Identification of Plant Transcription Factors that Play a Role in Triacylglycerol Biosynthesis

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1 East Tennessee State University Digital East Tennessee State University Electronic Theses and Dissertations Identification of Plant Transcription Factors that Play a Role in Triacylglycerol Biosynthesis Parker Dabbs East Tennessee State University Follow this and additional works at: Recommended Citation Dabbs, Parker, "Identification of Plant Transcription Factors that Play a Role in Triacylglycerol Biosynthesis" (2015). Electronic Theses and Dissertations. Paper This Thesis - Open Access is brought to you for free and open access by Digital East Tennessee State University. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Digital East Tennessee State University. For more information, please contact dcadmin@etsu.edu.

2 Identification of Plant Transcription Factors that Play a Role in Triacylglycerol Biosynthesis A thesis presented to the faculty of the Department of Biological Sciences East Tennessee State University In partial fulfillment of the requirements for the degree Master of Science in Biology by Parker Dabbs May 2015 Aruna Kilaru, Ph.D., Chair Lev Yampolsky, Ph. D. Cecilia McIntosh, Ph. D. Keywords: avocado, triacylglycerol, lipid biosynthesis, mesocarp, WRINKLED

3 ABSTRACT Identification of Plant Transcription Factors that Play a Role in Triacylglycerol Biosynthesis by Parker Dabbs This work identifies transcription factors (TF) controlling triacylglycerol (TAG) synthesis and accumulation in plant tissues. TAG plays vital role in plants and are used by humans. Most plants accumulate oil in the seed, but some species accumulate oil in other tissues. The Wrinkled1 (WRI1) TF has been shown to regulate oil accumulation in multiple species and tissues. Here, four WRI homologues in avocado were identified, their phylogeny was examined and three of them were cloned into expression vectors for further characterization. However, WRI1 likely does not act alone in regulation of TAG accumulation in plants. Additional candidate TFs were identified by using transcriptome data from a variety of species, and cloned into expression vectors. Future studies will be able to use this information to better understand regulation of TAG accumulation, which will allow increased oil accumulation in plants for various human uses. 2

4 ACKNOWLEDGEMENTS I would like to thank my advisor and thesis committee chair Dr. Aruna Kilaru for her support during my time in the lab. I would also like to thank my committee members Dr. Lev Yampolsky and Dr. Cecilia McIntosh for their valuable guidance and input on the thesis project. I would also like to thank my fellow lab members for their help and assistance within the lab. I thank Dr. John Ohlrogge, Michigan State University, for providing the transcriptome data, and also acknowledge all the other individuals who worked to collect these data. I would like to thank Dr. Mary Lu Arpaia, University of California at Riverside, for providing the avocado fruits, and Dr. Sanjaya, Michigan State University, for Arabidopsis seeds. I thank the support in the form of the Graduate Assistantship and the Fraley s Memorial Award from the Department of Biological Sciences, East Tennessee State University (ETSU). I would also like to thank the ETSU School of Graduate Studies for providing funding through the Student Research Grant and The James H. Quillen Scholarship. Additional funding for this work was provided by the Sigma-Xi Grants-in-Aid of Research Award (2014) and ETSU Research Development Committee. 3

5 TABLE OF CONTENTS Page ABSTRACT... 1 ACKNOWLEDGEMENTS... 3 LIST OF TABLES... 7 LIST OF FIGURES... 8 Chapter 1. INTRODUCTION... 9 Triacylglycerol Synthesis Fatty Acid Synthesis Triacylglycerol Assembly Triacylglycerol Storage Transcription Factors Controlling TAG Biosynthesis Master Regulators of Seed Maturation WRI1 Transcription Factor TAG Production and Function in Various Plant Tissues The Roles of TAG in Plants TAG in Seed Tissues TAG in Non-seed Tissues Avocado Model Human Uses of Plant Oils Hypothesis, Rationale, and Specific Aims MATERIALS AND METHODS

6 Plant Material Extraction of RNA Cloning of Avocado WRI Homologues Construction of WRI Gene Phylogeny Data Analysis for Identification of Candidate Transcription Factors Cloning of Candidate Genes RESULTS Identification of Avocado WRI Homologues A Homolog of AtWRI1 Shows High Expression During Oil Accumulation in Avocado Mesocarp Homologues of WRI Gene Family Are Also Highly Expressed in Avocado Mesocarp.. 33 Three Avocado WRI Homologues Were Cloned for Future Validation of Their Role in Oil Biosynthesis WRI Phylogeny Shows Multiple Gene Duplications with WRI2 Appearing to be the Oldest Form of the WRI Gene Identification of Transcription Factors That May Play a Role in Oil Accumulation Transcription Factors That Were Differentially Expressed Between Oil-rich Seed and Non-seed Tissues were Identified Three Transcriptions Factors Were Selected for Cloning and Future Validation of Their Role in Oil Biosynthesis Cloning of Candidates for Validation of Their Role in Oil Biosynthesis DISCUSSION Three WRINKLED Homologues are Highly Expressed in Avocado Mesocarp Tissue

7 The WRINKLED Gene Family Appears to Have Undergone Multiple Duplications and Likely has Conserved Function Three WRINKLED Genes Highly Expressed in Avocado Mesocarp Cloned into Gateway Cloning System Transcription Factor Candidates Identified that May Play a Role in TAG Accumulation Three Candidate Transcription Factors Cloned into Gateway Cloning System CONCLUSIONS AND FUTURE DIRECTIONS REFERENCES APPENDICES Appendix A: R Scripts Used in Analyzing Transcriptome Data Appendix B: Entry Clone Sequences Obtained from Molecular Biology Core Facility VITA

8 LIST OF TABLES Table Page 1. Gene Specific Primers Used for Gateway Cloning Candidate Transcription Factors Identified by Transcriptome Analysis

9 LIST OF FIGURES Figure Page 1. A Schematic Pathway of Fatty Acid Synthesis in Plants A Schematic Pathway of Triacylglycerol Assembly in Plants Transcriptional Regulation of Triacylglycerol Synthesis in Plants Phylogenetic Tree of Species for Which Transcriptome Data Has Been Obtained Expression Levels for Avocado WRI Homologues Cloning of PaWRI1 into Entry Vector Cloning of PaWRI2 into Entry Vector Cloning of PaWRI3 into Entry Vector Phylogenetic Analysis of the WRI Family of Genes Cloning of C3H into Entry Vector Cloning of NAC014 into Entry Vector Cloning of OZF into Entry Vector

10 CHAPTER 1 INTRODUCTION Triacylglycerols (TAG) are a class of lipid molecules composed of three fatty acyl chains esterified to a glycerol backbone. Many plants produce substantial amounts of these compounds in various tissues and for different purposes. Humans also utilize the TAG produced by plants for food products, supplements, and a multitude of industrial applications (Durrett et al. 2008;Carlsson 2009). Due to the increasing utilization of these plant products, many people today are interested in increasing TAG production in different plants to produce greater quantities. However in order to increase the yield of TAGs in plants, the pathway that produces these compounds and the mechanisms that control it must be better understood. Arabidopsis thaliana has been used as a model plant to study this pathway and its control mechanisms, and it has been utilized to understand many of the important factors (Harwood 2005;Baud andlepiniec 2009;Weselake et al. 2009). In Arabidopsis seeds the control of TAG production and storage is directly regulated by WRINKLED1 (WRI1) (Focks andbenning 1998;Cernac andbenning 2004), with master regulators of seed maturation such as FUSCA3 (FUS3), LEAFY- COTYLEDON 1 (LEC1) and 2 (LEC2), LEC1-Like (L1L), and ABSCIC ACID INSENSITIVE 3 (ABI3) acting upstream of WRI1 (Meinke et al. 1994;West et al. 1994;Kagaya et al. 2005;Mu et al. 2008;Yamamoto et al. 2010). WRI1-like genes have also been shown to have similar roles in regulating oil accumulation in other plant seeds, such as maize and Brassica napus (Liu et al. 2010;Pouvreau et al. 2011). Despite the absence of seed maturation transcription factors, a WRI1 homologue also exerts control over the TAG biosynthesis pathway in non-seed tissues 9

11 (Ma et al. 2013) and the factors that regulate WRI1 in such non-seed tissues are yet to be elucidated. Triacylglycerol Synthesis Triacylglycerol biosynthesis can be divided into two separate sets of reactions that primarily take place in two different cellular compartments. The first set involves the production of fatty acids from precursor carbon molecules. In plants, de novo fatty acid synthesis primarily takes place in the plastid (Harwood 2005). These fatty acids are then transported to the endoplasmic reticulum (ER) where they are sequentially esterified to a glycerol-3-phosphate backbone to form TAG (Weselake 2009). Fatty Acid Synthesis The fatty acid, or acyl, chains of TAG are primarily produced in the plastid. The reactions of this pathway have been well understood for decades and are highly conserved (Harwood 2005). The precursor of fatty acid synthesis, acetyl-coa, is produced from pyruvate through the glycolytic pathway. Evidence from a variety of sources suggests that the pyruvate used may be from glycolysis in the cytosol or the plastid, with cytosolic pyruvate or phosphoenolpyruvate being imported into the plastid and then converted to acetyl-coa (Harwood 2005). A pyruvate dehydrogenase enzyme complex converts the pyruvate to acetyl- CoA, which is then converted to malonyl-coa by a multi-subunit acetyl-coa carboxylase (ACCase) (Johnston et al. 1997;Mooney et al. 1999;Reverdatto et al. 1999). The ACCase enzyme of plastids is composed of multiple subunits encoded by different genes and is considered to catalyze the first committed step of fatty acid synthesis (Reverdatto et al. 1999;Harwood 2005). The ACCase complex of plastids contains biotin carboxylase, biotin 10

12 carboxyl carrier protein (BCCP), and carboxyl transferase subunits (Kannangara andstumpf 1972;Reverdatto et al. 1999). The ACCase has been considered a key regulatory enzyme in fatty acid synthesis and it has been shown that WRI1 acts to regulate the expression of the BCCP2 gene in A. thaliana (Baud et al. 2009) (Figure 1). Malonyl-CoA is converted to malonyl-acp by a malonyl-coa:acp acyltransferase (Harwood 2005). The malonyl-acp is then condensed with acetyl-coa in the first reaction of a Figure 1. A schematic pathway of fatty acid synthesis in plants. The reactions of fatty acid synthesis with enzymes and the structures of intermediates. The initial reactions up to pyruvate synthesis take place in either the cytoplasm or the plastid. However, only plastid pyruvate participates in subsequent reactions to generate Acyl-CoA. Note that KASI initiates fatty acid synthesis for chain lengths between 6 and 16 carbons while KASII initiates fatty acid synthesis of 18 carbon long fatty acids. 11

13 cycle that extends acyl chains of fatty acids. Subsequent reactions are catalyzed by a multitude of enzymes that form a large dissociable multi-enzyme complex known as fatty acid synthase (FAS) (Harwood 2005). The initial acylation is carried out by β-ketoacyl-acp synthase (KAS) III, which works specifically to acylate malonyl-acp with acetyl-coa (Clough et al. 1992;Jaworski et al. 1993). After this β-ketoacyl-acp reductase carries out the first reduction reaction, β-hydroxyacyl-acp dehydrogenase carries out the dehydration reaction, and then enoyl-acp reductase carries out the second reduction reaction (Harwood 2005). KAS I is then utilized for further acylations up to a 16 carbon fatty acid (Siggaard-Andersen et al. 1991;Harwood 2005). KASII acylates palmitoyl-acp (16:0) to form stearoyl-coa (18:0) and this enzyme is responsible for the ratio of 16C:18C fatty acids in plants (Harwood 1996). Termination of fatty acid synthesis can be carried out in various ways within plants, but for fatty acids bound for TAG production in the ER, the termination procedure involves hydrolysis by acyl-acp thioesterases to form acyl-coa moieties (Slabas et al. 1990;Hellyer et al. 1992;Harwood 2005). Flux and transcriptome analysis of oil palm mesocarp suggests that the reactions of fatty acid synthesis are more important in regulating oil accumulation than the later reactions of lipid assembly (Ramli et al. 2002;Bourgis et al. 2011). This suggests that these enzymes are likely targeted by regulatory mechanisms in order to control TAG levels in plants and WRI1 is the only known direct regulator of enzymes of late glycolysis and fatty acid synthesis (Baud et al. 2009). Triacylglycerol Assembly After synthesis of fatty acids in the plastid these compounds are exported to the ER where they act as precursors for production of both storage and membrane lipids. The first three reactions that lead to the production of diacylglycerol (DAG) are shared by the two pathways 12

14 Figure 2. A schematic pathway of triacylglycerol assembly in plants. The acyl-coa pool in the endoplasmic reticulum is primarily provided by de novo fatty acid synthesis in the plastid. Proteins in red show distinct expression profiles in TAG accumulating tissues or dramatically increase TAG accumulation when overexpressed. Abbreviations: G3P, glycerol-3-phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; MAG, monoacylglycerol; TAG, triacylglycerol; PC, phosphatidylcholine; LPC, lysophosphatidic acid. GPAT, G3P acyltransferase; LPAAT, LPA acyltransferase; PAP, PA phosphatase; DGAT, DAG acyltransferase; DGTA, DAG tranacylase; CPT, CDP-choline:DAG cholinephosphotransferase (Figure 2). First sn-glycerol-3-phosphate acyltransferase (GPAT), which many plants have a multitude of copies for, catalyzes the addition of an acyl chain from acyl-coa to the sn1 position of glycerol-3-phosphate to form lysophosphatidic acid (LPA) (Weselake 2009). This LPA intermediate is then acylated at the sn2 position through the action of LPA acyltransferase (LPAAT), another gene family with multiple homologues in plants, to form phosphatidic acid 13

15 (PA) (Weselake 2009). The PA is then dephosphorylated by PA phosphatase (PAP) to form DAG (Pierrugues et al. 2001;Weselake 2009). DAG can then be utilized to form triacylglycerol by a variety of enzymes or in the formation of phosphatidylcholine (PC) (Weselake 2009). The formation of PC from DAG is catalyzed by the enzyme cytidine diphosphate choline:1,2- diacylglycerol cholinephosphotransferase (CPT) (Weselake 2009). The most straightforward reaction of TAG synthesis is catalyzed by diacylglycerol acyltransferase (DGAT), which utilizes acyl-coa to acylate the sn-3 position of DAG to form TAG (Routaboul et al. 1999;Zou et al. 1999;Zhou et al. 2013). Another enzyme known as diacylglycerol transacylase (DGTA) acts on two different DAG molecules to produce TAG and monoacylglycerol (MAG) (Stobart et al. 1997). Finally, DAG and PC may act as substrates for phosphatidylcholine acyltransferase (PDAT) to produce TAG and LPA (Dahlqvist et al. 2000). The incorporation of PC acyl chains into the TAG pool is important because desaturases (other than the plastid desaturase that forms 18:1 fatty acids) and fatty acid elongases act on acyl chains attached to PC (Weselake 2009). Thus, the incorporation of most desaturated fatty acids or chain lengths over 18 into TAG requires the channeling of fatty acids through the PC pool. Currently it is not known how the enzymes of TAG assembly are actually regulated and which, if any, transcription factors might play a role in their control. However, some of the proteins and enzymes involved in these later steps show differential regulation in TAG accumulating tissue or cause increased TAG accumulation in overexpression lines (Jako et al. 2001;Banilas et al. 2011;Troncoso-Ponce et al. 2011). Triacylglycerol Storage Once TAGs have been assembled in the ER they must be stored in the cell. In plants, TAGs are stored in small compartments known as oil bodies. These compartments are different 14

16 from most other cellular compartments in that they have a single layer phospholipid membrane, with the polar head groups in contact with the cytosol and the non-polar tails in contact with the internal lipids (Yatsu andjacks 1972). Oleosins are small proteins unique to plants that coat the oil bodies, helping to determine size and shape and preventing coalescence of the oil bodies (Tzen andhuang 1992;Leprince et al. 1997;Hsieh andhuang 2004). Much like TAG assembly enzymes, it is not known what transcription factors directly regulate oleosin expression levels. However A. thaliana has at least 17 oleosin genes, which are differentially expressed in various tissues, suggesting that these genes are highly regulated even within a single plant species (Hsieh andhuang 2004). Oleosins are not highly expressed in non-seed oil accumulating tissues such as the mesocarp of olive, oil palm, and avocado (Murphy 2012). Recently however lipid droplet associated proteins have been identified in these tissues that may play a similar role (Horn et al. 2013). Transcription Factors Controlling TAG Biosynthesis The majority of research on transcription factors that control TAG biosynthesis has been conducted on seed tissues. In plants, the master regulators of embryogenesis and seed maturation such as LEC1, L1L, LEC2, FUS3, and ABI3 play a role in controlling oil accumulation, but these master regulators act to regulate TAG synthesis through downstream transcription factors (Kagaya et al. 2005;Mu et al. 2008;Baud et al. 2009). The most understood downstream regulator of lipid biosynthesis in seeds is the WRI1 protein, as discussed later. In seeds, WRI1 has been shown to play a role in controlling genes involved in late glycolysis and fatty acid synthesis (Baud et al. 2009). Studies have also shown that WRI1 homologues play roles in TAG accumulation outside of seed tissues in other plants (Ma et al. 2013). WRI1 homologues outside of seed tissues are likely regulated by currently unknown upstream transcription factors, since 15

17 these WRI genes are highly expressed but the master regulators of seed tissue are not expressed outside of seed tissue (Bourgis et al. 2011;Kilaru 2015). Other transcription factors are thought to play a role in regulating transcript levels involved in later steps of TAG accumulation and storage, such as the acyltransferases involved in TAG assembly and the oleosins needed to form oil bodies due to these genes often being differentially expressed during oil accumulation (Dussert et al. 2013;Kilaru 2015). Transcription factors that play a role in regulating TAG assembly enzymes are yet to be elucidated (Figure 3). Recent analysis showed that, in seeds, upstream regions of genes of TAG Figure 3. Transcriptional regulation of triacylglycerol synthesis in plants. It is currently not known what (if any) transcription factors regulate the proteins involved with TAG assembly and TAG storage. It is also not known what transcription factors might regulate WRI1 genes in non-seed tissues that accumulate oil. OPPP, oxidative pentose phosphate pathway 16

18 storage are overrepresented for binding motifs for B3-domain containing factors and bzip factors (Peng andweselake 2011). Additionally, in some plant species, such as A. thaliana and B. napus, increases in the activity of various acyltransferase enzymes has been shown to increase oil content (Zou et al. 1997;Weselake et al. 2008;Xu et al. 2008;Baud andlepiniec 2009). This evidence suggests that regulation of TAG assembly enzymes exerts control over TAG content. It is necessary to discover which transcription factors may be acting to alter expression levels of these TAG assembly enzymes in plants in order to better understand oil accumulation. Master Regulators of Seed Maturation While this project is primarily focused on regulation of oil accumulation in non-seed tissues, most of the regulatory factors currently known were discovered in oilseed plants. It is important to understand these factors since, much like WRI1, there may be some relationship between them and the control mechanisms in non-seed tissues. As stated before, a multitude of transcription factors affect oil accumulation in seed tissues. These transcription factors are often referred to as master regulators of seed maturation (LEC1, LEC2, FUS3, ABI3) and unlike WRI1, mutations in these genes often have a variety of effects beyond altered oil content, such as cotyledons displaying characteristics of leaves, lower desiccation tolerance, and accumulation of abnormal levels of pigments (Santos Mendoza et al. 2008). These transcription factors have been shown to regulate each other locally and their effects on seed development are dependent upon the downstream activity of other transcription factors (Kagaya et al. 2005;To et al. 2006;Mu et al. 2008). They together create a network of regulation, with each transcription factor regulating genes involved in a variety of seed developmental pathways, the other master regulators of seed maturation, and even themselves (Santos Mendoza et al. 2008). 17

19 Current evidence suggests that of the master regulators LEC2, and to an extent LEC1, exert direct control over WRI1 (Baud et al. 2007;Santos Mendoza et al. 2008). Consistent with their redundant regulatory actions, LEC2, FUS3, and ABI3 all belong to the B3 family of transcription factors and all bind to an RY motif present in the promoter of target genes, but other regulatory elements are required for proper expression patterns of the target genes (Santos Mendoza et al. 2008). LEC1 has homology with the CAAT box-binding factors (CBFs) (Lotan et al. 1998). The exact regulatory elements controlling WRI1 transcription have not been discovered in either seed or non-seed tissue. WRI1 Transcription Factor The WRI1 gene was originally discovered as a low seed oil mutant in Arabidopsis thaliana that showed impairment in seed development (Focks andbenning 1998). The homozygous wri1 mutant showed 80% lower seed oil content, wrinkled seed coats, and impairment in seed germination and seedling establishment (Focks andbenning 1998). The gene was later shown to code for a transcription factor of the plant specific AP2/EREBP family, which acts to regulate oil accumulation in seed tissues (Cernac andbenning 2004). Expression studies suggest that WRI1 primarily regulates genes involved in late glycolysis and fatty acid synthesis (Figure 1) such as BCCP (a subunit of ACCase), enoyl-acp reductase, β-ketoacyl-acp reductase, fatty acid desaturase 2, plastidial pyruvate kinase, sucrose synthase, pyruvate dehydrogenase, and acyl-carrier protein (Ruuska et al. 2002;Baud et al. 2007;To et al. 2012). WRI1 orthologues in Brassica napus and Zea mays have even been shown to regulate many of the same proteins involved in glycolysis and fatty acid synthesis (Liu et al. 2010;Pouvreau et al. 2011). 18

20 Currently four WRI genes (WRI1, WRI2, WRI3, and WRI4), all belonging to the AP2/EREBP family, have been identified in arabidopsis (To et al. 2012). Their exact functions, however, are not well understood; WRI3 and WRI4 are capable of complementing the wri1 mutant, but mutations in these two genes do not affect seed oil accumulation levels. WRI3 and WRI4 are expressed more highly in other tissues of the plant, such as flowers, stems and roots, and are thought to play a role in tissue specific synthesis of fatty acids (To et al. 2012). No function for WRI2 was discovered in the study (To et al. 2012). A similar phenomenon was seen in maize, where a duplication event has created two WRI1 genes referred to as ZmWRI1a and ZmWRI1b (Pouvreau et al. 2011). While the ZmWRI1a gene clearly regulates oil accumulation in seeds, and the ZmWRI1b gene rescues Arabidopsis wri1 mutants, differential expression patterns suggest unique roles for the two duplicates of the genes (Pouvreau et al. 2011). Their protein sequences are more closely related to WRI1 than to any of the other WRI family members in Arabidopsis; maize does have orthologues to WRI2, WRI3, and WRI4 (Pouvreau et al. 2011). Studies have also shown that unlike the other transcription factors mentioned above, WRI1 homologues likely play a role in directing TAG accumulation outside of the developing seed. A WRI1 homologue from oil palm has been shown to be highly expressed, compared to the closely related species date palm (which does not accumulate oil), and coordinately with an increase in TAG levels (Bourgis et al. 2011). The WRI1 homologue from oil palm has also been shown to rescue the wri1 mutant phenotype of A. thaliana (Ma et al. 2013). Interestingly the WRI homologues found in non-seed oil accumulating plants are most closely related to Arabidopsis WRI1 according to amino acid sequence and not to any of the other family members in 19

21 Arabidopsis (Bourgis et al. 2011). WRI1 genes play a key role in oil accumulation in a variety of plant tissues as well as a wide range of plant species. TAG Production and Function in Various Plant Tissues The Roles of TAG in Plants The primary role of TAGs in most plants is as a seed storage compound for energy through the process of germination. When the seed germinates it utilizes the stored TAG to provide necessary energy and as a carbon source until the seedling is capable of photosynthesis (Graham 2008). Storage oil, however, has also been detected in other tissues of plant species and may play a number of additional roles. For example, mutations in enzymes of the lipid biosynthesis pathway have been shown to affect pollen performance and tapetum development, suggesting a role for lipids in these processes (Zheng et al. 2003;Zhang et al. 2009). Recent studies have also indicated that TAG may be used as a diurnal photosynthetic store in the leaves of crabapple plants, and evidence suggests it may play a similar role in other plant species (Lin andoliver 2008). Various plant species accumulate different levels of oils in different tissues. This project primarily focuses on non-seed oil accumulation, but below is a description of the oil levels found in the various plants that accumulate oil in either seed or non-seed tissue that are used in the transcriptome analysis performed to determine candidates for this project. TAG in Seed Tissues While A. thaliana is not an oil crop, it has been utilized for the study of factors controlling TAG accumulation as it accumulates approximately 35% oil in the seed (Li et al. 20

22 2006). Arabidopsis is related to the oilseed crop B. napus, which accumulates up to 45% oil by dry weight (Goering et al. 1965). Ricinus communis (Castor) is another oilseed crop that produces around 60% oil in its seeds (Canvin 1963). Euonymus alatus and Tropaeolum majus are not oil crops but both are oilseeds, which accumulate 50% and 10% oil by dry weight, respectively (Troncoso Ponce et al. 2011). While this study primarily focusese on transcription factors regulating TAG accumulation outside of the seed tissue, it is important to include oil accumulating seed tissue as well in order to find transcription factors working to control oil levels solely in non-seed tissues. While overexpression of seed maturation transcription factors such as LEC1 have been shown to increase oil content in seeds of Zea mays, this can also cause detrimental side effects in the plant (Shen et al. 2010). However, in the same study overexpression of ZmWRI1 showed increased oil accumulation without the detrimental effects associated with ZmLEC1 overexpression such as poor germination and stunted growth. This suggests that targeted increases in fatty acid synthesis and TAG accumulation can create high yield oil crops without introducing negative agronomic traits into these plants. Studying oil accumulation in both seed and non-seed tissues may aid in teasing out the factors that are solely responsible for regulating oil accumulation. TAG in Non-seed Tissues Although the majority of what is currently known about TAG production and control over the TAG synthesis pathway is derived from studies involving arabidopsis and other oil seed species, many different plants accumulate TAG in other tissues as well. This includes the mesocarp of avocado and oil palm, which can accumulate up to 70% and 90% oil by dry weight, respectively (Platt-Aloia andthomson 1981;Ngando-Ebongue et al. 2012). The tubers of yellow 21

23 nutsedge (Cyperus esculentus) also accumulate high levels of TAG compared to other roots, with values between 20-36% oil by dry weight (Linssen et al. 1989). Bayberry plants are known to produce a unique waxy coating on leaves and fruit which is composed of TAG molecules (Harlow et al. 1965). While most of the studies analyzing transcriptional regulation of oil accumulation have involved seed tissues, recently some insight into control in non-seed tissues has been obtained. The transcriptome for oil palm mesocarp showed high expression levels of WRI1 homologues, and subsequent experiments demonstrated that the oil palm WRI1 homologue rescues the Arabidopsis wri1-1 (Bourgis et al. 2011;Ma et al. 2013). The studies also found that seedspecific transcription factors responsible for regulating WRI1 in the seed are not expressed in non-seed tissues even when WRI1 is highly expressed. The transcriptome data from these plant tissues has already aided in discovering what factors allow for increased oil content in these various tissues, and further analysis should reveal other factors playing a role in regulating oil content. Avocado Model While most of the work done with WRI1 so far has involved either seed tissue or nonseed tissue of more recently developed plant species (Figure 4), the avocado belongs to the group of basal angiosperm, the ancestors of all flowering plants (Heywood 1993). If the WRI1 gene serves the same function in avocado mesocarp as it does in the seeds and mesocarp of others, it would show that this mechanism of TAG regulation has been highly conserved in much of the plant kingdom. By analyzing the avocado transcriptome data and verification of candidates, we 22

24 Figure 4. Phylogenetic tree of species for which transcriptome data has been obtained. Constructed using NCBI Taxonomy Common Tree and visualized using Archaeopteryx ( The tree shows the relatively greater age of Persea americana compared to the other species; it being a basal angiosperm. may also elucidate what other regulatory elements of TAG accumulation may be conserved across the plant kingdom. Human Uses of Plant Oils Humans have developed a variety of uses for plant oils. The oldest human use of plant oils is for consumption by both humans and livestock, either by eating whole plants or harvesting oils directly. However humans have found a variety of other uses for plant oils as well. Today around 15% of plant oil production is used as industrial feedstock for the production of surfactants, soaps, detergents, lubricants, solvents, paints, cosmetics and chemical feedstocks 23

25 (Carlsson 2009). Plant oils have also recently attracted attention as a source of biofuels to help minimize human use of fossil fuels (Durrett et al. 2008). New interest has also been sparked in the production of unusual fatty acids as replacements for petroleum products in industrial and chemical feedstocks (Carlsson 2009). However, these new uses of plant oils could put greater strains on current production levels, meaning that oil production will need to be increased. Current interest lies in both increasing the yield of oil crops and increasing oil production from crops that are not currently considered as oil crops (Carlsson 2009). For either of these approaches to work, a greater understanding of the regulation of TAG biosynthesis will be required. Hypothesis, Rationale, and Specific Aims Using transcriptome data from a variety of plant species, candidate transcription factors will be identified that may play a role in TAG synthesis in plants. Transcriptome data from a variety of plant tissues from various species have been collected, including plants that accumulate oil in seed or non-seed tissue and also plants that do not accumulate significant levels of oil (Figure 4). Comparative analysis of transcript levels for about 1500 transcription factors, across species and tissues, is expected to allow us to identify candidates that are likely responsible for oil accumulation in seed and non-seed tissues and also those that may be tissuespecific. This study will show that transcriptional regulation of oil biosynthesis, in part, is likely to be tissue-specific, but WRI1 function is highly conserved across species. To show this, a comparative approach using in silico analysis will be taken to analyze transcriptome data for 10 different plant species and 14 different tissue types and generate a list of potential candidate transcription factors responsible for regulating oil accumulation outside of seed tissue. The 24

26 transcriptome data will also be used to identify WRI-like genes in avocado and examine the expression levels of these genes in avocado mesocarp to determine if the WRI gene function is likely conserved in this species. Finally the WRI-like genes expressed in avocado mesocarp and selected identified candidates will be cloned into entry vectors. These entry vectors will be used in future experiments to determine if these genes influence oil accumulation in plants. 25

27 CHAPTER 2 MATERIALS AND METHODS Plant Material Avocado fruits of the Hass cultivar harvested from California were used for all avocado tissue samples, which were collected during the mid to late stages of fruit development (Sung 2013;Kilaru 2015). For reverse transcription reactions of avocado genes, RNA previously extracted from late developmental stage of Hass mesocarp tissue was used. Arabidopsis Col-2 plants were germinated and grown in soil with 16/8 hour light/dark cycle at 20 C and watered regularly. Desired parts of the plant were removed and weighed before being used for RNA extraction. Extraction of RNA Total RNA was extracted from mature arabidopsis siliques. For each extraction, mg of tissue sample was ground by stainless steel beads of 3.2 mm diameter using a mini bead beater (Biospec). RNA was then extracted using the plant mini RNA kit (Qiagen) according to manufacturer protocols. Extracted RNA was examined using a Nanodrop-1000 to determine quality and quantity and stored at -80 C until further use. Cloning of Avocado WRI Homologues Avocado gene sequences were obtained from the avocado genome sequence project (Ibarra-Laclette 2013). Gateway compatible primers were designed based on instructions provided by Invitrogen. All avocado genes were cloned from late stage mesocarp RNA. Reverse 26

28 transcription reactions were performed using Gateway primers (Table 1) of the avocado gene and Table 1. Gene Specific Primers Used for Gateway Cloning agarose gels were run to determine proper size of the cdna. The cdna products were then cloned into the pentr/sd/d-topo Gateway entry vector from Invitrogen using the Gateway TOPO cloning reaction. Plasmids were then transformed into OneShot TOP10 chemically competent E. coli provided by Invitrogen. E. coli were plated on LB kanamycin (50 µg/ml) plates and allowed to grow overnight. Colony PCR using M13 forward primer with gene specific reverse or the M13 reverse primer with gene specific forward primer was done to confirm insert and determine size and direction. Inserts were also confirmed by sequencing using M13 forward and reverse primers at the ETSU Molecular Biology Core Facility. 27

29 Construction of WRI Gene Phylogeny The evolutionary relationship of WRI genes in a monocot (Oryza sativa), dicot (A. thaliana), basal angiosperm (P. americana) and bryophyte (Physcomitrella patens) was analyzed by construction of a phylogenetic tree. The protein sequences for four AtWRI genes were identified from the TAIR database and the avocado WRI-like amino acid sequence data was provided by the Avocado Genome Sequencing Project (Ibarra-Laclette 2013). The homologues of AtWRI genes in O. sativa and P. patens were identified using BLASTP (NCBI). In O. sativa, two sequences that were nearly identical in amino acid sequence to both AtWRI3 and AtWRI4 were referred to as WRI3/4-1 and WRI3/4-2. An AP2 transcription factor from Chlamydomonas reinhardi was used as the outgroup. A UPGMA tree was constructed with MEGA 6.0 using a ClustalW alignment of protein sequences (Larkin et al. 2007). The robustness of the tree was tested by bootstrap analysis with 1,000 replicates. Accession numbers: AtWRI1 (NP_ ); AtWRI2 (NP_ ); AtWRI3 (NP_ ); AtWRI4 (NP_ ); OsWRI1 (ABA ); OsWRI2 (BAO ); OsWRI3/4-1 (NP_ ); OsWRI3/4-2 (BAD ); PpWRI1-like (BAL ); PpWRI2-like (XP_ ); PpWRI3-like (XP_ ); PpWRI4-like (XP_ ); CrAP2 (XP_ ). Data Analysis for Identification of Candidate Transcription Factors Transcriptome data for a variety of species and tissue types was provided by Dr. Ohlrogge at Michigan State University (Bourgis et al. 2011;Troncoso Ponce et al. 2011). Data was provided for C. esculentus root tissue, E. guineensis leaf tissue and mesocarp tissue, P. dactylifera mesocarp tissue, T. majus seed tissue, B. napus seed tissue, A. thaliana seed tissue, E. 28

30 alatus seed tissue, R. communis seed tissue, Myrica pensylvanica mesocarp tissue, and P. americana mesocarp tissue. Over 1,500 putative transcription factors were included in the expression data. The expression profile data for transcription factors were imported to an excel file which was then read into the R programming suite for analysis. R scripts were written that allowed for the identification of transcription factor genes that met certain criteria (Appendix A). Two different criteria, given below, were used to identify the candidates using the R program. First, since WRI1 has been shown to regulate TAG accumulation in a variety of plants the first approach used its expression levels as a reference point in the various plants. The transcriptome data included developmental points for each tissue that coincided with oil accumulation. The temporal profile for WRI1 orthologues during development was used in each individual species to identify transcription factors expressed in a similar pattern. WRI1 orthologues were determined by the RNA-seq data. For non-seed oil accumulating plants such as oil palm and avocado, any transcription factors that showed similar expression levels in non-oil accumulating plants, such as date palm, were removed. After, genes generated by looking at individual species were examined and transcription factors that appeared in a multitude of species were retained. Second, as a more generalized approach, the average expression level for all genes was compared between oil accumulating non-seed tissue and all other tissues. The expression levels of transcription factors for all time points in avocado mesocarp, oil palm mesocarp, and nutsedge root were compared to the levels in all other tissues. Transcription factors that showed at least 2- fold greater expression in oil accumulating non-seed tissues were retained. All genes found through the initial analysis of the transcriptome were then examined by a variety of bioinformatics tools. Coexpression analysis was done using ATTED-II 29

31 ( to determine which genes the candidate transcription factors were coexpressed with in Arabidopsis thaliana. Genes of interest included enzymes of the fatty acid biosynthesis pathway, enzymes of the TAG assembly pathway, and proteins associated with lipid bodies. These genes were also examined using the Arabidopsis efp browser ( to determine expression levels of the transcription factors in various tissue types of arabidopsis. Genes of interest here were those highly expressed in arabidopsis seed tissue, when TAG is accumulating, but not highly expressed in other tissue types. The efp browser was also used to determine which tissue to use when extracting RNA from Arabidopsis. Cloning of Candidate Genes Three identified candidates were selected for validation and were isolated from arabidopsis and cloned into Gateway entry vector. These cloned genes will be cloned into a binary vector and used in later assays to determine if their expression increases oil accumulation. These genes were AT2G19810, referred to as C3H after the Cys3His motif found in members of this transcription factor family; AT1G33060, referred to as NAC014, a member of the large plant NAC transcription factor family; and AT1G04990, referred to as OZF for the oxidative zinc finger motif found in this transcription factor. To produce cdna of the C3H and OZF candidate genes, primers were designed for the Gateway system (Table 1), and a reverse transcription reaction was used on arabidopsis RNA samples. Both genes were cloned from RNA extracted from green siliques. RT-PCR reactions were run using gene-specific primers designed for Gateway cloning (Table 1) and examined on agarose gels to determine the product size. The cdna products were then cloned into the 30

32 pentr/sd/d-topo gateway plasmid from Invitrogen using the gateway TOPO cloning reaction. Plasmids were then transformed into OneShot TOP10 chemically competent E. coli provided by Invitrogen. E. coli were plated on LB kanamycin (50 µg/ml) plates and allowed to grow overnight. Colony PCR using M13 forward primer with gene specific reverse or the M13 reverse primer with gene specific forward primer was done to confirm insert and determine size and direction. Inserts were also confirmed by sequencing at the ETSU Molecular Biology Core Facility (Appendix B), except for the OZF insert, which could not be successfully sequenced. The puni51 plasmid bearing the NAC014 candidate gene was obtained from the Arabidopsis Biological Resource Center. Gateway primers were used to amplify the NAC014 gene from the puni51 plasmid (Table 1). After cdna of the gene was obtained the cloning steps were performed as outlined above to obtain expression vectors bearing the NAC014 gene. 31

33 CHAPTER 3 RESULTS Identification of Avocado WRI Homologues A Homolog of AtWRI1 Shows High Expression During Oil Accumulation in Avocado Mesocarp The avocado transcriptome was generated using Illumina RNA-seq. Data was collected for five different stages of mesocarp development. The expression levels for WRI1-like were high in avocado mesocarp, with an average expression level of 253 RPKM during the five developmental stages examined, which correspond to mid to late stages of fruit development (Figure 5). During these developmental stages the avocado fruit increases in overall weight and accumulates lipids in the mesocarp (Sung 2013). Expression levels of PaWRI1-like also shows a similar pattern to expression of genes it is known to regulate in Arabidopsis (BCCP2, KASII, pyruvate dehydrogenase components) (Kilaru 2015). Homologues of genes that regulate WRI1 in arabidopsis, such as the master regulators of seed development, however are not expressed in avocado mesocarp during oil accumulation (Kilaru 2015). Similar results were also found for oil palm (Elaeis guineensis) that accumulates high amounts of oil in non-seed tissue (Bourgis, Kilaru et al. 2011). WRI1 is likely regulated by a separate pathway in avocado mesocarp, but more research is needed to determine if this mechanism is conserved among different species that accumulate high levels of oil in the mesocarp tissue. 32

34 Transcript Levels(RPKM) I II III IV V Developmental Stage Wri2 Wri3 Wri1 Figure 5. Expression levels for avocado WRI homologues. Three homologues of the WRI gene family were identified in avocado and their transcript levels during mesocarp development were shown, as determined by RNA-seq analysis. RPKM: Reads per kilobase per million mapped reads. Homologues of WRI Gene Family Are Also Highly Expressed in Avocado Mesocarp Recently three WRI paralogues (WRI2, WRI3 and WRI4) were recognized in Arabidopsis, of which WRI3 and WRI4 compensated for the low fatty acid levels of the wri mutant (To, Joubès et al. 2012). WRI2, however, did not compensate for the loss of WRI1 in Arabidopsis thaliana plants. The study suggested that WRI3 and WRI4 might play a role in regulating oil content outside of seed tissues. Given the high expression levels of WRI3-like in avocado during oil accumulation in the mesocarp it is possible that WRI3 is playing a role in regulating oil content in the mesocarp of avocado (Figure 5). Also the relatively high expression levels for the 33

35 avocado WRI2 homologue suggest that this transcription factor might still function in regulation of genes associated with TAG accumulation in P. americana (Figure 5). PaWRI2-like and PaWRI3-like also show coordinated expression with genes of fatty acid synthesis during most of the developmental periods examined (Kilaru 2015). While a WRI4-like gene was detected in avocado mesocarp, the levels were extremely low when compared to the other WRI-like genes, and therefore the gene is not likely playing a role in oil accumulation in mesocarp. Three Avocado WRI Homologues Were Cloned for Future Validation of Their Role in Oil Biosynthesis To further study the functional role of the avocado WRI homologues expressed during mesocarp development, the genes were cloned into Gateway vector pentr/sd/d-topo (Invitrogen). Vectors bearing the genes of interest were examined by PCR (Figure 6-8) to determine if the cloning reaction was successful. A primer designed to anneal to the M13 priming site of the vector was used with a primer designed to adhere to the cdna insert of the plasmid to determine orientation. 34

36 Ladder WRI1-4 WRI1-5 WRI bp 1000 bp Figure 6. Cloning of PaWRI1 into Entry Vector. Agarose gel electrophoresis of PCR results for entry plasmids bearing WRI1 genes extracted from successful transformants. Six colonies were tested. Colony WRI1-6 showed a band of the expected size 1336 bp, indicated by an arrow. A 1000 bp ladder is used for comparison. For the entry vector bearing avocado WRI1-like gene, the band expected from the PCR reaction was 1336 base pairs. Figure 6 shows that a band of approximately that size resulted from one of the colonies tested with the PCR reaction when run on an agarose gel. This colony, referred to as WRI1-6, was confirmed by sequencing and utilized for further cloning procedures (Appendix B). For the entry vector bearing the avocado WRI2-like gene, the band expected from the PCR reaction was 1445 base pairs. Five colonies were tested after transformation, one of which 35

37 produced the expected product size. This colony was referred to as WRI2-1 (Figure 7). WRI2-1 was confirmed by sequencing and utilized for all further cloning experiments (Appendix B). For the avocado WRI3-like gene, the band expected from the PCR reaction was 1138 Ladder WRI bp 1000 bp Figure 7. Cloning of PaWRI2 into Entry Vector. Agarose gel electrophoresis of PCR results for entry plasmids bearing WRI2 genes extracted from successful transformants. Colony WRI2-1 showed a band of the expected size, 1445 bp, indicated by an arrow. A 1000 bp ladder is used for comparison. 36

38 base pairs. Five colonies were originally tested after transformation, of which one was demonstrated to be transformed with the plasmid bearing the gene. Figure 8 shows the PCR reaction run on the extracted plasmid from the colony. The colony was referred to as WRI3-5. This colony was confirmed by sequencing and used for further cloning procedures (Appendix B). WRI3-5 Ladder 1500 bp 1000 bp Figure 8. Cloning of PaWRI3 into Entry Vector. Agarose gel electrophoresis of PCR results for entry plasmids bearing WRI1 genes extracted from successful transformants. Colony WRI3-5 showed a band of the expected size, 1138 bp, indicated by an arrow. A 1000 bp ladder is used for comparison. 37

39 WRI Phylogeny Shows Multiple Gene Duplications with WRI2 Appearing to be the Oldest Form of the WRI Gene Studies of WRI homologues in arabidopsis showed that WRI3 and WRI4 can each rescue the low fatty acid phenotype of the wri1-1 mutant; WRI2, however, did not compensate for the loss of WRI1 (To et al. 2012). In avocado mesocarp, the overall expression pattern of WRI1, WRI2, and WRI3 orthologues was similar to that of genes WRI1 is known to regulate (Maeo et al. 2009) and the pattern of oil accumulation (Kilaru 2015). The high expression levels for avocado WRI2-like gene during oil accumulation in the mesocarp suggests a possible role for WRI2-like in regulation of fatty acid synthesis. To explore the relationship of the WRI family of genes a phylogenetic tree of WRI proteins from various plant families including dicots, monocots, and a basal angiosperm was constructed. The tree revealed a possible gene duplication event of WRI early in land plant evolution, as the WRI2 proteins formed a monophyletic group and separated from all other WRI genes (Figure 9). Other WRI genes formed three distinct groups with a clade of WRI genes belonging exclusively to P. patens, a bryophyte, separated from a monophyletic group of WRI1 homologues and a third group consisting of WRI3 and WRI4 genes (Figure 9). The tree constructed for the WRI genes of various species suggests that the PaWRI2-like and AtWRI2 are older than the other WRI genes and have also diverged a great deal from each other. The high expression levels for PaWRI2-like in avocado mesocarp that were not previously reported in any other oil-rich tissues, along with PaWRI1-like and PaWRI3-like but not PaWRI4-like, suggest that perhaps, the AtWRI2 may have lost its function in oil biosynthesis while the PaWRI2-like retained this function. Although AtWRI2 did not complement the wri1-1 mutant, complementation studies with PaWRI2-like are planned in future studies. Based on the gene 38

40 Figure 9. Phylogentic analysis of the WRI family of genes. Four monophyletic groups are present. One contains higher plant WRI3/4 genes. The second contains higher plant WRI1 genes. A third group consists of the WRI1, WRI3, and WRI4 genes within moss. Finally, all WRI2 genes from all examined species belong to a single monophyletic group. Arrows indicate likely gene duplication events. Scale represents number of nucleotide changes per site. expression data, it is predicted that the avocado WRI2 homologue may play an additional role in TAG accumulation in this basal angiosperm species. 39

41 Identification of Transcription Factors That May Play a Role in Oil Accumulation Transcription Factors That Were Differentially Expressed Between Oil-rich Seed and Non-seed Tissues were Identified Currently the only known transcriptional regulator of oil biosynthesis in plants is the WRI gene family. Because WRI1 has been associated with oil accumulation across species and tissue types, genes that showed similar expression patterns to WRI1 expression in multiple oil accumulating tissues would likely be associated with oil accumulation as well. This analysis of the transcriptome data revealed candidate transcription factors likely associated with oil accumulation (Table 2). The top ten transcription factors were chosen based on high expression levels in non-seed tissues that accumulate oil, especially compared to tissues that do not accumulate oil (Table 2). Transcription factors were anywhere from 2-12 times more highly expressed in oil accumulating non-seed tissue compared to non-oil accumulating tissue, and 2-10 times more highly expressed in oil accumulating non-seed tissue than oil accumulating seed tissues. Candidates also showed high expression in at least one non-seed tissue type that accumulates high amounts of oil. The genes listed in Table 2 were identified by both criteria previously described. 40

42 Table 2. Candidate Transcription Factors Identified by Transcriptome Analysis At = A. thaliana, Ce = C. esculentus, Eg = E. guineensis, Pa = P. americana Three Transcriptions Factors Were Selected for Cloning and Future Validation of Their Role in Oil Biosynthesis A C3H transcription factor homologue of AT2G19810 (C3H) showed 12 times higher expression levels in non-seed oil accumulating tissue compared to tissue that does not accumulate oil. Specifically, the C3H gene was highly expressed in the mesocarp of avocado and oil palm and also in the roots of nutsedge. It was also highly expressed in seed tissues that accumulate oil. However, the transcript levels in the non-seed oil accumulating tissue were on average higher by two-fold compared to oil-rich seed tissues (Table 2). A NAC014 transcription factor homologue of AT1G33060 was also highly expressed in non-seed tissues that accumulate oil compared to other tissue types, being nearly 10 times higher 41

43 than tissue that do not accumulate oil, and over 6 times higher compared to oil accumulating seed tissue (Table 2). Its highest expression level was shown to be in avocado mesocarp (Table 2). An OZF transcription factor, homologue of AT1G04990 (OZF), was the third candidate chosen for further examination in expression studies. While its average expression in oil accumulating tissue is not as great as the other two compared to other tissue types, just 2 times higher than non-oil accumulating tissue, coexpression analysis with the ATTED-II database revealed that in Arabidopsis the transcription factor is coexpressed with a variety of enzymes involved in fatty acid synthesis (Table 2). Since WRI1 exerts its control over fatty acid accumulation by regulating similar genes (Maeo et al. 2009), this candidate was chosen for further examination. Cloning of Candidates for Validation of Their Role in Oil Biosynthesis To further study the functional role of selected, identified transcription factors expressed during oil accumulation in a variety of plants, the genes were cloned from arabidopsis siliques or from provided vectors into Gateway vector pentr/sd/d-topo (Invitrogen). Vectors bearing the genes of interest were examined by PCR to determine if the cloning reaction was successful. A primer designed to anneal to the M13 priming site of the vector was used with a primer designed to adhere to the cdna insert of the plasmid. The entry vector bearing the C3H candidate was expected to produce a band of 1369 bp after the PCR reaction. Multiple colonies were tested after transformation, and one was demonstrated to bear the plasmid with the gene of interest. This colony was referred to as C3H-1 42

44 and a product of the expected size was produced when the PCR reaction was run on plasmids extracted from these E. coli. (Figure 10) This colony was confirmed by sequencing then used for further cloning experiments (Appendix B). For the entry vector bearing the NAC014 candidate, a band of 2113 base pairs was Ladder C3H bp 1000 bp Figure 10. Cloning of C3H into Entry Vector. Agarose gel electrophoresis of PCR results for entry plasmids bearing C3H genes extracted from successful transformants. Colony C3H-1 showed a band of the expected size, 1369 bp indicated by an arrow. A 1000 bp ladder is used for comparison. expected when the PCR was run. Multiple colonies were tested and two were shown to produce bands of the correct size. Of these two, only one referred to as NAC014-4 was confirmed by 43

45 sequencing and chosen to be used in further cloning experiments. Figure 11 shows the PCR product of plasmids extracted from this colony. The entry vector bearing the OZF candidate was expected to produce a band of 1238 base pairs. Of the five colonies examined one produced a band of the expected size, and was referred to as OZF-3. Figure 12 shows the results of PCR run on plasmids extracted from OZF-3. Ladder NAC bp 2000 bp Figure 11. Cloning of NAC014-4 into Entry Vector. Agarose gel electrophoresis of PCR results for entry plasmids bearing NAC014 genes extracted from successful transformants. Colony NAC014-4 showed a band of the expected size, 2113 bp, indicated by an arrow. A 1000 bp ladder is used for comparison. 44

46 Ladder OZF bp 1000 bp Figure 12. Cloning of OZF into Entry Vector. Agarose gel electrophoresis of PCR results for entry plasmids bearing OZF genes extracted from successful transformants. Colony WRI2-1 showed a band of the expected size, 1238 bp, indicated by an arrow. A 1000 bp ladder is used for comparison. Attempts to sequence the OZF colony using the M13 primers failed. This colony was used for further cloning procedures. 45