in cuticular waxes are generated by WSD1, a homologue of the bifunctional bacterial-type WS/DGAT (Li et al, 2008). Besides WSD1, there are 10 more WSD-type genes in A. thaliana and 12 genes encoding enzymes that share high amino acid sequence similarities with the jojoba-type WS, which gives rise to the question whether they are wax ester synthases and what their biological functions are. I am interested in the jojoba-type WS homologues, and they are abbreviated as WS1~WS12 (their corresponding gene loci are listed in table 1). WS1~3 are clustered on chromosome 1, WS6~12 are clustered on chromosome 5 within <1.5 kb from each other, whereas WS4 and WS5 are located on chromosome 3 and 5, respectively. All these genes share high nucleotide similarities (>54.1%), and several of them are expressed in reproductive organs, suggesting that they may have redundant functions (Table 1; Klypina and Hanson, 2008). With this in mind, I decided to use a biochemical approach to study the functions of these WS enzymes in A. thaliana. Accordingly, the second goal of my MSc project was to express the proteins in E. coli and yeast and investigate their enzymatic activities, in vitro and in vivo.  	
   44	
   4.2 Results and discussion 4.2.1 Bacterial expression To date, the expression of jojoba WS in E. coli has never been successful because the protein contains 9 transmembrane domains and the prokaryotic bacterial system does not have the sophisticated membrane system to accommodate this type of protein structure (Lardizabal et al, 2000). In an attempt to increase the expression of the membrane-associated jojoba-type WS and its Arabidopsis thaliana homologues in a bacterial system, we decided to make fusion proteins with maltose binding protein (MBP), a highly soluble protein that might solublize the highly membrane-associated protein in the bacterial cytoplasm (Fox and Waugh, 2003). Initially, the bacteria were grown at 37OC, but the MBP-jojoba WS fusion protein (positive control) did not accumulate (Figure 11). When the bacteria were grown at 16OC, the MBP-jojoba WS protein was present. However, the presence of multiple bands and smears in several different trials suggested that the fusion protein was produced in an aggregated form. 	
   45	
    Figure 11: Western blot of the soluble protein extract from BL21(DE3)codon+ cells containing the pMALc2x or pMALc2x-Jojoba WS  expression constructs grown at 37 and 16 OC. Anti-MBP antibody was used for fusion protein detection. The “-” indicates the non-induced sample while “+” indicates the induced sample by adding isopropyl β-D-1- thiogalactopyranoside (IPTG) into the medium. The band at 55 KDa (red dot) of the induced pMAL-c2x is the fusion protein of maltose binding protein (MBP) with the lacZ-alpha domain. The blue dot seems to correspond to the sum of the sizes of the jojoba WS and MBP (75KDa= 42KDa+ 33KDa).  In order to produce the fusion protein in a properly folded form, bacteria were grown in culture media containing sorbitol and betaine, glycerol, sucrose, tryptose phosphate, or an acidic medium. However, the protein expression profile appeared to be the same as when the cells were grown in a regular medium (Figure 12). 	
   46	
    Figure 12: Optimization of protein expression: Western blot of the soluble protein extract from BL21(DE3)codon+ cells containing the pMALc2x or pMALc2x-jojobaWS expression construct grown at 16OC. Anti-MBP antibody was used for fusion protein detection. Bacteria containing the jojoba WS expression construct were grown in different media, with added sorbitol and betaine, glycerol, and sucrose. They were also grown in a more acidic medium, and tryptose phosphate medium. The MBP-jojoba WS protein expression was not improved  To determine the WS activity of the jojoba WS and the jojoba-type WS enzyme family in A. thaliana, I adopted the 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) colorimetric assay. This assay is a safer and cheaper technique compared to using a radiolabelled substrate. During formation of a wax ester, the WS releases CoASH from acyl-CoA (Figure 13). This free CoASH forms a complex with one of the thio-nitrobenzoic parts and releases the 2-nitro-5-thiobenzoate (TNB-) ion (Srere et al, 1963). The TNB- anion ionizes to TNB2- in water which results in production of yellow colour, which can be quantified spectrophotometrically at 412 nm with an extinction coefficient of 13600 (Figure 13; Srere et al, 1963). 	
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    Figure 13: Chemical reaction catalyzed by wax ester synthase. Wax ester is formed by the esterification of acyl-CoA and primary alcohol and a free CoASH is released (a). The released free CoASH interacts with 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) and releases 2-nitro-5-thiobenzoic acid (TNB-). The TNB- ionizes in the water and forms TNB2-, the concentration of which can be measured at 412 nm with the coefficient at 13600  The biochemical colorimetric assay on the soluble cell fraction using DTNB as reagent showed that the soluble protein extracts had no enzyme activity (Figure 14). Based on combined data from the biochemical assay and western blotting, I conclude that the MBP-jojoba WS fusion protein was produced in bacteria in aggregated form with no enzyme activity. 	
   48	
    Figure 14: Biochemical colorimetric assay using DTNB as a reagent on the soluble protein extract from the bacteria transformed with pMALc2x or pMALc2x-jojobaWS expression construct. The reaction containing only fatty acyl-CoA (C16:0) serves as the negative control for the reaction containing both fatty acyl-CoA (C16:0) and alcohol (C16:0-OH). Colorimetric assay did not show any difference between the two samples.  4.2.2 S. cerevisiae expression Since the bacteria did not produce a soluble jojoba WS (positive control) with detectable enzyme activity, I decided to use a mutant yeast strain H1246, which has insertions disrupting the ARE1, ARE2, LRO1, and DGA1 genes (Sandager et al, 2002) as a heterologous system to produce WS proteins for enzyme activity assays. ARE1 and ARE2 encode acyl-CoA: sterol acyltransferases (ASATs), DGA1 encodes a diacylglycerol acyltransferase (DGAT) enzyme, and LRO1 encodes phospholipid:diacylglycerol acyltransferase (PDAT), an enzyme involved in acyl-CoA- independent TAG biosynthesis discovered in plants and yeast. Consequently, yeast mutant H1246 lacks all the major neutral lipids including triacyglycerols and sterol esters and represents an excellent system for detection of wax esters. The substrates chosen for the assay were palmitic acids (C16:0) and hexadecanol (C16:0-OH), because jojoba WS has been shown to have the enzyme activity towards these two substrates (Lardizabal et al, 2002) 	
   49	
   Initially, I had a problem with the activity assays, most likely because I used excessive amount of cells. As a result I detected wax ester bands even in H1246 mutant and the cells carrying empty pESC-URA vector, both of which serve as negative controls. A reduction in the amount of freeze-dried cells used for lipid extraction to 25 mg and changing the extraction system from using chloroform (Li et al., 2008) to using hexane allowed me to detect the enzyme activities of jojoba-type WS homologues (Figure 15). Using this assay, I showed for the first time that the jojoba WS is expressed as a functional enzyme in yeast (Figure 15). WS7 was the only homologue which accumulated wax esters to the level that can be detected by TLC (Figure 15). There were also bands detected in WS4, but this enzyme has been shown to produce lanosterol ester, which elutes at the same retention time as wax esters on TLC (Chen et al, 2007). The wax ester accumulation from the transgenic yeast mutants with pESC-URA-WS1, WS2, WS5, WS6, WS8, WS9, WS10, WS11, and WS12 was not detectable by TLC (Figure 15 and Supplemental figure 1). 	
   50	
    Figure 15: Neutral lipid analysis of mutant yeast strain H1246 and the H1246 strain transformed with pESC-URA, pESC-URA-WS2, WS4, WS5, WS7, WS12, and jojoba WS expression vectors. The “-” indicates that the cells were grown in the medium without the addition of fatty acid or alcohol, and the “+” indicates that the cultures were grown in the medium with fatty acid and alcohol. WS4 and WS7 appeared to have more activity than other homologues. The lipid fractions of transgenic yeast transformed with constructs pESC-URA-WS1, WS6, WS8, WS9, WS10, and WS11 were analyzed in the same way and showed no wax ester accumulation. TLC silica gel 60 was used. The solvent system was hexane:ethyl ester:acetic acid= 90:7.5:1. TLC data of pESC-WS1, 6, 8, 9, and 10 are in Supplemental figure 1.  The GC-FID showed the wax ester profiles of different jojoba-type WS homologues and the jojoba WS (Figure 16). Jojoba WS transfers endogenous C16:1, C18:1, and C18:0 acyl-chain to hexadecanol in addition to the C16:0 acyl-chain that was fed. This finding corresponded to the substrate specificities of jojoba WS (Lardizabal et al, 2000). There was no band in the WS12 sample, but GC-FID showed that the transgenic yeast carrying this gene accumulated more wax esters than the mutant H1246 or empty vector control (Figure 16). In addition to the lanosterol ester, WS4 also produced wax esters, showing the promiscuous enzyme activity of this protein. WS7 has the most obvious wax ester signal on the TLC assay and contain both saturated and unsaturated wax ester (Figure 16). 	
   51	
    Figure 16: GC-FID analysis on the wax ester fraction extracted from the TLC. Yeast strain H1246 expressing different enzymes produces different wax ester profiles even when fed with the same fatty acid and primary alcohol.  After the initial biochemical screening, WS4 and WS7 were picked for the detailed studies of their substrate specificities. WS12 was also selected because it is expressed in all the organs and more likely to have a biochemical function (Klypina and Hanson, 2008). When fed with primary 	
   52	
   alcohols of different chain lengths and palmitic acid, none of these three homologues showed the accumulation of wax esters (Figure 17). The bands shown at WS4 samples are lanosterol esters.  Figure 17: Neutral lipid accumulation in mutant yeast strain H1246 expressing WS4, WS7 and WS12 after feeding with palmitic (C16:0) fatty acid and primary alcohols of various chain lengths. Neither WS4 nor WS12 produce any wax esters with the alcohols tested. The bands in WS4 sample were identified as lanosterol esters by GC-FID. The data of WS7 was not shown because its TLC data was identical to that of WS12. TLC silica gel 60 was used. The solvent system was hexane:ethyl ester:acetic acid= 90:7.5:1.  WS7 was shown to accumulate wax esters when fed with fatty acids of different chain lengths, but the GC-FID analysis showed that in all cases they produced mainly the unsaturated wax ester with C16:1 as an acyl-chain (Figures 18 and 19). Palmitoleic acid (C16:1) is one of the major endogenous fatty acids in yeast, and WS7 seemed to have preferential enzyme activity toward this substrate. The endogenous C16:0, C18:1, and C18:0 fatty acid in yeast were also used by WS7 to make wax esters, but the enzyme activity of WS7 toward theses substrate is lower compared to C16:1 fatty acid. The endogenous C16:1, C16:0, C18:1, and C18:0 fatty acids were also used by both WS4 and WS12, but WS12 showed much weaker activity compared to WS4 (Figure 19). Both WS4 and WS12 can make a compound that has the same TLC retention time as wax ester when fed with C26:0 	
   53	
   fatty acid and C16:0 primary alcohol (Figure 19). This compound had the retention time of 40 minutes analyzed by GC-FID, but could not be identified by NISTO2 library.  Figure 18: Neutral lipid accumulation in mutant yeast strain H1246 expressing WS4, WS7 and WS12 after feeding with fatty acids of various chain lengths and hexadecanol (C16:0). There were wax ester bands observed in the in WS4 and WS7 expressing yeast. There was also a band observed when the yeast expressing WS12 was fed with C26:0 fatty acid and hexadecanol. TLC silica gel 60 was used. The solvent system was hexane:ethyl ester:acetic acid= 90:7.5:1.   Figure 19: GC-FID analysis of the wax ester fractions of three different transgenic yeast experiment samples extracted from the TLC. For WS7, the chromatogram of the sample that was fed with C30:0 fatty acid and C16:0 primary alcohol was shown. WS7 seemed to have preferential activity toward C16:1 fatty acids. WS4 and WS12 sample fed with C26:0 fatty acid and C16:0 alcohol were shown. In both WS4 and WS12, there was a compound that eluted at 40 minutes that could not be identified by NISTO2 library. WS4 and WS12 can also use endogenous fatty acids like WS7, but WS4 has more activity. 	
   54	
   The limitation of the feeding experiment is the accessibility of the wax ester synthase toward very long fatty acids or fatty alcohol. As the carbon chain lengths increase, the solubility of the substrates in the aqueous environment and the uptake by yeast decrease. This indicates that no accumulation of wax ester from the transgenic yeast carrying WS4, WS7, and WS12 fed with long fatty acid and primary alcohols could also be due to no accessibility of these enzymes to the substrates. Thus, it is hard to conclude that these three enzymes have no WS activities toward longer chain length of fatty acid and primary alcohols. This limitation can be overcome by using isolated micromal membrane fraction and adding the substrate of very long chain fatty acyl-CoA and primary alcohol (C22-30). The microsomal membrane preparation and the biochemical assway used in Lardizabal et al (2000) can be modified for yeast and considered to be a replacement of my yeast feeding experiment. 	
   55	
    5 Arabidopsis thaliana WS7 expression patterns 5.1 Introduction Because our results demonstrated that WS7 was a functional wax synthase, and that the WS7 (At5g55330) gene had a fairly narrow expression pattern with expression detected only in the stems, flowers and siliques (Klypina and Hanson, 2008), I was interested in determining more accurately its tissue specificity as a first step towards determining the biological function of this enzyme in the Arabidopsis.  A report by Wang et al, 2004 suggested that WS7 contains a homeodomain binding L1 box and a MYB binding motif that direct trichome-specific expression of this gene, but the supporting evidence for this claim has not been presented. I therefore decided to generate transgenic plants expressing the WS7promoter:GUS construct and carry out the histochemical analysis of GUS activity to define the expression domains of WS7. 5.2 Results and discussion With the time available for this experiment, I only had a chance to examine GUS activity in the T1 transgenic plants carrying the WS7promoter:GUS transgene. I collected rosette leaves, siliques, flowers, and the top 2 cm of the stems from 24 different T1 transgenic lines, and found 11 of them had consistent GUS expression pattern. My results show that WS7 is not expressed in the leaf trichomes as proposed by Wang et al. (2004), but in the leaf veins and hydathodes (Figure 20a). I observed the expression of WS7 in rosette leaves based on my GUS reporter gene assay, which was not seen in the previous RT-PCR data (Table 1: Klypina and Hansen, 2008). The different developmental stage of leaves used in my experiment could be the reason for the discrepancy. In addition, I detected WS7 expression in the floral organs and siliques, confirming the RT-PCR data of Klypina and Hanson, 2008 (Figure 20c and 20d). Specifically, the WS7 was 	
   56	
   only expressed in the sepals, and the gynoecium of the flower, at both ends of the young silique, and throughout the old silique. Interestingly, I also found WS7 to be expressed in the stem, but this result needs to be confirmed by additional experiments.  Figure 20: Organ and tissue-specific expression patterns of WS7 detected in WS7promoter:GUS transgenic lines. (a) leaves, (b), flower organs (c), siliques, and  (d), stems. 	
  Finding	
  by	
  the	
  GUS	
  expression	
  data,	
  WS7	
  is	
  expressed	
  in	
  the	
  expanding	
  area	
  of	
  the	
  leaves,	
  flowers	
   and	
   siliques,	
   all	
   of	
   which	
   were	
   not	
   known	
   to	
   accumulate	
   cuticular	
   wax	
   esters.	
  However,	
  as	
  theses	
  organs	
  are	
  expanding,	
   their	
  cells	
  might	
  require	
  acyltransferases,	
  such	
  as	
  the	
  gene	
  product	
  of	
  WS7,	
  to	
  transfer	
  acyl-­‐chains	
  from	
  acyl-­‐CoAs	
  to	
  the	
  alcohol	
  functional	
  group	
  during	
  cutin	
  biosynthesis.	
  The	
  protein	
  of	
  WS7	
  might	
  also	
  transfer	
  acyl-­‐chains	
  from	
  acyl-­‐CoAs	
  to	
  monoacylglycerol,	
  glycerol-­‐3-­‐phosphate,	
  and	
  lysophatic	
  acid	
  for	
  downstream	
  lipid	
  metabolism.	
   The	
  WS7	
   expression	
  was	
   also	
   observed	
   on	
   the	
   top	
   of	
   the	
   stem,	
  where	
  cuticular	
   wax	
   ester	
   biosynthesis	
   takes	
   place.	
   However,	
   the	
   enzyme	
   responsible	
   for	
   the	
   	
   57	
   cuticular	
   wax	
   ester	
   production	
   was	
   determined	
   to	
   be	
   WSD1	
   (Li	
   et	
   al,	
   2008).	
   My	
   GUS	
  expression	
  data	
  did	
  not	
  indicate	
  whether	
  WS7	
  was	
  specifically	
  expressed	
  in	
  the	
  epidermis,	
  so	
  sectioning	
  of	
  the	
  stem	
  coupled	
  with	
  GUS	
  assay	
  will	
  give	
  a	
  better	
  insight	
  into	
  the	
  tissue	
  specificity	
  of	
  WS7	
  in	
  the	
  stem.	
   	
   	
   58	
   	
   6 Conclusions and future directions The first objective of my MSc project was achieved. The ectopic expression of the jojoba wax ester biosynthetic genes KCS, FAR, and WS in A. thaliana seeds did result in the production of the long chain unsaturated jojoba-type wax esters  (C40:2, C40:1, C42:2, C42:1, C44:2, and C44:1), which are not found in A. thaliana seeds (Chapter 3). The findings of this experiment agree with the previously reported substrate specificity of the jojoba wax ester synthase and the jojoba wax ester compositional profile in jojoba seeds (Lardizabal et al, 2000; Spencer et al, 1997) The second objective of the project is ongoing. With the results that I generated so far I can conclude that: 1) WS4 is a bifunctional enzyme which has the ability to produce phytosterol esters, but can also produce wax esters when the primary alcohol is provided (Figure 15); 2) WS7 is an acyltransferase which can transfer C16:0 and C16:1 acyl-chains to C16:0 primary alcohol to make wax esters (Figures 15, 18, and 19). From the wax ester fractions of the transgenic yeast expressing the WS7 gene fed with different chain lengths of fatty acids (C16, C20, C28, and C30), the wax esters with the C16:1 acyl-chain were constantly observed (Figures 18 and 19). This suggests that WS7 utilizes the endogenous C16:1 acyl group. However, whether WS7 enzyme has preferential substrate specificity toward C16:1 acyl-chain still remains an open question due to the limitations of the in-vivo feeding experiment (Chapter 4). The in-vitro microsomal biochemical assay should be considered as an alternative to my yeast in-vivo feeding experiment. As discussed in chapter 4, the limitation of the yeast feeding experiment was the solubility of very long chain fatty acids and primary alcohols (C22-30) in aqueous growth medium, and the uptake of the substrates by the yeast. Another problem may be the specificity of the yeast long-chain acyl-CoA synthetase (LACS), which cannot use very long chain acyl-CoAs as substrates. This would preclude yeast from utilizing very long chain fatty 	
   59	
   acids for the production of wax esters. With an optimized biochemical assay which ensures the solubility of very long chain fatty acyl-CoAs and primary alcohols (Lardizabal et al, 2000), the tested WS will have better accessibility toward both substrates, so it will be possible to more accurately determine the substrate specificities of WS enzymes. The true biological function of WS7 remains to be determined. Preliminary substrate specificity assays and the GUS assay revealed that the enzyme can catalyze a transfer of C16:1 and C16:0 acyl-chains from acyl-CoAs to primary alcohols, and that it is expressed in the expanding regions of leaves, stem, flower and siliques. WS7 might be involved in lipid metabolism to fuel the cells in those tissues during organ expansion, or transfer acyl-chains to cutin matrices to ensure that the expanding cells are covered by cuticles. GUS expression data should be confirmed in T2 transgenic plants, and different developmental stages of plants should be examined for a better speculation. The ws7 mutant that has a T-DNA insertion in the At5g55330 gene has also been isolated and should be studied for morphological and lipid metabolic changes in the organs where the WS7 promoter activity was found through GUS assay. This work should allow us to understand the true function of WS7. Yeast enzyme assay established in this project coupled with Nile red staining can be used in the future to evaluate the activity levels of new wax ester synthases. A similar experiment using Nile red to test the function of different variants of DGAT enzymes has shown that the fluorescence intensity after the dye binds to triacylglycerols correlates well with the activity of the enzyme (Siloto et al, 2009). Nile red was proposed to stain neutral lipids, including wax esters, and the fluorescence staining can be observed at 505-555 nm when excited at 465 nm (Siloto and Weselake, 2010). A huge collection of transgenic yeast H1246 cell carrying putative WS enzymes can be grown in separate small volumes of growth medium as shown in my in-vivo feeding assay. Instead of harvesting the cells followed by lipid analysis to test the enzyme 	
   60	
   activity, a small volume of each sample can be placed on multi-well plate flouorimeter, incubated with Nile red, and scanned for the samples that have high fluorescence intensities. This method would provide a high-throughput system to search for the new wax ester synthases. Whether wax ester stained by Nile red has the same optimal excitation wavelength as triaclyglycerols is unknown, so determining the optimal excitation wavelength will help distinguish the different between WS and DGAT activity. Harvesting the cells followed by lipid analysis is still necessary to confirm the accumulation of wax esters. In addition, in an attempt to discover wax ester synthases with novel substrate specificities, the potential wax ester synthase activities of the known neutral lipid biosynthetic enzymes should also be explored. Based on the previous literature reviewed in Chapter 1 and the promiscuous enzyme activity of WS4 which can generate both phytosterol esters and wax esters, it is clear that the chemistry involved in transferring an acyl-chain to primary alcohols, and transferring an acyl-chain to diacylglycerols or sterols may be similar. It is therefore logical to suspect that the known acyl-CoA: diacylglycerol acyltransferase (DGAT), acyl-CoA: monoacylglycerol acyltransferase (MGAT), acyl-CoA: cholesterol acyltransferase (ACAT), and acyl-CoA sterol acyltransferase ASAT enzymes may also have wax ester synthase activities.   	
   61	
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   Appendix Supplemental table 1: Isomer composition of C40:2, 40:1, 42:2, 42:1, 44:2, and 44:1 wax esters from FWS1-B12, FWS1- B13, FWS3-UB4, and FWS3-UB10 transgenic lines. N.D. stands for not detected.  Fatty acid Alcohol FWS1B12 FWS1B13 FWS3UB4 FWS3UB10 C40:2 C16:1 C24:1 19.35 4.79 15.35 13.30   C18:1 C22:1 14.03 2.95 10.78 9.07   C20:1 C20:1 56.03 87.89 67.58 69.87   C22:1 C18:1 5.89 2.28 5.49 4.70   C24:1 C16:1 4.71 2.09 0.80 3.06 C40:1 C16:1 C24:0 43.36 17.16 33.82 21.28   C18:1 C22:0 2.80 5.80 4.22 5.17   C20:1 C20:0 14.08 34.36 29.32 39.53   C22:1 C18:0 3.32 0.67 1.88 1.21   C24:1 C16:0 0.88 0.60 0.03 0.00   C16:0 C24:1 22.71 15.90 27.13 15.92   C18:0 C22:1 4.05 1.01 0.00 0.71   C20:0 C20:1 3.32 14.52 0.58 10.62   C22:0 C18:1 3.92 8.02 1.27 5.26   C24:0 C16:1 1.56 1.95 1.75 0.29 C42:2 C16:1 C26:1 5.96 6.76 6.96 6.75   C18:1 C24:1 6.26 10.22 6.33 7.00   C20:1 C22:1 57.52 46.38 57.75 68.23   C22:1 C20:1 23.48 28.66 22.71 11.29   C24:1 C18:1 3.76 6.09 4.59 4.22   C26:1 C16:1 3.02 1.89 1.67 2.52 C42:1 C16:1 C26:0 0.37 2.91 3.19 3.17   C18:1 C24:0 14.26 9.22 14.92 17.01   C20:1 C22:0 38.54 38.87 44.00 40.73   C22:1 C20:0 8.48 0.35 1.42 0.49   C24:1 C18:0 1.81 3.68 2.04 0.08   C26:1 C16:0 1.38 0.30 0.24 0.43   C16:0 C26:1 3.76 0.42 1.82 1.32   C18:0 C24:1 0.00 1.08 1.06 0.70   C20:0 C22:1 0.00 0.31 0.00 0.00   C22:0 C20:1 17.79 22.50 17.22 24.39   C24:0 C18:1 12.38 16.49 10.94 11.07   C26:0 C16:1 1.23 3.88 3.16 0.60 C44:2 C16:1 C28:1 4.53 3.95 5.91 3.66   C18:1 C26:1 4.63 4.37 5.31 7.03   C20:1 C24:1 46.69 41.94 36.94 40.46   C22:1 C22:1 10.05 3.16 11.23 13.97   C24:1 C20:1 30.40 41.30 36.81 30.14   C26:1 C18:1 3.01 2.18 2.93 1.99   C28:1 C16:1 0.70 3.10 0.87 2.75 C44:1 C16:1 C28:0 0.23 1.42 1.75 2.79   C18:1 C26:0 0.82 1.17 2.54 1.67   C20:1 C24:0 46.60 16.78 41.96 40.84   C22:1 C22:0 0.00 0.33 0.00 0.00 	
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    Fatty acid Alcohol FWS1B12 FWS1B13 FWS3UB4 FWS3UB10   C24:1 C20:0 1.76 0.00 0.64 0.22   C26:1 C18:0 0.46 0.17 0.53 0.14   C28:1 C16:0 0.01 0.15 0.08 0.10   C16:0 C28:1 0.45 0.16 0.43 0.23   C18:0 C26:1 0.25 0.32 0.00 0.03   C20:0 C24:1 0.00 0.56 0.00 0.03   C22:0 C22:1 0.16 1.10 1.53 1.09   C24:0 C20:1 39.99 63.99 38.91 41.01   C26:0 C18:1 6.54 9.13 8.41 8.18   C28:0 C16:1 2.72 4.74 3.22 3.68 C46:2 C16:1 C30:1 5.56 N.D. 8.60 6.68   C18:1 C28:1 4.90 N.D. 4.82 3.28   C20:1 C26:1 7.41 N.D. 8.32 5.85   C22:1 C24:1 34.02 N.D. 34.01 35.83   C24:1 C22:1 36.30 N.D. 35.87 39.09   C26:1 C20:1 6.21 N.D. 3.10 4.24   C28:1 C18:1 2.75 N.D. 2.12 2.46   C30:1 C16:1 2.86 N.D. 3.16 2.57 C46:1 C16:1 C30:0 7.51 N.D. 4.84 4.42   C18:1 C28:0 3.69 N.D. 2.34 2.01   C20:1 C26:0 10.41 N.D. 7.26 6.03   C22:1 C24:0 6.48 N.D. 7.42 8.34   C24:1 C22:0 4.14 N.D. 3.48 2.29   C26:1 C20:0 1.01 N.D. 0.89 0.79   C28:1 C18:0 0.59 N.D. 0.20 0.09   C30:1 C16:0 0.70 N.D. 0.18 0.12   C16:0 C30:1 0.23 N.D. 0.14 0.21   C18:0 C28:1 0.23 N.D. 0.10 0.14   C20:0 C26:1 0.20 N.D. 0.18 0.21   C22:0 C24:1 3.73 N.D. 4.79 3.67   C24:0 C22:1 12.72 N.D. 20.75 28.83   C26:0 C20:1 39.82 N.D. 38.35 33.67   C28:0 C18:1 5.33 N.D. 5.29 5.29   C30:0 C16:1 3.23 N.D. 3.78 3.89 C48:2 C16:1 C32:1 N.D. N.D. 0.00 1.59   C18:1 C30:1 N.D. N.D. 23.20 10.90   C20:1 C28:1 N.D. N.D. 3.64 4.40   C22:1 C26:1 N.D. N.D. 8.37 5.35   C24:1 C24:1 N.D. N.D. 52.01 56.85   C26:1 C22:1 N.D. N.D. 1.62 9.94   C28:1 C20:1 N.D. N.D. 3.64 3.14   C30:1 C18:1 N.D. N.D. 4.47 4.69 	
   74	
    Fatty acid Alcohol FWS1B12 FWS1B13 FWS3UB4 FWS3UB10   C32:1 C16:1 N.D. N.D. 3.05 3.15 C48:1 C16:1 C32:0 N.D. N.D. 5.35 4.37   C18:1 C30:0 N.D. N.D. 3.76 3.80   C20:1 C28:0 N.D. N.D. 2.32 1.90   C22:1 C26:0 N.D. N.D. 1.73 1.32   C24:1 C24:0 N.D. N.D. 11.16 10.37   C26:1 C22:0 N.D. N.D. 1.58 1.46   C28:1 C20:0 N.D. N.D. 0.81 0.23   C30:1 C18:0 N.D. N.D. 0.36 0.15   C32:1 C16:0 N.D. N.D. 0.24 0.50   C16:0 C32:1 N.D. N.D. 0.03 0.34   C18:0 C30:1 N.D. N.D. 0.19 0.10   C20:0 C28:1 N.D. N.D. 0.50 0.33   C22:0 C26:1 N.D. N.D. 0.01 0.41   C24:0 C24:1 N.D. N.D. 40.55 36.61   C26:0 C22:1 N.D. N.D. 14.65 20.20   C28:0 C20:1 N.D. N.D. 11.59 12.39   C30:0 C18:1 N.D. N.D. 3.42 3.61   C32:0 C16:1 N.D. N.D. 1.73 1.92   Supplemental figure 1: Neutral lipid analysis of mutant yeast strain H1246 and the H1246 strain transformed with pESC- URA-WS1, WS6, WS8, WS9, WS10, and WS11 expression vectors. The “-” indicates that the cells were grown in the medium without the addition of fatty acid or alcohol, and the “+” indicates that the cultures were grown in the medium with fatty acid and alcohol. TLC silica gel 60 was used. The solvent system was hexane:ethyl ester:acetic acid= 90:7.5:1.

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