GENETIC AND BIOCHEMICAL ANALYSIS OF ESSENTIAL ENZYMES IN

TRIACYLGLYCEROL SYNTHESIS IN ARABIDOPSIS

By

KIMBERLY LYNN COTTON

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Molecular Plant Sciences

DECEMBER 2015

© Copyright by KIMBERLY LYNN COTTON, 2015 All Rights Reserved

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of

KIMBERLY LYNN COTTON find it satisfactory and recommend that it be accepted.

______John A. Browse, Ph.D., Chair

______Michael M. Neff, Ph.D.

______Thomas W. Okita, Ph.D.

______John J. Wyrick, Ph.D.

ii

ACKNOWLEDGMENTS

To my PI John Browse, thank you for your support, guidance and patience over the years.

To my committee, John Browse, Michael Neff, Thomas Okita, and John Wyrick, thank you for your insightful discussions and suggestions. To my collaborators Jay Shockey, Anushobha

Regmi, Neil Adhikari, John Browse, and Phil Bates, thanks for the great work and great times.

To Browse Lab members past and present, thanks for the help, guidance and wonderful memories. To the undergraduate students who have helped me over the years, thank you for all your help in making this project possible; and with a special thank you to Arlene, Tesa, Breana, and “no-longer-undergrads” David and Barbara Cotton for their particular dedication to this project. And finally, a huge thank you to my family and friends who have loved and supported me throughout this venture. Go Cougs!

iii

GENETIC AND BIOCHEMICAL ANALYSIS OF ESSENTIAL ENZYMES IN

TRIACYLGLYCEROL SYNTHESIS IN ARABIDOPSIS

Abstract

by Kimberly Lynn Cotton, Ph.D. Washington State University December 2015 Chair: John A. Browse

Plant oils are used in food, fuel, and feedstocks for many consumer products, and so understanding the process by which they are made and modified will help us to make plant oils more healthy, useful, and sustainable. While some of the genes encoding the ER-localized enzymatic steps to triacylglycerol (TAG) have been well understood and documented, several are still in need of study. The glycerol-3-phosphate acyl transferase (GPAT) enzymatic activity is the first step in the pathway to TAG, and it acylates glycerol 3-phosphate to produce lysophosphatidic acid. GPAT9 (AT5G60620) is conserved across land plants and is homozygous lethal, indicating an essential function. Transcript level in knockdown mutants correlates with GPAT activity and with oil levels, and the protein interacts with other enzymes in the TAG biosynthesis pathway.

These data suggest that GPAT9 encodes the main GPAT involved in membrane lipid and TAG synthesis. The phosphatidic acid phosphatase (PAP) step in TAG synthesis is responsible for the hydrolysis of inorganic phosphate from phosphatidic acid and creation of diacylglycerol (DAG).

There are 13 putative PAPs in Arabidopsis which are homologous to known PAPs. Most of these are involved in other processes, including the plastidial lipid synthesis pathway and signaling pathways. The Arabidopsis gene LPP β (At4g22550) is expressed in seed tissue, its protein product is localized to the ER, and it encodes PAP activity, indicating that it is a likely candidate for the

PAP involved in oil synthesis. At the conclusion of this work, questions remain about the role of

iv

LPP β in oil synthesis and which genes encode the major enzymes involved in the steps generating phosphatidylcholine and converting it back to DAG; but the main Kennedy Pathway enzymes generating TAG have been identified and characterized.

v

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... iii

ABSTRACT ...... iv

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

CHAPTER ONE . INTRODUCTION

GENERAL INTRODUCTION ...... 1

REFERENCES ...... 12

CHAPTER TWO . INITIAL ASSESSMENT OF POTENTIAL PAP GENES INVOLVED IN OIL SYNTHESIS IN

ARABIDOPSIS ...... 17

2.1 INTRODUCTION ...... 18

2.2 MATERIALS AND METHODS ...... 25

2.3 RESULTS AND DISCUSSION ...... 30

2.4 CONCLUSION ...... 41

2.5 REFERENCES ...... 54

CHAPTER THREE . GENETIC , MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF PUTATIVE

PAP GENES ...... 61

3.1 INTRODUCTION ...... 62

3.2 MATERIALS AND METHODS ...... 65

3.3 RESULTS ...... 73

3.4 DISCUSSION ...... 79

3.5 CONCLUSION ...... 83

vi

3.6 REFERENCES ...... 95

CHAPTER FOUR . IDENTIFICATION OF ARABIDOPSIS GPAT9 (A T5G60620) AS AN ESSENTIAL GENE

INVOLVED IN TRIACYLGLYCEROL BIOSYNTHESIS ...... 99

4.1 INTRODUCTION ...... 100

4.2 MATERIALS AND METHODS ...... 104

4.3 RESULTS ...... 112

4.4 DISCUSSION ...... 124

4.5 CONCLUSIONS ...... 131

4.6 REFERENCES ...... 148

4.7 SUPPLEMENTAL FIGURES , METHODS , AND REFERENCES ...... 158

4.8 SUPPLEMENTAL REFERENCES ...... 175

CHAPTER FIVE . CONCLUSIONS AND PERSPECTIVES ...... 176

REFERENCES ...... 185

vii

LIST OF TABLES

Page

CHAPTER 2

1. Literature review and protein information of putative PAPs ...... 45

2. Homologs of Arabidopsis putative PAP proteins in Castor Bean ...... 46

3. Homologs of Arabidopsis putative PAP proteins in Soybean ...... 47

4. Homologs of Arabidopsis putative PAPs in seed tissue of oilseeds ...... 48

5. Primers used in this work ...... 53

CHAPTER 3

1. Primers used in this work ...... 93

CHAPTER 4

1. Non- Mendelian segregation of GPAT9/gpat9-2 under sulfadiazine selection ...... 136

2. Transmittance of sulfadiazine resistance from reciprocal crosses … ...... 137

3. Ovule viability within GPAT9-2/gpat9-2 half siliques ...... 138

4. Pollen germination and tube growth of GPAT9-2/gapt9-2 within the qrt1 … ...... 140

S1. Primers utilized for PCR, RT-PCT, & qPCR ...... 173

S2. Gene identifiers for GPAT9, LPEAT1, and LPEAT2 sequences ...... 174

viii

LIST OF FIGURES

Page

CHAPTER 1

1. TAG biosynthesis in the eukaryotic pathway in Arabidopsis ...... 11

CHAPTER 2

1. Phylogenetic tree of LPP homologs ...... 42

2. Conserved domains in the PAP2 family of PAPs ...... 43

3. Bioinformatics Information on LPP β ...... 49

4. Tissue expression of genes of interest ...... 50

5. Subcellular localization of LPP β, SPP1 , and LPAP1 ...... 51

6. Non-Diurnal Expression Pattern of LPP β ...... 52

CHAPTER 3

1. RT-PCR of SALK Insertional Mutants...... 84

2. Oil Levels in LPP β RNAi Lines ...... 85

3. LPP β Expression in RNAi Plants ...... 87

4. LPP β Expression in Multiple Mutant Lines ...... 88

5. Seed Oil and Fatty Acid Content of Multiple Mutants ...... 89

6. PAP Complementation Assays ...... 90

7. PAP Activity Assays ...... 91

8. LPP β is Not Regulated by WRI1 at the Transcript Level ...... 92

CHAPTER 4

1. Sequence Comparison of Selected GPAT9 Enzymes from Plants...... 132

2. Phylogenetic Comparison of GPAT9… ...... 134

ix

LIST OF FIGURES (Continued)

Page

CHAPTER 4

3. Aborted Ovules in GPAT9/gpat9-2 Siliques...... 135

4. Reduced Pollen Size and Pollen Tube Growth of GPAT9-2/gpat9-2 Pollen … ...... 139

5. Genetic Complementation of gpat9-2 Mutants with Transgenic Plant GPAT9 ...... 141

6. Segregating Red and Brown T 2 GPAT9 -amiRNA Seed Oil Content and Size...... 142

7. Oil Quantity and Composition of Homozygous T 3 GPAT9 amiRNA Lines ...... 143

8. Developing Silique Microsome GPAT Activity ...... 144

9. Silique Microsome LPAAT Assays ...... 145

10. Testing of Protein:Protein Interactions Between AtGPAT9 and Other Lipid … .....146

11. Models of GPAT9-dependent Glycerol Flux, and Spatial Organization … ...... 147

S1. Sequence Alignment and Identity/Similarity Shading of Plant LPEAT1 Proteins ...158

S2. Sequence Alignment and Identity/Similarity Shading of Various Plant … ...... 161

S3. Characterization of GPAT9 T-DNA Insertions ...... 164

S4. Analysis of Viability in Wild-Type and GPAT9/gpat9-2 Pollen...... 165

S5. Lipid Characterization of Wild-Type and GPAT9-2/gpat9-2 Plants ...... 166

S6. Seed Fatty Acid Composition of GPAT9 amiRNA Knockdown Lines ...... 167

S7. Expression of GPAT9 in Developing Seeds of Wild-Type and GPAT9 … ...... 168

S8. Developing Seed Microsome GPAT Assays of Wild-Type and amiRNA … ...... 169

S9. Testing of Protein:Protein Interactions Between AtLPAAT2, AtDGAT1, and … ..170

S10. Sequence Identity Comparisons Between AtGPAT1 and AtGPAT9 to the … ...... 171

x

LIST OF FIGURES (Continued)

Page

CHAPTER 5

1. TAG Biosynthesis in the eukaryotic pathway of Arabidopsis ...... 184

xi

CHAPTER ONE

INTRODUCTION

PREFACE

Herein represents my contributions to the understanding of how oil is produced in plants. In

Chapters 2 and 3, I will discuss my work analyzing putative phosphatidic acid phosphatases

(PAPs) in Arabidopsis. To date, only two PAP genes, AtPAH1 and AtPAH2 , have been shown to affect oil synthesis: in plants containing a double null mutant at these loci, the fatty acid content of the seeds is reduced only 15% relative to wild type and each of the single mutants, therefore other PAPs must be present to generate the remaining oil [1]. In Chapter 2, I will identify genes as potential PAPs and then analyze them based on published literature, bioinformatics data, and subcellular localization to determine their fitness for study as PAPs involved in oil synthesis.

Chapter 3 will include work to characterize genes identified in Chapter 2, and whether or not they act redundantly with some of the other putative PAPs identified in Chapter 2. In Chapter 4, I describe my contributions to the identification and characterization of the main glycerol-3- phosphate acyltransferase (GPAT) in Arabidopsis, GPAT9. GPATs involved in cuticle formation and the plastidial lipid synthesis pathway have been identified, but this work represents the first identification and characterization of an essential GPAT involved in both oil and ER membrane synthesis.

1.1. INTRODUCTION TO LIPID METABOLISM

Lipids have diverse and vital roles in metabolism, signaling, and structure. Therefore, lipid biosynthesis has been an area of intense research for decades. Structural lipids form the barrier

1

between an organism and its environment, as well as partition organelles within the cell. As a compact source of energy and carbon for growth, the nonpolar lipid triacylglycerol (TAG) is vital to the developing plant embryo and pollen. Lipids also form signaling compounds and hormones for regulation of cellular responses.

Plant-based oils are used by humans both as a source of food calories and in commercial products. Oil from both plants and animals constitutes up to 40% of the caloric intake of a western pattern diet. Lipids are the main component of fats and oils, and as Americans increasingly suffer from obesity and related health problems, ingesting healthier fats by reducing saturated fats and increasing omega-3 fatty acids has become a priority among mainstream Americans. Additionally, as we reduce our dependence on non-renewable natural resources, it is increasingly important to engineer oilseed crops which generate fatty acids useful for industrial processes [2]. Commodities like cooking oil, plastics, paints, pharmaceuticals, lubricants, and dyes all rely on plant-derived oils. Many oil products could be sustainably and renewably sourced from plants, with the proper engineering and a keen eye to scale. Model systems such as Arabidopsis thaliana allow us to study how plants make oil, and then apply this knowledge to improve other plant species, in an effort to produce healthier foods or generate useful fuel alternatives.

In plants, TAG constitutes over 90% of the fatty acids in the seeds of Arabidopsis, and is an essential energy source for both germinating seeds and pollen [3]. A deficiency in TAG leads to sterile pollen and disruptions in embryo development, as seen in the dgat1-1 pdat1-2 double mutant [4]. While the pathways generating lipids are complex, many of the biochemical activities have been elucidated, and there is now a basic understanding of the reactions that generate galactolipids, phospholipids, and TAG in both plants and other organisms [3].

2

1.2. GLYCEROLIPID SYNTHESIS IN ARABIDOPSIS

1.2.1. LIPID BIOSYNTHESIS PATHWAYS AND TAG SYNTHESIS

Higher plants have two distinct pathways for the synthesis of glycerolipids [3]. The prokaryotic pathway is localized to the plastid and is responsible for the synthesis of the major plastidial membrane lipids phosphatidylglycerol (PG), monogalactosyl-diacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG) [3]. The eukaryotic pathway is localized to the endoplasmic reticulum (ER) and generates extraplastidial membrane lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI), as well as the storage lipid TAG [3].

Fatty acids are synthesized de novo in the plastid using acetyl-CoA as the initial substrate

[3]. They are then exported to the ER for the synthesis of lipids in the eukaryotic pathway [3].

This pathway of lipid production was first described by Kennedy and colleagues more than 60 years ago, and describes the steps of lipids synthesis from glycerol-3-phosphate to TAG [5]. As seen in Figure 1, fatty acids are esterified to the sn -1 and sn -2 positions of glycerol-3-phosphate to generate first lyso-phosphatidic acid (LPA) using the enzyme glycerol-3-phosphate acyl transferase (GPAT), and then phosphatidic acid (PA), by the activity of a lysophosphatidic acid acyl transferase (LPAT) [3]. Phosphatidic acid phosphatase (PAP) then dephosphorylates PA to generate diacylglycerol (DAG) [3]. The Kennedy pathway describes DAG as being converted directly to TAG; however more recent studies suggest that de novo DAG is converted to phosphatidylcholine (PC) using PC:DAG choline phosphotransferase (PDCT) [6]. Triacylglycerol can be generated directly from PC and DAG using PC:DAG acyl transferase (PDAT) [7], or either phospholipase C (PLC) or PDCT can generate PC-derived DAG which can be converted to TAG through diacylglycerol acyl transferase (DGAT).

3

Upon synthesis, TAG will form oil bodies, consisting of a monolayer phospholipid membrane filled with hydrophobic TAG. These oil bodies can bud off from the ER and be transported elsewhere for storage and eventual use as an energy source [3]. While each enzymatic function in this pathway is known to occur, many of the genes encoding these functions have not been identified. Chapters 2 and 3 in this dissertation will focus on identifying the PAP involved in oil synthesis, while Chapter 4 will focus on identifying and characterizing the GPAT.

The eukaryotic pathway is also used to generate membrane phospholipids. PC is the main structural lipid in the ER, and the desaturation and acyl editing pathways branch off from PC. PA can be used to make phosphatidyl serine (PS) directly, or can produce CDP-DAG, an activated form of DAG, which is then used to generate phosphatidyl glycerol (PG) and phosphatidylinositol

(PI). DAG can be converted to phosphatidylethanolamine (PE), which can also generate PS or

PC. These phospholipids function as membrane components in the plant, where PC is the main

ER structural lipid along with PE, and PS, PI, and PG make minor structural contributions. PI and its phosphorylated derivatives also play a major role in signaling. PA and DAG are present only in minute amounts in the ER membrane. Additionally, PC generated in the eukaryotic pathway can be converted to DAG and either used for TAG synthesis or transported back to the plastid to generate MGDG, DGDG, and SQDG

1.2.2. WRI1 CONTROL OF CARBON FLUX

Wrinkled1 (wri1 ) was first characterized in 1998 as a mutant whose seeds were impaired in the incorporation of carbohydrates into seed TAG, causing a wrinkled phenotype [8]. The wri1 mutant is compromised in glycolysis, rendering it deficient in converting sucrose into the precursors of TAG [9]. This is because WRI1 is a transcriptional activator which activates a subset of sugar responsive genes and controls the flow of carbon from sucrose import to oil accumulation

4

[10]. WRI1 is also controlled by the transcription factor LEC2 , which plays a central role in embryo development, and WRI1 is required for LEC2 to properly control FA metabolism [11].

WRI1 regulates the expression of genes involved in late glycolysis, fatty acid synthesis, biotin synthesis, and lipoic acid synthesis [12], but it is not known to control genes involved in oil synthesis or packaging. It is a member of the plant-specific transcription family AP2/EREBP family with two AP2 DNA-binding domains [9], and functions by binding the AW box domain

(CnTnGn 7CG) in the promoter [13]. Two enhancer domains have also been identified in the promoters of two targets of WRI1 [12], but it is not yet known whether these enhancer domains are found in promoters of other WRI1 targets.

1.3. THE ROLE OF GLYCEROL -3-PHOSPHATE ACYL TRANSFERASE IN LIPID SYNTHESIS

1.3.1. GPAT ACTIVITY IN LIPID SYNTHESIS

The sn-glycerol-3-phosphate acyltransferase activity is responsible for the first step in glycerolipid synthesis: the acylation of the glycolysis product glycerol-3-phosphate with an acyl-

CoA or acyl-ACP to produce lysophosphatidic acid. This begins the pathway of lipid synthesis which leads to structural lipids that form the membranes of a cell, triacylglycerol that is used as an energy reserve, and signaling molecules which regulate cellular processes. Lipid synthesis occurs mainly in the ER and plastid, although there is also a minor pathway in the mitochondria.

1.3.2. KNOWN GPAT S IN ARABIDOPSIS

The GPAT reaction, like most enzymatic activities in the Kennedy Pathway, was first characterized in animals over 60 years ago [5,14-16]. In mice, there are four homologous isoforms of GPAT, which are responsible for the synthesis of all glycerolipids and are the rate limiting step in TAG synthesis [17], but it has not been conclusively identified in humans. Arabidopsis contains

5

a plastid-localized GPAT (At1g32200; [18]), a plant-specific GPAT family consisting of 8 genes

[19-21], and the ER-localized GPAT9 [21], the focus of our studies. The plastidial GPAT is encoded by the ATS1 locus, and is involved in PG synthesis in the plastid. The ats1-1 mutation eliminated the prokaryotic pathway of lipid synthesis, and when it was combined with a mutant in tgd1-1, chloroplast membrane synthesis was abolished and resulted in lethality [22].

The GPAT1 to -8 family forms a clade distinct from the other two GPATs, and members are involved in the synthesis of cutin and suberin, not phospholipids [19-21,23]. Cutin and suberin serve as a barrier to prevent dehydration, uncontrolled gas exchange, and physical damage from pathogens and herbivores. GPAT4 and GPAT8 are required for the accumulation of C16 and C18 cutin monomers in stems and leaves [24], while GPAT6 is required for incorporation of C16 monomers, and functions in stamen development and pollen fertility [25,26]. These three genes form the first subclade of the family. GPAT5 and GPAT7 also form a subclade, where GPAT5 is responsible for accumulation of C22 and C24 suberin monomers in root and seed coat [20], and

GPAT7 appears to also produce C22 and C24 monomers for suberin synthesis in response to wounding [27]. GPAT1 to -3 form the final subclade, and GPAT1 displays sn -2 acyltransferase activity on saturated, mono unsaturated, and ω-oxidized acyl CoA’s which is essential for male fertility and tapetum differentiation. GPAT2 and GPAT3 have not yet been proven as acyltransferases in vitro [19,27].

While there has been significant study of Arabidopsis GPAT enzymes, the ER-localized isoform involved in structural, signaling, and storage lipid synthesis has not yet been identified. It is expected that a GPAT involved in these processes will be essential for viable plants.

6

1.4. THE ROLE OF PHOSPHATIDIC ACID PHOSPHATASE IN LIPID SYNTHESIS

Phosphatidic acid phosphatases (PAP) are also essential for three main functions in the plant: membrane lipid synthesis, regulation of the signaling functions of PA and DAG, and synthesis of storage lipids. Several of these functions have been elucidated, including the PAPs involved in the prokaryotic pathway of lipid synthesis in the thylakoid membranes. However, the main PAP which is involved in the eukaryotic pathway of the ER has not yet been discovered.

1.4.1. PAP FORMS (PAP1 AND PAP2)

Phosphatidic acid phosphohydrolases (PAH), lipid phosphate phosphatases (LPP), and diacylglycerol pyrophosphate phosphatases (DPP) all share a common enzymatic activity: the hydrolysis of a phosphate group from PA, resulting in DAG [28]. They are generally broken into two groups: the “PAP1” enzymes, which are specific for phosphatidic acid as a substrate, and include the PAHs in yeast and mammals, and the “PAP2” enzymes, including LPPs and DPPs, which are often not specific for a particular lipid substrate and are often involved in signaling functions [28]. These two enzyme classes have a single enzymatic activity: the cleavage of a single phosphate group from PA (or other substrates in the case of LPP) [28]. DPPs are more specific for diacylglycerol pyrophosphate (DGPP) than for PA and catalyze two reactions: first they remove the β-phosphate group from diacylglycerol pyrophosphate, and subsequently hydrolyze the α-phosphate to convert the intermediate PA to DAG [29]. All PAPs are localized to the membrane or are cytosolic proteins with a peripheral association with the membrane, since the substrate PA is almost exclusively found in the membrane [28]. The differentiation between PAHs and LPPs has been well documented in yeast [28]; however studies in plants have been less conclusive, mainly due to the lack of knowledge of the PAP involved in oil synthesis.

7

1.4.2. PAP S IN YEAST AND MAMMALS

Until recently, research on phosphatidic acid phosphatase (PAP) enzymes involved in lipid synthesis had been hampered by a lack of molecular information on the genes encoding this enzymatic activity [30]. LPPs and DPPs have been studied in yeast and mammals in some detail, but these enzymes have little effect on TAG synthesis [28]. The yeast gene Sc PAH1 allowed the molecular identification of the lipin family in mammals, which are PAPs involved in fat accumulation and regulation in mice, rats and humans. Its homologs in Arabidopsis, however, are involved in membrane remodeling and have only a minor effect on TAG. The final PAP in yeast is APP1 , which has no close homologs in Arabidopsis. The lipin family of proteins are the only

PAP enzymes in yeast and mammals which has been shown to affect TAG synthesis [1,31]. The main function of the proteins are the generation of the storage lipid TAG, although it also has a lesser effect on membrane growth, phosphatidic acid signaling, and regulation of overall lipid biosynthesis [1,32,33].

1.4.3. PAP S IN ARABIDOPSIS

Three PAPs in Arabidopsis (LPP γ, LPP ε1, and LPP ε2) have been identified and characterized as being involved in plastidial lipid synthesis, and are therefore not involved in oil synthesis [34]. Three other PAPs in Arabidopsis ( LPP1-3) have been characterized or implicated as being involved in signaling pathways, and are unlikely to be involved in oil synthesis [35,36].

Only two redundant PAP genes (PAH1 and PAH2 ) have been discovered to be involved in TAG synthesis, and these together only account for ~15% of TAG in the seed [1]. Arabidopsis must have other PAP genes which account for the remaining PAP activity. Other than pah1pah2 , no putative PAP mutants in Arabidopsis have been analyzed for a change in seed oil quantity. In this study, I will analyze all putative PAPs for possible involvement in the oil synthesis pathway.

8

1.4.4. PA AND DAG IN LIPID SIGNALING

In addition to their roles as Kennedy pathway intermediates and precursors for membrane lipids, PA, and DAG both have signaling functions, and therefore their accumulation is expected to be tightly regulated. Unlike structural lipids, signaling lipids are present in low quantities in the membrane until a stimulus causes their rapid accumulation; they are then turned over rapidly to attenuate their signaling functions.

Phosphatidic acid that is involved in signaling is generated not by the LPAAT activity mentioned above, but instead by two distinct pathways. In the first pathway, PLD removes the head group primarily from PC or PE to generate phosphatidic acid and the free head group [37].

In the second pathway, Phospholipase C removes both the head group and phosphate from phosphatidylinositol lipids to release DAG, which is then phosphorylated by DAG kinase to produce phosphatidic acid [37]. PA produced from different PLDs can be differentiated by the plant and have different roles in plant response, providing a means to specialize the response to different stresses. The PAP, diacylglycerol pyrophosphate phosphatase, or PA kinase activity may then attenuate the signaling functions of PA by converting it to DAG or DAG pyrophosphate [37].

Phosphatidic acid is produced as a signaling molecule in response to different conditions, including pathogen attack, ABA, osmotic stress, and temperature stress [37]. It acts through two main mechanisms: recruitment of proteins to the membrane and activation or deactivation of the protein’s activity [38-40].

The understanding of the role of DAG in signaling has lagged behind the understanding of

PA as a signaling molecule, but there is compelling evidence that it should not be discarded from consideration. The interacting proteins for DAG have not yet been identified, and those plant proteins homologous to known DAG interactors in animals appear to interact with protein kinases

9

instead of DAG [41]. Because of this, many believe that PA is the main signaling lipid in plants; and indeed the two are interconvertible, making discrimination difficult at times. However, there is important evidence arguing for DAG acting as a signaling molecule, including that DAG accumulates in a manner consistent with an activity in signaling pathways, under various conditions including salinity stress [42], phosphate starvation [43-45], pollen tube tip growth [46], and brassinolide treatment [47]. DAG is known to bind to the C1 domain in protein kinase C (PKC) proteins; and while there are no PKCs in plants, there remains the possibility that it binds to a protein that is not a PKC homolog. Thus, DAG appears to be involved in signaling pathways, and there are many possible targets whose DAG binding have not yet been explored.

1.5. CONCLUSION

Two enzymatic activities in the pathway to TAG and other lipids lack knowledge of their molecular basis. The glycerol-3-phosphate acyl transferase (GPAT) and phosphatidic acid phosphatase (PAP) enzymatic reactions are both known to occur in lipid synthesis in Arabidopsis, and are essential in generating structural, signaling, and storage lipids. In Chapter 2, I will describe my work identifying potential PAP candidates in Arabidopsis. Chapter 3 contains information characterizing the PAP candidates. In Chapter 4, I will describe the characterization of GPAT9 as the main GPAT in membrane and oil synthesis.

10

Figure 1: TAG biosynthesis in the eukaryotic pathway of Arabidopsis. Pictured is a simplified diagram of relevant pathways to TAG. The names of the intermediates and a cartoon of their structure are shown in black, as are the arrows showing flux into TAG. Enzymes are shown in red, and green highlights where membrane lipids diverge off from the oil synthesis pathway. The entire pathway occurs in the membrane of the endoplasmic reticulum, but after synthesis, TAG will exit the ER in oil bodies. [6,48,49]

11

REFERENCES

1. Eastmond PJ, Quettier AL, Kroon JT, Craddock C, Adams N, Slabas AR: Phosphatidic acid phosphohydrolase 1 and 2 regulate phospholipid synthesis at the endoplasmic reticulum in Arabidopsis . Plant Cell 2010, 22 :2796-2811. 2. Napier JA: The Production of Unusual Fatty Acids in Transgenic Plants . Annual Reiew of Plant Biology 2007, 58 :25. 3. Ohlrogge J, Browse J: Lipid biosynthesis . Plant Cell 1995, 7:957-970. 4. Zhang M, Fan J, Taylor DC, Ohlrogge J: DGAT1 and PDAT1 Acyltransferases Have Overlapping Functions in Arabidopsis Triacylglycerol Biosynthesis and Are Essential for Normal Pollen and Seed Development . The Plant Cell 2009, 21 :18. 5. Barron EJ, Stumpf PK: Fat metabolism in higher plants. XIX. The biosynthesis of triglycerides by avocado-mesocarp enzymes . Biochim Biophys Acta 1962, 60 :329-337. 6. Bates PD, Durrett TP, Ohlrogge J, Pollard M: Analysis of Acyl Fluxes through Multiple Pathways of Triacylglycerol Synthesis in Developing Soybean Embryos . Plant Physiology 2009, 150 :18. 7. Banas A, Dahlqvist A, Stahl U, Lenman M, Stymne S: The involvement of phospholipid:diacylglycerol acyltransferases in triacylglycerol production. . Biochemical Society Transactions 2000, 28 :3. 8. Focks N, Benning C: wrinkled1: A novel, low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism . Plant Physiol 1998, 118 :91-101. 9. Cernac A, Benning C: WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis . Plant J 2004, 40 :575- 585. 10. Masaki T, Mitsui N, Tsukagoshi H, Nishii T, Morikami A, Nakamura K: ACTIVATOR of Spomin::LUC1/WRINKLED1 of Arabidopsis thaliana transactivates sugar- inducible promoters . Plant Cell Physiol 2005, 46 :547-556. 11. Baud S, Mendoza MS, To A, Harscoet E, Lepiniec L, Dubreucq B: WRINKLED1 specifies the regulatory action of LEAFY COTYLEDON2 towards fatty acid metabolism during seed maturation in Arabidopsis . Plant J 2007, 50 :825-838.

12

12. Baud S, Wuilleme S, To A, Rochat C, Lepiniec L: Role of WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis . Plant J 2009, 60 :933-947. 13. Maeo K, Tokuda T, Ayame A, Mitsui N, Kawai T, Tsukagoshi H, Ishiguro S, Nakamura K: An AP2-type transcription factor, WRINKLED1, of Arabidopsis thaliana binds to the AW-box sequence conserved among proximal upstream regions of genes involved in fatty acid synthesis . Plant J 2009, 60 :476-487. 14. Kornberg A, Pricer WE, Jr.: Enzymatic esterification of alpha-glycerophosphate by long chain fatty acids . J Biol Chem 1953, 204 :345-357. 15. Weiss SB, Kennedy EP, Kiyasu JY: The enzymatic synthesis of triglycerides . J Biol Chem 1960, 235 :40-44. 16. Kennedy EP: Biosynthesis of complex lipids . Fed Proc 1961, 20 :934-940. 17. Wendel AA, Lewin TM, Coleman RA: Glycerol-3-phosphate acyltransferases: rate limiting enzymes of triacylglycerol biosynthesis . Biochim Biophys Acta 2009, 1791 :501-506. 18. Nishida I, Tasaka Y, Shiraishi H, Murata N: The gene and the RNA for the precursor to the plastid-located glycerol-3-phosphate acyltransferase of Arabidopsis thaliana . Plant Mol Biol 1993, 21 :267-277. 19. Zheng Z, Xia Q, Dauk M, Shen W, Selvaraj G, Zou J: Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility . Plant Cell 2003, 15 :1872-1887. 20. Beisson F, Li Y, Bonaventure G, Pollard M, Ohlrogge JB: The acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis . Plant Cell 2007, 19 :351-368. 21. Gidda SK, Shockey JM, Rothstein SJ, Dyer JM, Mullen RT: Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals: functional divergence of the dilysine ER retrieval motif in plant cells . Plant Physiol Biochem 2009, 47 :867-879.

13

22. Xu C, Yu B, Cornish AJ, Froehlich JE, Benning C: Phosphatidylglycerol biosynthesis in chloroplasts of Arabidopsis mutants deficient in acyl-ACP glycerol-3- phosphate acyltransferase . Plant J 2006, 47 :296-309. 23. Beisson F, Li-Beisson Y, Pollard M: Solving the puzzles of cutin and suberin polymer biosynthesis . Curr Opin Plant Biol 2012, 15 :329-337. 24. Li Y, Beisson F, Koo AJ, Molina I, Pollard M, Ohlrogge J: Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers . Proc Natl Acad Sci U S A 2007, 104 :18339-18344. 25. Li-Beisson Y, Pollard M, Sauveplane V, Pinot F, Ohlrogge J, Beisson F: Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester . Proc Natl Acad Sci U S A 2009, 106 :22008-22013. 26. Li XC, Zhu J, Yang J, Zhang GR, Xing WF, Zhang S, Yang ZN: Glycerol-3-phosphate acyltransferase 6 (GPAT6) is important for tapetum development in Arabidopsis and plays multiple roles in plant fertility . Mol Plant 2012, 5:131-142. 27. Yang W, Simpson JP, Li-Beisson Y, Beisson F, Pollard M, Ohlrogge JB: A land-plant- specific glycerol-3-phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution . Plant Physiol 2012, 160 :638-652. 28. Carman GM, Han G-S: Roles of phosphatidate phosphatase enzymes in lipid metabolism . TRENDS in Biochemical Sciences 2006, 31 :6. 29. Oshiro J, Han G-S, Carman GM: Diacylglycerol pyrophosphate phosphatase in Saccharomyces cerevisiae . Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids 2003, 1635 :9. 30. Han G-S, Carman GM: Characterization of the Human LPIN1 -encoded Phosphatidate Phosphatase Isoforms . The Journal of Biological Chemistry 2010, 285 :11. 31. Han GS, Wu WI, Carman GM: The Saccharomyces cerevisiae Lipin homolog is a Mg2+- dependent phosphatidate phosphatase enzyme . J Biol Chem 2006, 281 :9210-9218. 32. Han G-S, Wu W-I, Carman GM: The Saccharomyces cerevisiae Lipin Homolog is a Mg2+- dependent Phosphatidate Phosphatase Enzyme . The Journal of Biological Chemistry 2006, 281 :9.

14

33. Eastmond PJ, Quettier AL, Kroon JT, Craddock C, Adams N, Slabas AR: A phosphatidate phosphatase double mutant provides a new insight into plant membrane lipid homeostasis . Plant Signal Behav 2011, 6:526-527. 34. Nakamura Y, Tsuchiya M, Ohta H: Plastidic phosphatidic acid phosphatases identified in a distinct subfamily of lipid phosphate phosphatases with prokaryotic origin . J Biol Chem 2007, 282 :29013-29021. 35. Pierrugues O, Brutesco C, Oshiro J, Gouy M, Deveaux Y, Carman GM, Thuriaux P, Kazmaier M: Lipid phosphate phosphatases in Arabidopsis. Regulation of the AtLPP1 gene in response to stress . J Biol Chem 2001, 276 :20300-20308. 36. Katagiri T, Ishiyama K, Kato T, Tabata S, Kobayashi M, Shinozaki K: An important role of phosphatidic acid in ABA signaling during germination in Arabidopsis thaliana . Plant J 2005, 43 :107-117. 37. Testerink C, Munnik T: Phosphatidic acid: a multifunctional stress signaling lipid in plants . Trends Plant Sci 2005, 10 :368-375. 38. Zhang W, Qin C, Zhao J, Wang X: Phospholipase D alpha 1-derived phosphatidic acid interacts with ABI1 phosphatase 2C and regulates abscisic acid signaling . Proc Natl Acad Sci U S A 2004, 101 :9508-9513. 39. Zhang Y, Zhu H, Zhang Q, Li M, Yan M, Wang R, Wang L, Welti R, Zhang W, Wang X: Phospholipase dalpha1 and phosphatidic acid regulate NADPH oxidase activity and production of reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis . Plant Cell 2009, 21 :2357-2377. 40. Huang S, Gao L, Blanchoin L, Staiger CJ: Heterodimeric capping protein from Arabidopsis is regulated by phosphatidic acid . Mol Biol Cell 2006, 17 :1946-1958. 41. Dong W, Lv H, Xia G, Wang M: Does diacylglycerol serve as a signaling molecule in plants? Plant Signal Behav 2012, 7:472-475. 42. Kocourkova D, Krckova Z, Pejchar P, Veselkova S, Valentova O, Wimalasekera R, Scherer GF, Martinec J: The phosphatidylcholine-hydrolysing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress . J Exp Bot 2011, 62 :3753-3763.

15

43. Nakamura Y, Awai K, Masuda T, Yoshioka Y, Takamiya K, Ohta H: A novel phosphatidylcholine-hydrolyzing phospholipase C induced by phosphate starvation in Arabidopsis . J Biol Chem 2005, 280 :7469-7476. 44. Gaude N, Nakamura Y, Scheible WR, Ohta H, Dormann P: Phospholipase C5 (NPC5) is involved in galactolipid accumulation during phosphate limitation in leaves of Arabidopsis . Plant J 2008, 56 :28-39. 45. Jouhet J, Marechal E, Bligny R, Joyard J, Block MA: Transient increase of phosphatidylcholine in plant cells in response to phosphate deprivation . FEBS Lett 2003, 544 :63-68. 46. Helling D, Possart A, Cottier S, Klahre U, Kost B: Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling . Plant Cell 2006, 18 :3519-3534. 47. Wimalasekera R, Pejchar P, Holk A, Martinec J, Scherer GF: Plant phosphatidylcholine- hydrolyzing phospholipases C NPC3 and NPC4 with roles in root development and brassinolide signaling in Arabidopsis thaliana . Mol Plant 2010, 3:610-625. 48. Buchanan BB, Gruissem W, Jones RL (Ed): Biochemistry and Molecular Biology of Plants Rockville: American Society of Plant Physiologists; 2001. 49. Nakamura Y, Koizumi R, Shui G, Shimojima M, Wenk MR, Ito T, Ohta H: Arabidopsis lipins mediate eukaryotic pathway of lipid metabolism and cope critically with phosphate starvation . Proc Natl Acad Sci U S A 2009, 106 :20978-20983.

16

CHAPTER TWO

INITIAL ASSESSMENT OF POTENTIAL PAP GENES INVOLVED

IN OIL SYNTHESIS IN ARABIDOPSIS

ABSTRACT

Even though the enzymes involved in membrane lipid and oil synthesis have been studied for over 60 years, many of the genes associated with these enzymatic activities have yet to be determined. In plants, phosphatidic acid phosphatase (PAP) genes have been studied in plastidial lipid synthesis and in signaling, but the isoform involved in ER membrane lipid and oil synthesis has yet to be elucidated. Here, I use a bioinformatics approach to identify genes in the Arabidopsis genome which could be PAPs involved in membrane lipid and oil synthesis. Thirteen genes in the

Arabidopsis genome were identified using homology to known yeast, mammalian, and plant PAPs.

A PAP involved in these processes should show PAP activity, be expressed in the seed, and should not already be implicated in plastidial lipid synthesis or signaling. Most of the 13 candidate genes were either already shown to be involved in other pathways, or were not localized to the seed.

Only one gene, LPP β (At4g22550), showed characteristics warranting further study. Four other genes, PAH1, PAH2, SPP1 , and LPAP1 showed several characteristics indicating they could act redundantly with the PAP involved in oil synthesis, and therefore will be studied in that context.

At the end of this study, we have shown that LPP β is the only remaining PAP homolog in

Arabidopsis which could be the main PAP involved in oil and membrane lipid synthesis.

17

2.1. INTRODUCTION

While the enzymatic activities involved in lipid synthesis have been known for over 60 years, the genes encoding some of those functions still need to be elucidated. The phosphatidic acid phosphatase (PAP) reaction catalyzes hydrolysis of the phosphate group from phosphatidic acid (PA) to generate diacylglycerol (DAG). While PAP activity has been characterized in the plastidial prokaryotic pathway of lipid synthesis and in stress and signaling pathways, the gene (or genes) encoding the PAP involved in the ER-localized eukaryotic pathway of lipid synthesis has not yet been identified. This enzyme is expected to catalyze an essential step in lipid and oil synthesis, and is therefore expected to be essential and well conserved. Essential genes are often encoded redundantly in genomes to safeguard against possible mutations, deletions, or inactivation

(e.g. [1]); however, many highly conserved, essential housekeeping genes are actually maintained as single genes in a genome despite duplication [2]. Thus, either a single gene or several genes could encode the PAP enzymatic step in the eukaryotic pathway of lipid synthesis.

Two basic types of PAP activity are found in eukaryotic organisms: a “PAP1” type enzyme that is specific for PA, dependent on Mg 2+ , and containing a DXDX(T/V) motif; and a “PAP2” or lipid phosphate phosphatase (LPP) type activity that is not dependent on any divalent cation, is not specific for PA, and is directed by a three-motif domain (KX6RP, PSGH, and SR X5HX3D, reviewed by Carman and Han [3]). The PAP1 enzyme type is the only form that appears to be involved in phospholipid synthesis and oil accumulation; to date, no LPP type enzyme has been implicated in oil synthesis.

2.1.1. PAP HOMOLOGS IN YEAST

Yeast ( Saccharomyces cerevisiae ) has four genes encoding enzymes with PAP activity:

LPP1 [4], DPP1 [5], PAH1 [6], and APP1 [7]. Both the Pah1p and App1p proteins are Mg 2+ -

18

dependent and use a D XDX(T/V) motif for catalysis [8]. The two proteins are not homologous, however; Pah1p is a lipin homolog and the DXDX(T/V) motif is found within the haloacid dehalogenase-like (HAD-like) domain characteristic of the lipin family of proteins [6,9]. The

DXDX(T/V) motif of the App1p protein is found within a region of weak sequence similarity to the HAD-like domain, but the two protein sequences are not similar enough to allow alignment

[7]. In contrast, the Lpp1p and Dpp1p proteins use the PAP2 consensus sequences KX6RP, PSGH, and SR X5HX3D found in lipid phosphatases, mammalian glucose-6-phosphatases, and bacterial nonspecific acid phosphatases [10,11]. Additionally, Pah1p is specific for PA [6], while App1p,

Lpp1p and Dpp1p are not specific for PA and have been shown to also dephosphorylate DAG pyrophosphate, lysoPA, and, in the case of Lpp1p and Dpp1p, isoprenoid phosphates [4,5,7,12].

Lpp1p and Dpp1p both have six transmembrane domains, and are localized to the vacuole and golgi, respectively [4,5,13,14]. App1p is localized to the cytosol, microsome, and is most prominent in the mitochondria [7]. Pah1p exists mainly in the cytosol in its phosphorylated form, but for catalysis it is translocated to the nuclear/ER membrane through dephosphorylation [15].

The yeast Pah1p protein plays an important role in lipid metabolism and is the only PAP known to effect phospholipid and TAG synthesis. Compared to the ∆pah1 mutant, the ∆app1 single mutant and ∆lpp1 ∆dpp1 double mutant did not show significant changes in the DAG, TAG, or phospholipid levels which are characteristic of the ∆pah1 mutant (and likely contributes to its temperature-sensitive phenotype), indicating that PAH1 is the only PAP involved in mediating lipid synthesis in yeast [7]. In addition to the reduction in TAG and misregulation of phospholipid synthesis, the loss of Pah1p activity in the ∆pah1 mutant leads to abnormal expansion of the nuclear/ER membrane, susceptibility to fatty acid induced lipotoxicity, defects in lipid droplet formation and disruption of vacuole homeostasis [6,9,16-19].

19

Unlike Pah1p, the Lpp1p, Dpp1p, and App1p proteins are not involved in de novo lipid synthesis that occurs in the ER [3-5]. While the roles of these proteins have not been fully explored, Lpp1p and Dpp1p appear to play roles in signaling by regulating the relative amounts of

PA, DAG, diacylglycerol pyrophosphate, and lysoPA at the vacuole and golgi, respectively

[4,5,13]. Additionally, both PA and DAG are known to facilitate membrane fission and fusion events in model systems [20-23]. App1p localizes to cortical actin patches and could regulate the local concentrations of PA and DAG [24], although its nonspecific nature indicates it could also regulate concentrations of other phospholipids as well. App1p also shows interactions with endocytic proteins through P XX P domains, and could regulate the enzymes involved in vesicular trafficking [7,25,26].

A quadruple knockout of the four PAPs has no PAP activity, indicating that there are no remaining undiscovered PAPs in yeast [7]. Pah1p is essential for oil synthesis and is the only PAP involved in TAG synthesis or the regulation of phospholipid synthesis. Thus, the PAPs in yeast form two groups: a “PAP1” lipin homolog that is specific for PA and is involved in phospholipid and TAG synthesis, and substrate-nonspecific PAPs that are involved in signaling and other processes.

2.1.2. PAP HOMOLOGS IN MAMMALS

Mammals have two main forms of PAP activity, which generally follow the division in yeast: nonspecific lipid phosphate phosphatases (LPPs) involved in signaling, and the lipin family, which is homologous to ScPAH1 and is involved in TAG synthesis as well as the regulation of lipid metabolism.

The LPP family of proteins are integral membrane proteins that are capable of dephosphorylating a wide variety of substrates, including phosphatidic acid (PA), diacylglycerol

20

pyrophosphate (DGPP), lysoposphatidic acid (LPA), sphingosine 1-phosphate (S1P), and ceramide 1-phosphate (C1P) [27]. LPPs localize predominantly to the ER and golgi, but also localize to the plasma membrane and to other membrane organelles. There are three LPP proteins in both mice and humans, and they demonstrate overlapping catalytic activities and substrate specificities [28]. The LPP1 protein in mouse appears to be the main LPA phosphatase; although a knockout did not cause any strong visible phenotypes, multiple tissues showed reduced LPA phosphatase activity. The LPP2 mutants are similarly unremarkable, and its role in vivo has not yet been established. The LPP3 protein appears to be involved in vascular development in mice, and null mutants are lethal due to an inability to properly form vasculature in utero [28]. While the LPP family is important for the organism, they clearly are not the main PAPs involved in oil or phospholipid synthesis.

The lipin proteins were first studied when Langer and colleagues described a mutation in mice that caused fatty liver dystrophy (fld) [29]; later, the gene that encoded the mutant enzyme was identified and named lipin [30]. Like the yeast homolog, both the human and mouse isoforms contain a D XDX(T/V) motif in a C-terminal HAD-like domain [31]. Lipin proteins are PAPs specific for PA, and regulate fat metabolism in mammalian cells; lipin deficiency prevents normal development of adipose tissue and results in lipodystrophy and insulin resistance in mice, and an excess of lipin1 leads to obesity and insulin sensitivity [30,32].

There are three lipin genes each in mouse and human; the lipin1 gene is the most studied and has several splice variants (reviewed in [33]). The isoforms show differential transcript expression in tissues and across development, and the isoforms exhibit different levels of PAP activity and PA affinity [31,34]. Lipin1 contributes almost all of the PAP activity in skeletal muscle [34]. Deficiency causes accumulation of PA and phospholipids, possibly leading to the

21

severe cardiac muscle damage and rhabdomyolysis in early childhood that is seen in humans with mutations in lipin1 [35,36]. Lipin1 is also a transcriptional coactivator of lipid metabolism genes, and physically interacts with several receptors and transcription factors in different pathways to activate or repress gene expression (e.g. PGC-1α [37], HNF-4α [38]; reviewed in [33]). Unlike the yeast Pah1p, whose activity is regulated through phosphorylation at key phosphorylation sites, mammalian lipins do not have those sites and are not regulated by phosphorylation [39,40].

2.1.3. KNOWN PAP HOMOLOGS IN ARABIDOPSIS

At first glance, the PAP homologs in Arabidopsis follow the strict partitioning of yeast and mammals. There are two lipin homologs, and at least nine homologs of mammalian or cyanobacterial LPPs [41,42].

Of the nine LPP homologs that have been published so far, the biological functions of several have been well established in the literature. AtLPP δ, AtLPP ε1, and AtLPP ε2 are homologous to cyanobacterial LPP’s and are localized to the plastid, the site of the prokaryotic pathway of lipid synthesis [41]. While their specificity for PA was not analyzed, their PAP activity was assayed using the yeast ∆dpp1 ∆lpp1 ∆pah1 mutant, and all three were able to complement the mutant, indicating that they exhibit PAP activity [41]. A homozygous knockout of LPP δ could not be isolated, indicating that it is an essential gene, while a double knockout of lpp ε1lpp ε2 showed no significant phenotype [41]. This indicates that the LPP δ gene in Arabidopsis encodes the main plastidic PAP involved in the prokaryotic pathway of lipid synthesis.

Additionally, AtLPP1 and AtLPP2 are eukaryotic LPP homologs that have been implicated in signaling pathways in Arabidopsis. Both LPP1 and LPP2 proteins showed diacylglycerol pyrophosphate (DGPP) phosphatase activity and PAP activity; LPP2 did not show a preference for either substrate, and LPP1 preferred DGPP [43]. At LPP1 was differentially expressed in

22

response to stress conditions: expression was transiently induced after genotoxic stress and after elicitor treatments. Based on these results, LPP1 is expected to be a PAP involved in attenuating the signaling functions of PA in response to stress conditions [43]. Seeds mutant for AtLPP2 are more sensitive to ABA and the gibberellic acid biosynthesis inhibitor uniconazole during germination, though adult plants did not show this sensitivity [44]. Studies also suggest that LPP2 acts on the PA signaling pool which is downstream of ABA but upstream of ABI4, and parallel with ABI3 [44]. Thus, LPP2 is a negative regulator acting in the ABA signaling network regulating seed germination [44].

Based on the data from yeast and mammals, one would expect that one of the two lipin homologs would be involved in oil synthesis. However, this does not appear to be the case.

Individual knockouts of the At PAH1 and At PAH2 genes do not cause a phenotype, and only when both are knocked out is there a 15% decrease in seed oil [45]. The leaves and roots of pah1pah2 plants show an increase in total lipid and phospholipid content when measured based on fresh weight [45,46]. The mutant showed induced expression of phospholipid synthesis genes, leading to an increase in total ER membrane, similar to the yeast PAH1 mutant [45,46]. During phosphate starvation, the mutants are significantly affected and galactolipid biosynthesis was impaired, indicating that PAH1 and PAH2 could act as mediators of membrane lipid remodeling during phosphate starvation [42]. These data indicate that, similar to the yeast ScPAH1 , the Arabidopsis

AtPAH1 is involved in phospholipid synthesis and membrane management in the ER. However, the modest effect on oil synthesis starkly contrasts with the TAG synthesis phenotype in yeast and mammals, and indicates that the PAP involved in oil synthesis in Arabidopsis does not fit with the precedent set by yeast and mammals. There must be another non-lipin PAP in Arabidopsis that

23

accounts for the remaining 85% of the seed oil in pah1pah2 , which could be encoded either by an

LPP homolog or by a yet-unidentified PAP protein sequence.

In this paper, I will screen the Arabidopsis genome for genes that are homologous to known

PAPs, and analyze their known properties to determine if they could be PAPs involved in oil synthesis. I also confirm that expression of these genes is consistent with a PAP producing oil in the seed. Four genes have the potential to act redundantly with the main PAP involved in oil syntheses, and will be studied in that capacity. Only one of the genes homologous to known PAPs could still be the main PAP involved in oil synthesis, LPP β. However, there remains the possibility that the PAP involved in oil synthesis could be divergent from known sequences and therefore would not be identified in this project.

24

2.2. MATERIALS AND METHODS

2.2.1. PLANT MATERIALS AND GROWTH CONDITIONS

For tissue expression analysis, wild-type seeds of Arabidopsis (ecotype Columbia-0) were pl anted on soil and stratified for 2-3 d at 4°C in the dark. Plants were moved to a growth chamber and grown at 22°C under continuous light with a light intensity of 100-150 µmol m -2 s-1 light. For photoperiod expression analysis, wild type (WT) seeds were sterilized by mixing seeds in 70% ethanol for 10-15 minutes, then were washed with 100% ethanol and allowed to dry. Dry seeds were sprinkled on ½ MS + 1% sucrose plates and stratified for 4 d, then placed in a 22°C growth chamber with continuous light (100-150 µmol m -2 s-1 light). To explore subcellular localization of

LPP β, SPP1, and LPAP1, transient expression was performed in Nicotiana tabacum ‘Petit

Havana’. Seeds were sterilized and grown on 3x MS medium with 0.6% agar for two weeks, transferred to rockwool and grown two weeks more, then planted in soil. Plants were grown at

25°C under continuous light with light intensity of 120-150 µmol m -2 s-1 light.

2.2.2. GENE IDENTIFICATION AND LITERATURE REVIEW

Mouse, human, and yeast PAP, LPP, DPP, APP, and lipin protein sequences were used to search the Arabidopsis proteome using the protein Basic Local Alignment Search Tool (BLASTp)

[47]. Databases were also searched for genes annotated as PAPs, LPPs and DPPs, and the literature was searched for LPP, DPP, and PAP homologs which have been studied. Literature was screened to rule out PAPs which were involved in other pathways and to gain bioinformatics data detailed below.

Protein sequences of the 11 “PAP2” type genes were compared with other known PAPs; protein sequences were aligned with LPP protein sequences from human used by Nakamura et al.

25

[41]: human (“Hs”), mouse (“Mm”), Anabaena (Nostoc ) sp. PCC7120 (“Ana”), Chlorobaculum tepidum (“Ct”), Synechocystis sp. PCC6803 (“Syn”), Saccharomyces cerevisiae (“Sc”), and

Arabidopsis thaliana (“At”). In comparison with the previous study, we used the full protein sequence instead of only the domain sequences, two additional Arabidopsis sequences were used

(R1L and LPAP1), and the sequence for the Escherichia coli (“Ec”) PgpB protein was included.

A phylogenetic tree was constructed using ClustalW (www.genome.jp/tools/clustalw).

2.2.3. BIOINFORMATICS ANALYSIS

Known properties of all gene products were analyzed by searching relevant databases for experimental data first, and then examined for data generated by prediction programs.

Localization was examined using the Subcellular Localization of Proteins in Arabidopsis Database

(SUBA). This database includes data from published chimeric fusion studies and proteomic localization experiments, as well as predictive programs such as WoLF PSORT and TargetP [48-

50].

Tissue expression analysis was used to identify PAP candidates whose transcripts are differentially expressed in tissues where TAG synthesis is high, using the Electronic Fluorescent

Pictograph for visualization [51]. Expression of putative PAPs was analyzed in developing seed tissue in comparison with leaf and overall expression levels.

Coexpression with the known TAG synthesis genes in Arabidopsis could also indicate if the genes are expressed at the same time as TAG is being produced. Coexpression of PAP genes with those known to be involved in TAG synthesis, specifically DGAT1 and PDAT1, was analyzed using GeneCAT [52].

Expression patterns of a PAP involved in oil synthesis should be similar in Arabidopsis and other oilseed species because the PAP enzymatic function is expected to be required for

26

survival, and thus it is expected to be conserved among oilseed species. Therefore, transcript expression of the 13 PAP genes was analyzed on several platforms. Published pyrosequencing studies have examined expression of all 13 gene homologs in the oilseed species Brassica napus ,

Ricinus communis , Euonymus alatus and Tropaeolum majus [53]. Additionally, the 13

Arabidopsis protein sequences were searched against the transcriptomes of soybean and castor bean to determine if all of the genes have homologs and to better understand their function from their tissue expression. The soybean proteome at www.soybase.org and the castor proteome at www.phytozome.net were each searched using BLASTp with of the 13 putative PAP protein sequences. Top soybean and castor hits were then searched back against the Arabidopsis proteome at www.arabidopsis.org to determine best matches. Expression data was not available for any of the soybean PAP homologs.

To determine membrane integration and general protein structure, transmembrane regions in the protein were identified using TMHMM [54].

2.2.4. SILIQUE TISSUE EXPRESSION

To confirm seed transcript expression, 7-10 day old siliques and mature leaves were ground under liquid nitrogen, and RNA was isolated from tissue using the TRIzol Plus RNA Purification

Kit (Life Technologies). DNA contamination was removed using the DNA-Free RNA Kit (Zymo

Research), and complementary DNA generated using the SuperScript III First Strand Synthesis kit

(Life Technologies). LPP β full-length sequence was amplified using LBF1 and LBR1 primers and 35 cycles of PCR, with Actin8 as a control (see Table 5 for all primer sequences found in this chapter). Full length transcripts were amplified using LDF4 and LDR3 for SPP1 , and R1L F2 and

R1L R2 for R1L .

27

2.2.5. SUBCELLULAR LOCALIZATION

Coding sequence were amplified from silique cDNA ([55]; Quanta) and cloned into pENTR: LPP β was amplified using the LBF1-G and LBR1 primers; SPP1 was amplified using

LDF1-G and LDR3, and LPAP1 was amplified using UP1F1G and UP1R3. The sequenced gene was then cloned into pB7WGF2 using the LR reaction of Gateway cloning, to produce an N- terminal fusion under control of the strong, constitutive 35S cauliflower mosaic virus promoter.

BiP, a HSP70 molecular chaperone known to localize to the ER, was a kind gift from Dr. Hwang

(Pohang University, Korea [56]) and was sub-cloned into pB7WGR2 to create a fusion with RFP. pENTR-GUS was obtained from the Gateway cloning kit. BiP and GUS were then cloned into pB7WGR2 and pB7WGF2 as ER and cytoplasm controls, respectively [57]. Sequenced plasmids were transformed into Agrobacterium GV3101 using electroporation.

Tobacco leaves ( Nicotiana tabacum cv. Petit Havana) were infiltrated with a mixture of the experimental- and ER-control-containing bacteria in the presence of acetosyringone (100 uM final concentration). Epidermal peels were visualized on a confocal microscope 48 hours after infiltration. An argon laser (488 nm) and HeNe laser (584 nm) were used to excite GFP and RFP, respectively. Filters isolated 490-520 nm for GFP, 595-630 nm for RFP, and 650-750 nm for chlorophyll autofluorescence.

2.2.6. PHOTOPERIOD -INDUCED EXPRESSION ANALYSIS

To explore whether or not LPP β exhibits the same photoperiod expression as PnFL-1, transcript levels were examined after dark induction. Sterilized seeds were germinated on ½ MS plates and allowed to grow until full cotyledon expansion, about 4 days. Seedlings were placed in the dark, and tissue was harvested by freezing the cotyledons under liquid nitrogen; for samples harvested during dark induction, tissue was harvested in complete darkness. RNA was extracted

28

using the Trizol method (Invitrogen) and DNA contamination removed by digestion with DNaseI

(Zymo Research). Complimentary DNA was prepared using Quanta’s qScript DNA SuperMix cDNA synthesis kit. Expression level was quantified using the Quanta SYBR FastMix kit on the

MX3005P qPCR system (Stratagene). LPP β specific primers LB qPCR-F3 and LB qPCR-R2 amplified a ~200 bp segment of LPP β and the TUB2 qPCR-F and TUB2 qPCR-R primers amplified ~200bp of the Tubulin2 gene. LPP β gene expression was normalized to At TUB2 and the relative fold change between brown seeds and red seeds was calculated (=1/(2^exp-control)).

29

2.3. RESULTS AND DISCUSSION

2.3.1. GENE IDENTIFICATION

The LPP and DPP BLASTp sequence searches consistently resulted in eleven protein sequences (AtLPP β, AtLPP γ, AtLPP ε1, AtLPP ε2, AtSPP1, AtLPP1, AtLPP2, AtLPP3, AtLPP4,

LysoPhosphatidic Acid Phosphatase 1 (LPAP1), and Reduced Oleate Desaturation1-Like (R1L); see Table 1). When Saccharomyces cerevisiae PAH1 and Homo sapiens LPIN protein sequences were used as search queries against the Arabidopsis proteome, two proteins were identified

(AtPAH1 and AtPAH2). The fourth and final PAP in Saccharomyces cerevisiae is Actin Patch

Protein 1 (ScAPP1), which did not have any homologs in Arabidopsis. Through these combined approaches I identified a total of 13 putative Arabidopsis PAPs.

The 13 genes identified fall naturally into two groups. The PAP1 family is Mg 2+ dependent and, in this case, is comprised of only the two genes homologous to lipins from mammals and yeast: At PAH1 and At PAH2 . The second group contains the remaining 11 genes, which are part of the Mg 2+ -independent PAP2 (LPP) family. While PAH1 and PAH2 share high homology with each other and with other lipins, the LPP family of 11 genes shares much lower homology. This can be seen in an unrooted tree in Figure 1 and alignment in Figure 2. Indeed, Nakamura et al. used domain sequences instead of the full protein sequences to generate an unrooted phylogenetic tree, and separated the Arabidopsis sequences into the “prokaryotic” and “eukaryotic” sequences

[41]. They noted that the four “eukaryotic” LPP genes (LPP1-4) share more homology with mammalian and yeast LPP’s, while the “prokaryotic” LPP genes (LPP γ, LPP ε1, LPP ε2, and SPP1) form a second clade and share more homology with cyanobacterial LPP’s [41]. In their analysis,

LPP β was even more closely related to the cyanobacterial genes, and did not fall within either of the primarily-plant clades [41]. When the full protein sequences were used in my analysis, the

30

“prokaryotic” clade (LPP ε1, LPP ε2, LPP γ, SPP1, and LPAP1) was most divergent from known sequences, the “eukaryotic” clade (LPP1-4) was most similar to yeast and Chlorobaculum sequences, and the LPP β and R1L sequences were most similar to bacterial and mammalian clades

(Figure 1).

Most of the 13 genes identified also contained the expected conserved domains for their class of PAP. PAH1 and PAH2 shared a D XDXT catalytic motif as well as conserved N-terminal and C-terminal domains, as described in Eastmond et al. [45]. The PAP2 family has three conserved domains [10], which is present in most of the LPP family members identified. The E. coli protein PgpB is the only PAP2-like protein whose crystal structure has been solved. In PgpB, the three domains form a cluster on the periplasmic side of the membrane, just above the transmembrane domains, and at the top of a cleft where the substrate (such as PA) would sit, allowing access to and cleavage of the head group (phosphate, in the case of a true PAP; [58]).

While the PgpB is not highly conserved with LPP β or the other PAPs, this gives insight as to the mode of action of the PAP2 family. Of the Arabidopsis homologs identified, only 9 of the 11 genes identified by homology shared the three conserved domains (Figure 2). LPAP1 and R1L did not have any of the domains, while all of the remaining 9 genes had all three domains (See Figure

2). This was a definite indication that LPAP1 and R1L may not be prime candidates as PAPs; but since the homology to PgpB is so low, and so much is still to be learned about the mode of action in PAPs, I was hesitant to rule them out as possibly having PAP activity. Indeed, despite the lack of PAP2 domains, LPAP1 does show low PAP activity [59].

2.3.2. LITERATURE REVIEW

Three characteristics of the putative PAP genes excluded them from consideration in this study: a lack of PAP activity, localization to the plastid (or another location other than the

31

cytoplasm or ER), or lack of transcript expression in the seed. First, a protein that under rigorous scrutiny showed no PAP activity would be of little interest to this study, especially if it showed another enzymatic activity. However, a gene which showed even low PAP activity could still act redundantly with other PAPs and possibly compensate for a knockout of the main PAP. Second, the oil synthesis pathway in Arabidopsis is part of the eukaryotic pathway of lipid synthesis and is therefore localized to the ER membrane. It can involve proteins that are either membrane associated or cytoplasmic. A protein which localized to the plastid would likely be involved in the prokaryotic pathway of lipid synthesis, and would not be involved in oil synthesis.

Localization to organelles other than the ER or cytoplasm would also likely not be associated with oil synthesis. Lastly, we are interested in oil synthesis in the context of oilseeds that are harvested for food, fuel, or feedstocks. Genes which are not expressed in the seed are likely not involved in seed oil synthesis, and therefore were ruled out. A summary of bioinformatics and literature review results can be found in Table 1.

Several genes were ruled out summarily. AtLPP ɣ (At5g03080), AtLPP ɛ1 (At3g50920), and AtLPP ɛ2 (At5g66450) have all been shown to localize to the plastid, and are published as the primary plastidic PAPs forming thylakoid membrane lipids [41]. In their studies of Reduced

Oleate Desaturation 1 (ROD1 ), Lu et al [60] analyzed At3g15830. They found that this gene, which I am here calling ROD1-Like (R1L ), was not expressed in the silique; and my RT-PCR studies also confirmed that the gene is not expressed in the seed. (See “Seed Tissue Expression,” section 2.3.5.) Similarly, AtLPP4 (At3g18220), is expressed only in the pollen, indicating that it may be involved in oil synthesis in the pollen, but not in seeds.

AtLPP1 (At2g01180) and AtLPP2 (At1g15080) are not expressed in the seed, and both have roles in signaling processes: LPP1 is involved in PA and DGPP signaling [43] and LPP2 is

32

involved in ABA signaling [44]. AtLPP3 (At3g02600) is highly homologous to LPP1 and LPP2

[43]. These three genes form an LPP clade, which is further separated into two sub-groups: one composed of LPP1, and one composed of LPP2 and LPP3 [43]. The authors chose to study one gene from each sub group, and therefore chose LPP2 to represent both LPP2 and LPP3 [43]. I chose to follow the grouping set forth by Pierrugues et al., and assumed that LPP3 is also involved in lipid signaling [43].

Five genes remained after the exclusion of putative PAPs clearly not involved in oil synthesis. AtPAH1 (At3g09560) and AtPAH2 (At5g42870) were shown to have PAP activity and are expressed in the seed, but have been shown to be involved in membrane homeostasis and lipid remodeling under phosphate stress. While I did not characterize PAH1 or PAH2 except as controls to my experiments, I did use the pah1pah2 mutant background to determine if other putative PAPs were redundant with PAH1 and PAH2 .

Two genes, AtSPP1 and an unnamed lysophosphatidic acid phosphatase I am here calling

Lysophosphatidic acid phosphatase (AtLPAP1), showed interesting characteristics which warranted further study. AtLPAP1 (At3g03520) had lysophosphatidic acid phosphatase activity when expressed in yeast, but it also showed low levels of PAP activity [59]. This activity, coupled with its expression in the seed and lack of a clear physiological role, warranted further study. The sphingoid phosphate phosphatase 1 (SPP1) gene (At3g58490; formerly LPP δ) has slightly more information available, and is implicated in ABA-mediated stomatal closure [61]. Measurements of long-chain base-1-phosphate levels in yeast expressing SPP1 and mutant Arabidopsis suggested that it encodes a long-chain base-1-phosphate phosphatase, but rigorous enzyme assays, including

PAP activity, were not completed [61]. Due to the lack of molecular data on SPP1 ’s enzymatic activity, it also warranted further study. My goal with these two genes was to determine if they

33

were redundant with PAH1 and PAH2 and the remaining putative PAPs; and to confirm, if convenient, their lack of PAP activity.

The final and most promising candidate was AtLPP β (At4g22550). Information about expression was limited because there are no probes for it on the AffyMetrix chip used to determine tissue expression for most genes in Arabidopsis, and it had never been studied in depth. The only information available was that it was not found in the chloroplast [41]. Unpublished reports also suggest that LPP β encodes a protein that displays PAP activity [62]. A BLASTp analysis of putative homologs produced many putative or unknown proteins, but only one protein with any published information. PnFL-1 from the short day plant Japanese Morning Glory ( Ipomea nil , formerly Pharbitis nil ) has 58% homology to LPP β, and is expressed during the inductive dark period necessary for flowering, but expression does not occur if the plant is exposed to a night break (15 min of light during dark period) [63]. The two proteins are similar in size (LPP β is 213 amino acids; PnFL-1 209 aa); and a reciprocal BLASTp shows that LPP β is the top homolog of

PnFL-1 in the Arabidopsis genome (43% identity covering 93% of LPP β), followed by LPP γ (32% identity covering 60% of LPP γ). These reports have led some to believe that LPPβ is a photoperiodically expressed homolog of PnFL-1 [62], but this has not been experimentally confirmed. My further analysis of whether LPP β expression is diurnally controlled is described in

Section 2.3.7, below.

2.3.3 BIOINFORMATICS ANALYSIS

All of the bioinformatics data analyzed can be found in Tables 1-4, but my analysis will focus on those six genes which have not yet been excluded from consideration: LPP β, R1L,

LPAP1, SPP1, PAH1, and PAH2. Tissue expression was analyzed to determine if genes were expressed in developing seeds, the main site of oil synthesis. Online resources are largely based

34

on data produced from the Affymetrix ATH1 microarray chip which does not represent all the genes in Arabidopsis. Analysis of the database showed that LPAP1, PAH1 , and PAH2 are all expressed in the developing seed, but SPP1 and LPP β are not on the chip (Column 8, Table 1).

ROD1-Like also showed seed expression, which conflicted with the expression data in Lu et al.

[60]; therefore LPP β, SPP1 , and R1L required confirmation of their expression (See section 2.3.4,

“Seed Tissue Expression”).

Subcellular localization of the 13 identified putative PAPs was analyzed to determine if the identified genes localized to organelles other than the ER or cytoplasm, and therefore could be ruled out. Several PAPs have published localization data based on experimental evidence in conjunction with the data from the predictive programs found in the SUBA database. PAH1 and

PAH2 are localized to the cytoplasm [42], and SPP1 is localized to the ER [61] (see Table 1, column 5), consistent with possible involvement in the eukaryotic pathway of lipid synthesis.

None of these three proteins showed a consistent localization from the predictive programs. LPP β had 7 of 25 predictions for the ER, and only 1 of 25 predictions indicated plastidial localization.

With some data indicating ER localization and without a confident prediction to the plastid, LPP β remains a potential PAP involved in oil synthesis.

Coexpression with known Arabidopsis TAG synthesis genes was examined to determine if the putative PAPs are involved in oil synthesis. Although two genes may have similar expression patterns simply by chance, coexpression often indicates that the genes are involved in similar processes. DGAT1 (At2g19450) and PDAT1 (At5g13640) both produce TAG, and together represent the last step generating TAG [64]. Coexpression was examined using GeneCat, which queries published expression data based on the AffyMetrix ATH1 gene chip. As was seen in earlier analyses, LPP β and SPP1 are not on the ATH1 chip, and therefore data was not available

35

on these genes. Data from the remaining genes showed that they are not strongly coexpressed with

DGAT1 and PDAT1; coexpression values were between 0.22 and -0.10, with 1.00 being perfectly correlated and -1.00 being perfectly anti-correlated (See Table 1, column 9).

Other oilseed species were analyzed to determine identity and, if possible, expression levels of PAP homologs in those species. The castor bean ( Ricinus communis ) is an oilseed species which produces ricinoleic acid, a hydroxylated fatty acid important in manufacturing many items including lubricants, paints, plastics, and pharmaceuticals. When the thirteen PAPs of interest were searched against the Castor genome, many had multiple homologs (see Table 2). LPP β had only one primary homolog, meaning that when the homolog to LPP β was searched back against the Arabidopsis genome, LPP β was the top candidate. This homolog is annotated as a sphingosine-

1-phosphate phosphohydrolase, because of its (and LPP β’s) homology to SPP1; however this annotation, found in many species, is not based on experimental evidence.

Homologs of the identified putative PAPs were also analyzed in the soybean plant, Glycine max . Soybean is a crop plant grown for both its oil and protein in the seed; after extraction of the oil, the pressed meal is high in protein and is a common addition to human and animal food. As in Castor, LPP β only had one homolog in Soybean (see Table 3). LPP4 and LPP ε1 had no primary homolog, but the remaining 10 Arabidopsis genes all had 2 or 4 homologs.

Many gene expression analyses, even those done in species other than Arabidopsis, have been based on microarray data, and LPP β homologs are rarely on the microarrays. One data set that did include LPP β homologs is 454 pyrosequencing data done by Troncoso-Ponce and Kilaru and colleagues [53] (see Table 4). They sequenced transcripts from the oil producing seed tissues in the oilseeds Brassica napus, Tropaeolum majus, Ricinus communis, and Euonymus alatus. The oil-producing tissues of all four species showed low expression of LPP β homologs, but expression

36

was present. In the Brassicales species B. napus and T. majus , oil is stored in the embryo; but in the Fabids R. communis and E. alatus , oil is stored in the endosperm. R. communis and E. elatus provide an interesting opportunity to compare the oil producing tissue (endosperm) with non- producing tissue, the embryo. Interestingly, virtually all of the PAPs studied had higher in the embryo than in the endosperm, except SPP1 in E. alatus . This could indicate that none of the

PAPs studied are the main oil-producing PAP in these species, but a PAP that is involved in membrane lipid synthesis as well as oil synthesis may not be upregulated in oil producing tissues.

2.3.4. BIOINFORMATICS OF LPP β

After exploring data on all of the putative PAPs, it was clear that LPP β was the primary

PAP of interest for this study. Therefore, the remaining bioinformatics analyses focused only on

LPP β.

Since LPP β is not on the ATH1 microarray chip, we investigated other sources for tissue expression data in Arabidopsis. The AGRONOMICS tiling array is designed to allow expression profiling of more than 30,000 genes in Arabidopsis, with results that are comparable to the ATH1 chip [65]. LPP β is represented on the AGRONOMICS array and the four anatomical tissues tested

(juvenile and adult leaf, seedling, and flower) all show high expression levels (See Figure 3A).

Unfortunately, neither the seed nor silique were tested, so expression of LB in the seed was confirmed using RT-PCR (See “Seed Tissue Expression,” section 2.3.5).

The protein structure of LPP β was also investigated. Analysis using TMHMM indicated that LPP β has four transmembrane domains, and is probably an integral membrane protein. It was then compared with the only known PAP2-type protein whose crystal structure has been solved, the phosphatidylglycerol-phosphate phosphatase B in E. coli (ecPgpB) [58]. PgpB and LPP β share very little homology, but LPP β does share the three signature PAP2 motifs: C1, “KX 6RP”; C2,

37

“PSGH”; and C3, “SRX 5HX 3D” (Figure 2) [10]. The four transmembrane domains indicated by

TMHMM could be capable of forming a transmembrane core, as is seen in the PgpB structure

(Figure 3B). These data indicate an integral membrane structure and suggest PAP or related activity.

2.3.5. SEED TISSUE EXPRESSION

LPP β, SPP1, and R1L required confirmation that they actually were expressed in the seed, which could be accomplished by looking at expression from the entire silique using RT-PCR.

While the genomic reaction worked for R1L, indicating that the PCR reaction worked, the coding region of R1L was not able to be amplified from either leaf or silique cDNA, indicating that it is not expressed in these tissues (see Figure 4A). This is consistent with the data from Lu et al., which showed expression only in flowers and not in seedlings, young leaves, or siliques [60].

Therefore, R1L was conclusively dismissed from this study. SPP1 and LPP β both showed bands in the leaf and silique cDNA reactions, indicating that they are expressed in these tissues and warrant further study (See Figures 4). The LPP β gene does not have introns, so the genomic and cDNA bands are the same size, but the Actin8 reaction showed that the cDNA was not contaminated with genomic DNA. Therefore, after tissue expression analysis, LPP β remains the top PAP candidate, SPP1 remains a gene of interest because of its possible redundancy with LPP β, and R1L is no longer a gene of interest.

2.3.6. SUBCELLULAR LOCALIZATION

To confirm that the LPP β, SPP1, and LPAP1 proteins localize to the ER or cytoplasm, they were expressed in tobacco epidermal cells as N-terminal GFP fusions. GUS was fused to GFP as a cytosolic control, and a RFP-BiP fusion served as an ER control. Chlorophyll autofluorescence allowed visualization of the plastid. Plastidial localization would indicate involvement in the

38

prokaryotic pathway of lipid synthesis, as in the case of LPP γ, LPP ε1 and LPP ε2 [41] and would be cause to set that gene aside from consideration. Similarly, localization to other organelles such as the peroxisome, vacuole, mitochondrion or nucleus would indicate that the gene product is not involved in oil synthesis. However, PAPs involved in oil synthesis could be localized to either the cytoplasm or the endoplasmic reticulum. As seen in Figure 5, the GFP-GUS cytoplasmic control appears to co-localize to the RFP-BiP endoplasmic control, so discriminating between cytoplasmic and ER localization is not possible with this method. The four transmembrane domains in LPP β argues for an integral membrane association, so cytoplasmic localization is not expected. LPP β, as well as LPAP1 and SPP1, appear to localize to the cytoplasm/ER (Figure 5): the green gene fusion and the red ER appear to colocalize. The blue chlorophyll autofluorescence does not colocalize with the green fusions, and in some cases even fills in spaces in the ER occupied by the plastids. Thus, LPP β appears to localize to the ER and remains a strong candidate for the PAP involved in oil synthesis. LPAP1 and SPP1 also appear to localize to the ER/cytosol, and could still be acting redundantly with LPP β in oil synthesis.

2.3.7. PHOTOPERIOD EXPRESSION OF LPP β

The LPP β gene has not been carefully studied and there are few homologs of LPP β with any published data. The only published information on LPP β homologs is on the Pharbitis nil

(Ipomea nil ) gene FL-1, which is expressed during the flower-inducing dark period [63].

Expression during the light hours was negligible, but after 10 hours of darkness, Pn FL-1 expression was increased 100 fold. A night break (15 min light at the 8 hour time point) extinguished expression, and expression ended shortly after dawn. Thus, the authors surmised that

Pn FL-1 could be involved in inducing flowering in the short-day plant (see [63]).

39

Since the only homolog of LPP β that has been studied was published as a photoperiod- expressed gene involved in the inductive dark period, examining the photoperiod expression of

LPP β was warranted. Since morning glory is a short day plant and Arabidopsis is a long day plant, both long day and short day light conditions were tested; a continuous light condition was also tested as a control. Tissue from fully expanded cotyledons was harvested before light treatment, at the end of an 8 hour dark period and two hours after dawn, at the end of a 16 hour dark period and 2 hours after dawn, at the 24 hour time point after both treatments, and after 24 hours without any dark treatment. While the Pn FL-1 gene showed about a 100-fold increase in expression from

0h to 10h dark, LPP β showed less than a 1-fold decrease in all conditions tested (Figure 6). Thus, expression of LPP β does not appear to change enough to warrant involvement in any photoperiod- induced flowering pathway.

Photoperiod expression in a PAP involved in oil synthesis is not expected but also would not be unexpected. Many metabolic enzymes show diurnal expression patterns (e.g. see [66,67]), and one would expect that the substrates for fatty acid synthesis, and therefore lipid and oil synthesis, would also have similar regulation. Indeed, it has been known for many years that fatty acid synthesis levels and fatty acid composition undergo diurnal fluctuations [68], and more recently proteins involved in lipid synthesis (ACBP3) and oil synthesis (DGAT1 and DGAT3) have been shown to be diurnally regulated [69]. However, several oil synthesis genes do not show diurnal regulation (e.g.DGAT2 and PDAT1), indicating that not all steps in the pathway are similarly regulated. Thus, the consistent expression level of LPP β throughout the day does exclude it from being a photoperiod regulator, but it does not affect its status as a possible PAP involved in oil synthesis.

40

2.4. CONCLUSION

The Arabidopsis genome contains thirteen genes which are homologous to known PAP genes. Most have traits inconsistent with a PAP involved in oil synthesis, and have been set aside from consideration. AtLPP γ, AtLPP ε1, and AtLPP ε2 are localized to the plastid and are involved in the prokaryotic pathway of lipid synthesis. AtLPP1, AtLPP2 , and AtLPP3 are involved in signaling pathways. Neither AtLPP4 nor AtR1L are expressed in the seed. Several other genes display characteristics that indicate they could be redundant with the PAP involved in oil synthesis.

AtPAH1 and AtPAH2 are involved in oil synthesis, and together account for 15% of seed oil.

AtLPAP1 showed some PAP activity, and AtSPP1 was not assayed for PAP activity. The top candidate for a PAP involved in oil synthesis is AtLPP β, a gene whose tissue expression and subcellular localization are consistent with a PAP involved in oil synthesis in seeds. Therefore,

AtLPP β will be analyzed in Chapter 3 in conjunction with AtPAH1, AtPAH2, AtLAP1, and AtSPP1 to determine if they function redundantly .

41

AnaLPP1 AnaLPP2 Bacterial SynLPP EcPgpB HsPAP2A MmPpap2a HsPAP2C Mammalian MmPpap2c HsPAP2B AtLPP β AtR1L CtLPP2 AtLPP2 AtLPP3 “Eukaryotic” Arabidopsis proteins AtLPP1 AtLPP4 ScDPP1 CtLPP1 ScLPP1 AtLPP ε1 AtLPP ε2 AtSPP1 “Prokaryotic” Arabidopsis proteins AtLPPg AtLPAP1

Figure 1. Phylogenetic tree of LPP homologs. Rooted phylogenetic tree of human, mouse, yeast, bacterial, and plant LPPs drawn based on full protein sequences. Arabidopsis sequences are noted in bold, and LPP β is highlighted in yellow. The horizontal branch lengths are proportional to sequence divergence. Hs, Homo sapiens; Mm, Mus musculus ; Ana, Anabaena (Nostoc ) sp. PCC7120; Ct, Chlorobaculum tepidum ; Syn, Synechocystis sp. PCC6803; Ec, Escherichia coli ; Sc, Saccharomyces cerevisiae ; At, Arabidopsis thaliana .

42

1 10 20 30 40 50 60 | | | | | | | E. coli PgpB ------Yeast ScLPP ------MISVMADEKHKEYFKLYYFQYMIIGLCTILFLYSEI AtLPPb; AT4G22550.1 ------AtLPP1; AT2G01180.1 ------MTIGSFFSSLLFWRNSQDQEAQRGRMQEIDLSVHTIKSHGGRV AtLPP2; AT1G15080 ------MPEIHLGAHTIRSHGVTV AtLPP3; AT3G02600 MARFSFPCFPNFGGFNQAVTNRGPEISETADNWVSPSDIPLIEPNSKEHRMREAQLGGHTLRSHGMTV AtLPP4; AT3G18220.1 ------MAKIMLGSHSVKSHGWKV AtLPPe1; AT3G50920 ------MAASSSLLLLLHCPTCNFYFDSSSYLKRSRLSSYSSIISRGSP AtLPPe2; AT5G66450.1 ------MAASSSSLLLLHKPTYNFHFAASSVPTYINSARFRISSSIFPLDRRRRRR AtSPP1; AT3G58490.1 ------MKKILERSMEGSGTWQGLV AtLPPg; AT5G03080 ------

E. coli PgpB ---MRSIARRTAVGAALLLVMPVAVWISGWRWQPGEQSWLLKAAFWVTETVTQPWGVITHLILFGWFL Yeast ScLPP SLVPRGQNIEFSLDDPSISKRYVPNELVGPLECLILSVGLSNMVVFWTCMFDKDLLKKNRVKRLRERP AtLPPb; AT4G22550.1 ------MSPSTTTIFIGPILAVDAAVSHAIHTAAKPFLPPFVLLLLEISADFRFSFPV AtLPP1; AT2G01180.1 ASKHKHDWIILVILIAIEIGLNLISPFYRYVGKDMMTDLKYPFKDNTVPIWSVPVYAVLLPIIVFVCF AtLPP2; AT1G15080 ARFHMHDWLILLLLIVIEIVLNVIEPFHRFVGEDMLTDLRYPLQDNTIPFWAVPLIAVVLPFAVICVY AtLPP3; AT3G02600 ARTHMHDWIILVLLVILECVLLIIHPFYRFVGKDMMTDLSYPLKSNTVPIWSVPVYAMLLPLVIFIFI AtLPP4; AT3G18220.1 AREHLCDWLILVVLGLIDIVLNVIEPFHRYIGPDMLTDLTFPFYEDTIPMWAVPIICILVPICIFIVY AtLPPe1; AT3G50920 LFVSSFGSMTVKRFSSRVGSRSNDGNEQFGALEQESFINNSSEIRKDLVTGGGIEAIVNRLSKWVVSV AtLPPe2; AT5G66450.1 IWSVSGFKSMADLVKTNARRDGEDRFQALEQEAFISNSSSELQNELVSDAGDGIEAIANRLSKWIVAA AtSPP1; AT3G58490.1 LVGIVTWICASSYLKFTHKFRSLLQPWVARQVVGGVPLILRIQKCQNGVLDAFFSGLSCVVSVPFYTA AtLPPg; AT5G03080 ------MDLIPQQLKAVTLTHVRYRPGDQLGHFLAWISLVPVFISLG

E. coli PgpB WCLRFRIKAAFVLFAILAAAILVGQGVKSWIKDKVQEPRPFVIWLEKTHHIPVDEFYTLKRAERGNLV Yeast ScLPP DGISNDFHFMHTSILCLMLIISINAALTGALKLIIGNLRPDFVDRCIPDLQ------KMSD AtLPPb; AT4G22550.1 SLSFLLSPPLRSFLVPFLLGLLFDLIFVGIVKLIFRRARPAYNHPSMSA------AtLPP1; AT2G01180.1 YLKRTCVYDLHHSILGLLFAVLITGVITDSIKVATGRPRPNFYWRCFPD------G AtLPP2; AT1G15080 YFIRNDVYDLHHAILGLLFSVLITGVITDAIKDAVGRPRPDFFWRCFPD------GI AtLPP3; AT3G02600 YFRRRDVYDLHHAVLGLLYSVLVTAVLTDAIKNAVGRPRPDFFWRCFPD------G AtLPP4; AT3G18220.1 YYYRRDVYDLHHAILGIGFSCLVTGVTTDSIKDAVGRPRPNFFYRCFPN------GK AtLPPe1; AT3G50920 LFGSIILLRHDGAALWAVIGSISNSALSVVLKRILNQERPTTTLRSDP------AtLPPe2; AT5G66450.1 LFGSVLLLRHDGAALWAVIGSVSNSVLSVALKRILNQERPVATLRSDP------AtSPP1; AT3G58490.1 FLPLLFWSGHGRLARQMTLLIAFCDYLGNCIKDVVSAPRPSCPPVRRITAT------AtLPPg; AT5G03080 GFVSHFLFRRELQGIFFGIGLVISQFINEFIKTSVEQARPET------Domain 1

E. coli PgpB KEQLAEEKNIPQYLRSHWQKETGFAFPSGHTMFAASWALLAVGL------L Yeast ScLPP SDSLVFGLDICKQTNKWILYEGLKSTPSGHSSFIVSTMGFTYLW------QRV AtLPPb; AT4G22550.1 ------AVSADHYSFPSGHASRVFFVAASVHFFSAAAEASMTGPSYSFLDGWIRDHN AtLPP1; AT2G01180.1 KELYDALGGVVCHGKAAEVKEGHKSFPSGHTSWSFAGLTFLSLY------LSGKIKAFNNE AtLPP2; AT1G15080 GIFHNVTKNVLCTGAKDVVKEGHKSFPSGHTSWSFAGLGFLSLY------LSGKIRVFDQR AtLPP3; AT3G02600 KALYDSLGDVICHGDKSVIREGHKSFPSGHTSWSFSGLGFLSLY------LSGKIQAFDGK AtLPP4; AT3G18220.1 PKFHPDTKDVVCHGVKKIIKEGYKSFPSGHTSWSFAGLTFLAWY------LSGKIKVFDRR AtLPPe1; AT3G50920 ------GMPSSHAQSISFISVFAVLS------VMEWLG AtLPPe2; AT5G66450.1 ------GMPSSHAQSISFISVFSVFS------VMEWLG AtSPP1; AT3G58490.1 ------KDEEDNAMEYGLPSSHTLNTVCLSGYLLHY------VLSSLEYE AtLPPg; AT5G03080 ------CTLLEACDSHGWPSSHSQFMFFFATYFSLM------GCKGIGFWF Domain 2

E. coli PgpB WPRRRTLTIAILLVWATGVMGSRLLLGMHWPRDLVVATLISWALVAVATWLAQRICGPLTPPAEENRE Yeast ScLPP FTTRNTRSCIWCPLLALVVMVSRVIDHRHHWYDVVSGAVLAFLVIYCCWKWTFTNLAKRDILPSPVSV AtLPPb; AT4G22550.1 DGDVKVEVVVVVWIWATVTAISRILLGRHYVLDVAAGAFLGIVEALFALRFLRFDEMIFGR------AtLPP1; AT2G01180.1 GHVAKLCLVIFPLLAACLVGISRVDDYWHHWQDVFAGALIGTLVAAFCYRQFYPNPYHEEGWGPYAYF AtLPP2; AT1G15080 GHVAKLCIVILPLLVAALVGVSRVDDYWHHWQDVFGGAIIGLTVATFCYLQFFPPPYDPDGWGPHAYF AtLPP3; AT3G02600 GHVAKLCIVILPLLFAALVGISRVDDYWHHWQDVFAGGLLGLAISTICYLQFFPPPYHTEGWGPYAYF AtLPP4; AT3G18220.1 GHVAKLCLVFLPILISILIGISRVDDYWHHWTDVFAGAIIGIFVASFSYLHFFPYPYDENGWAPHAYF AtLPPe1; AT3G50920 TNGVSLFLSGLILALGSYFIRLRVSQKLHTSSQVVVGAIVGSLFCILWYTMWNSLLREAFEASLLVQI AtLPPe2; AT5G66450.1 TNVLSLFLSGFILALGSYFTWLRVSQKLHTTSQVVVGAIVGSVYSTLWYVTWNSLVLEAFTSTFSVQI AtSPP1; AT3G58490.1 SVSIQYYGFALACLLVALIAFGRVYLGMHSVVDIVSGLAIGVLILGLWLTVNEKLDDFITSKQNVSSF AtLPPg; AT5G03080 GLRSRWIMNLLHWSLAVVTMYSRVYLGYHTVAQVFAGAALGGIVGASWFWVVNSVLYPFFPVIEESVL Domain 3

43

E. coli PgpB IAQREQES------Yeast ScLPP ------AtLPPb; AT4G22550.1 ------AtLPP1; AT2G01180.1 KAAQERGVPVTSSQNGDALRAMSLQMDSTSLENMESGTSTAPR-- AtLPP2; AT1G15080 QMLADSRNDVQDSAGMNHLSVRQTELESVR- AtLPP3; AT3G02600 QVLEAARVQGAANGAVQQPPPQVNNGEEEDGGFMGLHLVDNPTMRREEDVETGRG-- AtLPP4; AT3G18220.1 RMLAERSTGRATTMTRTGSRGMLGNDVEPGNSASSPHDRHRESTDSDF------AtLPPe1; AT3G50920 SVFLFAATFALAFAAYVVLNWFKDER------AtLPPe2; AT5G66450.1 ALFLVAAASALGFAVYVLLNWFKDDR-- AtSPP1; AT3G58490.1 WTALSFLLLFAYPTPEHPTPSYEYHTAFNGVTLGIVTGVQQTYSQFHHEAAPRIFSPELPISSYLGRV AtLPPg; AT5G03080 GRWLYVKDTSHIPDVLKFEYDNARAARKDMDSAKSD-

E. coli PgpB ------Yeast ScLPP ------AtLPPb; AT4G22550.1 ------AtLPP1; AT2G01180.1 ------AtLPP2; AT1G15080 ------AtLPP3; AT3G02600 ------AtLPP4; AT3G18220.1 ------AtLPPe1; AT3G50920 ------AtLPPe2; AT5G66450.1 ------AtSPP1; AT3G58490.1 MVGIPTILLVKFCSKSLAKWTLPMVSNALGIPIRSSMYIPKLKGYASGKKTDEPKNSVGYLQKLCEFL AtLPPg; AT5G03080 ------

E. coli PgpB ------Yeast ScLPP ------AtLPPb; AT4G22550.1 ------AtLPP1; AT2G01180.1 ------AtLPP2; AT1G15080 ------AtLPP3; AT3G02600 ------AtLPP4; AT3G18220.1 ------AtLPPe1; AT3G50920 ------AtLPPe2; AT5G66450.1 ------AtSPP1; AT3G58490.1 SHDSFDIDTGIRFFQYAGLAWSVVDLVPSLFSYVNL AtLPPg; AT5G03080 ------

Figure 2. Conserved Domains in the PAP2 family of PAPs. Alignment of full protein sequences in known LPPs and newly identified LPPs. LPP β is highlighted in yellow. The three conserved PAP2 domains are highlighted in blue. Underlined regions indicate transmembrane domains in LPP β and in EcPgpB, the only PAP2 enzyme whose crystal structure has been solved.

44

1 2 3 4 5 6 7 8 9 Literature Review Coexpression*

AT2G19450

DGAT At AT5G13640 Accession Name aa PDAT PAP Activity? PAP Localization decreases KO levels? FA/lipid otherin Involved pathway? Tissue Expression

AtLPP β not At4g22550 213 chloroplast -- (LB) [41]

Expect ubiq, dry seed -- At3g02600 AtLPP3 364 signaling [43] [51]

Rod1Like Not in silique At3g15830 296 -0.103 (R1L) [60]

At3g18220 AtLPP4 308 pollen [44] 0.216

AtLPP1 DPP & PA At2g01180 327 Stress [43] leaves [51] 0.060 (PAP1) [43]

AtLPP2 DPP & ABA signaling SAM At1g15080 290 -0.091 (PAP2) PAP [43] [44] (embryo) [51]

Chloroplast chloroplast Yes [41] not seed [51] -0.069 At3g50920 AtLPP ε1 279 [41] PAP [41] No [41] Chloroplast chloroplast Yes [41] -- At5g66450 AtLPP ε2 286 [41] PAP [41]

Chloroplast chloroplast ubiq, dry seed Yes [41] Yes [41] -0.017 At5g03080 AtLPP γ 226 [41] PAP [41] [51]

Low PAP LPA P'ase [59] Seed [51] -0.071 At3g03520 AtLPAP1 523 [59]

AtSPP1 LCBP ABA signaling At3g58490 416 ER [61] -- (LPP δ) P'ase [61] [61]

At3g09560 AtPAH1 904 Yes [42] dry seed [51] 0.005 glycerolipid Yes [42] synthesis [42] At5g42870 AtPAH2 930 Yes [42] dry seed [51] -0.005

Table 1: Literature review and protein information of putative PAPs. LPP β, the main putative PAP of interest, is highlighted in yellow, while the four (putative) PAPs that could act redundantly with LPP β are highlighted in grey. Pink background indicates negative attributes; tan background indicates neutral attributes, and green background indicates positive attributes. * Column 9: Coexpression with DGAT and PDAT: value given is an average of the two coexpression values, as calculated by GeneCat [52].

45

Castor Bean ( Ricinus communis ) PAP Homologs At4g22550 At3G02600 At3G15830 At3G18220 At2G01180 At1g15080 At5G03080 At3g50920 At5g66450 At3g58490 At3G03520 At3G09560 At5G42870

Phytozome

Accession Name 1 2 γ ε ε β Number AtLPP3 Rod1Like AtLPP4 AtPAP1 AtPAP2 AtLPP AtLPP AtLPP AtSPP1 AtLPAP1 AtPAH1 AtPAH2 AtLPP 28455.m000359 sphingosine-1-phosphate phosphohydrolase, putative X 29586.m000620 phosphatidic acid phosphatase, putative X*** 29660.m000759 phosphatidic acid phosphatase, putative X*** 29747.m001075 phosphatidic acid phosphatase, putative ***X 29660.m000760 ER Phosphatidate Phosphatase ***X 29841.m002865 Phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT) * 29805.m001476 dolichyldiphosphatase, putative X 29634.m002101 conserved hypothetical protein *X 29634.m002100 PALMITOYL-PROTEIN THIOESTERASE/DOLICHYLDIPHOSPHATASE 1 *X 27574.m000218 sphingosine-1-phosphate phosphohydrolase, putative X 30148.m001447 Phospholipase C 3 precursor, putative X 30147.m014488 Phospholipase C 4 precursor, putative X 29681.m001334 hydrolase, acting on ester bonds, putative X 30142.m000628 hydrolase, acting on ester bonds, putative X 30170.m013896 PAH1 conserved hypothetical protein *X 30170.m013897 PAH2 conserved hypothetical protein *X

Table 2: Homologs of Arabidopsis putative PAP proteins in Castor Bean. LPP β, indicated in yellow, has only one homolog in Castor, while most other Arabidopsis putative PAPs have more than one. X: Primary homolog (reciprocal BLASTp results indicated Arabidopsis homolog as first candidate); *: Secondary homolog (reciprocal BLASTp results indicated Arabidopsis homolog as a candidate, but not the top choice).

46

Soybean ( Glycine max ) PAP Homologs At4g22550 At1g15080 At3g50920 At5g66450 At3g58490 At3G02600 At3G15830 At3G18220 At2G01180 At5G03080 At3G03520 At3G09560 At5G42870

Accession Number: 1 2 β γ ε ε JGI Soybean Name Gene Call AtLPP AtLPP3 Rod1Like AtLPP4 AtPAP1 AtPAP2 AtLPP AtLPP AtLPP AtSPP1 AtLAP1 AtPAH1 AtPAH2 Glyma.05G056700 Sphingosine-1-phosphate phosphohydrolase X Glyma.07G029800 phosphatidic acid phosphatase-related / PAP2-related X Glyma.08G213100 Sphingosine-1-phosphate phosphohydrolase X Glyma.02G089100 LIPID PHOSPHATE PHOSPHATASE X * * * Glyma.18G293800 LIPID PHOSPHATE PHOSPHATASE X * * * Glyma.10G270100 LIPID PHOSPHATE PHOSPHATASE * X * Glyma.20G121200 LIPID PHOSPHATE PHOSPHATASE * X * Glyma.09G115800 LIPID PHOSPHATE PHOSPHATASE * * X Glyma.10G270000 LIPID PHOSPHATE PHOSPHATASE * * X Glyma.11G165300 LIPID PHOSPHATE PHOSPHATASE * * X Glyma.18G058400 LIPID PHOSPHATE PHOSPHATASE * * X Glyma.20G121300 LIPID PHOSPHATE PHOSPHATASE * * X Glyma.10G050300 Palmitoyl-Protein Thioesterase/Dolichyldiphosphatase 1 X Glyma.13G137800 Palmitoyl-Protein Thioesterase/Dolichyldiphosphatase 1 X Glyma.01G171800 Palmitoyl-Protein Thioesterase/Dolichyldiphosphatase 1 * X Glyma.11G071400 Palmitoyl-Protein Thioesterase/Dolichyldiphosphatase 1 * X Glyma.03G207000 Sphingosine-1-phosphate phosphohydrolase X Glyma.19G204500 Sphingosine-1-phosphate phosphohydrolase X Glyma.03G092800 Phosphoesterase family X Glyma.16G081200 Phosphoesterase family X Glyma.18G064000 Phosphoesterase family X Glyma.20G040600 Phosphoesterase family X Glyma.03G174900 LIPIN X* Glyma.06G039200 LIPIN X* Glyma.10G046400 LIPIN X* Glyma.19G175600 LIPIN X* Glyma.04G038200 LIPIN *X Glyma.13G134500 LIPIN *X Glyma.17G242900 LIPIN *X Glyma.20G085600 LIPIN *X

Table 3. Homologs of Arabidopsis putative PAP proteins in Soybean. LPP β, indicated in yellow, has only one homolog in soybean while all the other putative PAPs have at least two homologs. X: Primary homolog (reciprocal BLASTp results indicated Arabidopsis homolog as first candidate); *: Secondary homolog (reciprocal BLASTp results indicated Arabidopsis homolog as a candidate, but not the top choice).

47

ESTs/100,000 B. napus T. majus Embryo R. communis Arabidopsis Name Em

and Locus ID 16 18 22 25 III IV VI VII- VIII Em Em Avg En Avg En Em Avg Em 14-20 21-25 26-30 31-35 Rc Rc Bn I II III IV I II III IVTm I II III IV AtLPP β AT4G22550 1 0 0 1 1 1 1 0 1 1 1 0 0 0 0 1 AtLPP3 AT3G02600 2 1 1 1 1 0 0 0 0 0 1 3 0 0 1 3 R1L AT3G15830 AtLPP4 AT3G18220 4 1 2 0 2 AtLPP1 AT2G01180 1 0 1 0 1 1 1 1 5 2 AtLPP2 AT1G15080 2 0 0 0 1 0 0 0 0 0 4 5 1 1 3 8 AtLPP γ AT5G03080 0 0 0 0 0 1 0 0 0 0 4 1 2 1 2 5 AtLPP ε1 AT3G50920 0 0 0 0 0 0 0 0 0 0 0 AtLPP ε2 AT5G66450 2 2 1 3 2 0 0 0 1 0 1 4 2 0 2 4 AtSPP1 AT3G58490 1 0 1 1 1 4 6 4 2 4 3 2 1 0 1 1 AtLPAP1 AT3G03520 0 0 0 0 0 1 1 0 0 0 AtPAH1 AT3G09560 9 3 3 1 4 2 2 1 2 2 AtPAH2 AT5G42870 2 2 2 1 2 0 0 0 1 0 3 2 1 1 2 4 ESTs/100,000 Microarray Signal E. alatus A. thaliana Seed b

Arabidopsis Name a

and Locus ID Em 8/22 8/29 9/06 9/19 En Avg En Ea seedling Stage6 Stage7 Stage8 Stage9 Ea Stage 10 A. thalianaA. I II III IV AtLPP β AT4G22550 2 1 1 3 2 4 1 AtLPP3 AT3G02600 0 1 1 0 0 0 9 338 309 436 513 550 R1L AT3G15830 AtLPP4 AT3G18220 0 4 4 5 9 5 AtLPP1 AT2G01180 0 0 0 1 0 0 3 117 69 62 60 141 AtLPP2 AT1G15080 0 0 1 0 0 0 0 34 28 8 12 12 AtLPP γ AT5G03080 2 0 1 2 1 1 1 356 456 562 456 562 AtLPP ε1 AT3G50920 0 0 0 0 0 0 1 134 129 70 60 74 AtLPP ε2 AT5G66450 0 0 0 0 0 0 0 AtSPP1 AT3G58490 1 0 2 4 2 1 4 AtLPAP1 AT3G03520 0 0 0 0 0 0 0 10 27 676 777 727 AtPAH1 AT3G09560 0 1 1 1 1 1 1 331 536 404 525 460 AtPAH2 AT5G42870 1 1 1 2 1 2 1 68 87 301 290 374

Table 4. Homologs of Arabidopsis putative PAPs in seed tissue of oilseeds . LPP β (highlighted in yellow) is expressed in all four species who were analyzed using 454 pyrosequencing. It is not on the microarray chip used to obtain the Arabidopsis data. Adapted from Troncoso-Ponce and Kilaru et al. [53].

48

A

B

Figure 3. Bioinformatics Information on LPP β. (A) Tissue expression of LPP β based on data from the AGRONOMICS whole genome tiling array [65] and visualized using Genevestigator (https://genevestigator.com/gv/index.jsp). LPP β shows high expression in all tissues tested, but seed or silique tissue was not examined. (B) Transmembrane domain prediction from TMHMM [54].

49

A

B

Figure 4. Tissue expression of genes of interest. (A) The first set of tissue expression experiments: Actin8 control; R1L is not expressed in the leaf or silique; and SPP1 is expressed in both the leaf and silique. (B) Second set of tissue expression experiments: Actin8 control; and LPP β is expressed in both the leaf and silique. LPP β has no introns, but the Actin8 control shows that there is no genomic contamination in the leaf or silique samples. G, Genomic DNA (PCR positive control); L, Leaf cDNA; S, Silique cDNA; --, water (PCR negative control).

50

GFP-Fusion* RFP-BiP (ER) Chloroplast Merged

GUS* - * * GFP β LPP - GFP

SPP1* SPP1* - GFP

* * LPAP1 -

GFP Figure 5. Subcellular Localization of LPP β, SPP1, and LPAP1 . Fusion proteins are shown in green, ER-localized BiP in red, and chloroplast autofluorescence in blue. The identity of the GFP-Fusion* is given in the row headings. GUS cytosolic localization is indistinguishable from BiP ER localization, but clearly different than chloroplast localization. LPP β, SPP1, and LPAP1 all appear to localize to either the ER or cytosol.

51

A Figure 6. Non-Diurnal Expression Pattern of LPP β. Grey boxes indicate dark periods. Error bars represent Standard Error of three biological replicates. qPCR data was normalized to Tubulin2 . (A) Short day expression level of LPP β. (B) Long day expression of LPP β. (C) Expression of LPP β under continuous light.

B

C

52

Name Primer Sequence Reference LBF1 ATGTCTCCGTCGACGACG This Work LB F1 -G CACC ATGTCTCCGTCGACGACG This Work LBR1 ACCTCCCAAAGATCATTTCA This Work LB qPCR -F3 TGCTGAAGCTTCCATGACTG This Work LB qPCR -R2 CTCAGAAATCGAAGAGCAAAGAGAGC This Work LDF1 -G CACC ATGAAGAAGATTCTAGAAAGATCAATG This Work LDR3 GGCACTGATTTTATATATCACACAATG This Work LDF4 AATCTACCAACTGTCTCGAAGTCTCT This Work LDR3 GGCACTGATTTTATATATCACACAATG This Work UP1F1G CACC ATGGTGGAGGAAACGAGCT This Work UP1R3 TCAATTATCACAAATCAAACACGAG This Work R1L F2 AGCATCTTGGCATCTTCACC This Work R1L R2 TCATTAAACTTCAACTAAATGTGTTTC This Work ACT8F ATGGCCGATGCTGATGAC Wayne 2012 [70 ] ACT8R TTAGAAGCATTTTCTGTGGACAATG Wayne 2012 [70 ] TUB2 qPCR -F ACTGTCTCCAAGGGTTCCAGGTTT Niu 2009 [71 ] TUB2 qPCR -R ACCGAGAAGGTAAGCATCATGCGA Niu 2009 [71 ]

Table 5. Primers used in this work. Gateway tags are indicated in bold .

53

2.5 REFERENCES

1. Enns LC, Kanaoka MM, Torii KU, Comai L, Okada K, Cleland RE: Two callose synthases, GSL1 and GSL5, play an essential and redundant role in plant and pollen development and in fertility . Plant Mol Biol 2005, 58 :333-349. 2. De Smet R, Adams KL, Vandepoele K, Van Montagu MC, Maere S, Van de Peer Y: Convergent gene loss following gene and genome duplications creates single-copy families in flowering plants . Proc Natl Acad Sci U S A 2013, 110 :2898-2903. 3. Carman GM, Han GS: Roles of phosphatidate phosphatase enzymes in lipid metabolism . Trends Biochem Sci 2006, 31 :694-699. 4. Toke DA, Bennett WL, Oshiro J, Wu WI, Voelker DR, Carman GM: Isolation and characterization of the Saccharomyces cerevisiae LPP1 gene encoding a Mg2+- independent phosphatidate phosphatase . J Biol Chem 1998, 273 :14331-14338. 5. Toke DA, Bennett WL, Dillon DA, Wu WI, Chen X, Ostrander DB, Oshiro J, Cremesti A, Voelker DR, Fischl AS, et al.: Isolation and characterization of the Saccharomyces cerevisiae DPP1 gene encoding diacylglycerol pyrophosphate phosphatase . J Biol Chem 1998, 273 :3278-3284. 6. Han GS, Wu WI, Carman GM: The Saccharomyces cerevisiae Lipin homolog is a Mg2+- dependent phosphatidate phosphatase enzyme . J Biol Chem 2006, 281 :9210-9218. 7. Chae M, Han GS, Carman GM: The Saccharomyces cerevisiae actin patch protein App1p is a phosphatidate phosphatase enzyme . J Biol Chem 2012, 287 :40186-40196. 8. Chae M, Carman GM: Characterization of the yeast actin patch protein App1p phosphatidate phosphatase . J Biol Chem 2013, 288 :6427-6437. 9. Han GS, Siniossoglou S, Carman GM: The cellular functions of the yeast lipin homolog PAH1p are dependent on its phosphatidate phosphatase activity . J Biol Chem 2007, 282 :37026-37035. 10. Stukey J, Carman GM: Identification of a novel phosphatase sequence motif . Protein Sci 1997, 6:469-472. 11. Toke DA, McClintick ML, Carman GM: Mutagenesis of the phosphatase sequence motif in diacylglycerol pyrophosphate phosphatase from Saccharomyces cerevisiae . Biochemistry 1999, 38 :14606-14613.

54

12. Faulkner A, Chen X, Rush J, Horazdovsky B, Waechter CJ, Carman GM, Sternweis PC: The LPP1 and DPP1 gene products account for most of the isoprenoid phosphate phosphatase activities in Saccharomyces cerevisiae . J Biol Chem 1999, 274 :14831- 14837. 13. Han GS, Johnston CN, Carman GM: Vacuole membrane topography of the DPP1-encoded diacylglycerol pyrophosphate phosphatase catalytic site from Saccharomyces cerevisiae . J Biol Chem 2004, 279 :5338-5345. 14. Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O'Shea EK: Global analysis of protein localization in budding yeast . Nature 2003, 425 :686-691. 15. Choi HS, Su WM, Han GS, Plote D, Xu Z, Carman GM: Pho85p-Pho80p phosphorylation of yeast Pah1p phosphatidate phosphatase regulates its activity, location, abundance, and function in lipid metabolism . J Biol Chem 2012, 287 :11290-11301. 16. Santos-Rosa H, Leung J, Grimsey N, Peak-Chew S, Siniossoglou S: The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth . Embo j 2005, 24 :1931-1941. 17. Fakas S, Qiu Y, Dixon JL, Han GS, Ruggles KV, Garbarino J, Sturley SL, Carman GM: Phosphatidate phosphatase activity plays key role in protection against fatty acid- induced toxicity in yeast . J Biol Chem 2011, 286 :29074-29085. 18. Adeyo O, Horn PJ, Lee S, Binns DD, Chandrahas A, Chapman KD, Goodman JM: The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets . J Cell Biol 2011, 192 :1043-1055. 19. Sasser T, Qiu QS, Karunakaran S, Padolina M, Reyes A, Flood B, Smith S, Gonzales C, Fratti RA: Yeast lipin 1 orthologue pah1p regulates vacuole homeostasis and membrane fusion . J Biol Chem 2012, 287 :2221-2236. 20. Liao MJ, Prestegard JH: Fusion of phosphatidic acid-phosphatidylcholine mixed lipid vesicles . Biochim Biophys Acta 1979, 550 :157-173. 21. Koter M, de Kruijff B, van Deenen LL: Calcium-induced aggregation and fusion of mixed phosphatidylcholine-phosphatidic acid vesicles as studied by 31P NMR . Biochim Biophys Acta 1978, 514 :255-263.

55

22. Weigert R, Silletta MG, Spano S, Turacchio G, Cericola C, Colanzi A, Senatore S, Mancini R, Polishchuk EV, Salmona M, et al.: CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid . Nature 1999, 402 :429-433. 23. Goni FM, Alonso A: Structure and functional properties of diacylglycerols in membranes . Prog Lipid Res 1999, 38 :1-48. 24. Weinberg J, Drubin DG: Clathrin-mediated endocytosis in budding yeast . Trends Cell Biol 2012, 22 :1-13. 25. Baron CL, Malhotra V: Role of diacylglycerol in PKD recruitment to the TGN and protein transport to the plasma membrane . Science 2002, 295 :325-328. 26. Roth MG: Molecular mechanisms of PLD function in membrane traffic . Traffic 2008, 9:1233-1239. 27. Sciorra VA, Morris AJ: Roles for lipid phosphate phosphatases in regulation of cellular signaling . Biochim Biophys Acta 2002, 1582 :45-51. 28. Ren H, Panchatcharam M, Mueller P, Escalante-Alcalde D, Morris AJ, Smyth SS: Lipid phosphate phosphatase (LPP3) and vascular development . Biochim Biophys Acta 2013, 1831 :126-132. 29. Langner CA, Birkenmeier EH, Ben-Zeev O, Schotz MC, Sweet HO, Davisson MT, Gordon JI: The fatty liver dystrophy (fld) mutation. A new mutant mouse with a developmental abnormality in triglyceride metabolism and associated tissue-specific defects in lipoprotein lipase and hepatic lipase activities . J Biol Chem 1989, 264 :7994-8003. 30. Peterfy M, Phan J, Xu P, Reue K: Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin . Nat Genet 2001, 27 :121-124. 31. Han GS, Carman GM: Characterization of the human LPIN1-encoded phosphatidate phosphatase isoforms . J Biol Chem 2010, 285 :14628-14638. 32. Phan J, Reue K: Lipin, a lipodystrophy and obesity gene . Cell Metab 2005, 1:73-83. 33. Chen Y, Rui BB, Tang LY, Hu CM: Lipin family proteins--key regulators in lipid metabolism . Ann Nutr Metab 2015, 66 :10-18. 34. Donkor J, Sariahmetoglu M, Dewald J, Brindley DN, Reue K: Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns . J Biol Chem 2007, 282 :3450-3457.

56

35. Zeharia A, Shaag A, Houtkooper RH, Hindi T, de Lonlay P, Erez G, Hubert L, Saada A, de Keyzer Y, Eshel G, et al.: Mutations in LPIN1 cause recurrent acute myoglobinuria in childhood . Am J Hum Genet 2008, 83 :489-494. 36. Bergounioux J, Brassier A, Rambaud C, Bustarret O, Michot C, Hubert L, Arnoux JB, Laquerriere A, Bekri S, Galene-Gromez S, et al.: Fatal rhabdomyolysis in 2 children with LPIN1 mutations . J Pediatr 2012, 160 :1052-1054. 37. Finck BN, Gropler MC, Chen Z, Leone TC, Croce MA, Harris TE, Lawrence JC, Jr., Kelly DP: Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway . Cell Metab 2006, 4:199-210. 38. Chen Z, Gropler MC, Mitra MS, Finck BN: Complex interplay between the lipin 1 and the hepatocyte nuclear factor 4 alpha (HNF4alpha) pathways to regulate liver lipid metabolism . PLoS One 2012, 7:e51320. 39. O'Hara L, Han GS, Peak-Chew S, Grimsey N, Carman GM, Siniossoglou S: Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+- dependent phosphatidate phosphatase . J Biol Chem 2006, 281 :34537-34548. 40. Grimsey N, Han GS, O'Hara L, Rochford JJ, Carman GM, Siniossoglou S: Temporal and spatial regulation of the phosphatidate phosphatases lipin 1 and 2 . J Biol Chem 2008, 283 :29166-29174. 41. Nakamura Y, Tsuchiya M, Ohta H: Plastidic phosphatidic acid phosphatases identified in a distinct subfamily of lipid phosphate phosphatases with prokaryotic origin . J Biol Chem 2007, 282 :29013-29021. 42. Nakamura Y, Koizumi R, Shui G, Shimojima M, Wenk MR, Ito T, Ohta H: Arabidopsis lipins mediate eukaryotic pathway of lipid metabolism and cope critically with phosphate starvation . Proc Natl Acad Sci U S A 2009, 106 :20978-20983. 43. Pierrugues O, Brutesco C, Oshiro J, Gouy M, Deveaux Y, Carman GM, Thuriaux P, Kazmaier M: Lipid phosphate phosphatases in Arabidopsis. Regulation of the AtLPP1 gene in response to stress . J Biol Chem 2001, 276 :20300-20308. 44. Katagiri T, Ishiyama K, Kato T, Tabata S, Kobayashi M, Shinozaki K: An important role of phosphatidic acid in ABA signaling during germination in Arabidopsis thaliana . Plant J 2005, 43 :107-117.

57

45. Eastmond PJ, Quettier AL, Kroon JT, Craddock C, Adams N, Slabas AR: Phosphatidic acid phosphohydrolase 1 and 2 regulate phospholipid synthesis at the endoplasmic reticulum in Arabidopsis . Plant Cell 2010, 22 :2796-2811. 46. Eastmond PJ, Quettier AL, Kroon JT, Craddock C, Adams N, Slabas AR: A phosphatidate phosphatase double mutant provides a new insight into plant membrane lipid homeostasis . Plant Signal Behav 2011, 6:526-527. 47. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool . J Mol Biol 1990, 215 :403-410. 48. Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, Nakai K: WoLF PSORT: protein localization predictor . Nucleic Acids Res 2007, 35 :W585-587. 49. Heazlewood JL, Verboom RE, Tonti-Filippini J, Small I, Millar AH: SUBA: the Arabidopsis Subcellular Database . Nucleic Acids Res 2007, 35:D213-218. 50. Emanuelsson O, Brunak S, von Heijne G, Nielsen H: Locating proteins in the cell using TargetP, SignalP and related tools . Nat Protoc 2007, 2:953-971. 51. Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ: An "Electronic Fluorescent Pictograph" browser for exploring and analyzing large-scale biological data sets . PLoS One 2007, 2:e718. 52. Mutwil M, Obro J, Willats WG, Persson S: GeneCAT--novel webtools that combine BLAST and co-expression analyses . Nucleic Acids Res 2008, 36 :W320-326. 53. Troncoso-Ponce MA, Kilaru A, Cao X, Durrett TP, Fan J, Jensen JK, Thrower NA, Pauly M, Wilkerson C, Ohlrogge JB: Comparative deep transcriptional profiling of four developing oilseeds . Plant J 2011, 68 :1014-1027. 54. Krogh A, Larsson B, von Heijne G, Sonnhammer EL: Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes . J Mol Biol 2001, 305 :567-580. 55. Onate-Sanchez L, Vicente-Carbajosa J: DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques . BMC Res Notes 2008, 1:93. 56. Jin JB, Kim YA, Kim SJ, Lee SH, Kim DH, Cheong GW, Hwang I: A new dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central vacuole in Arabidopsis. Plant Cell 2001, 13 :1511-1526.

58

57. Karimi M, Inze D, Depicker A: GATEWAY vectors for Agrobacterium-mediated plant transformation . Trends Plant Sci 2002, 7:193-195. 58. Fan J, Jiang D, Zhao Y, Liu J, Zhang XC: Crystal structure of lipid phosphatase Escherichia coli phosphatidylglycerophosphate phosphatase B . Proc Natl Acad Sci U S A 2014, 111 :7636-7640. 59. Reddy VS, Rao DK, Rajasekharan R: Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana . Biochim Biophys Acta 2010, 1801 :455-461. 60. Lu C, Xin Z, Ren Z, Miquel M, Browse J: An enzyme regulating triacylglycerol composition is encoded by the ROD1 gene of Arabidopsis . Proc Natl Acad Sci U S A 2009, 106 :18837-18842. 61. Nakagawa N, Kato M, Takahashi Y, Shimazaki K, Tamura K, Tokuji Y, Kihara A, Imai H: Degradation of long-chain base 1-phosphate (LCBP) in Arabidopsis: functional characterization of LCBP phosphatase involved in the dehydration stress response . J Plant Res 2012, 125 :439-449. 62. Nakamura Y, Ohta H: Phosphatidic Acid Phosphatases in Seed Plants . In Lipid Signaling in Plants . Edited by Munnick T: Springer; 2010:131-141. 63. Kim KC, Hur Y, Maeng J: Isolation of a gene, PnFL-1, expressed in Pharbitis cotyledons during floral induction . Mol Cells 2003, 16 :54-59. 64. Zhang M, Fan J, Taylor DC, Ohlrogge JB: DGAT1 and PDAT1 acyltransferases have overlapping functions in Arabidopsis triacylglycerol biosynthesis and are essential for normal pollen and seed development . Plant Cell 2009, 21 :3885-3901. 65. Rehrauer H, Aquino C, Gruissem W, Henz SR, Hilson P, Laubinger S, Naouar N, Patrignani A, Rombauts S, Shu H, et al.: AGRONOMICS1: a new resource for Arabidopsis transcriptome profiling . Plant Physiol 2010, 152 :487-499. 66. Harmer SL, Hogenesch JB, Straume M, Chang HS, Han B, Zhu T, Wang X, Kreps JA, Kay SA: Orchestrated transcription of key pathways in Arabidopsis by the circadian clock . Science 2000, 290 :2110-2113. 67. Schaffer R, Landgraf J, Accerbi M, Simon V, Larson M, Wisman E: Microarray analysis of diurnal and circadian-regulated genes in Arabidopsis . Plant Cell 2001, 13 :113-123.

59

68. Browse J, Roughan PG, Slack CR: Light control of fatty acid synthesis and diurnal fluctuations of fatty acid composition in leaves . Biochem J 1981, 196 :347-354. 69. Hsiao AS, Haslam RP, Michaelson LV, Liao P, Napier JA, Chye ML: Gene expression in plant lipid metabolism in Arabidopsis seedlings . PLoS One 2014, 9:e107372. 70. Wayne, L: "Investigating the electron transfer systems of the endoplasmic reticulum membrane in plants." Diss. Washington State U, 2012. Order No. 3517446 . ProQuest Dissertations & Theses. Web. 21 Oct. 2015. 71. Niu, Y: "Characterization of Key Transcriptional Regulators in Jasmonate Signaling." Diss. Washington State U, 2009. Order No. 3388523 . ProQuest Dissertations & Theses. Web. 21 Oct. 2015.

60

CHAPTER THREE

GENETIC, MOLECULAR AND BIOCHEMICAL CHARACTERIZATION

OF PUTATIVE PAP GENES

ABSTRACT

Despite recent advancements in the field, the identity of the phosphatidic acid phosphatase

(PAP) involved in oil synthesis in Arabidopsis still remains elusive. The PAP enzymatic function is known to be required for membrane lipid and triacylglycerol (TAG) synthesis, but the gene encoding that function is still unknown. Here, I use genetic and biochemical approaches to determine whether the Arabidopsis gene LPP β (At4g22550) could be the main PAP involved in

TAG synthesis. Attempts to knock down expression of LPP β using amiRNA and ihpRNA were largely unsuccessful, and attempts at a guided mutation using the CRISPR/Cas9 system have not yet succeeded. LPP β could also be redundant with other enzymes exhibiting PAP activity in the

ER, so multiple mutants of spp1, lpap1, pah1, and pah2 were pursed and tested for reductions in seed oil. None of the mutants exhibited phenotypes beyond what has been documented for pah1pah2 , indicating that SPP1 and LPAP1 are not redundant with PAH1 or PAH2 and are not involved in oil synthesis. In complementation assays with the yeast ∆pah1 mutant and in PAP radioactivity assays, LPP β showed modest activity for dipalmitoyl phosphatidic acid. Like other genes involved in oil synthesis, LPP β is not transcriptionally activated by WRI1, despite the presence of an AW box in its promoter. At the conclusion of this study, LPP β deomonstrates characteristics consistent with a PAP involved in oil synthesis, but still requires more study before placing it in the oil synthesis pathway.

61

3.1. INTRODUCTION

3.1.1. PAP ACTIVITY IN OIL SYNTHESIS

Phosphatidic acid phosphatase (PAP) enzymes catalyze the dephosphorylation of phosphatidic acid (PA) to diacylglycerol (DAG) in a hydrolysis reaction that releases inorganic phosphate. The substrate of the PAP reaction, PA, is used for the synthesis of the phospholipid phosphatidylserine, and is the precursor for CDP-DAG, which is used to make other phospholipids. The product of the PAP reaction, DAG, is utilized to make PC, which is the main

ER membrane lipid, the precursor for the acyl-editing cycle (the Lands cycle), and the precursor for a separate pool of DAG which can be exported to the plastid or used to make TAG. PA and

DAG are also signaling molecules in addition to their roles as intermediates in lipid synthesis.

Therefore, PAP activity is an essential step in the pathway to TAG, and it may also regulate the synthesis of phospholipids, TAG, and signaling molecules.

In yeast and mammals, the PAP enzyme involved in oil synthesis has been shown to not only affect oil accumulation, but also affect processes such as membrane homeostasis [1,2], lipid droplet formation [3], development of adipose tissue [4,5], and regulation of lipid metabolism genes [6]. (See Chapter 2, Sections 2.1.1 and 2.1.2, for more details on PAP activity in yeast and mammals.) In vegetative tissues of Arabidopsis, PAH1 and PAH2 function redundantly in ER membrane homeostasis and lipid remodeling during phosphate stress [7,8]. PAH1 and PAH2 also redundantly affect oil synthesis, but a double knockout still only results in a 15% decrease in oil

[7]. This indicates that another PAP(s) is involved in producing the remaining oil, and the pah1pah2 mutant provides a convenient background to determine other PAP genes that act redundantly with PAH1 and PAH2 to affect oil in the seed.

62

In Chapter 2, I screened the Arabidopsis genome for possible PAPs with homology to known yeast, mammalian, and plant PAPs. In addition to PAH1 and PAH2 , there are 11 genes in the Arabidopsis genome which are homologous to known PAPs. Of these genes, LPP β alone displayed characteristics which were consistent with the main PAP involved in oil synthesis. It is expressed in seed tissue, and the protein product is an integral membrane protein that localizes to the endoplasmic reticulum. Additionally, LPAP1 and SPP1 have been implicated in other functions, but they also displayed characteristics indicating that they could act redundantly with the main PAP involved in oil synthesis. LPAP1 does show some PAP activity in vitro , but its main activity is a lysophosphatidic acid (LPA) phosphatase [9]. However, its possible roles either in lipid synthesis, providing the intermediate monoacyl glycerol for membrane or storage lipid synthesis, or in attenuating the signaling functions of LPA, have not been characterized. Finally,

SPP1 displays activity as a long chain base phosphate phosphatase, and has a role in ABA- mediated dehydration stress response [10]. However, it was not assayed for PAP activity, and its possible involvement in oil synthesis remained unexplored. LPAP1 and SPP1 will be tested to determine if they act redundantly with LPP β, or with PAH1 and PAH2 in oil synthesis. (See

Chapter 2 for extensive background on PAPs, and the methodology used to determine PAPs that would be studied in this work.)

PAP activity is generally thought to be essential for TAG synthesis, and since TAG is an essential energy source for developing embryos and viable pollen [11], a complete knockout of all

PAPs involved in TAG synthesis may be lethal. In the case of a non-lethal mutant, it is expected that seed TAG will decrease, as is seen in other mutants in TAG biosynthesis, such as the dgat1 mutants AS11 and ABX45 [12,13]. The seeds of the AS11 and ABX45 lines show a 25% and

45% decrease in total oil, respectively, but are still able to germinate and produce viable plants

63

[12,13]. The activity could also be redundantly encoded in the genome, making multiple mutants necessary to determine which PAP(s) are involved in oil synthesis.

3.1.2. ∆PAH 1 YEAST MUTANTS

Yeast ( Saccharomyces cerevisiae) has four PAPs: APP1 , LPP1 , DPP1 , and PAH1 . APP1,

LPP1, and DPP1 are not specific for a particular lipid substrate, and are involved in regulation and endocytosis. PAH1 is the only PAP involved in de novo lipid synthesis and oil synthesis. While the mutants ∆app1, ∆lpp1, and ∆dpp1 do not show significant changes in TAG, ∆pah1 mutants show reduced DAG and TAG, as well as a temperature-dependent growth phenotype: they are unable to grow at 37°C unless complemented with a functional PAP [14]. This phenotype has provided a convenient assay for determining if genes display PAP activity [15].

In this work, I describe my attempts to knock down expression of LPP β in order to determine if it is involved in oil synthesis either alone or in conjunction with several other genes, and to determine if it has PAP activity. LPAP1 and SPP1 show conclusively that they do not function redundantly with PAH1 or PAH2 in oil synthesis, and are probably involved in signaling pathways as previously suggested. LPP β displays PAP activity when expressed in yeast, and its subcellular localization and tissue expression are consistent with a PAP involved in oil synthesis.

Continuing work is needed, however, to fully determine its biological role.

64

3.2. MATERIALS AND METHODS

3.2.1. PLANT MATERIALS AND GROWTH CONDITIONS

For oil quantification sets, seeds were germinated on ½ MS medium with 1% (w/v) sucrose and 2% agar in continuous light (100-150 umol m -2 s-1 light) at 22°C. Fourteen day old seedlings were transferred to soil and grown in a growth chamber under the same conditions, or transferred to a greenhouse with 16 h day 8 h night, 21-24°C day and 17-20°C nights and light intensity varying from 100-200 umol m -2 s-1 light. For each test, a minimum of 21 plants were planted per line and lines were randomized across flats.

3.2.2 IDENTIFICATION OF T-DNA INSERTIONAL MUTANTS

One homozygous T-DNA insertional mutant for LPP β (At4g22550) was available and was ordered from the Ohio State University (SALK_058344C; lpp β-1). Insertional mutants for SPP1

(At3g58490) and LPAP1 (At3g03520) were also ordered from The Ohio State University

(SALK_027084 for spp1-2 and SALK_065482 for lpap1-1) and the pah1pah2 mutant was requested from the Ohta lab to investigate any possible redundancy between LPP β and those genes .

Homozygous mutants of lpp β-1, spp1-2, and lpap1-1 were analyzed for full-length transcript. RNA was extracted from leaf tissue using the Trizol procedure, DNA removed with the DNA-free Kit, and cDNA synthesized using the SuperScript III kit. Full length transcript was amplified using the LBF4/LBR1 for LPP β, LDF4/LDR3 for SPP1 , or UP1F3/UP1R6 for LPAP1 . Genomic DNA was sequenced to confirm exact insert location.

3.2.3. OVERCOMING FUNCTIONAL REDUNDANCY : GENERATING MULTIPLE MUTANTS

Confirmed homozygous null lines were crossed in the following combinations: pah1pah2 x spp1 , pah1pah2 x lpap1 , spp1 x lpap1 . This generated pah1pah2spp1 , pah1pah2lpap1 , and spp1lpap1 . The triple mutants were then crossed, pah1pah2spp1 x pah1pah2lpap1 , to create

65

pah1pah2spp1lpap1 . PAH1 and LPAP1 are only 6.8 centimorgans apart on Chromosome 3, so F1 plants were screened for the pah1pah2 small phenotype first [8], and then genotyped for the lpap1 and spp1 mutations.

3.2.4. MUTANT LINE ANALYSIS : GENERATING A LPP Β MUTANT

Due to the unavailability of null insertional mutants, RNA silencing and CRISPR/Cas9 directed mutagenesis were pursued. Artificial micro RNA (amiRNA) constructs were designed using Detlef Weigel’s website (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi, [16]) to specifically target the LPP β gene. The amiRNA has a sequence of

“TAATCCCTAAAAAAGCACCCG” and targets the region targeted shown schematically in

Figure 1. PCR using KOD HotStart Taq (Novagen), the template plasmid pRS300, and the primers

Listed in Table 1 (amiRNA-A-G, I miR-s, II miR-a, III miR*s, IV miR*a, and amiRNA-B) was used to generate the amiRNA as described on the website. It was sequenced, then cloned into the pOEA vector [17] at the SwaI site using T4 DNA ligase to produce pKC06. This construct includes a Phaseolin promoter driving strong seed-specific expression of the amiRNA and DsRed as a visual marker [18].

Intron-containing hairpin RNA (ihpRNA) constructs were created by amplifying a 200 bp segment of LB using primers hp1F-G and hp1R (see Table X) and cloning into pENTR. The sequenced gene was then subcloned into the pB7GWIWG2(II) [19] plasmid using the Gateway

LR Reaction (Invitrogen), which inserted the gene in both directions on either side of an intron.

Unfortunately, the pB7GWIWG2(II) plasmid uses a 35S CaMV promoter, which has little activity in seeds; therefore the hairpin-intron-hairpin construct was cut out of pB7GWIWG2(II) using SwaI

(New England Biolabs) and ligated into the pOEA-DsRed vector at the SwaI site using T4 DNA

Ligase (New England Biolabs) to produce pKC05.

66

Both pKC05 and pKC06 were checked for correct orientation, then transformed into

Agrobacterium (GV3101) and the floral dip method (Clough and Bent, 1998) was used to transform WT Columbia-0, spp1-2, lpap1-1, and pah1pah2 plants.

When pKC05 and pKC06 failed to generate LPP β knockdowns, the CRISPR/Cas9 guided mutation system was employed. Three guide RNAs targeting the 5’ end of the gene were designed and constructed by overlapping PCR according to Li et al. [20], producing promoter-gRNA- terminator cassettes. Restriction sites were added by PCR using the U6-XhoI-F1 and 3’-PacI-R2 primers. These were digested by PacI and XhoI and cloned into pFGC-pcoCas9 (a kind gift from

Jen Sheen, AddGene #52256) to create pKC35 for the A cut site, pKC36 for B, and pKC37 for C.

The B cassette was also added to pFGC-DsRed, a version of pFGC created by Jeremy Jewell in our lab where the Cas9 enzyme appears to be nonfunctional (personal communication), but allows for red/brown screening of the seeds using the DsRed fluorophore; this generated pKC28.

Combinations of A/B and B/C were transformed into agrobacterium and into all eight backgrounds as described above: WT, spp1, lpap1, spp1 lpap1, pah1pah2, pah1pah2spp1, pah1pah2lpap1 , and pah1pah2spp1lpap1.

Additionally, pK7WGF2::hCas9 (a kind gift from Sophien Kamoun, Addgene plasmid #

46965 [21]) was digested with AatII and the C cut site gRNA cassette mentioned above was ligated into the plasmid. The resulting pKC41 plasmid was transformed into WT Arabidopsis using the floral dip method. Finally, a combination of the original pK7WGF2 plasmid and pKC35 were transformed into the WT background using floral dip. Kanamycin resistance (and/or silencing) already present in the SALK lines prevented these two methods from being used on any of the mutant lines.

67

CRISPR/Cas9 T1 plants were identified by red fluorescence (pKC28), Basta resistance

(pKC35-pKC37), Kanamycin resistance (pKC41), or a combination of Basta and Kanamycin

(pK7WGF2 and pKC35). The LB gene was amplified from T2 plants using LBF1/LBR1 primers, and first analyzed for a 200 bp deletion in the gene for those transformed with A/B or B/C “double cut” gRNA combinations. T2 mutants were identified by the Guide-It Mutation Detection Kit

(Clontech Laboratories, Cat #631443; using LBF1/LBR1 primers), and interesting amplicons were cloned into pCR using the ZeroBlunt kit (Invitrogen), then sequenced to determine the exact mutation(s) in each line.

3.2.5. SEED TISSUE EXPRESSION OF LPP Β IN WILD -TYPE AND MULTIPLE MUTANTS

Developing seed tissue was isolated according to Bates et al (2013) and stored at -80°C.

Samples were ground under liquid nitrogen using a handheld pestle spinner in a microcentrifuge tube, and RNA was extracted using a modified version of the Onate-Sanchez protocol [22]. After extracting with phenol, the aqueous layer was further extracted with another 500 uL of chloroform and separated using a pre-spun Phase-Lock Gel column (5Prime). Complimentary DNA was prepared using Quanta’s qScript DNA SuperMix cDNA synthesis kit. Quantitative RT-PCR was performed on the MX3005P qPCR system (Stratagene) using the Quanta SYBR FastMix kit.

Amplicons of roughly 200 bp were amplified, using LB qPCR F3 and LB qPCR R2. Data from

LPP β specific primers was normalized to the AtTUB2 gene, followed by calculation of the relative fold change (=1/(2^exp-control)).

3.2.6. TOTAL FATTY ACID CONTENT OF SEEDS

To quantify fatty acid content of seeds in the multiple knockout lines and in the LPP β

RNAi lines, all plants used in each test were grown together, and 20-30 individual plants from each line were randomized across all flats. Seeds were harvested at maturity, and dried under silica

68

gel for a minimum of 48 hours. Twenty to 40 mg of seed was weighed out and counted, then fatty acids were transmethylated according to Li et al. [23], extracted, and run on a polar GC column.

Chromatographs were analyzed using ChemStation software (Rev B.03.02[341])

3.2.7. COMPLEMENTATION OF THE YEAST ∆PAH 1 MUTANT

The coding sequences of LPP β, SPP1 , LPAP1, and AtPAH1 were amplified from silique cDNA using KOD Hotstart Polymerase (Toyobo) and the LBF1G/LBR1, LDF1G/LDR6,

UP1F1G/UP1R3 and PAH1FW/PAH1 RV primer pairs, respectively. ScPAH1 was amplified from yeast genomic DNA using ScPAHF1G/ScPAHR1 primers. Blunt-ended products were cloned into pENTR using the Gateway pENTR/D-TOPO Cloning Kit and were sequenced. All five genes were transferred into the destination vector using Gateway LR Clonase II (Invitrogen) and checked for orientation. The first of two destination vectors, pAG425GPD-ccdB (a kind gift from Susan Lindquist, Addgene plasmid # 14154), contains the strong, constitutive GPD promoter for expression of the gene of interest, and provides the LEU2 gene as a selectable marker.

pAG425GPD-ccdB was also modified to include the ScPAH1 promoter instead of the strong, constitutive GPD promoter. It was digested with SacI and SpeI, and the 8.8 kb fragment was purified; the ScPAH1 promoter was amplified from yeast genomic DNA using the PPF1 and

PPR1 primers. These were then assembled using the Gibson Assembly kit (Invitrogen) and the

PPCF1, PPCR1, PPCF1*, and PPCR1* primers, to create the pKC07 plasmid. The six genes above were also transformed into pKC07 using the LR Clonase II and checked for orientation.

Both the pAG425GPD and the pKC07 versions of the six genes were transformed into the

∆pah1 and ∆pah1 ∆lpp1 ∆dpp1 yeast mutants [14] using the One-Step transformation method described by Chen et al. [24]. All of the experiments described, except where noted, use the ∆pah1 mutant.

69

Complementation assays were conducted using freshly transformed cells. Cultures (2 mL) of each line were grown for 2 days, then a hemocytometer was used to count cells and determine exact density of the solution. Ten, 100, 10 2, 10 3, 10 4, and 10 5 cells were spotted onto media in duplicate. Standard Leucine dropout media was used for the plates that would be placed in the

30°C incubator, while YPD plates were used for those going in the 37°C incubator. Plates were grown 2 to 7 days more, and pictures from plates with visible colonies were analyzed for growth phenotype.

3.2.8. ENZYME ASSAYS : YEAST MICROSOMAL PAP ACTIVITY ASSAYS

The ∆pah1 yeast mutant expressing genes under control of the PAH1 promoter from the previous section were grown together 2 days at 30°C in 100 mL of standard dropout media without

Leucine. Cells were collected by centrifugation at 3500 rcf at 4°C, then washed in water and collected. Pellets were resuspended in 0.5 mL cold lysis buffer (20 mM Tris HCl, 1 mM EDTA,

5% glycerol, 10 mM MgCl2, 1 mM DTT, 300 mM NH 4SO 4) and cells were disrupted by vortexing with glass beads (10 x 30s). Supernatant was centrifuged at 10,000 g for 20 min at 4°C to remove intact organelles, and the resulting supernatant was centrifuged at 100,000 g for 60 min to isolate the microsomal fraction. The microsomal pellet was resuspended in 200 uL assay buffer (50 mM

Tris HCl, 50 mM NaCl, 5 % glycerol, 2 mM MgCl 2), and protein quantified using the Bradford

Assay.

To assay for PAP activity, 20 ug total protein was added to 100 uL substrate solution (20 mM Tris-HCl, pH 7.0, 0.1% (w/v) Triton X-100, and 45 nCi L-3-PA 1,2 dipalmitoyl) and enough assay buffer was added to make 200 uL total. The reaction proceeded for 60 min, then was quenched by vigorous vortexing with 2:1 chloroform:methanol. Products were separated using

0.88% NaCl, and the organic phase removed, then back-extracted with 2:1 chloroform:methanol.

70

In most cases, the combined fractions were cloudy, so all samples were extracted once more with

NaCl solution. The organic phase was dried down under a stream of nitrogen at RT, and resuspended in 2:1 chloroform:methanol.

Each sample was spotted on a silica gel TLC plate was developed halfway in 35:25:4:14:2 of chloroform:methanol:acetic acid:acetone:water. After drying, the plate was developed fully in

50:50:1 petroleum ether:ethyl ether:acetic acid. For samples quantified by GC, the plate was stained with 0.05% primulin in 8:2 acetone:water and marked with a soft pencil; for radioactivity the spots were quantified using the Typhoon FLA7000 laser scanner system and ImageQuantTL software(GE Healthcare); for unquantified samples the plate was stained with iodine vapor.

3.2.9. ENZYME ASSAYS : E. COLI EXPRESSION AND PAP ACTIVITY ASSAYS

The coding sequences of LPP β, SPP1 , and LPAP1 were amplified from their pENTR plasmids (see 2.7 above) using Ligation Independent Cloning (LIC) compatible primers: LBF1-

Lic/LBR1-NsLic, LDF1-Lic/LdR6-NSLic, and UP1F1-Lic/UP1R3-NsLic, respectively. The E. coli expression vector pET-GFP-LIC (AddGene #29772) was a gift from Scott Gradia. The vector was linearized by digestion with EcoRV. Vector and inserts were treated with T4 DNA polymerase in the presence of dCTP in the case of pET-GFP-LIC or in the presence of dGTP in the case of the inserts, LPP β, SPP1 , and LPAP1 . Inserts were annealed into LIC vectors in the presence of EDTA and transformed into E. coli. This cloning resulted in a C-terminal protein fusion of the gene of interest ( LPP β, SPP1 , or LPAP1) with eGFP [25], driven by the inducibleT7 bacterial promoter.

Sequenced plasmids were then transformed into Rosetta 2(DE3)pLysS cells for protein expression.

For protein expression, saturated cultures were used to inoculate 100-mL LB cultures.

Cultures were allowed to grow at 37°C to an OD 600 of 0.4, and then were induced with 25 uL of

0.4 M IPTG and grown for three hours at 37°C. Cells were harvested by centrifugation at 4300 x

71

g for 20 min at 4°C, and cell pellets were lysed with the addition of 1mg/mL lysozyme in lysis buffer (25 mM TrisHCl pH 7.0, 150 mM NaCl, 0.1% (w/v) Triton X-100, 1 unit/20 uL DNaseI).

Fractions of the crude lysate were run on 10% BisTris SDS-PAGE gels for Western Blotting.

Proteins were transferred to a nitrocellulose membrane and were probed with 1:5000 rabbit anti-

GFP primary antibody (ab290) and 1:20,000 goat anti-rabbit secondary antibody conjugated to

Horse Radish Peroxidase (ab97080). Blots were visualized using ECL Western Blotting Substrate

(Pierce).

Protein was quantified using the Bradford Assay, and 20 ug total protein was used for PAP activity assay. The assay and TLC plate were run as noted above for the yeast microsomal assay

(see 2.8 above), except for the composition of the assay buffer (20 mM Tris-HCL, pH 7.0, 150 mM NaCl, 15% (v/v) glycerol, 0.1% (w/v) Triton X-100).

3.2.10 WRI 1 CONTROL OF LPP Β

Segregating T2 seeds expressing WRI1 under control of the strong seed-specific Phaseolin promoter were obtained from Neil Adhikari (unpublished data). Red (containing transgene) and brown (isogenic segregants) seeds were planted on soil and stratified for 4 days at 4°C. Developing seeds from five segregating mutant plants and four isogenic segregants were harvested according to Bates et al [26] at 7-9 days after flowering. RNA was extracted using a modified protocol from

Onate-Sanchez. After extracting with phenol, the aqueous layer was further extracted with another

500 uL of chloroform and separated with the help of a pre-spun Phase-lock Gel column (5Prime).

Complimentary DNA was prepared using Quanta’s qScript DNA SuperMix cDNA synthesis kit, and LPP β expression was quantified as described above. WRI1 expression was also quantified in relation to TUB2 using qWRI1F(3) and qWRI1R(3) primers from Neil Adhikari (personal communication).

72

3.3. RESULTS

3.3.1. MUTANT LINE ANALYSIS : IDENTIFYING SINGLE KO’ S

Putative T-DNA insertional mutant lines were obtained from ABRC: lpp β-1 for LPP β

(At4g22550), spp1-2 for SPP1 (At3g58490), and lpap1-1 for LPAP1 (At3g03520). The insertion location of each line was sequenced, and their relative locations can be seen in Figure 1. The insertions in both spp1-2 and lpap1-1 were in exons, but the insertion in lpp β-1 was upstream of the 5’UTR, indicating it may not be a true knockout. RT-PCR was pursued to determine if the T-

DNA insertion lines encoded null mutations. Amplification of the Actin8 gene from two plants from each background showed similar amounts of cDNA was loaded on each sample, and RT control shows no genomic contamination. The lpp β1-1 line, a “confirmed” homozygous line, showed amplification at similar levels as WT in both of the plants tested, indicating that the insertion in the promoter does not affect expression levels. Both the spp1-2 and lpap1-1 mutants showed expression in the WT line but no amplification in the mutant lines, indicating that they are most likely null mutations.

3.3.2. MUTANT LINE ANALYSIS : CHARACTERIZING LPP β RNA I MUTANTS

Since I could not isolate a null mutant of LPP β from the available T-DNA lines, I attempted to reduce LPP β expression using both an amiRNA and an ihpRNA approach. Second generation

(T2) segregating seeds were separated into red (expressing the amiRNA or ihpRNA transgene) and brown (WT isogenic segregants), and total seed fatty acid levels were quantified in the WT, spp1-2, lpap1-1, and pah1pah2 backgrounds (Figure 2). While it is well established that

Arabidopsis seed oil varies widely between plants [23], using LPP β-knockdown and segregating

WT seeds off the same plant compensates for that variability. Out of the roughly 80 independent

73

lines (~20 per background) analyzed, only seven showed a statistically significant change in oil between the knockdown and WT segregants.

Recognizing the relatively low number of RNAi lines with reduced oil, I set out to confirm that the RNAi lines actually showed reduced expression of LPP β. Unfortunately, qPCR showed that none of the lines tested showed a reduction in expression beyond 50% of WT; instead, many of the lines had increased LPP β expression (Figure 3). Basal levels of LPP β expression were increased in the lpap1 and spp1 mutant, but the RNAi constructs did not decrease these levels much below WT when introduced to the mutants. Thus, the RNAi constructs did not appear to work, and are not a viable option to study the effects of LPP β on oil synthesis.

3.3.3. MUTANT LINE ANALYSIS : INVESTIGATING REDUNDANCY

To investigate redundancy between the identified genes and prepare for eventual introduction of an lpp β mutant, crosses were made between lpap1, spp1-2, and pah1pah2. All of the genes were unlinked except LPAP1 and PAH1, which are located 6.98 cM apart (based on 1 cM equivalent to 300,000 nucleotides) on chromosome three. In the lpap1 x pah1pah2 cross, the

F2 generation plants were screened first for the pah1pah2 visual phenotype and then genotyped for lpap1 , and mutants were isolated which were homozygous mutant at all three loci. Since the other crosses did not include linked genes, spp1 pah1 pah2, spp1 lpap1, and spp1 lpap1 pah1 pah2 were all isolated in the F 2 generation.

As I noted in the initial studies of the LPP β RNAi lines, some of the mutants appeared to have increased expression of LPP β. I investigated this further, by analyzing LPP β expression using qPCR in all of the multiple mutant lines that I had generated. Indeed, all seven lines showed increased LPP β expression relative to WT (Figure 4). This indicates that LPP β likely exhibits

74

PAP activity and could be compensating for the mutations, especially in the pah1pah2 line where a known PAP activity has been knocked out.

I had hoped to introduce an LPP β knockdown into all of the combinations of spp1, lpap1, and pah1pah2 , so I prepared for these experiments by characterizing the backgrounds for changes in seed oil and fatty acid composition. Trials were grown in both the greenhouse and growth chambers, but the data from the two growth conditions was largely similar. The visible growth phenotype of all plants was very similar to WT, or to pah1pah2 in that background (the pah1pah2 phenotype includes shorter plants; smaller, greener, rounder leaves; and shorter siliques [7,8]).

Seed oil was investigated as well, and Figure 5 shows a representative sample of seed oil measurements, from growth chamber-grown plants. In all cases, the mutants in the WT background were similar to WT (Figure 5A), and those in the pah1pah2 background showed the characteristic pah1pah2 phenotype (a ~15% reduction in seed oil; Figure 5B). There was also no statistically significant change in fatty acid composition of the seeds in the WT background (data not shown), and most of the changes in fatty acid composition in the pah1pah2 background were consistent with the published pah1pah2 phenotype, including a decrease in 18:2 and 20:1 (Figure

5C) [7]. The increase in 20:0 and 22:1 in the pah1pah2 background has not been examined before, but all four mutants in the pah1pah2 background showed a very similar phenotype. The only irregularity with published data was seen in 18:3, which Eastmond et al. reported as decreasing in the double mutant, while my studies indicate it increases [7]. This could be due to growth conditions, as all four lines in the pah1pah2 background show the same increase. There were also minor changes in the triple and quadruple mutants in 18:2 and 18:3 relative to WT and pah1pah2 : the addition of the spp1 and lpap1 mutations seemed to recapitulate the WT phenotype.

Additionally, pah1pah2spp1lpap1 showed a lower 20:1 level than either pah1pah2 or WT.

75

However, these are relatively minor changes, and are not indicative of any redundancy between the genes tested. Thus, the SPP1, LPAP1, PAH1, and PAH2 genes do not exhibit redundancy with each other beyond that which is characterized for PAH1 and PAH2 .

3.3.4. ENZYME ASSAYS : COMPLEMENTATION OF THE YEAST ∆PAH 1 MUTANT

In yeast, ScPAH1 is the main PAP involved in oil synthesis, and the null mutant ∆pah1 has reduced oil levels and displays a temperature sensitive phenotype. Both the single mutant and the triple mutant ∆pah1 ∆lpp1 ∆dpp1 have been used to characterize PAPs, including AtPAH1 and

AtPAH2 [8,14,15], by providing a complementation assay to confirm PAP activity. Genes with

PAP activity which complement the mutant allow growth at 37°C. The Arabidopsis LPP β protein was expressed in both yeast mutants, under control of the strong, constitutive ADH promoter, as well as under control of the native ScPAH1 promoter. The four expression conditions provided comparable assay results, but the ∆pah1 single mutant grew more consistently. As shown in Figure

6, LPP β usually grew to similar levels as AtPAH1 and ScPAH1, and grew more than the negative control. LPAP1 grew to similar levels as LPP β and AtPAH1, but SPP1 grew noticeably less. This indicates that LPP β, LPAP1, and SPP1 all display some PAP activity, although LPP β and LPAP1 are better able to complement the mutant. Additionally, in some assays, the negative control also grew, indicating that the assay is not consistent. More studies are needed to confirm or refute the

PAP activity of LPP β.

3.3.5. ENZYME ASSAYS : MICROSOMAL YEAST AND E. COLI PAP ACTIVITY ASSAYS

PAP assays using 14 C-PA (dipalmitoyl) were undertaken. The ∆pah1 yeast expressing

LPP β under control of the strong, constitutive ADH promoter from the complementation assays were used. The inconsistent growth phenotypes affected these assays as well, which is why there is no PAP assay data for SPP1 – too few cultures grew for analysis. However, I was able to assay

76

LPPβ and LPAP1 for activity, and LPPβ showed an average of 4 pmol/min/ug protein, while

LPAP1 showed virtually no activity (see Figure 7A). The yeast expressing PAH1 showed an average activity of 32 pmol/min/ug protein, indicating that LPP β is not as active as PAH1 under these assay conditions. These data do indicate, however, that LPP β has PAP activity.

LPP β, LPAP1, and SPP1 were also expressed in E. coli as C-terminal GFP fusions. This allowed confirmation that the proteins were expressed, by Western Blotting with Anti-GFP polyclonal antibodies. Western Blots showed that, indeed, the fusions were expressed, although the GFP tag was cleaved from the LPP β and SPP1 proteins (data not shown). However, the enzyme assays showed no PAP activity at all (see Figure 7A), possibly indicating that these proteins undergo post translational modifications which are not available in E. coli .

While neither the complementation assays nor the PAP radioactivity assays were conclusive alone, together they indicate that LPP β does exhibit PAP activity. Further studies to determine the lipid and fatty acid specificities of the protein are needed.

3.3.6 WRI1 CONTROL OF LPP β

The WRI1 transcription factor has long been associated with oil synthesis, and the wri1-1 mutant has a compromised glycolysis pathway making the developing embryo unable to effectively convert sugar precursors into the fatty acids required for TAG synthesis, and leaving the mutant with an 80% reduction in seed TAG [27]. WRI1 has been shown to control transcript levels of enzymes involved in the generation of TAG precursors, but not the TAG assembly pathway itself [27-30]. It acts by binding to the AW Box domain in the promoter of its targets and directly activates them. LPP β has an AW Box sequence roughly 117 bp upstream of the translation start site, indicating that like other genes with the AW Box, it may be regulated by WRI1 (see

Figure 8A). To determine if this is the case, developing seeds were harvested from a segregating

77

population of plants overexpressing WRI1 . Expression levels of WRI1 were increased 4-6 fold in overexpressor plants compared to their isogenic segregants, but LPP β expression decreased about

20% (Figure 8B). Although I was unable to measure oil in these lines, other lines overexpressing the same construct of WRI1 which had only 2.5 or 3 fold increase in expression still showed a 19% and 15% increase in oil, respectively (Neil Adhikari, personal communication). This indicates that the 4-6 fold increase seen in WRI1 is enough to affect oil synthesis, and should have had an effect on LPP β if it was a target. Since LPP β expression in WRI1 overexpressors does not show a significant change from WT, it appears that it is not regulated by WRI1 , despite the presence of the AW Box in its promoter.

78

3.4 DISCUSSION

3.4.1 LPP β SHOWS MODEST PAP ACTIVITY

The PAP activity assays and yeast complementation assays indicate that LPP β displays

PAP activity and that LPAP1 showed less activity. LPAP1 was published to show low PAP activity, but much stronger activity towards the substrate lysophosphatidic acid (LPA) [9]. In the complementation assays, it appeared to complement the mutant, but in my radioactivity tests,

LPAP1 averaged nearly zero PAP activity. This supports the conclusion that it acts as an LPA phosphatase with only minor PAP activity. In radioactivity assays, LPP β showed roughly one tenth the activity of PAH1; this indicates that it is a PAP, but the low activity could be due to fatty acid substrate specificity, assay or buffer conditions, or lipid substrate specificity. Coupled with the complementation assays which actually showed similar levels of complementation to AtPAH1 and ScPAH1, this presents solid evidence that LPP β does indeed encode PAP activity. It is also consistent with unreviewed reports (e.g. not published in a peer reviewed journal) that the protein is a PAP [31].

In order to attempt to identify other possible reactions of the enzyme, the TLC separation protocol allowed for separation of nonpolar lipids (TAG, DAG, free fatty acids), as well as polar lipids (including PA, PC, PE, PI; see Figure 7B). This allowed tracking of 14 C PA not only into

DAG, but also into side reactions that use PA as a substrate, such as PS. There were no radioactive side products seen in any of the assays conducted with PA, indicating that DAG is the main product when dipalmitoyl PA is used. More analyses are needed to determine the specificity of LPP β for other molecular species of PAP and to other lipids in vitro.

79

3.4.2 ATTEMPTS TO KNOCK DOWN LPP β EXPRESSION HAVE FAILED

There is only one T-DNA line available for LPP β and it is not a null mutant, which perhaps is not surprising given the small size of the gene (642 bp). However, the amiRNA and ihpRNA data were interesting in that many showed an increase in the transcript level instead of a decrease.

Additionally, a CRISPR/Cas9-induced mutation in LPP β has not yet been identified at the time of this publication. Over 100 lines with two gRNA cassettes have been screened, and none have the

200-bp deletion which would have resulted from both of gRNA’s successfully guiding Cas9 to a cut site in the same primary transgenic, despite reports that in stably transformed lines, 20-100% of lines have resulted in a double cut (deletion) [32-34].

One reason that three separate attempts to knock down expression have failed could be that a null mutation in LPP β is lethal. It is indeed possible that all three failed due to the technical limitations of each method, especially since CRISPR is a new, unproven technology and RNAi techniques are sometimes limited by endogenous feedback mechanisms. But lethal mutations are some of the most difficult to characterize, because the phenotype is a lack of mutated progeny. A

CRISPR/Cas9 system that is effectively mutating an essential gene and causing lethality is very difficult to distinguish from a system that is not working at all. Until a mutant with a significant decrease in can be isolated for LPP β, perhaps in the form of a heterozygous mutation, the lack of a genetic test will prevent our full understanding of the role of LPP β in the plant.

3.4.3 SPP1 AND LPAP1 ARE NOT INVOLVED IN OIL SYNTHESIS

PAH1 and PAH2 are the only PAPs in Arabidopsis to date to show any effect on oil synthesis. They act as a convenient background to determine which other PAPs act redundantly in oil synthesis; while the main PAP involved in TAG synthesis may show a strong phenotype alone, there is also the possibility that it will act redundantly with PAH1 and PAH2 or other genes, making

80

a multiple mutant necessary to determine its involvement. LPAP1 has shown modest PAP activity, both in my experiments and in published reports [9], indicating that it could be redundant with

PAH1 and PAH2 or the main PAP involved in oil synthesis. Additionally, SPP1 showed minor complementation of the ∆pah1 yeast mutant, and it displayed enough interesting characteristics to warrant testing its redundancy with the other putative PAPs. Neither had been assayed for a decrease in seed oil in the mutant, so a possible involvement in oil synthesis, even if it didn’t encode a PAP, could not be ruled out.

However, none of the combinations of mutants ( spp1, lpap1, spp1lpap1, pah1pah2spp1, pah1pah2lpap1, or pah1pah2spp1lpap1 ) showed a total seed oil phenotype (or a growth phenotype) beyond what has already been published for pah1pah2 . This indicates several things.

First, neither SPP1 nor LPAP1 are involved in oil synthesis alone. Second, since they don’t show an additional phenotype in combination with pah1pah2 , they are probably not redundant with

PAH1 or PAH2 in any of their known phenotypes, including oil synthesis. This clearly shows that

SPP1 and LPAP1 can be conclusively dismissed from the conversation of which gene encodes the main PAP involved in oil synthesis.

3.4.4 LPP β IS NOT TRANSCRIPTIONALLY REGULATED BY WRI1

Even though the promoter of LPP β contains an AW Box indicating it may be regulated by

WRI1 , transcript levels of LPP β do not increase in WRI1 overexpressors (Figure 8). The lack of regulation at the transcript level of LPP β is consistent with the fact that the other known TAG synthesis genes GPAT9 , LPAAT4 , DGAT1 , and PDAT1 are also not regulated transcriptionally by

WRI1 [30]. It may seem contradictory that WRI1 controls the amount of precursors available to

TAG synthesis, and eventually affects the amount of oil produced but does not directly target the oil synthesis pathway. However, it could be that TAG synthesis is not the rate-limiting step, and

81

that the increased substrate flux dictates the eventual levels of oil. Since most heterozygous mutations in oil synthesis and other pathways show a WT phenotype, I expect that most enzymes in TAG synthesis are in excess and able to handle the increased substrate load (see Chapter 4 sections 4.3.6 and 4.4.2, regarding GPAT9 as the rate-limiting step of oil synthesis).

82

3.5 CONCLUSION

PAP activity is necessary for lipid synthesis in the plant, but much remains to be understood about the gene and protein responsible for the step. I was able to confirm that SPP1 and LPAP1 are not redundant with PAH1 and PAH2 in oil synthesis, and their functions are likely confined to those already published. LPP β exhibits at least modest PAP activity in multiple assays, indicating that it does indeed encode PAP activity. Since I was unable to isolate a null mutant, its involvement in oil synthesis cannot be confirmed or rejected. Thus, a genetic test to determine its role in oil synthesis would clarify the physiological role of LPP β in Arabidopsis.

83

A

B

C

D

E

Figure 1. RT-PCR of SALK Insertional Mutants. (A), (B), and (C) Gene schematic of the LPP β, SPP1 , and LPAP1 genes, respectively. The T-DNA insertion is denoted with an empty triangle. The two regions of LPP β targeted for RNA interference are noted with green arrows; regions targeted by the CRISPR/Cas9 guided mutation system are indicated with blue arrows. (D) Actin8 RT-PCR control. Similar amounts of cDNA were loaded in all samples. Numbers represent the line tested; C: Cauline leaf on WT plant #1, L: rosette leaf on WT plant #2. (E) RT-PCR analysis of lpp β T-DNA mutants indicate that it is expressed, but spp1-2 and lpap1-1 are both null mutants.

84

*

85

*

*

* * * *

Figure 2 : Oil Levels in LPP β RNAi lines Paired red and black bars represent seeds off the same plant, from one independent transformation event. Standard error bars represent standard error of three samples of 30 seeds each. Red bars, seeds containing the LPP β (LB) RNAi construct; Black bars, isogenic segregants. “hpi”, ihpRNA construct; “ami”, amiRNA construct. * - statistically significant decrease in oil in KD seeds. (A) WT background; (B) lpap1 background; (C) spp1 background; (D) pah1pah2 background.

86

Figure 3: LPP β expression in RNAi Plants Expression was normalized to the Tubulin2 gene. Error bars represent standard error of three biological replicates. Grey line represents WT level of expression for comparison.

87

Figure 4. LPP β Expression in Multiple Mutant Lines. Error bars represent standard error of three biological replicates. Grey line represents WT level of expression for comparison.

88

A B

C 30%

25%

20%

15%

10%

5%

0% 16:0 % 16:1 % 18:0 % 18:1 % 18:2 % 18:3 % 20:0 % 20:1 % 20:2 % 22:1 %

WT pah1/2 PL PS PSL

Figure 5: Seed Oil and Fatty Acid Content of Multiple Mutants. Data is from plants grown in a growth chamber, but is largely similar to those grown in a greenhouse. Error bars represent standard error of 15-85 plants. (A) Oil levels in the seed, WT background mutants. (B) Oil levels in the seed, pah1pah2 background mutants plus WT for comparison. (C) Fatty acid composition of seed oil, a representative chart (growth chamber grown). PL: lpap1 pah1pah2 ; PS: spp1 pah1pah2 ; PSL: spp1 lpap1 pah1pah2

89

A B 6 5 4 3 2 1 6 5 4 3 2 1 10 10 10 10 10 10 10 10 10 10 10 10 ScPAH1

AtPAH1

LPP β

LPAP1

SPP1

GUS

Figure 6. PAP Complementation Assays. A representative set of plates, using ∆pah1 ∆lpp1 ∆dpp1 yeast and expressing genes under control of the PAH1 promoter. (A) Growth of complemented yeast at 30°C. (B) Growth of complemented yeast at 37°C.

90

A B

Figure 7. PAP Activity Assays. (A) PAP activity in yeast microsomes and E. coli crude extract, normalized to negative control (GUS for Yeast and GFP for E. coli) and background subtracted. (B) Iodine-stained TLC plate of PAP activity assay showing separation of both polar and neutral lipids. Shown is an experiment using yeast microsomes. BHT, butylated hydroxytoluene, (an antioxidant used to protect the fatty acids from oxidative degradation).

91

A Gene Location AW Box Sequence PI-PK β1 -2 TGCTTAGTTCGTATCGAA 21 GCCTTCGTAAGTTCCGCC BCCP2 -12 TTCTTCGTAAGCATCGAA 84 TTCCTCGGTTTCATCGTC KAS1 -54 TTCCTCGAATAATTCGCT 48 AACATCGTACTTATCGCC ENR1 2 AGCTTCGATGAGATCGAG ACP1 -890 CTCTTTCTACACTCCGCC FATA -27 GTCCTCGGTCTCATCGTC -12 GTCGTTGACAAATACGAA SUS2 -238 CACTTAGAGCGTATCGCC LPP β -117 CTCTTAGAAAGCGACGTC

B

Figure 8 . LPP β is not regulated by WRI1 at the transcript level. (A) The promoter of LPP β contains an AW box, a known WRI1 - binding domain found in genes activated by WRI1 . Location is relative to ATG start codon. Underlined: Activation by WRI1 was verified in Maeo et al. [35]. Bold : Activation verified in To et al. [30]. Red text: Required for fatty acid synthesis. Blue text: Involved in glycolysis. Blue highlighting: conserved motif of the AW Box domain. (B) LPP β transcript level is not increased in WRI1 overexpression plants. Values are the means and standard error of four or five individual plants. Grey line: WT level of expression of both WRI1 and LPP β.

92

Name Sequence Reference LBF1 ATGTCTCCGTCGACGACG This Work LBF1G CACC ATGTCTCCGTCGACGACG This Work LBF4 GACAGAGATCTGTTACGACAGTTATG This Work LBR1 ACCTCCCAAAGATCATTTCA This Work LB qPCR F3 TGCTGAAGCTTCCATGACTG This Work LB qPCR R2 CTCAGAAATCGAAGAGCAAAGAGAGC This Work LD F1 ATGAAGAAGATTCTAGAAAGATCAATG This Work LD F1G CACC ATGAAGAAGATTCTAGAAAGATCAATG This Work LDF4 AATCTACCAACTGTCTCGAAGTCTCT This Work LDR3 GGCACTGATTTTATATATCACACAATG This Work LDR6 CTACAAGTTAACGTAAGAGAATAGTGAAGG This Work UP 1 F1 ATGGTGGAGGAAACGAGCT This Work UP1 F1G CACC ATGGTGGAGGAAACGAGCT This Work UP1 F3 AAGGCTACCATTTAGACGGACA This Work UP1 R3 TCAATTATCACAAATCAAACACGAG This Work UP1 R6 CAAATTTCAAACGCTCTTCACA This Work PAH1FW GCGGCCGCATGAGTTTGGTTGGAAGAGTTGGGAG [8] PAH1RV ACGCGTTCATTCAACCTCTTCTATTGGCAGTTTCC [8] ScPAHF1G CACC ATGCAGTACGTAGGCAGAGC This Work ScPAHR1 TTAATCTTCGAATTCATCTTCGTC This Work amiRNA -A-G CACC CTGCAAGGCGATTAAGTTGGGTAAC [16 ], This Work I miR -s GA TAATCCCTAAAAAAGCACCCG TCTCTCTTTGTA [16 ], This Work TTCC II miR -a GA CGGGTGCTTTTTTAGGGATTA TCAAAGAGAAT [16 ], This Work CAATGA III miR*s GA CGAGTGCTTTTTTTGGGATTT TCACAGGTCGTG [16 ], This Work ATATG IV miR*a GA AAATCCCAAAAAAAGCACTCG TCTACATATATA [16 ], This Work TTCCT amiRNA -B GCGGATAACAATTTCACACAGGAAACAG [16 ], This Work hp1F -G CACC CGTTGCTGCATCTGTTCACT This Work hp1R CACCAGCAGCTACATCGAGA This Work U6 -XhoI -F1 CGTGA CTCGAG AGAAATCTCAAAATTCCG [20 ], This Work 3’ -PacI -R2 CGTGA TTAATTAA TAATGCCAACTTTGTACA [20 ], This Work R1 -A TCTTAGCGGTGGACGCCGCC AATCACTACTTCGTC [20 ], This Work TCT F2 -A GGCGGCGTCCACCGCTAAGA GTTTAGAGCTAGAA [20 ], This Work ATAGC R1 -B CCATTACGCTCTTTCCTCGT AATCACTACTTCGTCT [20 ], This Work CT F2 -B ACGAGGAAAGAGCGTAATGGAGG GTTTAGAGCTA [20 ], This Work GAAATAGC R1 -C CCGTGTGAATCGCGTGAGAT AATCACTACTTCGTC [20 ], This Work TCT F2 -C ATCTCACGCGATTCACACGG GTTTAGAGCTAGAA [20 ], This Work ATAGC

93

Name Sequence Reference PPC F1 CTAAAGGGAACAAAAGCTGGAGGACGTCgcggtacctag This Work agtccaaactcaacagcc PPC R1 TGATGGGGGATCCACTAGTTCTAGAATCCGCATATG This Work gccggaattcataatcgaccgatgtgtc PPC F1* GGCTGTTGAGTTTGGACTCTAGGTACCGCGACGTCC This Work TCCAGCTTTTGTTCCCTTTAG PPC R1* GACACATCGGTCGATTATGAATTCCGGCCATATGCG This Work GATTCTAGAACTAGTGGATCCCCCATCA LBF1 -Lic TTTAAGAAGGAGATATAGATC ATGTCTCCGTCGAC This Work GACG LBR1 -NsLic GTTGGAGGATGAGAGGATCCC CCTCCCAAAGATC This Work ATTTCATCA LDF1 -Lic TTTAAGAAGGAGATATAGATC ATGAAGAAGATTCT This Work AGAAAGATCAATG LDR6 -NsLic GTTGGAGGATGAGAGGATCCC CAAGTTAACGTAA This Work GAGAATAGTGAAGGAA UP1 F1 -Lic TTTAAGAAGGAGATATAGATC ATGGTGGAGGAAA This Work CGAGCT UP1 R3 -NsLic GTTGGAGGATGAGAGGATCCC ATTATCACAAATC This Work AAACACGAGAATA P1F1 GTGCTCGATTTGACAATCTAAATG This Work P1R1 TAGATTCCGATCCTCAAAAGTAGG This Work P2F1 CTGTTGTTATCTCTCCTGATGGTC This Work PAH2Rv CTGCAGTCACATAAGCGATGGAGGAGGCAG Nakamura et al 2009 pROK2 deep CTTTAGGGTTCCGATTTAGTGCTTTA This Work qWRI1 (3)F GACCCGACACCATCTTGAAT Neil Adhikari qWRI1 (3)R GGCGGAGAGAAGCCAAAT Neil Adhikari

Table 1. Primers used in this work. Blue Bold : Gateway cloning tags. Red Bold : amiRNA targeting LPP β. Green Bold : LIC cloning tags. Orange Bold : gRNA targeting LPP β in CRISPR/Cas9 system. Purple Bold : Restriction Site

94

3.6 REFERENCES

1. Han GS, Siniossoglou S, Carman GM: The cellular functions of the yeast lipin homolog PAH1p are dependent on its phosphatidate phosphatase activity . J Biol Chem 2007, 282 :37026-37035. 2. Santos-Rosa H, Leung J, Grimsey N, Peak-Chew S, Siniossoglou S: The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth . Embo j 2005, 24 :1931-1941. 3. Adeyo O, Horn PJ, Lee S, Binns DD, Chandrahas A, Chapman KD, Goodman JM: The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets . J Cell Biol 2011, 192 :1043-1055. 4. Peterfy M, Phan J, Xu P, Reue K: Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin . Nat Genet 2001, 27 :121-124. 5. Phan J, Reue K: Lipin, a lipodystrophy and obesity gene . Cell Metab 2005, 1:73-83. 6. Chen Y, Rui BB, Tang LY, Hu CM: Lipin family proteins--key regulators in lipid metabolism . Ann Nutr Metab 2015, 66 :10-18. 7. Eastmond PJ, Quettier AL, Kroon JT, Craddock C, Adams N, Slabas AR: Phosphatidic acid phosphohydrolase 1 and 2 regulate phospholipid synthesis at the endoplasmic reticulum in Arabidopsis . Plant Cell 2010, 22 :2796-2811. 8. Nakamura Y, Koizumi R, Shui G, Shimojima M, Wenk MR, Ito T, Ohta H: Arabidopsis lipins mediate eukaryotic pathway of lipid metabolism and cope critically with phosphate starvation . Proc Natl Acad Sci U S A 2009, 106 :20978-20983. 9. Reddy VS, Rao DK, Rajasekharan R: Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana . Biochim Biophys Acta 2010, 1801 :455-461. 10. Nakagawa N, Kato M, Takahashi Y, Shimazaki K, Tamura K, Tokuji Y, Kihara A, Imai H: Degradation of long-chain base 1-phosphate (LCBP) in Arabidopsis: functional characterization of LCBP phosphatase involved in the dehydration stress response . J Plant Res 2012, 125 :439-449. 11. Zhang M, Fan J, Taylor DC, Ohlrogge J: DGAT1 and PDAT1 Acyltransferases Have Overlapping Functions in Arabidopsis Triacylglycerol Biosynthesis and Are Essential for Normal Pollen and Seed Development . The Plant Cell 2009, 21 :18.

95

12. Katavic V, Reed DW, Taylor DC, Giblin EM, Barton DL, Zou J, MacKenzie SL, Covello PS, Kunst L: Alteration of Seed Fatty Acid Composition by an Ethyl Methanesulfonate- Induced Mutation in Arabidopsis thaliana Affecting Diacylglycerol Acyltransferase Activity . Plant Physiology 1995, 108 :11. 13. Routaboul J-M, Benning C, Bechtold N, Caboche M, Lepiniec L: The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase . Plant Physiology and Biochemistry 1999, 37 :10. 14. Han GS, Wu WI, Carman GM: The Saccharomyces cerevisiae Lipin homolog is a Mg2+- dependent phosphatidate phosphatase enzyme . J Biol Chem 2006, 281 :9210-9218. 15. Mietkiewska E, Siloto RM, Dewald J, Shah S, Brindley DN, Weselake RJ: Lipins from plants are phosphatidate phosphatases that restore lipid synthesis in a pah1Delta mutant strain of Saccharomyces cerevisiae . Febs j 2011, 278 :764-775. 16. Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D: Highly specific gene silencing by artificial microRNAs in Arabidopsis . Plant Cell 2006, 18 :1121-1133. 17. Lu C, Fulda M, Wallis JG, Browse J: A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis . Plant J 2006, 45 :847-856. 18. Stuitje AR, Verbree EC, van der Linden KH, Mietkiewska EM, Nap JP, Kneppers TJ: Seed- expressed fluorescent proteins as versatile tools for easy (co)transformation and high- throughput functional genomics in Arabidopsis . Plant Biotechnol J 2003, 1:301-309. 19. Karimi M, Inze D, Depicker A: GATEWAY vectors for Agrobacterium-mediated plant transformation . Trends Plant Sci 2002, 7:193-195. 20. Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J: Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 . Nat Biotechnol 2013, 31 :688-691. 21. Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S: Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease . Nat Biotechnol 2013, 31 :691-693. 22. Onate-Sanchez L, Vicente-Carbajosa J: DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques . BMC Res Notes 2008, 1:93.

96

23. Li YH, Beisson F, Pollard M, Ohlrogge J: Oil content of Arabidopsis seeds: The influence of seed anatomy, light and plant-to-plant variation. Phytochemistry 2006, 67 :904-915. 24. Chen DC, Yang BC, Kuo TT: One-step transformation of yeast in stationary phase . Curr Genet 1992, 21 :83-84. 25. Pedelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS: Engineering and characterization of a superfolder green fluorescent protein . Nat Biotechnol 2006, 24 :79-88. 26. Bates PD, Jewell JB, Browse J: Rapid separation of developing Arabidopsis seeds from siliques for RNA or metabolite analysis . Plant Methods 2013, 9:9. 27. Focks N, Benning C: wrinkled1: A novel, low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism . Plant Physiol 1998, 118 :91-101. 28. Cernac A, Andre C, Hoffmann-Benning S, Benning C: WRI1 is required for seed germination and seedling establishment . Plant Physiol 2006, 141 :745-757. 29. Cernac A, Benning C: WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis . Plant J 2004, 40 :575- 585. 30. To A, Joubes J, Barthole G, Lecureuil A, Scagnelli A, Jasinski S, Lepiniec L, Baud S: WRINKLED transcription factors orchestrate tissue-specific regulation of fatty acid biosynthesis in Arabidopsis . Plant Cell 2012, 24 :5007-5023. 31. Nakamura Y, Ohta H: Phosphatidic Acid Phosphatases in Seed Plants . In Lipid Signaling in Plants . Edited by Munnick T: Springer; 2010:131-141. 32. Mao Y, Zhang H, Xu N, Zhang B, Gou F, Zhu JK: Application of the CRISPR-Cas system for efficient genome engineering in plants . Mol Plant 2013, 6:2008-2011. 33. Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N, et al.: The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation . Plant Biotechnol J 2014, 12 :797-807. 34. Brooks C, Nekrasov V, Lippman ZB, Van Eck J: Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system . Plant Physiol 2014, 166 :1292-1297.

97

35. Maeo K, Tokuda T, Ayame A, Mitsui N, Kawai T, Tsukagoshi H, Ishiguro S, Nakamura K: An AP2-type transcription factor, WRINKLED1, of Arabidopsis thaliana binds to the AW-box sequence conserved among proximal upstream regions of genes involved in fatty acid synthesis . Plant J 2009, 60 :476-487.

98

CHAPTER FOUR

IDENTIFICATION OF ARABIDOPSIS GPAT9 (AT5G60620) AS AN ESSENTIAL GENE

INVOLVED IN TRIACYLGLYCEROL BIOSYNTHESIS

ABSTRACT

The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER) localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this

GPAT activity has remained elusive. Here we present the results of a series of in vivo, in vitro, and in silico experiments designed to assign this essential function to AtGPAT9 . This gene has been highly conserved throughout evolution, and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of AtGPAT9 in developing seed is required for wild-type levels of triacylglycerol accumulation, and transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together these results suggest that

GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis.

99

PREFACE

This chapter of my dissertation is a manuscript accepted for publication in Plant Physiology and includes data from Jay Shockey *1, Anushobha Regmi *2, Kimberly Cotton 3, Neil Adhikari 3,

John Browse 3, and Philip Bates 1†. I was responsible for the data obtained in the qrt1 mutant background (crossing, tetrad analysis, pollen germination) and assisting in manuscript preparation.

This data is found in Table 4 (“Pollen germination and tube growth of GPAT9/gpat9-2 within the qrt1 background”) and Figure 4 (“Reduced Pollen Size and Pollen Tube Growth of GPAT9/gpat9-

2 Pollen in the qrt1 Background”).

4.1 INTRODUCTION

Glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) was first characterized biochemically in extracts from animal and plant tissues in a series of reports beginning more than

60 years ago [1-3]. GPAT transfers an acyl moiety from either acyl-CoA or acyl-acyl carrier protein (ACP) to the sn -1 position of glycerol-3-phosphate to produce 1-acyl-2-lyso-glycerol-3- phosphate (or lysophosphatidic acid). It catalyzes the first step in the synthesis of various types of glycerolipids including membrane lipids and triacylglycerol (TAG) in all living organisms, and thus plays an extremely important role in basic cellular metabolism. In addition, the enzymes which esterify fatty acids (FAs) to the glycerol backbone of TAG control the TAG FA composition, and thus the functional value of plant oils. Acyl selective isozymes for the last step in TAG assembly have demonstrated to be crucial for enhancing the control of seed oil content

* These co-authors contributed equally to this work. 1 United States Department of Agriculture – Agricultural Research Service, Southern Regional Research Center, New Orleans, LA 70124 2 Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, MS 39406 3 Institute of Biological Chemistry, Washington State University, Pullman, WA 99163 † Corresponding author. [email protected]

100

through bioengineering [4]. Therefore, our ability to produce designer plant oils that will meet the nutritional or industrial needs of the future will require identifying the genes which encode all of the acyltransferase steps in TAG production, including the essential first step catalyzed by GPAT.

Early in vitro biochemical characterization of GPAT activities from various plant tissues demonstrated multiple sub-cellular locations for GPAT activity including endoplasmic reticulum

(ER) [3,5,6], plastid [7], and mitochondria [8]. These, and other gene-independent biochemical analyses of plant lipid metabolic activity have in part contributed to the “two-pathway” hypothesis for plant glycerolipid synthesis that has stood the test of time and has been reviewed extensively

[9-11]. Plants contain two parallel lipid biosynthetic pathways each containing distinct GPAT enzymes to produce glycerolipids. The ER localized “eukaryotic pathway” utilizes a membrane- bound acyl-CoA dependent GPAT for synthesis of lysophosphatidic acid (LPA). LPA is subsequently acylated to produce phosphatidic acid (PA) by an acyl-CoA dependent lysophosphatidic acid acyltransferase (LPAAT). PA, or its dephosphorylated diacylglycerol

(DAG), provide the glycerolipid backbones for ER localized phospholipid or triacylglycerol

(TAG) synthesis. The three consecutive acyl-CoA dependent acylations of the glycerol backbone to produce TAG in the ER is commonly called the Kennedy pathway [2,12].

In contrast to the eukaryotic glycerolipid assembly pathway, the plastid-localized

“prokaryotic pathway” utilizes a soluble acyl-ACP dependent GPAT, and an acyl-ACP dependent

LPAAT to produce PA for phosphatidylglycerol synthesis in plastids of all plants, and the DAG for galactolipid synthesis in some plants. The prokaryotic pathway GPAT has been characterized extensively through both biochemical and molecular genetic approaches demonstrating species- specific acyl selectivities which control the composition of prokaryotic glycerolipids [13-17]. In addition to prokaryotic galactolipid synthesis in the plastid, plants also utilize DAG derived from

101

the eukaryotic pathway synthesized membrane lipid phosphatidylcholine (PC) for galactolipid synthesis. The PC-derived DAG is imported into the plastid for galactolipid production, and contains a distinctive eukaryotic FA composition [18]. Therefore the ER-localized GPAT of the eukaryotic pathway is vital for lipid assembly within both the ER and the plastid.

The essential nature of the ER GPAT for membrane lipid biosynthesis, and its role in controlling one-third of the FA composition within economically valuable plant oils, has inspired many studies focused on characterizing the ER GPAT enzyme. Microsomal extracts from a wide range of plant tissues have demonstrated a variety of in vitro ER GPAT activities utilizing various common and unusual FAs as substrates [19-24]. However, dissection of the range of potential effects of the ER-localized GPAT on seed oil content and FA composition has still remained unclear, awaiting the identification and molecular characterization of the requisite gene(s) encoding the ER localized GPAT.

Zheng et al. (2003) was first to classify a family of genes as ER and mitochondrial localized

GPAT s in plants. This eight gene family was identified based on homology to the acyltransferase domains of GPATs from yeast, and most members of this group demonstrated GPAT activity when heterologously expressed. Genetic analysis of Arabidopsis mutants defective in the first of these genes, designated AtGPAT1 , indicated altered tapetal differentiation and frequent abortion of microspores impacting male fertility, and suggesting that AtGPAT1 may be involved in essential membrane lipid production [25]. However, more recent biochemical and genetic characterization has revealed that AtGPAT1 -AtGPAT8 are a land plant-specific family of sn -2 GPATs involved in cutin and suberin synthesis [26-29]. These GPATs predominantly utilize acyl groups not found in membranes or TAG (e.g. ω-hydroxy and α,ω-dicarboxylic FAs) to produce the sn -2 monoacylglycerol precursors utilized during extracellular polymerization of both the cutin and

102

suberin barriers of land plants [30]. Therefore, the GPAT1-GPAT8 family is likely not involved in membrane lipid and triacylglycerol biosynthesis required in all living organisms.

Another gene, designated AtGPAT9 , previously has been identified as a candidate for the

ER membrane and oil biosynthetic GPAT activity based on substantial sequence identity to mouse and human GPAT3 [31,32]. The expression profiles and biochemical properties of mGPAT3 and hGPAT3 strongly support a role for these enzymes in TAG biosynthesis within lipid-rich mammalian tissues. However, definitive determination of the roles of AtGPAT9 in plant lipid metabolism has remained elusive. Complete understanding of plant lipid biosynthesis, and the ability to predictably engineer designer plant oils, awaits identification of all the component enzymes (and associated genes), and determination of how each one functions within the lipid metabolic network to produce the thousands of different possible molecular species of membrane and storage lipids.

The molecular identification of an ER-localized GPAT involved in membrane lipid and/or

TAG biosynthesis in plants has not been accomplished until now. Here we present the results of a series of experiments designed to address the role of AtGPAT9 in lipid biosynthesis. We present strong evidence that: (A) AtGPAT9 is a highly conserved, single copy, and essential gene which when knocked out is homozygous lethal and causes both male and female gametophyte lethalities;

(B) significant expression of AtGPAT9 is required for normal levels of microsomal GPAT enzymatic activity in seeds, and is required for wild-type levels of TAG accumulation in vivo ; (C)

AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and

TAG biosynthesis.

103

4.2 MATERIALS AND METHODS

4.2.1 PHYLOGENETIC ANALYSES

Plant GPAT9 and LPEAT protein sequences were identified from the species-specific searches of the Phytozome version 10.3 server (http://phytozome.jgi.doe.gov/pz/portal.html#).

Phylogenetic comparisons were carried out using alignment (.aln) files generated using the default settings provided in CLUSTALX (v1.8) [33]. Neighbor joining dendograms were created in

TreeView program [34,35], using the Boxshade tool at the ExPASy Bioinformatics Resource

Portal (http://www.ch.embnet.org/software/BOX_form.html).

4.2.2 GENOTYPING OF T-DNA MUTANTS

Mutant genotyping utilized gene and T-DNA specific primers (Supplemental Table S1), and GoTaq Green (Promega, www.promega.com) with manufacturers recommended protocol. For insertion site sequencing the left border-gene junction was amplified by PCR with GoTaq® Flexi

(Promega), gel-purified by QIAquick Gel Extraction Kit (QIAGEN and sequenced from both the left border primer and a gene-specific primer in the flanking sequence by Eurofins

(http://www.eurofinsgenomics.com/).

4.2.3 GENE EXPRESSION ANALYSIS

Leaf RNA was extracted with RNeasy Plant Mini Kit (QIAGEN, www.qiagen.com) and treated with RNase-free DNase (QIAGEN) using on-column DNase digestion. RNA was quantified using a Nanophotometer (Implen Inc). cDNA was synthesized using SuperScript III

First-Strand Synthesis System for RT-PCR (Life Technologies). RT-PCR utilized standard conditions as per the GoTaq® Flexi (Promega) protocol with 30 cycles of amplification. Transcript levels were analyzed by quantitative PCR (qPCR) using Platinum SYBR Green qPCR SuperMix-

UDG (Life Technologies) and the Stratagene Mx3005P Quantitative PCR system (Agilent

104

Technologies Inc). For developing seed transcript analysis, frozen developing seeds (9-10 DAF) were collected from liquid N 2 frozen siliques [36]. Approximately 50-100 mg of frozen seeds were mechanically pulverized to a fine powder with steel beads (TissueLyser LT, QIAGEN), RNA extracted [37], and DNA removed (DNA-Free RNA Kit™, Zymo Research, www.zymoresearch.com/). cDNA synthesis and quantification were performed as described above. Normalized Relative Quantity (NRQ) calculated [38] against TIP41-like [39].

4.2.4 MATERIALS , PLANTS , AND GROWTH CONDITIONS

Unless specified all chemicals are from Fisher Scientific (https://www.fishersci.com), and solvents are HPLC grade or higher. Arabidopsis thaliana lines gpat9-1, Salk_052947C [40] and qrt1-4 CS25041 [41] were obtained from ABRC (https://abrc.osu.edu/), and gpat9-2

GABI_867A06 [42] were obtained from Gabi-kat (https://www.gabi-kat.de/). Control plants were the Arabidopsis Col-0 ecotype. Seeds were surface sterilized (30% ethanol, 10% bleach, 0.1%

SDS) for 5 min, rinsed 5 times with sterile water and plated onto germination media (2.5 mM MES pH 5.7, 1% sucrose, 1x Murashige and Skoog Plant Salt Mix, (MP Biomedicals, http://www.mpbio.com/), 0.8% agar). Seeds from GPAT9/gpat9-2 plants were always germinated on the plate media containing an additional 5.25 mg/L sulfadiazine (Sigma, www.sigmaaldrich.com/) to select for the heterozygotes. Plated seeds were stratified for 4 days at

4 °C, prior moving to a growth chamber. After 10 days on the plate seedlings were transferred to soil. Plants were grown in growth chambers under continuous white light ~130-170 µmol photons m-2 sec -1, at 22-24 °C. Silique ages were determined by trimming each plant to one main shoot and counting the new open flowers/siliques produced each day.

105

4.2.5 DNA MANIPULATION AND PLASMID CONSTRUCTION

Binary complementation plasmids were generated as follows. The phaseolin promoter from cloning vector pK8 [43] was removed by Kpn I and Not I digestion and replaced with an 1594 bp portion of the 5’ upstream region of the AtGPAT9 gene, resulting in the synthesis of cloning vector pK51. The open reading frames of AtGPAT9 and tung GPAT9 (VfGPAT9 ) were cloned into the Not I and Sac II sites of pK51, resulting in plant shuttle plasmids pB447 and pB448, respectively. The Asc I fragments bearing the respective AtGPAT9 promoter: GPAT9 ORF:35S terminator cassettes from pB447 and pB448 were purified by gel electrophoresis and ligated into the Asc I site of the DsRed-selectable binary vector pB110 [43], resulting in the final plant transformation binary plasmids pE434 and pE437. Binary plasmids were transformed into

Agrobacterium tumefaciens strain GV3101 via electroporation and selection on solid media containing 50 µg/ml each of kanamycin and gentomycin. Individual A. tumefaciens colonies were inoculated into liquid media containing the same antibiotics and cultured overnight at 28 °C prior to plant transformation by floral dip, as described previously [44].

4.2.6 MUTANT COMPLEMENTATION

Approximately 100,000 T 1 seeds from each of the three transformations were sown on flats of soil for approximately 10 days, then heavily misted with a solution of 500 mg/L sulfadiazine containing 0.03% of the surfactant Silwet L-77, as described in Thomson et al. [45]. Plants that survived the sulfadiazine application and displayed red fluorescence were transferred to pots of untreated soil and grown to maturity. At the end of this experiment, nine, twenty-five, and thirty individual T 1 transgenic plants were identified for B110 control, E434, and E437, respectively.

Representative lines from each transgenic genotype were propagated to the T 3/T 4 generations, with sulfadiazine selection, until plants producing homozygous red fluorescent seeds were identified.

106

Batches of seeds from these plants, representing empty vector control B110 and GPAT9 overexpressor E434 and E437 lines, were surface-sterilized and sown on sulfadiazine agar plates for complementation testing.

4.2.7 GPAT9/GPAT 9-2 GAMETOPHYTE VIABILITY ANALYSIS

To determine the rate of gpat9-2 ovule abortion, aborted ovules were counted in opened half siliques from Col-0 and GPAT9/gpat9-2, and imaged under a Leica DM2000 microscope equipped with a DFC295 camera. Alexander staining of aborted pollen, and Nitro Blue

Tetrazolium (NBT) staining for pollen viability was done by previously optimized methods

[46,47].

4.2.8 ANALYSIS OF QRT 1-4 GPAT9/GPAT 9-2 POLLEN

GPAT9/gpat9-2 was crossed with qrt1-4 homozygous mutant as the pollen donor. F1 seeds were germinated on sulfadiazine to identify gpat9-2 heterozygotes. F1 plants were selfed, and F2 seeds again selected on sulfadiazine. Pollen from F2 plants was examined visually for the tetrad pollen phenotype to identify those homozygous for the qrt1-4 mutation, and genotype was confirmed with PCR. Pollen tube germination was measured by dusting pollen from qrt1

GPAT9/gpat9-2 and qrt1 plants onto one of two different optimized pollen germination media

[48,49], except that the pH of Boavida and McCormick media was adjusted with 1M KOH instead of NaOH. Pollen was then prehydrated and allowed to germinate for 8 hours at 22°C as described previously [48]. Images of germinated quartets were captured using Leica Application Suite

(version 4.2.0) software using a Leica DM2000 microscope equipped with a DFC295 camera.

Pollen size was measured using ImageJ software (National Institutes of Health; http://rsb.info.nih.gov/ij). To analyze the small pollen phenotype, tetrads were analyzed only if all four pollen grains were in the plane of focus. The longest apparent diameter (a) and the diameter

107

perpendicular to the longest diameter (b) of each pollen grain of 100 tetrads was measured, and

the apparent 2-D area of the pollen grain was calculated using the formula for an ellipse, = .

4.2.9 SEED AND LEAF LIPID COMPOSITION AND QUANTIFICATION

Whole seed (or leaf) lipids were converted to fatty acid methyl esters (FAMEs) in 5% (v/v) sulfuric acid in methanol, to which was added 0.2 ml toluene containing 20 µg tri-17:0 TAG

(Nucheck Prep Inc., www.nu-chekprep.com/) as internal standard, for 1.5 h at 85°C. FAMEs were quantified on an Agilent gas chromatograph with flame ionization detection on a wax column (EC wax; 30 m X 0.53 mm i.d. X 1.20 µm; Alltech).

4.2.10 SPLIT UBIQUITIN -BASED MEMBRANE YEAST TWO -HYBRID ASSAY

Protein:protein interactions between AtGPAT9 and other lipid metabolic enzymes, including those representing other steps in the Kennedy pathway, were characterized using the

DUALmembrane split-ubiquitin system (Dualsystems Biotech, Schlieren, Switzerland), essentially as described in Gidda et al. [50]. Plasmids were constructed for AtGPAT9, AtLPAAT2

[51], AtDGAT1 [52] and AtLPCAT2 [53]. Primarily, only bait fusion proteins containing Cub-

LexA fused to the C-terminus of proteins, and prey fusion proteins containing NubG fused to the

N-terminus of proteins were used in this studies to avoid false negative results, as explained in

Gidda et al. [50]. Basic analysis for interactions was conducted via dilution assays on solid agar media lacking leucine, histidine, tryptophan and adenine, using multiple serial 1:5 dilutions of cell cultures starting from an OD 600nm value of 0.5. The strength of specific bait-prey combinations was measured by quantitative β-galactosidase activity from cell lysates using the β-gal assay kit from

Pierce Protein Research Products (Thermo Scientific).

108

4.2.11 ARTIFICIAL MICRO RNA KNOCKDOWN OF ATGPAT9 EXPRESSION

Artificial microRNAs were designed using Web microRNA Designer

(http://wmd3.weigelworld.org). AT5G60620.1 ( GPAT9 ) was input as the target gene into the form on the “Designer” section of WMD3 and compared against Arabidopsis thaliana cDNA TAIR8 to get recommendations of amiRNAs. The minimum number of targets were 1, and accepted off-site targets 0. We selected nucleotide region +191-211 (5’-TAGATGTCTAGCAAATCGCGC-3’) for cloning. Using the GPAT9-specific amiRNA sequences, oligonucleotide sequences were generated by WMD3 for site-directed mutagenesis to introduce the sequences of interest in the microRNA precursor gene MIR319a. PCR was conducted using these oligos and pRS300

(MIR319a) as template and following instructions described on WMD3. Using Gateway cloning, the final PCR products were cloned into the pENTR vector and eventually introduced into the pDS-Red-PHAS binary vector under control of the P. vulgaris phaseolin promoter [54]. Both constructs were transformed into Agrobacterium tumefaciens strain GV3101 which was used for transforming Col-0 WT plants.

4.2.12 GPAT ENZYME ACTIVITY ASSAYS

GPAT assays were performed with microsomes collected from 9-11 DAF developing seeds, or whole siliques, from wild-type and amiRNA knockdown lines. Microsomes were prepared as described previously [55]. In brief, approximately 100 whole siliques, or 0.1 ml volume of developing seeds dissected from siliques, were homogenized in 0.1 M potassium phosphate buffer pH 7.2, 1% BSA, 1000 units of catalase/mL, 0.33 M sucrose and 1x Thermo

Scientific Halt TM Protease Inhibitor Cocktail, EDTA-free (100X) with a Kinematica AG

Polytron® PT-MR 2100 on ice. The homogenate was filtered through two layers of buffer soaked

Miracloth (EMD Millipore Corporation), and centrifuged at 15,000g for 10 min at 4 °C. The

109

supernatant was centrifuged at 105,000g for 90 min at 4 °C. The pellet was rinsed with phosphate buffer pH 7.2 then resuspended in 0.1 ml of phosphate buffer pH 7.2 containing 1000 units of catalase. Microsomes were quantified using Thermo Scientific Pierce™ BCA Protein Assay Kit.

Approximately, 0.3-0.6 mg total protein of isolated microsomes were utilized for GPAT assays and performed in 0.1 ml of 0.1 M Tris, pH 7.2, 4 mM MgCl 2, 1% BSA and 1 mM DTT [29], with

3.55 nmol [ 14 C]-glycerol 3-phosphate ([ 14 C]G3P, 161mCi/mmol, www.perkinelmer.com) as acyl acceptor and 25 nmol palmitoyl-CoA (Sigma, http://www.sigmaaldrich.com/) as acyl donor at

24 °C with constant mixing (1250 rpm) for 15 minutes. The reaction was terminated by adding 0.12 ml of 0.15 M acetic acid with 400 nmol unlabeled G3P and 1.2 ml CHCl 3:MeOH (1:2).

Approximately, 25 µg of carrier LPA and PA in 0.4 ml CHCl 3 were added as a carrier and lipids extracted [56]. In brief, the organic layer was collected after phase separation, followed by two back-extractions of the aqueous phase with CHCl 3. The combined chloroform extracts were rinsed with H 2O:MeOH (1:1) to remove any remaining labeled G3P, and evaporated under N 2. The lipids were resuspended in 0.1 ml CHCl 3:MeOH (9:1), total radioactivity in 10 µl was quantified by liquid scintillation counting on a Beckman Coulter™ LS 6500 Multi-Purpose Scintillation

Counter, and the remaining extract was loaded onto Merck 20x20 cm silica gel 60 plates developed with solvent system CHCl 3:MeOH:acetic acid:H 2O, 75:15:10:3.5 (v/v/v/v). Radioactivity on TLC plates was quantified with a GE Typhoon FLA 7000, and ImageQuant™ TL Image Analysis

Software v8.1.

4.2.13 SOFTWARE AND STATISTICS

Graphs and statistical analysis indicated in each figure were produced with GraphPad

Prism (http://www.graphpad.com/).

110

4.2.14 ACCESSION NUMBERS

Sequence data from this article can be found in The Arabidopsis Information Resource

(http://www.arabidopsis.org/) under the following accession numbers:

AtGPAT9 (At5g60620), AtLPEAT1 (At1g80950), and AtLPEAT2 (At2g45670). The Genbank accession numbers for RcGPAT9 and VfGPAT9 are ACB30546 and FJ479751, respectively. All other gene identifiers are as described in the species-specific data subsets in the Phytozome database (version 10.3, http://phytozome.jgi.doe.gov/pz/portal.html#). All identifiers are listed individually in Supplemental Figure S2.

111

4.3 RESULTS

4.3.1 GPAT9 PROTEIN SEQUENCES HAVE BEEN STRONGLY CONSERVED ACROSS PLANT EVOLUTION

Genome evolution often gives rise to large, complex gene families [57]. Duplicated genes can either be maintained and achieve new function, or lost, causing one of the genes in question to revert to ‘singleton’ status, which often occurs with genes that encode essential functions [58,59].

A single GPAT responsible for the majority of extra-plastidial membrane lipid and TAG biosynthesis would fit this essential housekeeping role, and is supported by digital northern analyses of AtGPAT9 [32,60], which suggests a ubiquitous expression pattern. From this perspective, we analyzed the evolution of plant GPAT9 genes, by comparing the predicted protein sequences of GPAT9s from representative species from each major taxonomic grouping of plants.

These included Arabidopsis thaliana (Brassicacea), a bryophyte moss ( Physcomitrella patens ), a lycophyte ( Selaginella moellendorffii ), a core eudicot ( Aquilegia coerulea ), a grass

(Brachypodium distachyon ), and representatives from the dicot families Pentapetalae, Malvidae, and Fabidae ( Solanum tuberosum, Salix purpurea, Gossypium raimondii, and Cucumis sativus ).

The divergence and speciation events separating these plants cover at least 400 million years of evolution. As shown in Figure 1, GPAT9 proteins from this diverse group of plants have maintained a remarkable level of sequence identity, with approximately 55% identity and 65% similarity overall, with several individual cross-species pairs at >80% identity and >90% similarity.

To add perspective to these findings, GPAT9 sequences from each of these plants were compared to the corresponding LPEAT1 and LPEAT2 proteins from each species. GPATs and lysophosphatidylethanolamine acyltransferases (LPEATs) utilize the same acyl donor and similar

112

acyl acceptors (in the case of LPEAT1/2, lysophospholipids including LPA, lysophosphatidylcholine, and lysophosphatidylethanolamine), and the sequences of GPAT9,

LPEAT1, and LPEAT2 are homologous themselves [61], making phylogenetic comparisons between them useful. As members of a larger gene family derived at least in part from gene duplication events, LPEAT1 and LPEAT2 display more sequence divergence and are much less likely to encode essential housekeeping functions. LPEAT1 and LPEAT2 sequences from several plant species were aligned and analyzed in a fashion similar to the GPATs (Supplemental Figures

S1 and S2, respectively). The input protein sequences for the two LPEAT groups derive from a much less diverse range of species (including sequences from only three monocots and nine dicots) than those in the GPAT9 group shown in Figure 1, yet the sequences within the LPEAT1 and

LPEAT2 groups are significantly more diverged, sharing only 34% identity/47% similarity and

31% identity/43% similarity, respectively (Supplementary Figures S1 and S2). The contrast between the relative rates of evolution amongst these three families of genes can be seen directly in Figure 2. GPAT9, LPEAT1, and LPEAT2 protein sequences from the three monocot species

(Zea mays, Setaria italica, and Oryza sativa ) and nine dicot species ( Arabidopsis thaliana, Citrus clementina, Glycine max, Prunus persica, Populus trichocharpa, Ricinus communis, Solanum tuberosum, Theobroma cacao, and Vitis vinifera ) were aligned and compared phylogenetically.

Each of the three gene families form distinct clades, containing smaller subclades specific to monocots and dicots. However, in all cases the branch lengths for the GPAT9 family are significantly shorter than that of the members of either LPE acyltransferase clade, indicating less evolutionary drift of protein coding sequences in the GPAT9 s relative to either the LPEAT1 or

LPEAT2 gene families. Significantly higher constraint on sequence divergence is consistent with the hypothesis that plant GPAT9 s encode an essential housekeeping function (Figure 2).

113

4.3.2 A GPAT 9 T-DNA KNOCKOUT IS HOMOZYGOUS LETHAL

The homology of AtGPAT9 to GPATs in lipid-rich mammalian tissues alludes to a possible important role in TAG synthesis, but does not directly indicate its function within lipid metabolism. Therefore, we initially set out to investigate if T-DNA insertional mutants of GPAT9 display an altered glycerolipid phenotype. The Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/) indicates only two available T-DNA insertion lines targeted to exons in the GPAT9 locus, and both were obtained. The gpat9-1 line was a “confirmed” homozygous Salk line (Salk_052947C) putatively containing a T-DNA insertion the first exon

[40]. Initial plantings of gpat9-1 indicated no obvious growth phenotypes, and we confirmed the presence of a homozygous T-DNA insertion near the 5’ end of GPAT9 by PCR of genomic DNA with T-DNA and gene-specific primers (primer sequences are shown in Supplemental Table S1).

The gpat9-2 (GABI_867A06) line [42] indicated a T-DNA insertion in the fourth exon, and contains a sulfadiazine resistance selectable marker. Germination of gpat9-2 seeds on sulfadiazine-containing media over several generations produced only heterozygous

GPAT9/gpat9-2 plants as confirmed by segregation of resistant and susceptible seedlings, and by

PCR. Once transferred to soil there was no obvious difference in growth of GPAT9/gpat9-2 plants from wild-type. The quantitation of seed germination and seedling viability on sulfadiazine (Table

1) indicated that at each generation ~6-26% of seeds from GPAT9/gpat9-2 plants do not germinate, and over 84% of germinated seedlings do not survive on sulfadiazine, and thus are wild-type. All sulfadiazine-resistant seedlings continue to produce sulfadiazine-susceptible offspring, indicating the parental lines were heterozygous GPAT9/gpat9-2. Together, the inability to isolate a homozygous line, and the non-Mendelian segregation of sulfadiazine resistance suggests the gpat9-2 T-DNA insertion line is homozygous lethal.

114

The contradictory phenotypes of the gpat9-1 and gpat9-2 insertion lines necessitated further characterization of each mutation. We identified the actual T-DNA insertion locations by sequencing of the T-DNA insertion regions. PCR products were obtained with T-DNA left border primers paired with both upstream and downstream gene-specific GPAT9 primers, using genomic

DNA from both gpat9-1 and GPAT9/gpat9-2 plants; the results indicated the presence of multiple linked inverted T-DNAs at the GPAT9 insertion site in each line. The gpat9-1 T-DNA insertions were located 46 bp upstream of the GPAT9 coding sequence start site, and the gpat9-2 T-DNA insertions were located at the end of exon 4 (Supplemental Figure S3 A). RT-PCR of GPAT9 mRNA from leaves of gpat9-1 indicated that full length coding sequence was expressed at similar levels to wild-type (Supplemental Figure S3 B). Quantitative RT-PCR of GPAT9 mRNA from leaves of GPAT9/gpat9-2 demonstrated that GPAT9 expression was reduced to ~50% of wild-type

(Supplemental Figure S3 C). Given the minor effect on transcription of GPAT9 in gpat9-1, we concentrated on the sole exon-targeted mutant, gpat9-2 (GABI_867A06) for the remainder of our studies.

4.3.3 THE GPAT 9-2 T-DNA MUTATION PRODUCES POLLEN LETHALITY AND PARTIAL FEMALE GAMETOPHYTE LETHALITY PHENOTYPES

The expected Mendelian segregation ratios from a self-fertilized heterozygous parent that produces embryonic lethal mutants is ~67% heterozygous and ~33% wild-type. Single gametophyte lethality mutants are expected to produce 50% heterozygous, and 50% wild-type offspring. That GPAT9/gpat9-2 plants produced <30% heterozygous seeds and >70% wild-type

(Table 1) suggests the gpat9-2 mutation may have reduced viability of both gametophytes.

Therefore, we analyzed the transmittance of the gpat9-2 mutation (by sulfadiazine resistance)

115

through reciprocal crosses with wild-type (Table 2). When GPAT9/gpat9-2 flowers were used as the pollen donor with Col-0 pistils as the pollen acceptor no sulfadiazine resistant seedlings were obtained. However, when Col-0 was the pollen donor with GPAT9/gpat9-2 pistils, ~14-22% of the germinated seedlings were resistant to sulfadiazine, similar to the transmittance of sulfadiazine resistance from selfed GPAT9/gpat9-2 in Table 1. These results suggest that gpat9-2 pollen is not viable, and that most gpat9-2 female gametophytes do not survive until fertilization with pollen containing wild-type GPAT9 .

In support of a partial female gametophyte lethality phenotype of gpat9-2, aborted ovules were observed in siliques of G PAT9/gpat9-2 but not in Col-0 (Figure 3). Quantitation of the developing seeds and aborted ovules indicated that GPAT9/gpat9-2 siliques contained ~28-43% aborted ovules, with an average of 35% (Table 3). Siliques from heterozygous plants should produce wild-type and mutant ovules at a ratio of 50:50. If 35 of the 50 gpat9-2 ovules abort, the segregation of gpat9-2:WT ovules is 15:50, or ~23% gpat9-2. Considering that no pollen from a selfed GPAT9/gpat9-2 plant can transmit the gpat9-2 mutation (Table 2), then all viable ovules will be fertilized with wild-type pollen, producing ~23% heterozygotes and ~77% wild-type seeds.

Therefore the proportion of aborted ovules in GPAT9/gpat9-2 siliques also supports the segregation results obtained from selfing GPAT9/gpat9-2 (Table 1) and the reciprocal crosses

(Table 2).

4.3.4 THE GPAT 9-2 POLLEN ARE SMALLER AND DO NOT PRODUCE POLLEN TUBES

The reciprocal crosses (Table 2) suggested a male gametophyte defect. To investigate pollen lethality we utilized histochemical staining for pollen abortion and pollen viability with

Alexander [46] and NBT [47] staining, respectively (Supplemental Figure S4). To our surprise we

116

did not detect any quantitiative differences in histochemical staining from pollen of GPAT9/gpat9-

2 plants compared to that of wild-type. TAG production in developing pollen is required for fertilization [62]. Therefore, we hypothesized that the gpat9-2 mutation may produce pollen with viable cells, however if TAG production is reduced due to the gpat9-2 mutation it may limit pollen tube growth and the transmittance of the gpat9-2 genotype. To investigate this hypothesis, we crossed the GPAT9/gpat9-2 into the qrt1-4 background, where microspores from a single meiosis event remain attached throughout development [63,64]. The qrt1 mutant background facilitates analysis of pollen defects because a heterozygous parent will produce tetrad pollen containing two affected and two unaffected pollen grains. Tetrads from qrt1 GPAT9/gpat9-2 displayed two larger and two smaller pollen grains (Figure 4A-B), consistent with the segregation of the mutant gpat9-

2 allele producing aberrant pollen. While all the pollen from qrt1 parent was close to the same size and followed a Gaussian distribution, the pollen from qrt1 GPAT9/gpat9-2 plants showed a bimodal distribution, with the small pollen roughly half the size of the larger pollen (Figure 4C).

To determine if gpat9-2 pollen can produce pollen tubes, we analyzed tetrad pollen germination (Table 4). In the qrt1 background, 5.5% of ~4800 tetrads produced 3 or 4 pollen tubes

(Figure 4A, Table 4). However, when over 6800 tetrads from the qrt1 GPAT9/gpat9-2 background were analyzed, no tetrads with 3 or 4 pollen tubes were observed (Table 4, Figure 4B). These results indicate that the gpat9-2 pollen grains do not produce pollen tubes, and supports the lack of transmittance of gpat9-2 through pollen in the reciprocal crosses (Table 2).

117

4.3.5 FUNCTIONAL PLANT GPAT9 GENES COMPLEMENT THE GAMETOPHYTIC LETHALITY PHENOTYPES OF THE HETEROZYGOUS GPAT9/ GPAT 9-2 MUTANT

Wild-type copies of GPAT9 ORFs from Arabidopsis and tung tree ( Vernicia fordii ) driven by the AtGPAT9 promoter were used to test for complementation of the GPAT9/gpat9-2 phenotypes. We used the AtGPAT9 promoter in an attempt to match the tissue- and cell-type specific transgenic expression of GPAT9 s as closely as possible to that of the native gene, instead of using other, more well-known constitutive promoters (e.g. cauliflower mosaic virus 35S) which does not express well in pollen or developing seeds [65,66] and would therefore be unlikely to complement the gpat9-2 phenotypes. Transgenic plants containing the complementation constructs were selected using cassava vein mosaic virus (CVMV) promoter-driven expression of the DsRed fluorescent protein as a selectable marker [67,68].

A population of sulfadiazine-resistant GPAT9/gpat9-2 seedlings were transformed with

Agrobacterium bearing either empty binary plasmid (B110), or the complementation plasmids

E434 or E437, which contained the ORFs for AtGPAT9 or VfGPAT9 , respectively. Very few red seeds from the empty vector B110 line were sulfadiazine resistant (approximately 4%, Figure 5A,

D) similar to the segregation analysis of GPAT9/gpat9-2 in Table 1. However, the homozygous red fluorescent seeds from the E434 (Figure 5B, 5E) and E437 (Figure 5C, 5F) lines effectively germinated and established photosynthetic competency on sulfadiazine agar media, at about 60-

70% of all seeds sown. Therefore, the complementation constructs clearly function to complement the rare occurrence of sulfadiazine-resistant GPAT9/gpat9-2 progeny (Table 1). This result confirms that the AtGPAT9 gene is essential and responsible for the observed segregation and gametophytic lethality phenotypes in mutant plants. Additionally, GPAT9 genes from multiple plant species can complement the Arabidopsis mutant lethality phenotype.

118

4.3.6 SEED -SPECIFIC KNOCKDOWN OF GPAT9 REDUCES OIL CONTENT AND ALTERS FATTY ACID COMPOSITION

Our analysis of GPAT9/gpat9-2 suggests that AtGPAT9 is an essential gene and a homozygous gpat9 knockout cannot be obtained. Analysis of lipid content in heterozygous

GPAT9/gpat9-2 plants indicated no change from wild-type in leaves (Supplemental Figure S5A), or seeds (Supplemental Figure S5B-C). The latter was as expected since most seeds produced from

GPAT9/gpat9-2 are wild-type (Table 1). Therefore, a single copy of GPAT9 is sufficient for vegetative growth, and a different approach must be taken to investigate the role of GPAT9 in lipid metabolism. A strong, constitutive knockdown of GPAT9 expression would be expected to severely reduce plant growth, complicating characterization of a lipid phenotype associated with aberrant GPAT9 expression. Therefore, we chose to investigate the role of GPAT9 in TAG biosynthesis in seeds (which has high flux through ER GPAT activity compared to other tissues) by creating seed-specific GPAT9 knockdown Arabidopsis lines. An artificial microRNA

(amiRNA) construct for GPAT9 was expressed under the seed specific Phaseolus vulgaris phaseolin promoter [54], with the fluorescent protein DsRed as a selectable marker [68].

Heterozygous transformed (red) T 1 seeds were planted, and the corresponding segregating T 2 seeds were harvested from 20 individual T 1 transformants. Red T 2 seeds were separated from the segregating untransformed brown seeds, and each set analyzed for oil content (Figure 6). Oil content varies considerably between individual Arabidopsis plants [69], and the co-segregating non transformed seed acts as a plant specific wild-type control for seed oil levels. Red seeds from each individual transformed line had a 15-75% reduction in total lipid relative to the plant-specific brown seeds (Figure 6A). In the lines with the largest reduction in oil content, the red seeds were

119

smaller and displayed a wrinkled phenotype compared to the corresponding brown seeds (Figure

4 B-C). A wrinkled seed phenotype is typical for Arabidopsis seeds with very low oil content [70].

Eight GPAT9 amiRNA lines with low oil content were identified to have single T-DNA inserts based on 3:1 segregation ratios of red and brown seeds, and were propagated further.

Homozygous red T 3 seeds were analyzed for seed oil content and composition (Figure 7). Oil content was reduced by 26-44% (Figure 7A). Seed lipids from all eight amiRNA lines also contained altered fatty acid composition (Supplemental Figure S6). The FA composition of two

GPAT9 amiRNA knockdown lines chosen for further experiments (#2 and #12) is demonstrated in Figure 7B. Significant changes in all lines included an average increase in 18:3 from 19.8% to

28.9%, and in 20:3 from 0.5% to 0.9%. Correspondingly, 20:1 was decreased from an average of

20.6% to 13.1%, and 18:2 was decreased from 26.6% to 24.3%. Analysis of GPAT9 gene expression by qPCR in developing T 4 seeds from lines #2 and #12 indicated that GPAT9 transcript is reduced >88% from wild-type in each line (Supplemental Figure S7). Together these results indicate that reduced GPAT9 expression impacts the amount and composition of TAG in seeds.

4.3.7 SEEDS FROM GPAT9 KNOCKDOWN LINES HAVE REDUCED GLYCEROL -3-PHOSPHATE ACYLTRANSFERASE ACTIVITY

The sequence of AtGPAT9 closely resembles mammalian GPATs that contribute to TAG synthesis [31]. However, AtGPAT9 displays high sequence homology to multiple classes of plant lysophospholipid acyltransferases (including LPEAT1 and LPEAT2 which also use LPA as a substrate, Figure 2). Additionally, plant GPAT9s in general more closely match plant LPAATs in protein length (~375-390 amino acid residues for both enzyme classes) [71] than they do the land plant specific sn -2 GPAT family (500-585 residues). Reduction of either GPAT or LPAAT activity in the amiRNA knockdown lines has the possibility to affect seed oil content. Therefore to

120

determine which enzymatic activity is affected in the knockdown lines, we performed in vitro

GPAT and LPAAT assays from microsomes isolated from developing siliques of wild-type and homozygous T 4 GPAT9 amiRNA lines (Figures 8 and 9). Figure 8A demonstrates that silique microsome GPAT assays utilizing [ 14 C]G3P and palmitoyl-CoA as substrates produce LPA and

PA as the major products. For both wild-type and amiRNA lines, [ 14 C]G3P distribution between

LPA and PA was approximately 3:7 LPA/PA at 5-7 days after flowering (DAF), and 7:3 LPA/PA at 9-11 DAF (Fig. 8A). However, the total amount of [ 14 C]G3P incorporated into lipids differed between the wild-type and knockdown lines. [ 14 C]G3P is incorporated into PA only after LPA has been formed therefore the quantification of both LPA and PA represents total GPAT activity. Total

GPAT activity was reduced ~19-49% at the two different stages of silique development (Fig. 8B-

C). To confirm that the reduced GPAT activity measured from whole silique microsomes is due to the seed specific knockdown of ER GPAT activity in GPAT9 amiRNA lines, we also performed

GPAT assays on microsomes isolated from developing seeds dissected out of 9-11 DAF siliques

(Supplemental Figure S8). Similar to the whole silique results, the amiRNA lines had ~25-55% reduction in seed microsome GPAT activity from wild-type.

LPAAT assays were performed with the same set of whole silique microsomes as for

GPAT assays (Figure 8) utilizing LPA and [ 14 C]oleoyl-CoA as substrates. The [ 14 C]oleoyl-CoA can also be used by other acyltransferases and endogenous lipid acceptors found within the microsomes. However, since only LPA was added as an exogenous acyl acceptor, PA was the major product of the assay in all samples (Figure 9A-B). No differences in LPAAT activity between wild-type and the GPAT9 amiRNA lines were detected (Figure 9C-D). The reduction of

GPAT enzymatic activity in seeds of GPAT9 amiRNA knockdown lines (Figures 8, S8), and the corresponding lack of a change in LPAAT activity (Figure 9), together supports the conclusion

121

that the reduced oil accumulation in these transgenic lines (Figures 6, 7) is due to a reduction in seed GPAT activity, specifically caused by the GPAT9 mRNA knockdown.

4.3.8 ATGPAT9 PHYSICALLY INTERACTS WITH OTHER ENZYMES IN THE KENNEDY PATHWAY AND ACYL EDITING CYCLE

Enzymes that make up various biochemical pathways, including some lipid biosynthetic pathways, often form multi-component complexes [72,73]. Such complexes allow for rapid and efficient transfer of metabolites to downstream enzymes in the pathway. Given the possibility of metabolic networking between the enzymes of the Kennedy pathway to assemble glycerolipids, and the enzymes of acyl editing which provides polyunsaturated fatty acids for lipid assembly

[11,18,74], we sought to explore potential protein:protein interactions between AtGPAT9 and other potential partners using the split-ubiquitin yeast two hybrid (Y2H) system [75]. The split- ubiquitin system eliminates the need for importation of target enzymes and proteins into the nucleus of the yeast cell as in traditional Y2H systems [76], which is especially problematic for integral membrane proteins such as GPAT9 and other ER-localized acyltransferases. This system previously has been used to search for interacting partners of glycerolipid synthesis enzymes from tung tree [50]. As shown in Figure 10, AtGPAT9 interacted with itself, AtLPAAT2, and

AtLPCAT2, but not with AtDGAT1, the dominant TAG biosynthetic DGAT isozyme in

Arabidopsis [52]. AtDGAT1 did weakly interact with both AtLPAAT2 and AtLPCAT2 however, as shown in Supplementary Figure S9. The homomeric and heteromeric interactions between

AtGPAT9 and AtLPAAT2 or AtLPCAT2 were ~8-20-fold stronger than the corresponding interactions with AtDGAT1. Together these results suggest that AtGPAT9 likely interacts in vivo with AtLPAAT2, the next step in glycerolipid assembly after GPAT; and with AtLPCAT2, the

122

main enzyme involved in acyl editing which provides polyunsaturated FAs to the acyl-CoA pool for incorporation into de novo glycerolipid synthesis [74,77-81].

123

4.4 DISCUSSION

The genetic dissection of the Kennedy pathway with the ultimate goal for complete biotechnological control plant oil synthesis began in earnest more than 15 years ago, with the identification of the genes that encode what are now known as AtDGAT1 [52,82] and AtDGAT2

[83]. Meaningful progress has been made towards identification and functional characterization of these and many other enzymes that contribute to plant membrane phospholipid and storage triacylglycerol biosynthesis in the years since. However, despite such progress on many fronts, definitive isolation of the gene or genes that encode for the initial G3P acylation reaction that feeds both the ER localized membrane lipid and TAG biosynthesis pathways has eluded the plant lipid biotechnology community. Evidence is presented here that firmly supports the assignment of

AtGPAT9 to that role.

4.4.1 ATGPAT9 IS THE GPAT INVOLVED IN TAG BIOSYNTHESIS

The results presented here establish that AtGPAT9 is a single copy and essential gene, demonstrated by both male and female gametophyte lethality phenotypes of the gpat9-2 mutant, and the inability to obtain a homozygous mutant (Figures 1-5, Tables 1-4). The reduction in oil content of seed specific GPAT9 knockdowns (Figures 6, 7), and the corresponding reduction in

GPAT activity (Figures 8), but not LPAAT activity (Figure 9), demonstrates that AtGPAT9 is a glycerol-3-phosphate acyltransferase involved in seed oil accumulation. This result could not be demonstrated with any of the genes in the AtGPAT1-AtGPAT8 family, which further supports their vastly different roles within plant metabolism. Since GPAT9 is essential for gametophyte function, the GPAT activity involved in seed TAG biosynthesis is likely also required for ER membrane lipid synthesis in other tissues.

124

4.4.2 ATGPAT9 KNOCKDOWN SEED OIL PHENOTYPE , AND PROTEIN INTERACTORS HELPS TO FURTHER DEFINE THE PLANT OIL SYNTHESIS METABOLIC NETWORK

Protein:protein interaction analysis (Figure 10) provided insights that strongly suggest the proper placement of AtGPAT9 at an intersection between the ‘Kennedy’ de novo glycerolipid biosynthetic pathway, and the acyl editing cycle, supporting current models of the Arabidopsis lipid biosynthetic network (Figure 11A) [11,18,74]. In the current study, AtGPAT9 was found to interact with itself, AtLPAAT2, and AtLPCAT2, but not AtDGAT1. How each interaction fits into current models of TAG biosynthesis is discussed below.

Self-interaction of AtGPAT9 is consistent with discovery of oligomerization of yeast- expressed Erysimum asperum plastidial GPAT [84]. The self-associating properties of EaGPAT may suggest that it undergoes self-allosteric regulation, since many allosteric enzymes exhibit quaternary structure. Future studies will address possible correlations between homomeric assembly of AtGPAT9 subunits and allosteric regulation of GPAT activity. The interaction between AtGPAT9 and AtLPAAT2 (the activity expected to occur immediately downstream of

GPAT in the lipid biosynthetic pathway) suggests metabolic channeling between Kennedy pathway enzymes within lipid biosynthesis (Figure 11), and supports previous studies that found lipid biosynthetic enzymes co-localized to specific microdomains of the ER [50,85].

LPCAT works in both the forward and reverse directions [80,81]. The interaction of

AtGPAT9 with AtLPCAT2 fits with current metabolic models from in vivo lipid flux analysis which indicates that the acyl groups removed from PC by acyl editing are the major source of acyl-

CoA for de novo glycerolipid synthesis by Kennedy pathway enzymes [11,86-88]. The combined loss of AtLPCAT1 and AtLPCAT2 stops acyl editing in Arabidopsis seeds, and causes a dramatic shift in acyl flux such that newly synthesized FAs are directly incorporated into de novo

125

glycerolipid synthesis through GPAT and LPAAT rather than direct incorporation into PC through acyl editing as in the wild-type [77,78]. The interaction of AtGPAT9 and AtLPCAT2 demonstrated here supports that nascent acyl groups destined for AtLPCAT2-mediated acyl editing in wild-type can be readily shifted to AtGPAT9 within the lpcat1/2 double mutant for enhanced de novo glycerolipid synthesis.

The lack of interaction between AtGPAT9 and AtDGAT1 importantly fits with current models of TAG biosynthesis from in vivo labeling studies that indicate a kinetic separation of de novo glycerolipid synthesis and TAG synthesis. [ 14 C]glycerol kinetic labeling in Arabidopsis and soybean indicates that DAG synthesized de novo from the GPAT, LPAAT, and PA phosphatase activities of the Kennedy pathway is not directly utilized for TAG synthesis. Instead de novo DAG is utilized to synthesize PC, and DAG for TAG synthesis is later derived from PC [87,89]. This flux of DAG through PC, combined with the acyl editing cycle, enhances the residence time of acyl groups in PC for production of polyunsaturated FAs [11,86]. In Arabidopsis the fluxes of de novo DAG into PC, and PC-derived DAG out of PC are predominantly controlled by phosphatidylcholine:diacyglycerol cholinephosphotransferase (PDCT) [90]. Since PDCT activity does not involve the net synthesis or turnover of PC, only a headgroup exchange reaction, a reduction in glycerol flux into the TAG biosynthetic pathway by reduced GPAT activity should not affect the rate of PDCT action (Figure 11).

AtLPCAT2 interacted with both AtGPAT9 and AtDGAT1 (Figures 10, S9). LPCAT activity has been demonstrated within multiple cellular localizations [91,92]. Together these results suggest that there may be multiple sites for acyl editing within the cell (Figure 11), and strongly suggest that both the first and last glycerol acylation reactions may be fed at least in part with acyl-CoA derived directly from PC by AtLPCAT2. These results are also consistent with

126

recent co-expression and biochemical analyses using flax ( Linum usitatissimum ) DGAT1 and

LPCAT [79]. These authors showed that LPCAT likely mediates direct channeling of PC-derived acyl-CoA to DGAT1, thus accounting for the elevated polyunsaturated FA content in flax oil, while also helping to overcome the thermodynamically unfavorable reverse reaction of LPCAT

[79]. The results demonstrated here (Figures 10, S9) strongly suggest that a high degree of channeling also occurs in Arabidopsis (and likely many other plants as well), with at least two entry points for PC-derived acyl-CoA into glycerolipid assembly (Figure 11).

Finally, a consideration of TAG biosynthesis as a metabolic network involving multiple highly active exchange reactions in/out of PC that are independent of total acyl flux through the network (Figure 11, large black arrows), can be used to explain how reduced GPAT activity leads to increased levels of polyunsaturated FAs in the seed oil of GPAT9 amiRNA knockdown lines

(Figure 6B, S6). Acyl editing involves a cycle of PC deacylation and LPC reacylation by LPCAT enzymes in which the rate of acyl exchange on/off PC can be up to 15 times the rate of de novo glycerolpid synthesis [74,87,88]. Likewise PDCT activity can be independent of the total rate of

DAG and PC synthesis and turnover, leading to rapid exchange of DAG in/out of PC. The reduction of GPAT activity in GPAT9 amiRNA lines slows the total rate of glycerol flux (thus

DAG flux) through the lipid metabolic network into oil (Figure 11B, smaller purple arrows).

However, the flux through the acyl and headgroup exchange reactions with PC are independent of the total rate glycerolipid biosynthesis, allowing FAs and DAGs to continually cycle in/out of PC, enhancing the total residence time in PC prior to incorporation into TAG. The longer time an acyl group spends within the PC pool allows for greater access to the desaturases, and the higher the probability that oleate will be fully desaturated to linolenate prior to incorporation into TAG

[11,86]. Therefore, a slower rate of TAG assembly, combined with no change in the exchange

127

reactions explains the significant increase in 18:3 content of TAG in the GPAT9 amiRNA knockdown lines.

4.4.3 STRICT MAINTENANCE OF SINGLE COPY GPAT9 GENES IN PLANTS REINFORCES THEIR ESSENTIALITY

Plant genomes have been subjected to enormous evolutionary pressures over time, including whole- or partial-genome duplications (GD) [93], and small-scale duplication events

[57] which often result in sets of duplicated genes that help an organism to meet new metabolic requirements. Some duplicated genes are retained and ultimately achieve specialized function via changes in temporal or tissue/organ-specific gene expression profile, subcellular targeting, enzyme substrate specificity, etc. [59]. However, duplicated genes from genome duplication events can revert back to singleton status due to pressures from factors such as functional redundancy, epigenetic silencing, and chromosomal instability [58]. Many such ‘singleton’ genes have been found to encode for essential housekeeping functions [58,59], and the same appears to be true for

AtGPAT9 .

The various functional analyses shown here reveal that AtGPAT9 (and likely, by extension,

GPAT9 genes from other plants in general), is not genetically redundant (Figures 1-5, Tables 1-4), unlike DGATs, LPAATs, and several other classes of plant lipid metabolic enzymes [18]. Most plants contain a single copy of GPAT9 , or at least a single copy per diploid genome, and appear to have been subject to selection pressures that maintain the respective GPAT9 s genes as singletons

[58,59]. Searches across a wide array of sequenced plant genomes, covering many of the major branch points in plant evolution, found a very high number of single-copy GPAT9 genes (Figure

1). Pima cotton ( Gossypium raimondii ) is the only one of nine diverse species shown in Figure 1 that contained two copies of GPAT9 , as per the data available in Phytozome 10.3. The ancestor to

128

G. raimondii and other Gossypium sp. underwent a cotton-specific whole genome duplication event ~16-17 MYA [94]; the existing tetraploid status of cotton, and the relative recency of this duplication event may not yet have allowed for enough selection pressure, or provided enough evolutionary time for resolution of the ultimate fate of the second copy of GraGPAT9 .

GPAT9 s are also highly conserved across broader sections of the tree of life, suggesting a role in essential cellular metabolism. The homology between AtGPAT9 and mouse GPAT3 (which contains minimal sequence identity to any of the other eight known Arabidopsis extra-plastidial

GPATs) was one of the initial indicators that AtGPAT9 might be an actual sn -1 glycerol-3- phosphate acyltransferase [31,32]. Also unlike the larger family of sn -2 GPATs, AtGPAT9 is highly conserved across Animalia in general. AtGPAT9 is closely related to representative proteins from hundreds of animal species, the closest of which is shown in Supplementary Figure S10. A

GPAT from the orca whale ( Orcinus orca ) shares 39% identity and 58% similarity with the amino acid sequence of AtGPAT9 over a 360 amino acid region of the protein (nearly the full length of

AtGPAT9). In contrast, the top Animalia BLASTP hit for AtGPAT1 is an uncharacterized protein from moonfish ( Xiphophorus maculates ) which is only 27% identical and 42% similar, over a 256 residue portion of the protein (less than half of the AtGPAT1 protein). Conversely, O. orca does not possess a GPAT1, while X. maculates GPAT9 still retains 36% identity and 54% amino acid similarity to AtGPAT9 (Supplementary Figure S10).

Many of the genes in the ‘duplication-resistant’ and ‘mostly single-copy’ categories described by De Smet et al. [95] encode essential housekeeping functions. That AtGPAT9 is essential was proven by our inability to recover a homozygous gpat9-2 mutant plant, even after germination of heterozygous mutant seeds on solid media containing vitamins and sugars. The abnormally low recovery of sulfadiazine-resistant progeny from heterozygous gpat9-2 parents also

129

indicates that AtGPAT9 mutants are gametophytic lethal. The incomplete gametophytic lethality of the gpat9-2 female gamete may arise from minimally sufficient transfer of residual GPAT9 protein and GPAT9 mRNA between cells during cell division to maintain viability of a few eggs until fertilization with wild-type pollen, as has been demonstrated for other genes [96]. The complementation of the low penetrance of the GPAT9/gpat9-2 heterozygous phenotype with both transgenic Arabidopsis and tung tree GPAT9 constructs (Figure 5 and Supplementary Figure S5) proved that the gpat9-2 mutation was indeed responsible for the observed mutant phenotypes, and that somewhat distantly related plant GPAT9s can functionally complement for one another. This latter result may be key to finally controlling the FA composition at each position within the TAG backbone by isolating acyl selective GPAT enzymes from different species.

130

4.5 CONCLUSIONS

Taken together, the results presented here confirm that AtGPAT9 is an ancient gene that is essential in Arabidopsis, and likely other plants as well. Experimental evidence suggests that

GPAT9 fulfills an indispensable role in catalysis of the first step in the synthesis of storage and membrane lipids required for life. This role may help to explain why GPAT9 genes have been maintained throughout most of the course of evolution of life on earth and why the sequences of

GPAT9s have evolved so conservatively. Finally, the characterization of GPAT9 at the intersection of de novo glycerolipid synthesis and acyl editing, and our demonstration that a distantly related plant GPAT9 can replace the essential AtGPAT9, suggests that bioengineering strategies around GPAT9 may be valuable for production of the designer vegetable oils of the future.

131

GraGPAT9A 1 ------MNSSEG-KLKSSSSELDLDRPN IEDYLPSGSSIQEP----HGK LRLRDL GraGPAT9B 1 ------MSSGKGGELIPSSSELDLDRPN IEDYLPSGSSIHEP----LGK LRLCDL SpuGPAT9 1 ------MDTPG-NLKTSISELDLDRPN IEDYLPSGTSIQEP----LDK LSLRDL StuGPAT9 1 ------MS-NLKSSSSELDFDRPN LEDYLPTG-SIQEP----HGK LRLRDL AthGPAT9 1 ------MSSTAG-RLVTSKSELDLDHPN IEDYLPSGSSINEP----RGK LSLRDL CsaGPAT9 1 ------MSGAA-LLKSSASELDLDRPN IEDYLPSGSSIQQP----TAK LRLRDL AcoGPAT9 1 ------MDIDRPN IEEYLTTD-SIHKP----QEK LLLRDL BdiGPAT9 1 ------MASS-----LDAPN LDDYLPTDSLPQEP----PRS LNLRDL SmoGPAT9 1 MGIQRTGVKDSNQLRDSSLFRPSQSEVDLYQHD MEDFLLDSAAEPAAPH--QNK LLLCDL PpaGPAT9 1 ------MEGDQFKGSADGINAGEAGRGICNEV VADFLNTNVEEDSSEAYLDNK MQLRDL

GraGPAT9A 45 LDIS PA LTEAAGAIVDDSFTRCFKSN PPEPWNWNVYL FPLWCC GV VF RY LI LFPMRALV L GraGPAT9B 46 LDIS PT LTEAAGAIVDDSFTRCFKSN PPEPWNWNVYL FPLWCC GV VI RY LI LFPVRVIV L SpuGPAT9 44 LDIS PT LTEAAGAIVDDSFTRCFKSN PPEPWNWNVYL FPLWCC GV VI RY GI LFPVRVLV L StuGPAT9 40 LDIS PT LTEAAGAIIDDSFTRCFKSN PPEPWNWNIYL FPLWCL GV VV RY GI LFPIRVIV L AthGPAT9 45 LDIS PT LTEAAGAIVDDSFTRCFKSN PPEPWNWNIYL FPLYCF GV VV RY CI LFPLRCFT L CsaGPAT9 44 LDIS PT LTEAAGAIVDDSFTRCFKSN PPEPWNWNIYL FPLWCC GV VI RY LF LFPARVLI L AcoGPAT9 30 LDIS PA LTEAAGAIIDDSFTRCFKSN PPEPWNWNVYL FPLWCF GV VI RY GI LYPVRVLV L BdiGPAT9 33 LDIS PV LTEAAGAIVDDSFTRCFKSN SPEPWNWNIYL FPLWCF GV VV RY GL LFPLRVLT L SmoGPAT9 59 LDIS GV LSEAASAIVDDSFTRCFKSN IPEPWNWNIYL LPLWCL GV IV RY CI LFPVRVLL L PpaGPAT9 54 LDIS QV LSEAGSAIIDDSFTRCFKSN APEPWNWNIYL FPLWVM GV AV RY LI LFPIRVIL L

GraGPAT9A 105 TI GWIIFLSC FIP VHFL LKGNDNLRKK MER ALVE LICSF FVASWTGVVKYHGPRPS MRPK GraGPAT9B 106 TV GWIIFLSC FIP VHLL LKGHDKLQKN MER ALVE LICSF FVASWTGVINYHGPRPS MRPK SpuGPAT9 104 AI GWIIFLSS YIP VHLL LKGHDKLRKK IER SLVE VICMF FVASWTGVVKYHGPRPS RRPK StuGPAT9 100 TI GWIIFLSC YIP VHLL LKGHDKFRKK LER CLVE LICSF FVASWTGVVKYHGPRPS IRPK AthGPAT9 105 AF GWIIFLSL FIP VNAL LKGQDRLRKK IER VLVE MICSF FVASWTGVVKYHGPRPS IRPK CsaGPAT9 104 TI GWIIFLST FIP VNLL LKGHPKLRAK LER FLVE LICSF FVASWTGVVKYHGPRPS IRPK AcoGPAT9 90 TI GWIIFLSS FTA VHFL LKGNDKWRRQ MER YLVE LICSF FVASWTGVVKYHGPRPS MRPK BdiGPAT9 93 GL GWMVFFAA FFP VHFL LKGQNKLRSK IER KLVE MMCSV FVASWTGVIKYHGPRPS SRPY SmoGPAT9 119 TV GWIIFLGA FIP VHFI LRKHDHMRRK IER GLVE FICSV FVASWTGVVKYHGPRPS RRPR PpaGPAT9 114 AL GWIIFLSL FFP LHFA LKNHDQLRRQ IER GLVE FMCSV FVASWTGVVKYHGPRPS RRTK

GraGPAT9A 165 QVFVANHTSMIDFIILEQMTAFAV IMQKHPGWVG LLQSTILE SVGCIWF NRSEAK DR EI V GraGPAT9B 166 QVFVANHTSMIDFIILEQMSSFAV IMQKHPGWVG LLQSTILE SVGCIWF NRTEAK DR ET V SpuGPAT9 164 QVFVANHTSMIDFIVLEQMTPFAV IMQKHPGWVG LLQSTILE SVGCIWF HRSEAK DR EI V StuGPAT9 160 QVFVANHTSMIDFIVLEQMTAFAV IMQKHPGWVG LLQSTILE GVGCIWF NRSEAK DR EI V AthGPAT9 165 QVYVANHTSMIDFIVLEQMTAFAV IMQKHPGWVG LLQSTILE SVGCIWF NRSEAK DR EI V CsaGPAT9 164 QVFVANHTSMIDFIVLEQMTAFAV IMQKHPGWVG LLQSTILE SIGCIWF NRTELK DR EI V AcoGPAT9 150 QVFVANHTSMIDFIVLEQMTAFAV IMQKHPGWVG LLQSTILE SVGCIWF NRTEAK DR EI V BdiGPAT9 153 QVFVANHTSMIDFIILEQMTAFAV IMQKHPGWVG FIQKTILE SVGCIWF NRNDLK DR EV V SmoGPAT9 179 QVFVANHTSMIDFIILEQMTAFAV IMQKHPGWVG LLQNTVLE SLGCIWF NRTESK DR HV V PpaGPAT9 174 QVFVANHTSMIDFVILEQMTGFSA IMQKHPGWVG FLQTTVLE SLGCIWF NRTEAN DR HA V

GraGPAT9A 225 TR KL RE HSQGA DNNPLLIFPEGTCVNN QYSVMFKKGAFEL GCT VCPIAIKYNKIFVDAFW GraGPAT9B 226 TK KL RE HSQGV DNNPLLIFPEGTCVNN QYSVMFKKGAFEL GCT ICPIAIKYNKIFVDAFW SpuGPAT9 224 AK KL RD HVQGA DNNPLLIFPEGTCVNN HYTVMFKKGAFEL DST VCPIAIKYNKIFVDAFW StuGPAT9 220 AK KL RQ HVEGA DNNPLLIFPEGTCVNN HYTVMFKKGAFEL GCT VCPVAIKYNKIFVDAFW AthGPAT9 225 AK KL RD HVQGA DSNPLLIFPEGTCVNN NYTVMFKKGAFEL DCT VCPIAIKYNKIFVDAFW CsaGPAT9 224 AK KL ND HVQGA DNNPLLIFPEGTCVNN HYSVMFKKGAFEL GCS VCPIAIKYNKIFVDAFW AcoGPAT9 210 AR KL RD HVQGA DNNPLLIFPEGTCVNN HYTVMFKKGAFEL GCT VCPIAIKYNKIFVDAFW BdiGPAT9 213 GR KL RD HVQRP DNNPLLIFPEGTCVNN QYTVMFKKGAFEL GCA VCPIAIKYNKIFVDAFW SmoGPAT9 239 GE KL RK HVIDP ESNLLLIFPEGTCVNN EYIVMFKKGAFEL DCT VCPVAIKYNKIFVDAFW PpaGPAT9 234 AQ KL KN HVNDP DANPLLIFPEGTCVNN EYTVMFKKGAFEL DCV VCPIAIKYNKIFVDAFW

132

GraGPAT9A 285 NSRKQSFT MHL LQ LMTSWAVVCDVWYLEPQ NLRPG ET PIEFAERIRDIISV RAGLKKV PW GraGPAT9B 286 NSRKQSFT MHL LQ LMTSWAVVCDVWYLEPQ NLRPD ET PIEFAERVRDIISV RAGLKKV PW SpuGPAT9 284 NSRKQSFT THL LQ LMTSWAVVCDVWYLEPQ NLRPG ET PIEFAERVRGIISD RAGLKKV PW StuGPAT9 280 NSKKQSFT THL LQ LMTSWAVVCDVWYLEPQ NIRPG ET PIEFAERVRGIISV RAGLKKV PW AthGPAT9 285 NSRKQSFT MHL LQ LMTSWAVVCEVWYLEPQ TIRPG ET GIEFAERVRDMISL RAGLKKV PW CsaGPAT9 284 NSRKQSFT MHL LQ LMTSWAVVCDVWYLEPQ VLKPG ET PIEFAERVRDIICA RAGLKKV PW AcoGPAT9 270 NSRQQSFT MHL LR LMTSWAVVCDVWYLEPQ NLMRG ET SIEFAERVRDIISV RAGLKKV PW BdiGPAT9 273 NSKKQSFT MHL GR LMTSWAVVCDVWFLEPQ YLREG ET SIAFTERVRDMIAA RAGLKKV LW SmoGPAT9 299 NSRKQSFT MHL LR LMTSWAVVCDVWYLEPQ TIRPN ET PIEFAERVRDMIAK RAGIKKV AW PpaGPAT9 294 NSKKQSFT MHL VR LMTSWAVVCDVWYLEPQ TIKKG ET PIEFSERVRDLICT RAGIKKV PW

GraGPAT9A 345 DGYLK YS RPS PKHR ERK QQS FAESVL RGLE LEEK------GraGPAT9B 346 DGYLK YS RPS PKHR EQK QQS FAESVL R--RLEEN------SpuGPAT9 344 DGYLK YS RPS PKHR ERK QQS FAESVL R--SLEEK------StuGPAT9 340 DGYLK YS RPS PKHR ERK QQS FAESVL R--RLEEK------AthGPAT9 345 DGYLK YS RPS PKHS ERK QQS FAESIL A--RLEEK------CsaGPAT9 344 DGYLK HS RPS PKYR ERK QQS FAESVL Q--LLDNK------AcoGPAT9 330 DGYLK YS RPS PKLT EHK QQI FAESVL Q--RLEEK------BdiGPAT9 333 DGYLK HN RPS PKHT EEK QRI FAESVL K--RLEES------SmoGPAT9 359 DGYLK YY RPS SKLT EKM QQK FAESML R--RLRTKTSDEDLSTPMEG PpaGPAT9 354 DGYLK YH RPS PKLT EKK QQN FSEAVI R--RLNATAQNQ------

Figure 1 . Sequence Comparison of Selected GPAT9 Enzymes from Plants.

Amino acid sequences from Aquilegia coerulea, Arabidopsis thaliana, Brachypodium distachyon, Cucumis sativus, Gossypium raimondii, Physcomitrella patens, Salix purpurea, Selaginella moellendorffii, and Solanum tuberosum were aligned using the ClustalX algorithm [33]. Amino acids identical in all ten proteins are shaded in black, similar residues are shaded in grey. Name abbreviations indicate the first letter of the genus, and the first two letters of the species.

133

Figure 2. Phylogenetic Comparison of GPAT9, LPEAT1, and LPEAT2 from Various Monocot and Dicot Plant Species.

Protein sequences were aligned using ClustalX (version 1.8.1, [33]). An unrooted phylogenetic tree was created from the alignment, using TREEVIEW (version 1.6.6, http://taxonomy.zoology.gla.ac.uk/rod/rod.html, Page 1996). The branch lengths are proportional to the degree of divergence, with the scale of “0.1” representing 10% change.

134

Figure 3. Aborted Ovules in GPAT9/gpat9-2 Siliques White arrows indicate aborted ovules in GPAT9-2/gpat9-2 siliques. The Col-0 silique did not contain aborted ovules.

135

Table 1. Non-Mendelian segregation of GPAT9/gpat9-2 under sulfadiazine selection Generation Seeds Germinated Sulfadiazine % resistant of % not sown seeds resistant germinated germinated F3 n.d. 425 66 15.5 n.d. F4 (F3 -1) 376 276 35 12.7 26.6 F4 (F3 -2) 126 100 8 8.0 20.6 F4 (F3 -3) 285 221 26 11.8 22.5 F4 (F3 -4) 327 253 33 13.0 22.6 F4 ave. ± S.D. 11.4 ± 2.3 23.1 ± 2.5 F5 (F3 -1-1) 255 228 26 11.4 10.6 F5 (F3 -2-1) 207 194 17 8.8 6.3 F5 (F3 -3-1) 242 223 19 8.5 7.9 F5 (F3 -4-1) 223 203 26 12.8 9.0 F5 ave. ± S.D 10.4 ± 2.1 8.4 ± 1.8

136

Table 2. Transmittance of sulfadiazine resistance from reciprocal crosses of GPAT9/gpat9-2 and Col-0 Cross Male gametophyte Female Germinated Sulfadiazine % gametophyte seedlings resistant resistant 1-1 Col -0 GPAT9 -2/gpat9 -2 90 20 22.2 1-2 GPAT9 -2/gpat9 -2 Col -0 113 0 0 2-2 Col -0 GPAT9 -2/gpat9 -2 139 20 14.4 2-2 GPAT9 -2/gpat9 -2 Col -0 242 0 0

137

Table 3. Ovule viability within GPAT9/gpat9-2 half siliques

Plant – Silique Developing seed Aborted ovules % aborted 1-1 21 9 30.0 1-2 21 8 27.6 1-3 18 11 37.9 1-4 20 11 35.5 1-5 20 14 41.2 2-1 22 14 38.9 2-2 20 15 42.9 2-3 23 10 30.3 2-4 20 9 31.0 2-5 21 13 38.2 Ave. ± S.D. 20.6 ± 1.3 11.4 ± 2.5 35.4 ± 5.3

138

Figure 4. Reduced Pollen Size and Pollen Tube Growth of GPAT9-2/gpat9-2 Pollen in the qrt1 Background. (A) Tetrad pollen in the qrt1 mutant can germinate to produce up to four pollen tubes, one from each attached pollen grain. (B) Tetrad pollen in the qrt1 GPAT9-2/gpat9-2 line does not produce more than two pollen tubes per tetrad. (C) Size of individual tetrad pollen grains from qrt1 and qrt1 GPAT9-2/gpat9-2 plants. Pollen grain length and width was measured to calculate the apparent surfact area of each pollen grain.

139

Table 4. Pollen germination and tube growth of GPAT9/gapt9-2 within the qrt1 background. qrt1 qrt1 GPAT9-2/gpat9-2 Pollen 3-4 tubes/tetrad Pollen 3-4 tubes/tetrad tetrads n % tetrads Predicted a n % 1b 1285 52 4 1256 50 0 0 2b 694 15 2.2 1380 30 0 0 3c 991 52 5.2 1830 95 0 0 4c 608 108 17.8 836 149 0 0 5c 1282 41 3.2 1543 49 0 0 a Predicted tube growth based on percent growth in qrt1 during that experiment. Germination medias: b Zhu et al., [49]; c Boavida & McCormick [48].

140

Figure 5. Genetic Complementation of gpat9-2 Mutants with Transgenic Plant GPAT9. Hemizygous gpat9-2 plants were transformed with different binary vectors, with or without functional full- length plant ORFs driven by the AtGPAT9 promoter. T4 generation seeds from DsRed-fluorescent and sulfadiazine-resistant representative lines from: negative control B110 (A, D); AtGPAT9prom::AtGPAT9 (pE434, B, E); or AtGPAT9prom::VfGPAT9 (pE437, C, F) were sown on solid agar media containing sulfadiazine. Wide-angle views of the agar plates are shown in the top panels, close views of the same plates are shown in the bottom panels. Note the high proportion of stunted and aborted seedlings on the negative control plate compared to the high number of healthy, complemented seedlings established on both the E434 and E437 plates.

141

Figure 6. Segregating Red and Brown T 2 GPAT9 -amiRNA Seed Oil Content and Size.

(A) Oil content of segregating brown (not transformed, black bar) and red (transformed, red bar) T 2 seed from 20 individual T 1 plant lines. Twenty red or brown seeds were used per analysis. (B) Line 7 seed under white light. (C) Same seed as (B) under green light with red filter. (B-C) The red GPAT9 amiRNA seed are smaller than the brown untransformed seed.

142

Figure 7. Oil Quantity and Composition of Homozygous T 3 GPAT9 amiRNA Lines. (A) Distribution of whole seed FAME content. Each sample contained 50 seeds from an individual plant. Red bar indicates average. (B) Fatty acid composition of seeds from part A. Lines Col-0, and amiRNA knockdowns 12 and 2. Average ± S.D.

143

Figure 8. Developing Silique Microsome GPAT Activity. GPAT assays with microsomes isolated from whole developing siliques 5-7 and 9-11 days after flowering (DAF), from Col-0 and GPAT9 amiRNA knockdown lines 2 and 12. The assay utilized 3.55 nmol [14 C]glycerol 3-phosphate and 25 nmol palmitoyl-CoA, for 15 min at 24 °C. (A) Phosphor image of TLC plate indicating that [ 14 C]LPA and [ 14 C]PA are the major products of whole silique microsome GPAT assays utilizing palmitoyl-CoA as an acyl donor. (B, C) Quantification of total products from GPAT activity in whole silique microsomes.

144

Figure 9. Silique Microsome LPAAT Assays. LPAAT assays with microsomes isolated from whole developing siliques of 5-7 (left side) and 9-11 (right side) days after flowering (DAF), from Col-0 and GPAT9 amiRNA knockdown lines 2 and 12. The assay utilized the same conditions as the GPAT assays in the manuscript text except 3.33 nmol [ 14 C]oleoyl-CoA was the acyl donor and 25 nmol18:1-LPA was used as acyl acceptor for 15 min at 24°C. The [ 14 C]oleoyl- CoA can also be used by other acyltransferases and endogenous lipid acceptors found within the microsomes. However PA was the major product of the assay in all samples. (A-B) Phosphor image of TLC separation of products. (C-D) Quantitation of PA within each lane of the TLC image.

145

Figure 10. Testing of Protein:Protein Interactions Between AtGPAT9 and Other Lipid Acyltransferases. Coding sequences for each of two different membrane proteins of interest are ligated in-frame to either the C-terminal half of ubiquitin (Cub)-LexA transcription factor protein fusion or the N-terminal half of ubiquitin (Nub). Nub may be represented as either native polypeptide sequence (NubI), or a mutant form of Nub containing an isoleucine/glycine conversion point mutation (NubG). NubI strongly interacts with Cub. NubG has very weak affinity for Cub, and must be brought into close proximity to Cub to allow for interaction of the two halves of ubiquitin, release of the LexA transcription factor and finally, activation of the reporter genes (histidine and adenine prototrophic markers and β-galactosidase, for quantitative analysis). (A-B) Prototrophic growth assay of yeast strains containing various combinations of AtGPAT9 bait plasmid co-expressed with potential prey NubG-acyltransferase plasmids. Serial dilutions of cells expressing different bait-prey combinations were plated on non-selective (A) or selective (B) media conditions. (C) Quantitative measurement of β-galactosidase activity from cell lysates of the strains used in the serial dilution assays.

146

Figure 11 . Models of GPAT9-dependent Glycerol Flux, and Spatial Organization of TAG Biosynthesis. (A) Wild-type TAG biosynthesis is dependent on the flux of G3P through GPAT9 for the initial incorporation of the glycerol backbone into glycerolipids. GPAT9, LPAAT2, and LPCAT2 are localized together for efficient flux of acyl groups out of PC into de novo glycerolipid synthesis. De novo DAG [DAG(1)] is utilized to produce PC, and eventually PC-derived DAG [DAG(2)] is incorporated into TAG. DGAT1 is spatially separated from GPAT9 and de novo DAG, and is associated with PC-derived DAG. The amount of 18:2/18:3 in TAG is dependent on the residence time of acyl groups in PC for desaturation vs the flux out of PC for incorporation into TAG. (B) In GPAT9 amiRNA lines the flux of G3P into glycerolipid synthesis is reduced (smaller purple arrows), lowering total TAG accumulation. However, the rate of acyl exchange, and DAG exchange into/out of PC is not changed (large black arrows). Therefore, the residence time of acyl groups in PC for desaturation increases, leading to higher overall levels of polyunsaturated fatty acids in the knockdown lines. (A-B) The differences in arrow width between A and B indicate changes in flux.

147

4.6 REFERENCES

1. Kornberg A, Pricer WE: Enzymatic esterification of alpha-glycerophosphate by long chain fatty acids . Journal of Biological Chemistry 1953, 204 :345-357. 2. Weiss SB, Kennedy EP, Kiyasu JY: Enzymatic synthesis of triglycerides . Journal of Biological Chemistry 1960, 235 :40-44. 3. Barron EJ, Stumpf PK: Fat metabolism in higher plants. XIX. The biosynthesis of triglycerides by avocado-mesocarp enzymes . Biochim Biophys Acta 1962, 60 :329-337. 4. Liu Q, Siloto RMP, Lehner R, Stone SJ, Weselake RJ: Acyl-CoA:diacylglycerol acyltransferase: Molecular biology, biochemistry and biotechnology . Progress in Lipid Research 2012, 51 :350-377. 5. Griffiths G, Stobart AK, Stymne S: The acylation of sn-glycerol 3-phosphate and the metabolism of phosphatidate in microsomal preparations from the developing cotyledons of safflower (Carthamus-tinctorius l) seed . Biochemical Journal 1985, 230 :379-388. 6. Ichihara Ki: sn-Glycerol-3-phosphate acyltransferase in a particulate fraction from maturing safflower seeds . Archives of biochemistry and biophysics 1984, 232 :685-698. 7. Joyard J, Douce R: Site of synthesis of phosphatidic acid and diacyglycerol in spinach chloroplasts . Biochimica et Biophysica Acta (BBA)-Lipids and Lipid Metabolism 1977, 486 :273-285. 8. Sparace SA, Moore TS: Phospholipid Metabolism in Plant Mitochondria Submitochondrial Sites of Synthesis . Plant physiology 1979, 63 :963-972. 9. Roughan PG, Slack CR: Cellular-organization of glycerolipid metabolism . Annual Review of Plant Physiology and Plant Molecular Biology 1982, 33 :97-132. 10. Ohlrogge J, Browse J: Lipid biosynthesis . Plant Cell 1995, 7:957-970. 11. Allen DK, Bates PD, Tjellström H: Tracking the metabolic pulse of plant lipid production with isotopic labeling and flux analyses: Past, present and future . Progress in Lipid Research 2015, 58 :97-120. 12. Kennedy EP: Biosynthesis of complex lipids . Federation proceedings 1961, 20 :934-940. 13. Frentzen M, Heinz E, McKeon TA, Stumpf PK: Specificities and Selectivities of Glycerol- 3-Phosphate Acyltransferase and Monoacylglycerol-3-Phosphate Acyltransferase

148

from Pea and Spinach Chloroplasts . European Journal of Biochemistry 1983, 129 :629- 636. 14. Frentzen M, Nishida I, Murata N: Properties of the plastidial acyl-(acyl-carrier-protein): glycerol-3-phosphate acyltransferase from the chilling-sensitive plant squash (Cucurbita moschata) . Plant and cell physiology 1987, 28 :1195-1201. 15. Cronan JE, Roughan PG: Fatty Acid Specificity and Selectivity of the Chloroplast sn- Glycerol 3-Phosphate Acyltransferase of the Chilling Sensitive Plant, Amaranthus lividus . Plant Physiology 1987, 83 :676-680. 16. Ishizaki O, Nishida I, Agata K, Eguchi G, Murata N: Cloning and nucleotide sequence of cDNA for the plastid glycerol-3-phosphate acyltransferase from squash . FEBS Letters 1988, 238 :424-430. 17. Weber S, Wolter F-P, Buck F, Frentzen M, Heinz E: Purification and cDNA sequencing of an oleate-selective acyl-ACP: sn-glycerol-3-phosphate acyltransferase from pea chloroplasts . Plant molecular biology 1991, 17 :1067-1076. 18. Li-Beisson Y, Shorrosh B, Beisson F, Andersson MX, Arondel V, Bates PD, Baud S, Bird D, DeBono A, Durrett TP, et al.: Acyl-Lipid Metabolism . the Arabidopsis Book 2013, 11 :e0161. 19. Bafor M, Smith MA, Jonsson L, Stobart K, Stymne S: Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor-bean (Ricinus-communis) endosperm . Biochemical Journal 1991, 280 :507-514. 20. Bafor M, Jonsson L, Stobart AK, Stymne S: Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea- lanceolata . Biochemical Journal 1990, 272 :31-38. 21. Stobart AK, Stymne S: The regulation of the fatty-acid composition of the triacylglycerols in microsomal preparations from avocado mesocarp and the developing cotyledons of safflower . Planta 1985, 163 :119-125. 22. Rutter AJ, Sanchez J, Harwood JL: Glycerolipid synthesis by microsomal fractions from Olea europaea fruits and tissue cultures . Phytochemistry 1997, 46 :265-272.

149

23. Manaf AM, Harwood JL: Purification and characterisation of acyl-CoA: glycerol 3- phosphate acyltransferase from oil palm (Elaeis guineensis) tissues . Planta 2000, 210 :318-328. 24. Ruiz-López N, Garcés R, Harwood JL, Martínez-Force E: Characterization and partial purification of acyl-CoA:glycerol 3-phosphate acyltransferase from sunflower (Helianthus annuus L.) developing seeds . Plant Physiology and Biochemistry 2010, 48 :73-80. 25. Zheng ZF, Xia Q, Dauk M, Shen WY, Selvaraj G, Zou JT: Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility . Plant Cell 2003, 15 :1872-1887. 26. Beisson F, Li YH, Bonaventure G, Pollard M, Ohlrogge JB: The acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis . Plant Cell 2007, 19 :351-368. 27. Li YH, Beisson F, Koo AJK, Molina I, Pollard M, Ohlrogge J: Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin- like monomers . Proceedings of the National Academy of Sciences of the United States of America 2007, 104 :18339-18344. 28. Yang W, Pollard M, Li-Beisson Y, Beisson F, Feig M, Ohlrogge J: A distinct type of glycerol- 3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol . Proceedings of the National Academy of Sciences 2010, 107 :12040- 12045. 29. Yang W, Simpson JP, Li-Beisson Y, Beisson F, Pollard M, Ohlrogge JB: A Land-Plant- Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution . Plant Physiology 2012, 160 :638-652. 30. Beisson F, Li-Beisson Y, Pollard M: Solving the puzzles of cutin and suberin polymer biosynthesis . Current Opinion in Plant Biology 2012, 15 :329-337. 31. Cao JS, Li JL, Li DM, Tobin JF, Gimeno RE: Molecular identification of microsomal acyl- CoA : glycerol-3-phosphate acyltransferase, a key enzyme in de novo triacylglycerol synthesis . Proceedings of the National Academy of Sciences of the United States of America 2006, 103 :19695-19700.

150

32. Gidda SK, Shockey JM, Rothstein SJ, Dyer JM, Mullen RT: Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals: Functional divergence of the dilysine ER retrieval motif in plant cells . Plant Physiology and Biochemistry 2009, 47 :867-879. 33. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools . Nucleic acids research 1997, 25 :4876-4882. 34. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees . Mol Biol Evol 1987, 4:406 - 425. 35. Page RD: TreeView: an application to display phylogenetic trees on personal computers . Comput Appl Biosci 1996, 12 :357-358. 36. Bates P, Jewell J, Browse J: Rapid separation of developing Arabidopsis seeds from siliques for RNA or metabolite analysis . Plant Methods 2013, 9:9. 37. Suzuki Y, Kawazu T, Koyama H: RNA isolation from siliques, dry seeds, and other tissues of Arabidopsis thaliana . Biotechniques 2004, 37 :542, 544. 38. Rieu I, Powers SJ: Real-time quantitative RT-PCR: design, calculations, and statistics . The Plant Cell Online 2009, 21 :1031-1033. 39. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W-R: Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis . Plant Physiology 2005, 139 :5-17. 40. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al.: Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana . Science 2003, 301 :653-657. 41. Francis KE, Lam SY, Copenhaver GP: Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectin methylesterase gene . Plant physiology 2006, 142 :1004-1013. 42. Kleinboelting N, Huep G, Kloetgen A, Viehoever P, Weisshaar B: GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database . Nucleic Acids Res 2012, 40 :D1211-1215.

151

43. Shockey J, Mason C, Gilbert M, Cao H, Li X, Cahoon E, Dyer J: Development and analysis of a highly flexible multi-gene expression system for metabolic engineering in Arabidopsis seeds and other plant tissues . Plant molecular biology 2015, 89 :113-126. 44. Clough S, Bent A: Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana . Plant J 1998, 16 :735 - 743. 45. Thomson JG, Cook M, Guttman M, Smith J, Thilmony R: Novel sulI binary vectors enable an inexpensive foliar selection method in Arabidopsis . BMC Res Notes 2011, 4:44. 46. Peterson R, Slovin JP, Chen C: A simplified method for differential staining of aborted and non-aborted pollen grains , vol 1; 2010. 47. Regan SM, Moffatt BA: Cytochemical Analysis of Pollen Development in Wild-Type Arabidopsis and a Male-Sterile Mutant . The Plant Cell Online 1990, 2:877-889. 48. Boavida LC, McCormick S: TECHNICAL ADVANCE: Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana . The Plant Journal 2007, 52 :570-582. 49. Zhu L, Zhang Y, Kang E, Xu Q, Wang M, Rui Y, Liu B, Yuan M, Fu Y: MAP18 regulates the direction of pollen tube growth in Arabidopsis by modulating F-actin organization . The Plant Cell 2013, 25 :851-867. 50. Gidda SK, Shockey JM, Falcone M, Kim PK, Rothstein SJ, Andrews DW, Dyer JM, Mullen RT: Hydrophobic-Domain-Dependent Protein–Protein Interactions Mediate the Localization of GPAT Enzymes to ER Subdomains . Traffic 2011, 12 :452-472. 51. Kim HU, Li Y, Huang AH: Ubiquitous and endoplasmic reticulum-located lysophosphatidyl acyltransferase, LPAT2, is essential for female but not male gametophyte development in Arabidopsis . Plant Cell 2005, 17 :1073-1089. 52. Zou JT, Wei YD, Jako C, Kumar A, Selvaraj G, Taylor DC: The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene . Plant Journal 1999, 19 :645-653. 53. Stahl U, Stalberg K, Stymne S, Ronne H: A family of eukaryotic lysophospholipid acyltransferases with broad specificity . FEBS Lett 2008, 582 :305-309.

152

54. Sengupta-Gopalan C, Reichert NA, Barker RF, Hall TC, Kemp JD: Developmentally regulated expression of the bean β-phaseolin gene in tobacco seed . Proceedings of the National Academy of Sciences 1985, 82 :3320-3324. 55. Guan R, Lager I, Li X, Stymne S, Zhu L-H: Bottlenecks in erucic acid accumulation in genetically engineered ultrahigh erucic acid Crambe abyssinica . Plant Biotechnology Journal 2014, 12 :193-203. 56. Bligh E, Dyer W: A rapid method of total lipid extraction and purification . Can J Biochem Physiol 1959, 37 :911 - 917. 57. Shockey J, Browse J: Genome ‐level and biochemical diversity of the acyl ‐activating enzyme superfamily in plants . The Plant Journal 2011, 66 :143-160. 58. Paterson AH, Chapman BA, Kissinger JC, Bowers JE, Feltus FA, Estill JC: Many gene and domain families have convergent fates following independent whole-genome duplication events in Arabidopsis, Oryza, Saccharomyces and Tetraodon . Trends in Genetics 2006, 22 :597-602. 59. De Smet R, Adams KL, Vandepoele K, Van Montagu MC, Maere S, Van de Peer Y: Convergent gene loss following gene and genome duplications creates single-copy families in flowering plants . Proceedings of the National Academy of Sciences 2013, 110 :2898-2903. 60. Toufighi K, Brady SM, Austin R, Ly E, Provart NJ: The Botany Array Resource: E ‐ Northerns, Expression Angling, and promoter analyses . The Plant Journal 2005, 43 :153-163. 61. Stalberg K, Stahl U, Stymne S, Ohlrogge J: Characterization of two Arabidopsis thaliana acyltransferases with preference for lysophosphatidylethanolamine . Bmc Plant Biology 2009, 9. 62. Zhang M, Fan J, Taylor DC, Ohlrogge JB: DGAT1 and PDAT1 Acyltransferases Have Overlapping Functions in Arabidopsis Triacylglycerol Biosynthesis and Are Essential for Normal Pollen and Seed Development . Plant Cell 2009, 21 :3885-3901. 63. Preuss D, Rhee SY, Davis RW: Tetrad analysis possible in Arabidopsis with mutation of the QUARTET (QRT) genes . Science 1994, 264 :1458-1460.

153

64. Rhee SY, Somerville CR: Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall . The Plant Journal 1998, 15 :79-88. 65. Wilkinson JE, Twell D, Lindsey K: Activities of CaMV 35S and nos promoters in pollen: implications for field release of transgenic plants. Journal of Experimental Botany 1997, 48 :265-275. 66. Kay R, Chan A, Daly M, McPherson J: Duplication of CaMV 35S Promoter Sequences Creates a Strong Enhancer for Plant Genes . Science 1987, 236 :1299-1302. 67. Verdaguer B, De Kochko A, Beachy RN, Fauquet C: Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter . Plant molecular biology 1996, 31 :1129-1139. 68. Stuitje AR, Verbree EC, Van Der Linden KH, Mietkiewska EM, Nap J-P, Kneppers TJA: Seed-expressed fluorescent proteins as versatile tools for easy (co)transformation and high-throughput functional genomics in Arabidopsis . Plant Biotechnology Journal 2003, 1:301-309. 69. Li YH, Beisson F, Pollard M, Ohlrogge J: Oil content of Arabidopsis seeds: The influence of seed anatomy, light and plant-to-plant variation. Phytochemistry 2006, 67 :904-915. 70. Focks N, Benning C: wrinkled1: A novel, low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism . Plant Physiology 1998, 118 :91-101. 71. Maisonneuve S, Bessoule J-J, Lessire R, Delseny M, Roscoe TJ: Expression of Rapeseed Microsomal Lysophosphatidic Acid Acyltransferase Isozymes Enhances Seed Oil Content in Arabidopsis . Plant Physiology 2010, 152 :670-684. 72. Roughan PG, Ohlrogge JB: Evidence that isolated chloroplasts contain an integrated lipid- synthesizing assembly that channels acetate into long-chain fatty acids . Plant physiology 1996, 110 :1239-1247. 73. Mo C, Bard M: A systematic study of yeast sterol biosynthetic protein–protein interactions using the split-ubiquitin system . Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 2005, 1737 :152-160.

154

74. Bates PD, Stymne S, Ohlrogge J: Biochemical pathways in seed oil synthesis . Current Opinion in Plant Biology 2013, 16 :358-364. 75. Johnsson N, Varshavsky A: Split ubiquitin as a sensor of protein interactions in vivo . Proceedings of the National Academy of Sciences 1994, 91 :10340-10344. 76. Chien C-T, Bartel PL, Sternglanz R, Fields S: The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest . Proceedings of the National Academy of Sciences 1991, 88 :9578-9582. 77. Bates PD, Fatihi A, Snapp AR, Carlsson AS, Browse J, Lu C: Acyl Editing and Headgroup Exchange Are the Major Mechanisms That Direct Polyunsaturated Fatty Acid Flux into Triacylglycerols . Plant Physiology 2012, 160 :1530-1539. 78. Wang L, Shen W, Kazachkov M, Chen G, Chen Q, Carlsson AS, Stymne S, Weselake RJ, Zou J: Metabolic Interactions between the Lands Cycle and the Kennedy Pathway of Glycerolipid Synthesis in Arabidopsis Developing Seeds . The Plant Cell Online 2012, 24 :4652-4669. 79. Pan X, Chen G, Kazachkov M, Greer MS, Caldo KM, Zou J, Weselake RJ: In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase . J Biol Chem 2015, 290 :18068-18078. 80. Lager I, Yilmaz JL, Zhou X-R, Jasieniecka K, Kazachkov M, Wang P, Zou J, Weselake R, Smith MA, Bayon S, et al.: Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions . Journal of Biological Chemistry 2013, 288 :36902-36914. 81. Stymne S, Stobart AK: Evidence for the reversibility of the acyl-coA- lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus-tinctorius L) cotyledons and rat-liver . Biochemical Journal 1984, 223 :305-314. 82. Routaboul J-M, Benning C, Bechtold N, Caboche M, Lepiniec L: The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase . Plant Physiology and Biochemistry 1999, 37 :831-840.

155

83. Lardizabal K, Mai J, Wagner N, Wyrick A, Voelker T, Hawkins D: DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity . J Biol Chem 2001, 276 :38862 - 38869. 84. Chen X, Miles R, Snyder C, Truksa M, Zhang J, Shah S, Weselake RJ: Possible allostery and oligomerization of recombinant plastidial sn-glycerol-3-phosphate acyltransferase . Archives of biochemistry and biophysics 2014, 554 :55-64. 85. Shockey J, Gidda S, Chapital D, Kuan J, Dhanoa P, Bland J, Rothstein S, Mullen R, Dyer J: Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum . Plant Cell 2006, 18 :2294 - 2313. 86. Bates PD, Browse J: The significance of different diacylgycerol synthesis pathways on plant oil composition and bioengineering . Frontiers in Plant Science 2012, 3:147. 87. Bates PD, Durrett TP, Ohlrogge JB, Pollard M: Analysis of Acyl Fluxes through Multiple Pathways of Triacylglycerol Synthesis in Developing Soybean Embryos . Plant Physiology 2009, 150 :55-72. 88. Bates PD, Ohlrogge JB, Pollard M: Incorporation of Newly Synthesized Fatty Acids into Cytosolic Glycerolipids in Pea Leaves Occurs via Acyl Editing . Journal of Biological Chemistry 2007, 282 :31206-31216. 89. Bates PD, Browse J: The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds . The Plant Journal 2011, 68 :387-399. 90. Lu C, Xin Z, Ren Z, Miquel M, Browse J: An enzyme regulating triacylglycerol composition is encoded by the ROD1 gene of Arabidopsis . Proceedings of the National Academy of Sciences 2009, 106 :18837-18842. 91. Tjellström H, Yang Z, Allen DK, Ohlrogge JB: Rapid Kinetic Labeling of Arabidopsis Cell Suspension Cultures: Implications for Models of Lipid Export from Plastids . Plant Physiology 2012, 158 :601-611.

156

92. Larsson KE, Kjellberg JM, Tjellstrom H, Sandelius AS: LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum . Bmc Plant Biology 2007, 7:12. 93. Simillion C, Vandepoele K, Van Montagu MC, Zabeau M, Van de Peer Y: The hidden duplication past of Arabidopsis thaliana . Proceedings of the National Academy of Sciences 2002, 99 :13627-13632. 94. Wang K, Wang Z, Li F, Ye W, Wang J, Song G, Yue Z, Cong L, Shang H, Zhu S: The draft genome of a diploid cotton Gossypium raimondii . Nature genetics 2012, 44 :1098-1103. 95. De Smet R, Adams KL, Vandepoele K, Van Montagu MC, Maere S, Van de Peer Y: Convergent gene loss following gene and genome duplications creates single-copy families in flowering plants . Proc Natl Acad Sci U S A 2013, 110 :2898-2903. 96. Muralla R, Lloyd J, Meinke D: Molecular Foundations of Reproductive Lethality in Arabidopsis thaliana . PLoS ONE 2011, 6:e28398.

157

4.7 SUPPLEMENTAL FIGURES AND METHODS

Supplementary Figure S1. Sequence Alignment and Identity/Similarity Shading of Plant LPEAT1 Proteins. Phylogenetic tree of this alignment makes up part of Figure 2 in the main body of the text.

TcLPEAT1 1 -MESELKDLNSKPAKTTQKPDPTH---DDGSTKDD RPLL KSD------SSSADT-NIQ VvLPEAT1 1 -MESELKDLD------PRSNS---QDVSSKDD RPLL KSD------STVVSQDNLQ GmLPEAT1 1 -MESELKDLNSKPPN-----GNGN------SVRDD RPLL KPE------PPVSADSIA PpLPEAT1 1 -MESELKDLNSKPAKPVADDGSGEPAHDDASGKDD RPLL KPDL------APQVSTEELQ CcLPEAT1 1 -MESELKDLNSKQLKSTASSDDGG------SAKDD RPLL KPD------AAENIQ PtLPEAT1 1 -MDTELKSMNPDPPKPE-QPDPASR-DDGSNSKDD RPLL KSDSN-----RISSTTGESIE RcLPEAT1 1 -MDTELKDINPEPSKSKPEPEPAP--DDGS-SKDD RPLL KSDSN-----RMST---ENIE StLPEAT1 1 -MESELQKLPQPETTRPSQPESID----EGSTKDD RPLL KPDPTNPQLQVQSQSSSPSIE AtLPEAT1 1 -MESELKDLNSNSNPP------SSKED RPLL KSE------SDLAAAIE OsLPEAT1 1 MALPLHDATTSPSDPDDLGGGGEEEE----ERLAS KPLL SSPST-YPSAGTEEGVEE--L SiLPEAT1 1 -MAPNDAATTAPSEPESVGG-RMSSE----DTVAT RPLL SSPST-SPSAASTAPVQESIE ZmLPEAT1 1 -MALDTADADAP------RLRLD----ASAAV HPLL SDDGS-TAAGETAE------

TcLPEAT1 48 E LE KKFAAYVRNDVYG TMG RGKLPLKEKLL LG IALVT LLPVRIVLGMT ILVFYY LICRVC VvLPEAT1 40 E LE KKFAAYVRSDAYG PMG CGELPLKEKLL LA FALVT LVPIRLVVAFT ILVVYY LICRVC GmLPEAT1 40 D ME KKFAAYVRRDVYG TMG RGELPTKEKLL LG FALVT LLPIRVVLAVT ILLFYY LICRVC PpLPEAT1 53 E LE KKCAAYVRRDAYG TMG RGELTVKEKVL LG LALVT LVPIRVVLAMT ILVIYY LICRIC CcLPEAT1 42 E LE KKFAPYVRNDVYG TMG RGELPLAEKFL IG IAMVT LLPIRVVLAMT VLVIYY LICRVC PtLPEAT1 53 E LE KKFAAYVRNDVYG PMG RGELPLVEKVL LG IAVVT LVPIRFVLALI ILVVYY IICRVC RcLPEAT1 49 E LE KKFAAYVRNDVYG PMG RAELPLAEKLL LG IALVT LVPIRTLLAIT VLLVYY AICRIC StLPEAT1 56 E LE KKYAPYVRHDVYG IMG RGELPWTEKVL LG IALVT LVPMRVIGATT VLVVYY LICKIC AtLPEAT1 36 E LD KKFAPYARTDLYG TMG LGPFPMTENIK LA VALVT LVPLRFLLSMS ILLLYY LICRVF OsLPEAT1 54 E LD RRYAPYARRDAYG AMG RGPLGAAGAGR LA VGAAV LFPLRLAAGVL VLVAYY LVCRVC SiLPEAT1 54 E LD RRYAPYARRDAYG PMG LGPVGAAEAFR LA FAAVV LIPLRVVAGML VLVVYY LVCRVC ZmLPEAT1 39 Q LD ARYAPYARRDAYG TMG RGPLPAAQAVR LA LAAAV LLPLRFVAGML VLLLYY LVCRAC

TcLPEAT1 108 TLFLAPNRE--DE------QEDYA HMG GWRRAV IV RS GRLFS RVMLFLV GFYWI NETH VvLPEAT1 100 TLFSAPNREGEDE------QEDYA HMG GWRRAV IV QC GRFLS RALLFTL GFYWI NVTY GmLPEAT1 100 TLFSAPT--GEEE------QEDYA HMS GWRRTI IV SC GRALS RLMLFIF GFYWI PESN PpLPEAT1 113 TLFSVPN--RDEE------QEDYA HMG GWRRAV IV QC GRSLS RLMLFVL GFYWI NETY CcLPEAT1 102 TLFSAPNR-GEDE------QEDYA HMG GWRRSV IV VS GRFLS RVMLSVL GFYWI TETF PtLPEAT1 113 TLFSAPNRDEEEE------QEDFA HMG GWRRAV IV WC GRFLS RMLLFVL GFYWI SVSY RcLPEAT1 109 TLFSAPNRDEEEE------QEDFA HMG GWRRAV IV WC GRFLS RVMLFVF GFYWI KETY StLPEAT1 116 TAFWAPNR--EDE------QEDYA HTG GWRRTV MM QS GMFLS RVMLFVF GFYWI HETY AtLPEAT1 96 TLFSAPYRGPEEEEDEGGVVFQEDYA HME GWKRTV IV RS GRFLS RVLLFVF GFYWI HESC OsLPEAT1 114 TLRVEEEEREGGGGGAAGEVEGDGYA RLE GWRREG VV RC GRALA RAMLFVF GFYWI REYD SiLPEAT1 114 TLRVEEE-REGG------EGDGYA RLE GWRREG VV WC GRALA RAMLFVF GFYWI REYD ZmLPEAT1 99 TLFVDAD------GGRP RLA GWRRKA VL RS GCALS RVMLFVF GFYWI RETR

158

TcLPEAT1 158 R------DSANTQENSKTEGINQSEEQE RPGAIVSNHVSYLDILYH MSSS FPSFVAK VvLPEAT1 152 R------DPLTTED----EGKDEDEEPE RPGAIISNHVSYLDILYH MSSS FPSFVAK GmLPEAT1 150 ------SASQED-----KSRQPEE-LR RPGVIISNHVSYLDILYH MSSS FPSFVAK PpLPEAT1 163 RIP------SDAQPNPTTDCKDEGEEEGTE RPGAIISNHVSYLDILYH MSSS FPSFVAK CcLPEAT1 153 RILDVQEKSENEAKNQSKDEDEAKDQDEESG RPGAIISNHVSYLDILYH MSSS FPSFVAK PtLPEAT1 165 R------DIELPDQNKSSSQNEGKDQSEEPE RLGAIISNHVSYLDILYH MSAS FPSFVAK RcLPEAT1 161 R------ILEPP------QDEGKDQSEDPE RPGAIISNHVSYLDILYH MSSS FPSFVAK StLPEAT1 166 QP------INLNGNSNNEDGSKLQAEELE RPGAIVSNHISYLDILYH KSSS FPSFVAK AtLPEAT1 156 PDRD-SDMDSNPKTTSTEINQKGEAATEEPE RPGAIVSNHVSYLDILYH MSAS FPSFVAK OsLPEAT1 174 C------RFPDAEDEHQEQSKELG RPGAVVSNHVSYVDILYH MSSS FPSFVAK SiLPEAT1 165 C------RFPDAEVEHVDQSKEME RPGAIVSNHVSYVDILYH MSAF FPSFVAK ZmLPEAT1 144 R------RSTNAKGLNQDQFEESQ RPGAIVSNHVSYVDILYH MSAS FPSFVAK

TcLPEAT1 209 R SV AKIPLVGLIS KCLGCVYVQRE SKS SDFKGV AGVVTERVCEAHQNESA PMMMLFPEGT VvLPEAT1 199 R SV AKLPLIGLIS KCLGCVYVQRE SKS SDFKGV AGVVTERVCEAHQNKFA PMMMLFPEGT GmLPEAT1 194 R SV AKLPLVGLIS KCLGCVYVQRE SRS SDFKGV SAVVTDRIREAHQNESA PLMMLFPEGT PpLPEAT1 216 R SV AKLPLVGLIS KCLGCVYVQRE SKS SDFKGV SGVVTERVKEAHQNNSA PPMMLFPEGT CcLPEAT1 213 R SV AKLPLVGLIS KCLGCVYVQRE SKS SDFKGV SGVVTERVREAHRDKSA PMMMLFPEGT PtLPEAT1 219 R SV AKLPLVGLIS KCLGCVYVQRE SKS SDFKGV SGIVTERVKESHENSSA PMMMLFPEGT RcLPEAT1 208 R SV AKLPLVGLIS KCLGCVYVQRE SKS SDFKGV AGVVTERVREAHQNKSA PIMMLFPEGT StLPEAT1 218 R SV AKLPLVGLIS KCLGCVYVQRE SKS PDFKGV SGVVNERIREAHQNKSA PIMLLFPEGT AtLPEAT1 215 R SV GKLPLVGLIS KCLGCVYVQRE AKS PDFKGV SGTVNERVREAHSNKSA PTIMLFPEGT OsLPEAT1 221 R SV ARLPMVGLIS KCLGCIFVQRE SKT SDFKGV SGAVTERIQRAHQQKNS PMMLLFPEGT SiLPEAT1 212 R SV ARLPLVGLIS KCLGCIFVQRE SKT SDFKGV SGAVTERIQRAHQQKNA PMMLLFPEGT ZmLPEAT1 191 E SV SRLPLIGLIS NCLGCIFVQRE SKS SEAKGV SGAVTERIQDVCQDKNT PMMLLFPEGT

TcLPEAT1 269 TTNG DFLL PFKTGAFL ARA PV VPVIL RYPYQRFSV AWDSI SGLR HVVF LLCQ FVNRMEVT VvLPEAT1 259 TTNG GFLL PFKTGAFL AKA PV LPVIL RYPYQRFSP AWDSI SGVR HVIF LFCQ FVNHIEVT GmLPEAT1 254 TTNG EFLL PFKTGGFL AKA PV LPVIL QYHYQRFSP AWDSI SGVR HVIF LLCQ FVNYMEVI PpLPEAT1 276 TTNG DFLL PFKTGAFL AKA PV LPVIL RYPYERFSP AWDSI SGVR HVIF LFCQ FVNHIEVT CcLPEAT1 273 TTNG DYLL PFKTGAFL ARA PV LPVIL RYPYQRFSP AWDSI SGAR HVFF LLCQ FVNHIEVT PtLPEAT1 279 TTNG DFLL PFKTGAFL ATA PV RPVIL RYPYQRFSP AWDSI SGAL HVFY LFCQ FINHMEAV RcLPEAT1 268 TTNG DFLL PFKTGAFL AGA PV LPVIL RYPYQRFSP AWDSI SGVR HVIF LLCQ FVNCIEVT StLPEAT1 278 TTNG DFLL PFKSGAFL SGA PV QPVIL RYPYQRLSP AWDSI SGAR HVIL LLCQ FVNYLEAT AtLPEAT1 275 TTNG DYLL TFKTGAFL AGT PV LPVIL KYPYERFSV AWDTI SGAR HILF LLCQ VVNHLEVI OsLPEAT1 281 TTNG DYLL PFKTGAFL AKA PV KPVIL RYPYKRFSP AWDSM SGAR HVFL LLCQ FVNNLEVI SiLPEAT1 272 TTNG DYLL PFKTGAFL AKA PV QPVIL RYPYKRFNP AWESM SGAR HVFL LLCQ FVNYVEVT ZmLPEAT1 251 TTNG DYLL PFKTGAFL AGA PV QPVIL KYPYRRFSP AWDSM DGAR HVFL LLCQ FVNHMEVV

TcLPEAT1 329 W LPVY YPS QQ EKDDQKLYA NNVRRLMANEGN LIL SDIGL AEKR TYH AA LNG------LFC VvLPEAT1 319 R LPVY IPS QQ EKDDPKLYA NNVRKLMASEGN LIM SDIGL AEKR IYH AA LNG------LFC GmLPEAT1 314 R LPVY HPS QQ EMDDPKLYA NNVRRLMATEGN LIL SDIGL AEKR IYH AA LNGNNSLPSVLH PpLPEAT1 336 R LPVY YPS QQ EKDDPKLYA TNVRRLMASEGK MTQ SDIGL AEKR VYH AA LNGNNSRPSVLH CcLPEAT1 333 S LPVY HPS QQ EKDDPKLYA ENVRRLMASERN LTV SDIGL AEKR IYH AA LNGNNSLPSVLH PtLPEAT1 339 W LPVY YPS QE EKDDPKLYA SNVRRLMAREGN LKM SDIGL AEKR IYH TA LNGNISLPSVLH RcLPEAT1 328 R LPVY YPS QE EKDDPKLYA NNVRRLMAHEGN LLM SDIGL AEKR VYH AA LNGLF------StLPEAT1 338 W LPVY YPS QQ EKDDPRLYA ENVRRLMAHEGN LLL SDIGL AEKR VYH AA LNG------LFC AtLPEAT1 335 R LPVY YPS QE EKDDPKLYA SNVRKLMATEGN LIL SELGL SDKR IYH AT LNGNLSQTRDFH OsLPEAT1 341 H LPVY YPS EQ EKEDPKLYA NNVRKLMAVEGN LIL SDLGL AEKR VYH AA LNGNNSLPRALH SiLPEAT1 332 H LPVY YPS EQ EKDDPKLYA NNVRKLMAVEGN LIL SDLGL AEKR VYH AA LNGN-SLPRALH ZmLPEAT1 311 R LPVY YPS QL EKEDPKLYA NNVRKLIAMEGN LVL SNIGL AEKR VYH AA LTGS-SLPGARH

159

TcLPEAT1 383 QS-- VvLPEAT1 373 QR-- GmLPEAT1 374 QKDE PpLPEAT1 396 QKDD CcLPEAT1 393 QKDD PtLPEAT1 399 QKDD RcLPEAT1 ---- StLPEAT1 392 QQ-- AtLPEAT1 395 QKEE OsLPEAT1 401 QKDD SiLPEAT1 391 QKDD ZmLPEAT1 370 EKDD

160

Supplementary Figure S2. Sequence Alignment and Identity/Similarity Shading of Various Plant LPEAT2 Proteins. Phylogenetic tree depiction of this alignment makes up part of Figure 2 in the main body of the text.

PpLPEAT2 1 MADN------DLAS PLISSPPSDHP------HLILTVQDDTDTD-HNNHNGNHG VvLPEAT2 1 MAGTG----AD LIT PLISSQPSDQP------ELILTVDDRPGFESFSDHCSSNG PtLPEAT2 1 MAN------HD LES PLLSSQPSDPP------HIILNVHDSDS------SIQQS RcLPEAT2 1 MTD------DS LSS PLLQPQPSDHP------PVILSIRDNHN------HYYHR CcLPEAT2 1 MADHHRDHDHD LSS PLLQSPRSDHS------PVIISIEADGNDTVSDPATQDQL TcLPEAT2 1 MAE------HD LTS PLLSPRSSDQP------QTVLIVSDDEDSEPSDRPPSNQQ AtLPEAT2 1 MADP------DLSS PLIHHQSSDQP------EVVISIADDDDDESGLNLLPAVV GmLPEAT2 1 MADD-----SD LTS PLLSSSSSSSSD------HVVVTVHPSAAP------StLPEAT2 1 MSDH------SIFA PLLPSDHLPHKSNGDDQEHAPEPHVILTVEDDGVQH-----QLSNG SiLPEAT2 1 MASTSPSP-AS LFT PLLSGSVAPAR------AANGHANNHR--HHGDSDG ZmLPEAT2 1 MASTCLAP-AS LST PLLSDSIARAP------AANGHATNHRHHHHDDSDG OsLPEAT2 1 MASRNPSP-AS LST PLLSDSISPTP------TTNGHAGHHN--HHDDDEE

PpLPEAT2 42 S------NSTHHHFR--NPYAF LGSDG-----FTVPGSTTAD PF RNHTL VvLPEAT2 45 T------KSDHTQPESDN PFEF LGSAG-----FSVPGTPTVD PF RNNTP PtLPEAT2 36 N------NHKPNPNTSSRN PFEF IGSDG-----LSVPAPSTLD PF RNDTP RcLPEAT2 36 H------NSNSSSHN-LRN PFEF LGSDF-----LSVPPPSTVD PF RNNTP CcLPEAT2 49 N------NHDTRYHNNPGN PYWF IGSDG-----LSVPGPNTAN PF LNDTP TcLPEAT2 43 SSLQTGIPRQNHNQSYGNGNGNSQVSSRN PYEF LGSDG-----FSVPAPTTID PF RNGTP AtLPEAT2 43 DP------RVSRGFEFDHLN PYGF LSESE-----PPVLGPTTVD PF RNNTP GmLPEAT2 34 ------PRN PFRV LGTDDDDDDDLSVPPPSTLD PF RNRTP StLPEAT2 50 D------HLGTHISEVDDN PYAF LGANR-----FDMPGSTTVD PF RNNTP SiLPEAT2 42 AA------AVSVCDDGGG-DPFAF LSEDRPP----QDRGPSPAD PF RNGTP ZmLPEAT2 44 AA------AVSVCDDGGG-DPFAF LSEDRPP----RDRGPSPAD PF RNGTP OsLPEAT2 42 SP------TVCGGDGGGGGD PFAF LSEDRPAW--WSPRGVSPAD PF RNGTP

PpLPEAT2 78 EIR G-LYEWL KIG ICL PIALA RL VLFGAS LLI GFVAT KL AL QGW KDK------KN PMP VvLPEAT2 83 KID G-FYEWF KIL VCV PIAAI RL VLFGLC LLV GYLAT KF AL QGW KDK------QN PMP PtLPEAT2 75 DIE G-LYELI KIV ICL PIAIV RL VLFGVC LAT GYVAT KI AL LGW RDK------HN PMP RcLPEAT2 74 KIE G-VYEVL KSL ICL PIALA RL VLFGAC LLV GYLAT KL AL GGW KDK------NN PMP CcLPEAT2 88 HVV G-VYEFV KIV VCF PIVLI RL VLFGFC LLV GYLAT KL AL EGW KDK------QN PMP TcLPEAT2 98 FVS G-VYEVI KIL LCL PIALA RL VLFGVC LAV GYIAT RI AL EGW KDK------QN PMP AtLPEAT2 83 GVS G-LYEAI KLV ICL PIALI RL VLFAAS LAV GYLAT KL AL AGW KDK------EN PMP GmLPEAT2 68 AIE G-LYEWA KTV LCL PLAAL RL AIFGLC LAL GYVAT KV AL QGW KDK------EN PMP StLPEAT2 89 RVE G-VYEWL KIV VCL PITLV RL VLFGLA LMI GYVAT RT AL LGW KDR------SS PMP SiLPEAT2 82 AWG GGV YAWA RTL LLA PVAAV RL VLFGLA IAI GYAAT WV AL RGW ADVQDRP-REGAG PMP ZmLPEAT2 84 AWG GGV YAWS RTL LLL PVALV RL ALFGLS IAI GYAAT WV AL RGW ADTHGRP-RVGGG PMP OsLPEAT2 85 GWC G-AYELV RAL VCA PVAAA RL VLFGLS IAV GYAAT WV AL RGW VDVRERAAQEGAG PMP

161

PpLPEAT2 129 K WR CRIMWITRVCTRCILFSFG YHWIRRKGKP APREI APIVVSNHVSFIEPIFYFYELF P VvLPEAT2 134 K WR CRVMWVTRICSRCILFSFG YHWIKRRGRP ASRET APIVVSNHVSYVEPIFFFYELF P PtLPEAT2 126 K WR SRLMWLTRGCTRCILFSFG YHWIKRKGKL APREI APIVVSNHVSYIDPIFYFFEFF P RcLPEAT2 125 K WR S------SYHWIKRKGNP APREI APIVVSNHVSYIEPIFYFYELF P CcLPEAT2 139 V WR SRLMWVTRVCSRCILFSFG YHWIRRKGKP APRQI APIVVSNHISYIEPIFFFYELF P TcLPEAT2 149 K WR SRIMWVTRVCARFILFSFG YQWIRRKGKP APRDV APIVVSNHVSYIEPIFYFYELF P AtLPEAT2 134 L WR CRIMWITRICTRCILFSFG YQWIRRKGKP ARREI APIVVSNHVSYIEPIFYFYELS P GmLPEAT2 119 K WR CRVMWITRLCARCILFSFG YQWIKRKGKP APREI APIIVSNHVSYIEPIFYFYELF P StLPEAT2 140 K WR SRLMWVTRMSARTILFSFG YQWIRRKGKP APREI APIVVSNHVSYIDPIFFFYELF P SiLPEAT2 141 A WR RRLMWITRISARCILFSFG YHWIRKKGRP APREL APIVVSNHVSYIEPIFFFYELF P ZmLPEAT2 143 A WR RRLMWITRISARCILFSFG YHWIRKKGRP ARREL APIVVSNHISYIEPIFFFYELF P OsLPEAT2 144 A WR RRLMWITRISARCILFSFG YHWIRRKGKP APREL APIVVSNHVSYIEPIYFFYELF P

PpLPEAT2 189 TIV AS ESHD SLPLVGTIIRAMQVIYV NRF SAS SRK HAVSEIKRKASCDR FPRVLLFPEGT VvLPEAT2 194 TIV AS ESHD SLPFVGTIIRAMQVIYV NRF SQS SRK QAVNEIKKKASCER YPRVLLFPEGT PtLPEAT2 186 TIV AA ESHD SMPFVGTIIRAMQVIYV NRF SPS SRK LAVNEIKRKASCGR FPRVLLFPEGT RcLPEAT2 168 TIV AA ESHD SIPFVGTIIRAMQVIYV NRF SQS SRK LAVNEIKRKASCDR FPRVLLFPEGT CcLPEAT2 199 TIV AS ESHD SIPFVGTIIRAMQVIYV DRF SQS SRK NAVSEIKRKASCDR FPRVLLFPEGT TcLPEAT2 209 TIV AS ESHD SIPFVGTIIRAMQVIYV NRF SQA SRK NAVNEIKRRASCDT FPRLLLFPEGT AtLPEAT2 194 TIV AS ESHD SLPFVGTIIRAMQVIYV NRF SQT SRK NAVHEIKRKASCDR FPRLLLFPEGT GmLPEAT2 179 TIV AA ESHD SIPFVGTIIRAMQVIYV NRF LPS SRK QAVREIKRRASCNR FPRVLLFPEGT StLPEAT2 200 TIV AS ESHD SMPFVGTIIRAMQVIYV NRF SPT SRK HAINEIKRKASCDQ FPRVLLFPEGT SiLPEAT2 201 TIV AS ESHD ALPFVGTIIRAMQVIYV DRF SPA SRK AAVNEIKRKAACNS FPRVLLFPEGT ZmLPEAT2 203 TIV SS ESHD ALPFVGTIIRAMQVIYV DRF SPA SRK AAVNEIKRKAACNS FPRVLLFPEGT OsLPEAT2 204 TIV SS DSHD SIPFVGTIIRAMQVIYV DRF SPA SRK SAVNEIKRKAACNS FPRVLLFPEGT

PpLPEAT2 249 TTNGRFLISF EL GAFIPGFP IQPV TVRYPHVHFDQSWG HISL AK LM FRMFTQFHNF MEVE VvLPEAT2 254 TTNGRVIISF QL GAFIPGYP IQPV VVRYPHIHFDQSWG HISL GR LM FRMFTQFHNF MEVE PtLPEAT2 246 TTNGKVLISF QL GAFIPGYA VQPV IVRYPHVHFDQSWG NVSL GM LM FRMFTQFHNF MEVE RcLPEAT2 228 TTNGKVVISF QL GAFIPGYA IQPV IVRYPHVHFDQSWG YISL AK LM FRMFTQFHNF MEVE CcLPEAT2 259 TTNGKFLISF QL GAFIPAYP IQPV IVRYPHVHFDQSWG DVSL GK LM FRMFTQFHNF MEVE TcLPEAT2 269 TTNGKVLISF QL GAFIPGHP IQPV VVRYPHVHFDQSWG LISL AK LM LRMFTQFHNF MEVE AtLPEAT2 254 TTNGKVLISF QL GAFIPGYP IQPV VVRYPHVHFDQSWG NISL LT LM FRMFTQFHNF MEVE GmLPEAT2 239 TTNGRNLISF QL GAFIPGYP IQPV IVRYPHVHFDQSWG HVSL GK LM FRMFTQFHNF FEVE StLPEAT2 260 TTNGRSIISF QL GAFIPGYP IQPV IVRYPHVHFDQSWG NVSL AM LM FRMFTQFHNF MEVE SiLPEAT2 261 TTNGRFLISF QH GAFIPGYP VQPV VVRYPHVHFDQSWG NISL LK LM FKMFTQFHNF MEVE ZmLPEAT2 263 TTNGRFLISF QH GAFIPGYP VQPV VVRYPHVHFDQSWG NISL LK LM FKMFTQFHNF MEVE OsLPEAT2 264 TTNGRFLISF QH GAFIPGYP VQPV IVRYPHVHFDQSWG NISL GK LM FKMFTQFHNF MEVE

PpLPEAT2 309 YLP VVSPLDNKKESAV RFSER TCHA MATS LN VVQTSHS YGDLML LM KATQSKSKLERPAS VvLPEAT2 314 YLP VISPLENKKENAV HFAKR TSHA IATA LN VVQTSHS YGDLML LT RASQSKQ--EKPSS PtLPEAT2 306 YLP IVSPLDNCKENPS HFAKR TSHA IASA LN VVQTNHS YGDLML LM KASELKQ--EKPSS RcLPEAT2 288 YLP IVSPLDNCKENPV HFAKR TSHS IASA LN VVQTFHS YGDLML LM KASHSKQ--EKPSS CcLPEAT2 319 YLP VVFPSDNQKENAL RFAER TSHA MASA LN AVQTSHA YGDLML LM KASELKE--ENASS TcLPEAT2 329 YLP TIMPPDHQKQNAV HFAER TGQA MASA LN VVQTSHS YGDLML LM KAAQLQK--EKPWS AtLPEAT2 314 YLP VIYPSEKQKQNAV RLSQK TSHA IATS LN VVQTSHS FADLML LN KATELKL--ENPSN GmLPEAT2 299 YLP VIYPLDD-KETAV HFRER TSRD IATA LN AVQTGHS YGDIML HM KAQEAKQ--ENPSS StLPEAT2 320 YLP VITPHENRKESAV RLSQR TGHA VASA LN VVQTSHS YGDVLL LA KALEANQ--ENPSL SiLPEAT2 321 YLP VVYPPEIKQENAL HFAEN TNYA MARA LN VLPTSYS YGDSMI MA RAVEAGK--ANCSN ZmLPEAT2 323 YLP VVYPPEIKQENAL HFAED TSYA MARA LN VLPTSYS YGDSMI MA RAIEAGK--VNGSN OsLPEAT2 324 YLP VVYPPEIKQENAL HFAEN TSYA MAHA LN VIPTSYS YGDSMI MA RAVEDGK--VNCSN

162

PpLPEAT2 369 YMVEM AT VKSLLH ISSMEA VDF LDKFLSMNPD PR GHVNYSG FLRVLR LKACTFSEE IFAF VvLPEAT2 372 YMVEM AR VESSFH LSTLEA VDF LDTFLSMNPD PS GCVKIHD FFRVLR LKPCYLSEK IFGF PtLPEAT2 364 YMVEM AK VESLFH INSLEA VNF LDKFLSMNPD AS GRVKFND FLRAFR LRTCTLSEE LFGF RcLPEAT2 346 YMVEM AK VESLYH ISSLEA VDF LDKFLSMNPD PS GRVKFHD FLRAMR LRTCSLLEE IFGF CcLPEAT2 377 YMVEM AR VGSIFH ISSLEA VNF LEKFLSMNPD PS GCVKLLD FLSVLR LKTCPLSDE IFGF TcLPEAT2 387 YMVEM AR IESLYH ISSLEA VDF LDKFLSMNPD TS GCVKLHD FLRVLR LKACTLSEE IFGF AtLPEAT2 372 YMVEM AR VESLFH VSSLEA TRF LDTFVSMIPD SS GRVRLHD FLRGLK LKPCPLSKR IFEF GmLPEAT2 356 FMVEM TK VESLFH ISSMEA VDF LDKFLAMNPD SS GRVQYHD FLRVLR LKACPLSAK IFSF StLPEAT2 378 YLVEM AG VEAEFH LSSLEA VDF LDVFLSMNPD SR GQVEIHH FLKVLR LKPSTLSEK IFGF SiLPEAT2 379 YMVEM AW VKDMYG VSTAEA MEL LEHFLAMNPD SD GRVKAQD FWAYFG LDCSPLCKK IFHY ZmLPEAT2 381 YMVEM AW VKDIYG VSTAEA MDL LEHFLAMNPD ND GRVKAQD FWAHFG LDCSPLCKK IFHY OsLPEAT2 382 YMVEM AW VKETYG VSTSEA MAL LEDFLCMSPD KD GRVNAQD FWAHFG LNCTPLCKK IFQY

PpLPEAT2 429 I DVEKSGS ITFKQFL FGSV HVLKQ PLFRRA CE LVFSEYVSGENDYISEQEFGES VRPA IP VvLPEAT2 432 I DVDKSGR VTFKQFL FGSA HVMKQ PLFRQA CE LAFAECDSDGDLYISEQELGDS IRPV IP PtLPEAT2 424 L DVEKNGS ITFKQFL YGSA HVMKR PLFHQS CE LAFAQCDTRGHNQISEQELGET IRHA IP RcLPEAT2 406 V DVEKIGS ITFKQFL YGSA HVMKQ PLFRQT CE LACTRCSGGEDNLISKEQLGDT IRLA IP CcLPEAT2 437 I DVDKNGS ITFKQFL YASA HVMKL PLFWQA CE LAFAECDPDGNGFISENQLEVT IRPA IP TcLPEAT2 447 L DVEKNGS ITFKQFL FGSA HVLKQ PLFRQA CE LAFAECDVEGENYFMEKDLADI LRHA IP AtLPEAT2 432 I DVEKVGS ITFKQFL FASG HVLTQ PLFKQT CE LAFSHCDADGDGYITIQELGEA LKNT IP GmLPEAT2 416 I DVEKSGT ITFRQFL YGSA HVMSQ PGFHQA CE EAFAGCGGAVKAYVVEQELRDF IQPV IL StLPEAT2 438 I DVQKSGK ITFKQFL VGSA HILKQ PLFHQA CE SAFTACDGDGKNYIMEKEFGDS LMLS IP SiLPEAT2 439 F DFSIKES ITFRQFL VGCA HLRKQ PLFQGA CE TAFEKCRDPETSEISRGQLADI LRLS ML ZmLPEAT2 441 F DLGIKES ITFRQFL VGCA HLRKQ PLFQGA CE TAFEKCRDPETSEISRAQLADV LRLS ML OsLPEAT2 442 F DFEAKES ITFRQFL IGCA HLRKQ PSFQDA CE TAFERCRNPLTSHIGREQLADV LRSS ML

PpLPEAT2 489 DLNED-EVHE LF NLFDADG DGRISKDEFWTC LKRN PLLIAL FSPC LLNKDISQDGNRLEE VvLPEAT2 492 DLNED-EIQE MF NLFDTDK DGRVSKDDFSNC LRRH PLLIAL FSPS LLHNA------PtLPEAT2 484 DFDED-EIHE LF SIFDMDG DGSVSKDNFLYC LRQN PLLIAL FKPC LVHKDSSQVGQGILE RcLPEAT2 466 DLDDA-EIHE LF KLFGGND DGRASKETFMCC LKKN PLLIAL FWPC LVYKDSLECGGRMLE CcLPEAT2 497 DLNKY-EIDS LF RLFDSDG DGRVSRDDFICC LRKN PLLIAI FSPT LLHTDLSEARNRMPG TcLPEAT2 507 ELNED-EIHG LF NLFDTDK DGRISRDDFFSC LRKN PLLISL FSPR LLHRDTSKAGDRMLE AtLPEAT2 492 NLNKD-EIRG MY HLLDDDQ DQRISQNDLLSC LRRN PLLIAI FAPD LAPT------GmLPEAT2 476 NWSED-EVHE LF MVFDNDN DGRIDKNDFLSC LRKT PLLIAF FTLQ LQQKEFE--GNGVIE StLPEAT2 498 GLSNN-EIRG LF TLFDIDR DGKMSKDDFIGC LRRY PLLIAL FSPC VLQQICP------SiLPEAT2 499 LPSDD-GMQK LF KTFDVDG DEKISRDDFITC LGRF PFLIAF FAAP INGEVYIEIV----- ZmLPEAT2 501 LPSGD-RMLD LF KTFDIDG DEKISKDDFMTC LGRF PFLIAF FAAP INGEVYIEIV----- OsLPEAT2 502 ELMTDNG MMK LF KTLDVDD DDGISKDDLMAS LRKL PFMIAL FARR INGEVYIEIV-----

PpLPEAT2 548 IV- VvLPEAT2 --- PtLPEAT2 543 EIV RcLPEAT2 525 ELV CcLPEAT2 556 DVI TcLPEAT2 566 EIV AtLPEAT2 --- GmLPEAT2 533 IV- StLPEAT2 --- SiLPEAT2 --- ZmLPEAT2 --- OsLPEAT2 ---

163

Figure S3 . Characterization of GPAT9 A gpat9-1 Salk_052947C gpat9-2 GABI_867A06 T-DNA Insertions LB LB LB LB ATG (A) Representation of GPAT9 T-DNA insertion locations. For both lines T- DNA left border primers will generate ~ amplicons with gene specific primers B flanking either side of the insertion suggesting there are multiple insertions with inverse orientation. The gpat9-1 insertions are 46 bp upstream of the coding sequence. The gpat9-2 insertion is at the end of the 4 th exon. Pictured is Col-0 gpat9-1 just the first four exons out of 12 in AtGPAT9.

C 0.006 (B) RT-PCR of full length GPAT9 coding sequence from leaves of homozygous gpat9-1. 0.004 (C) qPCR results for GPAT9 wild-type (Col-0) and GPAT9/gpat9-2 leaves. 0.002

NRQ (TIP41-like) NRQ 0.000 Col-0 GPAT9-2/gpat9-2

Methods: (A) For insertion site sequencing the left border-gene junction was amplified by PCR with GoTaq® Flexi (Promega), gel purified by QIAquick Gel Extraction Kit (QIAGEN) and sequenced from both the left border primer and a flanking gene-specific primer by Eurofins (http://www.eurofinsgenomics.com/). (B-C) Leaf RNA extraction with RNeasy Plant Mini Kit (QIAGEN) and treated with RNase-free DNase (Qiagen) and on-column DNase digestion. RNA was quantified using a Nanophotometer (Implen Inc). cDNA was synthesized using SuperScript III First-Strand Synthesis System for RT-PCR (Life Technologies). RT-PCR utilized standard conditions as per GoTaq® Flexi (Promega) protocol with 30 cycles of amplification. For qPCR transcript levels were analyzed by quantitative PCR (qPCR) using Platinum SYBR Green qPCR SuperMix-UDG (Life Technologies) and the Stratagene Mx3005P Quantitative PCR system (Agilent Technologies Inc). Transcript was normalized to the TIP41-like gene [1]. (A-C), the sequences for primers utilized are shown in supplemental table S1.

164

Figure S4. Analysis of Viability in Wild-Type and GPAT9/gpat9-2 Pollen.

(A-B) Alexander staining [2] for pollen abortion. Purple = non-aborted pollen, green pollen (black arrows) = aborted pollen. Pollen from wild-type (A), and GPAT9/gpat9-2 (B) plants.

(C-D) Nitro blue tetrazolium staining for pollen viability [3]. Viable pollen stain black. Anthers from wild- type (C), and GPAT9/gpat9- 2 (D) plants.

165

A 60 Figure S5 . Lipid Col-0 Characterization of Wild-Type GPAT9-2/gpat9-2 and GPAT9-2/gpat9-2 Plants.

40 (A) The relative fatty acid composition of leaves. Average

wt % wt and standard deviation. n = 5 for wild-type (Col-0), and 13 for 20 GPAT9-2/gpat9-2.

(B) Total seed fatty acid content from seeds of plants grown at the 0 same time under 24 hr light :0 :3 :0 :1 :2 :3 2 6 d9 d3 6 8 8 8 8 1 :1 :1 1 1 1 1 1 ~150-170 µmol/m /sec white 6 6 1 1 light. 30 seeds per FAME analysis from a single plant. B 15 n = 19-20 individual plants per line.

(C) The average fatty acid 10 composition of seeds from part C. Bars are average and standard deviation.

5 g FAME/ seed

µ

0 Col-0 GPAT9-2/gpat9-2

C 30 Col-0

GPAT9-2/ gpat9-2 20

wt % wt 10

0 :0 :1 :0 :1 :2 :3 :0 :1 :2 :1 6 8 8 8 8 0 0 0 2 1 16 1 1 1 1 2 2 2 2

166

Supplemental Figure S6. Seed Fatty Acid Composition of GPAT9 amiRNA Knockdown Lines. Fatty acid compositions are from seed samples analyzed for total oil content in Figure 7A of the main text. Average ± S.D.

35

Col-0 30 12 10 25 7 2 20 11 3

mol % mol 15 16 20

10

5

0

:0 :1 :0 :1 :2 :3 :0 :1 :2 :3 :1 6 6 8 8 8 8 0 0 0 0 2 1 1 1 1 1 1 2 2 2 2 2

167

Supplemental Figure S7 . Expression of GPAT9 in Developing Seeds of Wild-Type and GPAT9 amiRNA Knockdown Lines. GPAT9 transcript was normalized to TIP41-like [1].Two biological replicates for Col-0, and three biological replicates for each amiRNA knockdown line. Each biological replicate is the average of three technical replicates.

10

8

6

4

NRQ (TIP41-like) NRQ 2

0

-0 2 ol ine C ine 12 L L

Methods: Frozen developing seeds (9-11 DAF) were separated from silique tissue by silique popping method [4]. ~50 µL volume of frozen seeds per biological replicates were mechanically pulverized to a fine powder with steel beads (TissueLyser LT, QIAGEN), RNA was extracted and used for cDNA synthesis using SuperScript III First-Strand Synthesis System for RT-PCR (Life Technologies). RT-PCR utilized standard conditions as per Maxima SYBR Green/ROX qPCR Master Mix (Thermo) and BioRad CFX96™ Real- Time System and C1000™ Thermal Cycler with 45 cycles of amplification. The sequences for primers utilized are shown in supplemental table S1.

168

Supplemental Figure S8. Developing Seed Microsome A GPAT Assays of Wild-Type and amiRNA Knockdown TAG Lines. GPAT assays with developing 9-11 DAF seed 14 MAG microsomes utilizing 3.55 nmol [ C]glycerol 3- phosphate and 25 nmol palmitoyl-CoA, for 15 min at 24°C. (A) Phosphor image of TLC plate indicating that PA [14 C]LPA was the major product of developing seed microsome activity with palmitoyl-CoA. (B) Percent of wild-type (Col-0) GPAT activity in GPAT9 amiRNA knockdown lines 2 and 12. Mean and S.D. of two replicate assays.

LPA

2 Col-0 12 B Origin

B 100

80

60

40

20

Percent of Col-0 activity 0 2 2 1 ne e Li n Li

169

Supplementary Figure S9. Testing of Protein:Protein Interactions Between AtLPAAT2, AtDGAT1, and AtLPCAT2.

Testing of Protein:Protein Interactions Between AtLPAAT2, AtDGAT1, and AtLPCAT2. Cub-LexA bait fusions and Nub prey fusions were produced as described in Figure 4 and in the main text. (A-B) Prototrophic growth assay of yeast strains containing various combinations of Arabidopsis acyltransferase bait plasmids co-expressed with potential NubG-fusion acyltransferase prey plasmids. Serial dilutions of cells expressing different bait-prey combinations were plated on non-selective (A) or selective (B) media. (C) Quantitative measurement of β-galactosidase activity from cell lysates of the strains used in the serial dilution assays. Cultures used for serial dilutions shown in A. and B. contained bait and prey combinations as numbered in C.

170

Supplementary Figure S10 . Sequence Identity Comparisons Between AtGPAT1 and AtGPAT9 to the Animalia Subset of the NCBI Protein Database.

The top hits to AtGPAT9 and AtGPAT1 are shown first, followed by the best hits from within the individual species that provided the top hits to the alternate AtGPAT sequence. See text for additional details.

>AtGPAT9 MSSTAGRLVTSKSELDLDHPNIEDYLPSGSSINEPRGKLSLRDLLDISPTLTEAAGAIVDDSFTRCFKSNPPEPWNW NIYLFPLYCFGVVVRYCILFPLRCFTLAFGWIIFLSLFIPVNALLKGQDRLRKKIERVLVEMICSFFVASWTGVVKY HGPRPSIRPKQVYVANHTSMIDFIVLEQMTAFAVIMQKHPGWVGLLQSTILESVGCIWFNRSEAKDREIVAKKLRDH VQGADSNPLLIFPEGTCVNNNYTVMFKKGAFELDCTVCPIAIKYNKIFVDAFWNSRKQSFTMHLLQLMTSWAVVCEV WYLEPQTIRPGETGIEFAERVRDMISLRAGLKKVPWDGYLKYSRPSPKHSERKQQSFAESILARLEEK vs Metazoa/Animalia: PREDICTED: glycerol-3-phosphate acyltransferase 3 [Orcinus orca] Sequence ID: ref|XP_004282203.1| Length: 438Number of Matches: 1 Gene -associated gene details Map Viewer -aligned genomic context Range 1: 75 to 429 GenPeptGraphics Next Match Previous Match First Match

Alignment statistics for match #1 Score Expect Method Identities Positives Gaps Frame 259 2e- Compositional 139/360(39%) 211/360(58%) 17/360(4%) bits(661) 82() matrix adjust. Features: Query 24 DYLPSGSSINEPRGK-LSLRDLLDISPTLTEAAGAIVDDSFTRCFKSNPPEPWNW----N 78 D P ++ RG+ L D+ S EA IV+D T+ F S WN N Sbjct 75 DESPMEKGLSGLRGRDFELSDVFYFSKKGLEA---IVEDEVTQRFSSEELVSWNLLTRTN 131

Query 79 I---YLFP----LYCFGVVVRYCILFPLRCFTLAFGWIIFLSLFIPVNALLKGQDRLRKK 131 + Y+ P ++ GV+VRYC+L PLR TLAF I FL + + L + LR + Sbjct 132 VNFQYISPKLTMVWVLGVIVRYCVLLPLRV-TLAFVGISFLVIGTTLVGQLP-ESSLRNR 189

Query 132 IERVLVEMICSFFVASWTGVVKYHGPRPSIRPKQVYVANHTSMIDFIVLEQMTAFAVIMQ 191 + ++ C V + +G + YH + + + VANHTS ID ++L +A++ Q Sbjct 190 LSELVHLTCCRICVRALSGTIHYHNKQYRPQKGGICVANHTSPIDVLILTTDGCYAMVGQ 249

Query 192 KHPGWVGLLQSTILESVGCIWFNRSEAKDREIVAKKLRDHVQGADSNPLLIFPEGTCVNN 251 H G +G++Q +++S +WF RSE KDR +V K+LR+H+ P+LIFPEGTC+NN Sbjct 250 VHGGLMGIIQRAMVKSCPHVWFERSEMKDRHLVTKRLREHIADKKKLPILIFPEGTCINN 309

Query 252 NYTVMFKKGAFELDCTVCPIAIKYNKIFVDAFWNSRKQSFTMHLLQLMTSWAVVCEVWYL 311 +MFKKG+FE+ T+ P+AIKYN F DAFWNS K + +LL++MTSWA+VC+VWY+ Sbjct 310 TSVMMFKKGSFEIGGTIYPVAIKYNPQFGDAFWNSSKYNMVSYLLRMMTSWAIVCDVWYM 369

Query 312 EPQTIRPGETGIEFAERVRDMISLRAGLKKVPWDGYLKYSRPSPKHSERKQQSFAESILA 371 P T GE ++FA RV+ I+++ GL ++PWDG LK ++ E +Q+++++ I+ Sbjct 370 PPMTREEGEDAVQFANRVKSAIAIQGGLTELPWDGGLKRAKVKDTFKEEQQKNYSKMIVG 429

171

>AtGPAT1 MVLPELLVILAEWVLYRLLAKSCYRAARKLRGYGFQLKNLLSLSKTQSLHNNSQHHLHNHHQQNHPNQTLQDSLDPL FPSLTKYQELLLDKNRACSVSSDHYRDTFFCDIDGVLLRQHSSKHFHTFFPYFMLVAFEGGSIIRAILLLLSCSFLW TLQQETKLRVLSFITFSGLRVKDMDNVSRSVLPKFFLENLNIQVYDIWARTEYSKVVFTSLPQVLVERFLREHLNAD DVIGTKLQEIKVMGRKFYTGLASGSGFVLKHKSAEDYFFDSKKKPALGIGSSSSPQDHIFISICKEAYFWNEEESMS KNNALPRERYPKPLIFHDGRLAFLPTPLATLAMFIWLPIGFLLAVFRISVGVFLPYHVANFLASMSGVRITFKTHNL NNGRPEKGNSGVLYVCNHRTLLDPVFLTTSLGKPLTAVTYSLSKFSEFIAPLKTVSLKRDRKKDGEAMQRLLSKGDL VVCPEGTTCREPYLLRFSPLFAELTEDIVPVAVDARVSMFYGTTASGLKCLDPIFFLMNPRPVYCLEILKKLPKEMT CAGGKSSFEVANFIQGELARVLGFECTNLTRRDKYLVLAGNEGIVR vs Metazoa/Animalia: PREDICTED: ancient ubiquitous protein 1-like isoform X1 [Xiphophorus maculatus] Sequence ID: ref|XP_005811174.1| Length: 415Number of Matches: 1 Gene -associated gene details Map Viewer -aligned genomic context Range 1: 20 to 262 GenPeptGraphics Next Match Previous Match First Match Alignment statistics for match #1 Score Expect Method Identities Positives Gaps Frame 59.3 3e- Compositional 69/256(27%) 109/256(42%) 31/256(12%) bits(142) 10() matrix adjust. Features: Query 336 LATLAMFIWLPIGFLLAVFRISVGV------FLPYHVANFLASMSGVRITFKTH 383 +A L + I+ P+G L + RI +GV F+ V ++S+ G+ + + Sbjct 20 VALLLLLIYSPVGVCLMLMRIFIGVHVFLVSCAIPDSFVRRFVVRVMSSVLGMHVRQR-- 77

Query 384 NLNNGRPEKGNSGVLYVCNHRTLLDP--VFLTTSLGKPLTAVTYSLSKFSEFIAPLKTVS 441 N R N+ L VCNH T D + L T P + ++ + + S Sbjct 78 ---NPRSRDKNTK-LCVCNHVTEFDHNIINLLTPFNTPQLEGSTGFMCWARGFMEIHSAS 133

Query 442 LKRDRKKDGEAMQRLLSKGD----LVVCPEGTTCREPYLLRFSPLFAELTEDIVPVAVDA 497 ++ GE++QR S LV E TT LL+FS LTE I PVA+ Sbjct 134 ---SQEAVGESLQRYCSADGAPPLLVFPEEDTTNGRAGLLKFSSWPFTLTESIQPVALRV 190

Query 498 RVSMFYGTTASGLKCLDPIFFLMNPRPVYCLEILKKLPKEMTCAGGKSSFEVANFIQGEL 557 + +T D ++ +P VY + L + ++ G+S+ E AN +Q L Sbjct 191 SRPLLSLSTVESSWLTDLLWTFFSPCTVYHVSWLPPVSRQ----DGESTQEFANKVQELL 246

Query 558 ARVLGFECTNLTRRDK 573 A LG T +T+ DK Sbjct 247 AIELGLVSTKITKADK 262

172

Supplemental Table S1 . Primers utilized for PCR, RT-PCT, & qPCR.

Purpose Primer Name Sequence (5’ to 3’) Genotyping gpat9-1 (Salk_052947C)

Gene specific GPAT9 salk F1 ACTTTCTTTGGAGAGTCGCTGT

Gene specific GPAT9 salk R1 CATCAACAATGGCACCAGCA

T-DNA left border LBb1.3 ATTTTGCCGATTTCGGAAC

Genotyping gpat9-2 (GABI_867A06) Gene specific GPAT9 GK F1 GCAGGCGTGATTTGCTAGAC

Gene specific GPAT9 GK R1 AATGCGGTCATCTGCTCCAA

T-DNA left border o8409 ATATTGACCATCATACTCATTGC

Genotyping qrt1-4 (CS25041)

Gene specific Qrt F2 TTTGCCTCTCAGGGTGTTTC

Gene specific Qrt R1 AATGAGAGCTATGCGGGAGA

T-DNA left border LBb1.3 ATTTTGCCGATTTCGGAAC

GPAT9 RT-PCR (Fig. S1) GPAT9 ORF start ATGAGCAGTACGGCAGGGAGGC GPAT9 ORF end TGCAAAAGTGAGTTATGTTTATTGAGA qPCR of GPAT9 (Fig. S1) GPAT9LP1046 AGAGAGGGTCAGAGACATGA G9 qPCR R 1172 CGACTCTGCGAAACTCTGTTG qTIP41-like829F ACATTTCAGTCTCTATCTGCGAAAGG

qTIP41-like946R GGATCTTCAGTTTCTGTGTCGTATGC

173

Supplemental Table S2 . Gene identifiers for GPAT9, LPEAT1, and LPEAT2 sequences used in Figures1 and 2 in the main text. AGI locus identifiers for the three Arabidopsis genes are described at The Arabidopsis Information Resource (TAIR) homepage ( https://www.arabidopsis.org/ ). The Genbank accession numbers are shown for tung tree GPAT9 ( Vernicia fordii , Gidda et al., 2009) and Ricinus communis GPAT9 [originally annotated as a putative LPAAT, Burgal et al., (2008)]. All other identifiers are as listed for each species in the Phytozome 10.3 database (http://phytozome.jgi.doe.gov/pz/portal.html# ).

*: not analyzed in this study.

Species GPAT9 LPEAT1 LPEAT2 Arabidopsis thaliana At5g60620 At1g80950 At2g45670 Vernicia fordii FJ479751 * * Physcomitrella patens Phpat.011G104300 * * Selaginella moellendorffii 152980 * * Aquilegia coerulea Aquca_047_00038.1 * * Brachypodium distachyon Bradi1g25790 * * Solanum tuberosum PGSC0003DMT400032021 * * Salix purpurea SapurV1A.0039s0700 * * Gossypium raimondii Gorai.012G158300 and * * Gorai.005G258500 Cucumis sativus Cucsa.099550 * * Zea mays GRMZM2G165681 GRMZM2G096010 GRMZM2G116243 Setaria italica Si030220m Si022261m Si006175m Oryza sativa LOC_Os07g34730 LOC_Os05g28960 LOC_Os06g49790 Citrus clementina Ciclev10028668m Ciclev10001396m Ciclev10019536m Glycine max Glyma.09G119200 Glyma.04G033600 Glyma.03G019200 Prunus persica ppa007262m ppa006689m ppa003784m Populus trichocharpa Potri.004G183300 Potri.014G042200 Potri.014G074300 Ricinus communis ACB30546 30174.m008937 30170.m014002 Theobroma cacao Thecc1EG006479t1 Thecc1EG033997t1 Thecc1EG005455t1 Vitis vinifera GSVIVT01023935001 GSVIVT01009661001 GSVIVT01027580001

174

4.8 SUPPLEMENTAL REFERENCES

1. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W-R: Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis . Plant Physiology 2005, 139 :5-17. 2. Peterson R, Slovin JP, Chen C: A simplified method for differential staining of aborted and non- aborted pollen grains , vol 1; 2010. 3. Regan SM, Moffatt BA: Cytochemical Analysis of Pollen Development in Wild-Type Arabidopsis and a Male-Sterile Mutant . The Plant Cell Online 1990, 2:877-889. 4. Bates P, Jewell J, Browse J: Rapid separation of developing Arabidopsis seeds from siliques for RNA or metabolite analysis . Plant Methods 2013, 9:9.

175

CHAPTER FIVE:

CONCLUSIONS AND PERSPECTIVES

Triacylglycerol (TAG) is one of the most energy-rich molecules made by plants, and it serves as an important source of calories and essential fatty acids in the human diet. TAG also serves many non-biological roles for humans, and the long hydrocarbons derived from the fatty acids in TAG are of particular interest for fuels, chemical feedstocks, and additives to many consumer products. The bulk of oil produced by most plants, including Arabidopsis, is made in the seed, where is serves the plant as a source of energy for the germinating seed or an attractant for seed dispersers.

The importance of oils both for plant biology and for human uses has led to over 60 years of study, and the Kennedy Pathway of TAG synthesis has driven our knowledge of fat and oil synthesis ever since. It describes how glycerol-3-phosphate, derived from the glycolysis intermediate dihydroxyacetone phosphate, is acylated at the sn -1 and then sn -2 positions to produce first lysophosphatidic acid (LPA) and then phosphatidic acid (PA). The dephosphorylation of phosphatidic acid produces the nonpolar lipid diacylglycerol (DAG), which is then acylated at the sn-3 position to produce TAG. The acyl groups are provided to the enzymes as acyl-CoA, exported from the plastid after fatty acid synthesis or cleaved from phosphatidyl choline (PC) during the acyl editing cycle. Today we know that the Kennedy Pathway acts slightly differently in Arabidopsis than it does in mammals and some plants. Flux to TAG actually goes through PC, the main membrane lipid in the ER and the site of acyl editing.

The fatty acid chains esterified to the three positions of the glycerol backbone are the main contributor to the properties of the oil. For example, a high proportion of saturated fatty acids

176

encourages a solid state at room temperature, while unsaturated fatty acids lead to a more liquid state. Monounsaturated and omega-3 fatty acids are desirable in food oils because of the health benefits to humans, while polyunsaturates such as α-linolenic acid can be used as drying oils in paints and varnishes. These fatty acids are modified in the acyl editing cycle by elongation, desaturation, or specialized modifications (e.g. hydroxylation, conjugation, etc.), and then esterified to the glycerol backbone using acyltransferases. The fatty acid composition of the oil

(and therefore its properties) is dependent on the overall composition of the fatty acids in the acyl-

CoA pool as well as the specificity of the acyltransferases.

The importance of acyltransferase specificity has been demonstrated in the attempts to produce ricinoleic acid in Arabidopsis. Castor bean ( Ricinus communis ) accumulates up to 90% of its fatty acids in seed oil as hydroxylated fatty acids (HFAs) such as ricinoleac acid, which are useful industrially in the manufacturing of products such as lubricants, paints, plastics, and soaps.

The castor plant is a poor agronomic crop, and so attempts have been made to produce the fatty acid in other oilseed plants, starting with Arabidopsis. When the FAH12 enzyme, responsible for generating ricinoleic acid, was heterologously expressed in Arabidopsis, only 17% of the fatty acids in the seed oil were hydroxy-fatty acids [1]. However, when castor isoforms of diacylglycerol acyltransferase (DGAT, RcDGAT2 ), and phospholipid:diacylglycerol acyltransferase (PDAT,

RcPDAT1A ), were expressed in the transgenic Arabidopsis, HFA levels rose to 20-30% and 27%, respectively [2,3]. Additionally, while the oil of castor accumulates up to 90% HFA, there is almost no HFA in the membranes of the seed. A castor phospholipase A2, RcsPLA2 α, appears to be involved in specifically cleaving HFA from PC [4], removing it from membrane lipids and encouraging incorporation into TAG. These examples indicate that acyl-selective enzymes do control the fatty acid content, and therefore the properties, of seed oil.

177

As our society attempts to become more sustainable and rely less on non-renewable resources, we have been working to produce plant oils that meet the needs of a growing population.

We aim to produce food oils that are healthier, with higher levels of monounsaturated and omega-

3 fatty acids and lower levels of saturated fatty acids, and both without the need for chemical hydrogenation which generates unhealthy trans fats. We are working to produce industrially- and commercially-useful oils that could be used in the generation of plastics, coatings, and dyes, in a renewable, domestically grown crop, while preserving land for food crop growth. Finally, while trying to replace all petroleum products with plant-grown fuels would be impossible due to the scale needed, certain niche markets of fuel or fuel additives could benefit from production in plants. But before any of these goals can become reality, we must understand the steps generating oil. Identifying and characterizing the genes and enzymes involved in the biosynthesis of TAG is the first step to being able to generate healthier, more renewable oils for food, fuel, and chemical feedstocks.

GPAT9 IS AN ESSENTIAL GENE AND SHOWS GPAT ACTIVITY

The first step of lipid biosynthesis is catalyzed by a glycerol-3-phosphate acyltransferase

(GPAT), which transfers an acyl group from acyl-CoA to the sn -1 position of glycerol-3- phosphate. While eight GPAT homologs are involved in suberin or cutin synthesis, the identity of the main GPAT involved in membrane lipid and oil synthesis has not yet been determined. GPAT9 is homologous to known mammalian and yeast GPATs which are involved in generating TAG, and indeed it appears to be highly conserved throughout evolution, a hallmark of genes with essential housekeeping function. The gpat9 mutant must be maintained as a heterozygote, and it shows no transmission of the mutant allele through the male germ line and reduced transmission in the female germ line. This lethality is consistent with an essential role in producing the

178

membranes and oil necessary for sustaining life. In amiRNA knockdown lines, only lines with high GPAT9 transcript levels accumulated WT oil levels, and transcript level was directly related to the level of GPAT activity in developing seeds. This indicates that GPAT9 encodes GPAT activity, and is the main GPAT responsible for oil accumulation in the seed. The GPAT9 protein also interacts with other ER glycerolipid biosynthetic enzymes, indicating that the proteins form complexes to funnel substrate into glycerolipids. Together, these results indicate that GPAT9 is the main GPAT involved in ER lipid biosynthesis, and it plays an essential role in the generation of membrane and storage lipids.

LPP β MAY BE A PAP INVOLVED IN OIL SYNTHESIS

The phosphatidic acid phosphatase (PAP) step is the third step in the TAG synthesis pathway, and catalyzes the hydrolysis of inorganic phosphate from phosphatidic acid (PA) to produce DAG. Several PAPs have been identified in the prokaryotic pathway of lipid synthesis in chloroplasts, and several others have been implicated in signaling pathways; however the main

PAP involved in oil synthesis in the ER remains elusive. After analyzing all the genes in

Arabidopsis for homologs to known PAPs, only one homolog displayed characteristics consistent with the PAP involved in oil synthesis. LPPβ is expressed in developing seed tissue and is localized to the ER, probably as an integral membrane protein. Attempts to decrease LPPβ transcript using amiRNA and ihpRNA resulted in increased or no change in transcript level, and attempts to mutate the gene using the CRISPR/Cas9 system have so far been ineffective. However, LPPβ shows PAP activity when expressed in yeast. The pah1pah2 knockout line showed increased LPPβ transcript level, indicating that it may act redundantly with PAH1 and PAH2 in oil synthesis. More studies, particularly genetic analysis of mutants, are still needed to confirm its role in the plant.

179

SPP1 AND LPAP1 ARE NOT INVOLVED IN OIL SYNTHESIS

In Chapter 2, I discussed my rationale in determining which PAP homolog(s) could possibly be involved in oil synthesis. Of the 13 genes in Arabidopsis homologous to known PAPs, most were set aside due to involvement in signaling pathways or the plastidial pathway of lipid synthesis. While LPPβ was the only PAP that could potentially be the main PAP involved in oil synthesis, SPP1 and LPAP1 also showed enough characteristics to warrant determining if they were involved in oil synthesis through redundancy. Single mutants showed no oil phenotype, and multiple mutants with each other and pah1pah2 also showed no oil phenotype, and only minor changes in fatty acid composition. This indicates that they are not involved in oil synthesis, and is consistent with their placement in the signaling pathway for ABA-mediated dehydration stress response in the case of SPP1 [5], and in the case of LPAP1, as a lysophosphatidic acid phosphatase which is highly expressed during stress conditions [6]. Since it is unusual for PAPs which are involved in signaling to also be involved in de novo lipid synthesis, it is not unexpected to find that neither gene is involved in oil synthesis.

THE STATE OF KNOWLEDGE OF TAG SYNTHESIS GENES

As described in Chapter Four, GPAT9 is the main GPAT involved in oil synthesis, and the first step of oil and membrane lipid synthesis. It provides LPA for the lysophosphatidic acid acyltransferase (LPAAT) enzyme. There are five LPAATs in Arabidopsis, but LPAT2 encodes the ubiquitous, ER-localized LPAAT [7]. It has a female-gametophyte lethality phenotype, due to the fact that the pollen is complemented by the LPAT3 isoform, which is mainly expressed in that tissue. This lethality phenotype suggests an essential function similar to what is seen in GPAT9 .

Recombinant LPAT2 and LPAT3 both showed LPAAT activity in vitro , but LPAT4 and LPAT5

180

did not. This indicates that LPAT2 is the main LPAAT involved in membrane lipid and oil synthesis, and LPAT3 is an active LPAAT in the pollen.

The next enzyme in the pathway is PAP, discussed in Chapters Two and Three. There are

13 PAPs in Arabidopsis that are homologous to known PAPs, and all have been dismissed from consideration as the PAP involved in oil synthesis, except LPPβ, which encodes PAP activity and is expressed in seed tissue. However, work remains to characterize mutants and place it in the oil synthesis pathway, and there remains the possibility that a PAP with no homology to known PAPs could be encoding this step.

After the PAP reaction, the oil synthesis pathway in Arabidopsis diverges from the traditional Kennedy pathway, and DAG used for TAG synthesis is actually derived from PC [8].

There are two distinct DAG pools: a de novo pool resulting from PAP activity and one derived from PC. Thus, de novo DAG is converted to PC probably through headgroup exchange catalyzed by the ROD1 gene encoding a phosphatidylcholine:diacylglycerol cholinephosphotransferase

(PDCT) enzyme [9], or possibly through the action of CDP-choline:DAG cholinephosphotranferase (CPT) encoded by AAPT1 and/or AAPT2 [10]. Conversion of PC back to DAG could be achieved by PDCT or CPT. Additionally, a non-specific phospholipase C

(nsPLC;), or a combination of a phospholipase D (PLD ζ) and a PAP could encode the conversion of PC to DAG; however, PLC and PLD enzymes are normally found in signaling pathways and not de novo lipid synthesis, and of the 6 nsPLC’s and 2 PLD ζ’s in Arabidopsis, none have been implicated in oil synthesis. Thus, the understanding of which enzyme contributes most to this flux remains to be explored, but genes have been identified for both of the enzymatic steps in generating

PC then DAG for oil synthesis.

181

This second DAG pool can be converted to TAG by one of two enzymatic functions: the acylation of DAG by diacylglycerol acyltransferase (DGAT) using acyl-CoA as a substrate, or the acylation of DAG using PC as the substrate by phosphatidylcholine:diacyglycerol acyltransferase

(PDAT). A double homozygous mutant of pdat1 and dgat1 is lethal in Arabidopsis, indicating that these may be the main isoforms, but combinations of other DGATs and PDATs have yet to be studied [11]. There are two more DGATs, DGAT2 and DGAT3: DGAT2 has even higher activity than DGAT1 does, and different substrate specificity, indicating that it could still contribute strongly to TAG synthesis [12]. The role of DGAT3 is less clear, but it appears to be involved in oil synthesis in sucrose-rescued seedlings [13]. PDAT2 has not been specifically characterized, but its expression is strongest in developing seed (compared to PDAT1 which is expressed constitutively), indicating that it may also be involved in oil synthesis.

This also highlights the growing body of data that indicates that plant oil synthesis does not contain a single Kennedy pathway generating oil as is seen in mammals. Instead the first portion of the pathway (GPAT, LPAAT, PAP) are likely involved in de novo lipid synthesis and provide substrate to the PC pool; DAG is then derived from the PC pool specifically for oil synthesis, and possibly for export to the plastid. This is supported not only by flux analysis indicating two pools of DAG, but also by interaction data indicating that GPAT and DGAT do not interact, but enzymes of the membrane synthesis (GPAT, LPAAT, and LPCAT, which provides substrate), do interact. This is not to say that the early steps are not involved in oil synthesis; indeed, decreased levels of GPAT9 show reduced oil. However, the idea of a linear pathway from

G3P to oil appears to be evolving in Arabidopsis to a pathway of membrane lipid synthesis followed by the generation of TAG.

182

Many of the genes involved in oil synthesis in Arabidopsis have now been identified.

GPAT, LPAAT, DGAT, and PDAT enzymatic activities in oil synthesis have all been assigned to at least one gene. The PAP step has one gene with several promising indicators of its function.

The identification of the genes involved in this pathway will allow further characterization and manipulation of the pathway, which will eventually lead to more valuable, healthier, more sustainable plant oils.

183

Figure 1: TAG biosynthesis in the eukaryotic pathway of Arabidopsis. Recent data suggests that GPAT, LPAAT, and LPCAT proteins interact, and DGAT and LPCAT interact, but GPAT and DGAT do not interact, indicating that the pathway can be roughly partitioned into two portions of the ER lipid synthesis pathway.

184

REFERENCES

1. Broun P, Somerville C: Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean . Plant Physiol 1997, 113 :933-942. 2. Burgal J, Shockey J, Lu C, Dyer J, Larson T, Graham I, Browse J: Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil . Plant Biotechnol J 2008, 6:819-831. 3. van Erp H, Bates PD, Burgal J, Shockey J, Browse J: Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis . Plant Physiol 2011, 155 :683-693. 4. Bayon S, Chen G, Weselake RJ, Browse J: A small phospholipase A2-alpha from castor catalyzes the removal of hydroxy fatty acids from phosphatidylcholine in transgenic Arabidopsis seeds . Plant Physiol 2015, 167 :1259-1270. 5. Nakagawa N, Kato M, Takahashi Y, Shimazaki K, Tamura K, Tokuji Y, Kihara A, Imai H: Degradation of long-chain base 1-phosphate (LCBP) in Arabidopsis: functional characterization of LCBP phosphatase involved in the dehydration stress response . J Plant Res 2012, 125 :439-449. 6. Reddy VS, Rao DK, Rajasekharan R: Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana . Biochim Biophys Acta 2010, 1801 :455-461. 7. Kim HU, Li Y, Huang AH: Ubiquitous and endoplasmic reticulum-located lysophosphatidyl acyltransferase, LPAT2, is essential for female but not male gametophyte development in Arabidopsis . Plant Cell 2005, 17 :1073-1089. 8. Bates PD, Durrett TP, Ohlrogge JB, Pollard M: Analysis of Acyl Fluxes through Multiple Pathways of Triacylglycerol Synthesis in Developing Soybean Embryos . Plant Physiology 2009, 150 :55-72. 9. Lu C, Xin Z, Ren Z, Miquel M, Browse J: An enzyme regulating triacylglycerol composition is encoded by the ROD1 gene of Arabidopsis . Proc Natl Acad Sci U S A 2009, 106 :18837-18842. 10. Goode JH, Dewey RE: Characterisation of aminoalcoholphosphotransferases from Arabidopsis thaliana and soybean . Plant Physiology and Biochemistry 1999, 37 :13.

185

11. Zhang M, Fan J, Taylor DC, Ohlrogge JB: DGAT1 and PDAT1 acyltransferases have overlapping functions in Arabidopsis triacylglycerol biosynthesis and are essential for normal pollen and seed development . Plant Cell 2009, 21 :3885-3901. 12. Zhou XR, Shrestha P, Yin F, Petrie JR, Singh SP: AtDGAT2 is a functional acyl- CoA:diacylglycerol acyltransferase and displays different acyl-CoA substrate preferences than AtDGAT1 . FEBS Lett 2013, 587 :2371-2376. 13. Hernandez ML, Whitehead L, He Z, Gazda V, Gilday A, Kozhevnikova E, Vaistij FE, Larson TR, Graham IA: A cytosolic acyltransferase contributes to triacylglycerol synthesis in sucrose-rescued Arabidopsis seed oil catabolism mutants . Plant Physiol 2012, 160 :215- 225.

186