Science, Faculty of
Botany, Department of
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UBCV
Liu, Mingzhe
Master of Science - MSc
University of British Columbia
Fatty acids (FAs) are building blocks for different glycerolipids, including phospholipids and galactolipids that serve as major structural components of cell membranes, as well as triacylglycerols (TAGs), which are the main form of energy and carbon storage in plants. They are also precursors of the plant surface lipids: cutin, cuticular waxes, and suberin, which play roles during the plant’s resistance to drought, pathogens, or insects. In-plant cells, de novo fatty acid synthesis happens exclusively within plastids with the growing fatty acyl chain attached to the acyl carrier protein (ACP). Once completed, the majority of these fatty acids are removed from ACP by thioesterases and re-attached to coenzyme A (CoA) under the activities of long-chain acyl-CoA synthetase (LACS) to make membrane lipids, storage or surface lipids in the ER, the major site for lipid assembly. Since the production of fatty acids and the production of complex lipids happen in different organelles, a transport system between plastids and other subcellular compartments is required in order to deliver FAs from the plastid to the ER. In Arabidopsis, neither the lacs9 single knock-out mutants which removed all plastidial LACS activities, nor the lacs4 single mutants which removed the activity of an ER LACS showed any visible phenotype while the lacs4lacs9 double mutants are dwarf, and with decreased seed oil and stem wax content. These not only suggest the functional overlap between the plastidial-LACS9 and ER-LACS4 but also suggest proposed access for the ER-LACS to the fatty acids from the plastid envelopes. In my research, I have proved such access directly by re-targeting the plastidial-LACS9 to the ER and complemented the dwarf, seed oil, and stem wax content phenotypes of the lacs4lacs9 double mutants. Besides, I also showed that the outer-envelope LACS9 and inner-envelope FAX1 could form a complex, which play roles during fatty acid export from the plastid, while the ER-LACS4 is not associate with FAX1 even it is functionally equivalent with LACS9 in such pathway.
THE MECHANISM FOR FATTY ACIDS TRAFFICKING FROM PLASTIDS TO THE ENDOPLASMIC RETICULUM by Mingzhe Liu B. Sc., Northwest A&F University, 2014 M. Sc., Northwest A&F University, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (BOTANY) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2021 © Mingzhe Liu, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: The mechanism for fatty acids trafficking from plastids to the endoplasmic reticulum Submitted by Mingzhe Liu in in partial fulfillment of the requirements for the degree of Master of Science in Botany Examining Committee: Dr. George W. Haughn, Professor, Botany, UBC Co-supervisor Dr. Ljerka. Kunst, Professor, Botany, UBC Co-supervisor Dr. Yuelin Zhang, Associate Professor, Botany, UBC Supervisory Committee Member Dr. Abel Rosado, Associate Professor, Botany, UBC Supervisory Committee Member Dr. Lacey Samuels, Professor, Botany, UBC Additional Examiner iii Abstract Fatty acids (FAs) are building blocks for different glycerolipids, including phospholipids and galactolipids that serve as major structural components of cell membranes, as well as triacylglycerols (TAGs), which are the main form of energy and carbon storage in plants . They are also precursors of the plant surface lipids: cutin, cuticular waxes, and suberin, which play roles during plant’s resistance to drought, pathogens, or insects. In plant cells, de novo fatty acid synthesis happens exclusively within plastids with the growing fatty acyl chain attached to the acyl carrier protein (ACP). Once completed, majority of these fatty acids are removed from ACP by thioesterases and re-attached to coenzyme A (CoA) under the activities of long-chain acyl-CoA synthetase (LACS) to make membrane lipids, storage or surface lipids in the ER, the major site for lipid assembly. Since the production of fatty acids and the production of complex lipids happen in different organelles, a transport system between plastids and other subcellular compartments is required in order to deliver FAs from the plastid to the ER. In Arabidopsis, neither the lacs9 single knock-out mutants which removed all plastidial LACS activities, nor the lacs4 single mutants which removed the activity of an ER LACS showed any visible phenotype while the lacs4lacs9 double mutants are dwarf, and with decreased seed oil and stem wax content. These not only suggest the functional overlap between the plastidial -LACS9 and ER-LACS4, but also suggest a proposed access for the ER-LACS to the fatty acids from the plastid envelopes. In my research, I have proved such access directly by re-targeting the plastidial-LACS9 to the ER and complemented the dwarf, seed oil and stem wax content phenotypes of the lacs4lacs9 double mutants. Besides, I also showed that the outer-envelope LACS9 and inner-envelope FAX1 could form a complex, which play roles during fatty acid export from the plastid, while the ER-LACS4 is not associate with FAX1 even it is functionally equivalent with LACS9 in such pathway. iv Lay summary My research provides a better understanding on how fatty acids are transported from their site of synthesis in the plastid to the ER where they are assembled into more complex lipids. By re-targeting a plastid localized enzyme, LACS9, to the ER and demonstrating that it can function normally, I provided evidence that LACS9 and ER-localized LACS4 enzymes are functionally redundant. However, I also showed that the LACS9 enzyme but not the LACS4 interacts with the plastid fatty acid transporter FAX1 demonstrating that the two enzymes are not biochemically identical. My results provide information on the enzymes involved in fatty acid trafficking between plastids and the ER and paves the way for potential applications in the genetic engineering of seed oil and cuticular wax in oilseed crops. v Preface The original idea to re-target the plastidial LACS enzyme to the ER was devised by Dr. George Haughn and Dr. Ljerka Kunst. The lacs4lacs9 double mutants used in this research were generated by Dr. Lifang Zhao, a former post-doctoral researcher in the Kunst laboratory. The experiments and data analysis in Chapter 3 were done by myself. Vectors of epitope tagged TGD proteins, including HA-TGD4 and TGD5-GFP used in co-immunoprecipitations in Chapter 4 were gifts from Dr. Changcheng Xu from Brookhaven National Laboratory, NY, USA. Assistance with the co-immunoprecipitation techniques was provided by Dr. Di Wu, a former member of Dr. Yuelin Zhang’s laboratory. The experiments and data analysis in Chapter 4 were done by myself. The fluorescence microscopy work described in this thesis was conducted using the Nikon C1 confocal laser scanning microscope from Dr. Abel Rosado’s lab and the Perkin-Elmer spinning-disk confocal microscope from the UBC bio-imaging (BIF) facility. Dr. Eunkyoung Lee helped with crossing Arabidopsis LACS8ss-LACS9(-cTP)-GFP-KKXX transgenic line 10 with the RFP-HDEL marker line. She also helped with generating the figures for the co-localization analysis of the LACS8ss-LACS9(-cTP)-GFP-KKXX recombinant protein (Figures 3.1A and 3.1B). vi Table of contents: Abstract ........................................................................................ iii Lay summary ................................................................................. iv Preface .......................................................................................... v Table of contents ........................................................................... vi List of tables .................................................................................. ix List of figures .................................................................................. x List of abbreviations....................................................................... xi Acknowledgements ...................................................................... xii Chapter 1: Introduction ...................................................................1 1.1 Fatty acids and lipids.......................................................................................................1 1.2 Arabidopsis as a model organism to study lipid metabolism ..............................................2 1.3 Fatty acid synthesis in plant cells .....................................................................................3 1.4 Proteins involved in fatty acid export from the plastid ......................................................6 1.4.1 Acyl-ACP thioesterases (FATs) .....................................................................................6 1.4.2 Fatty acid exporters (FAXs) ..........................................................................................8 1.4.3 Long chain Acyl-CoA synthetases (LACSs) ................................................................... 10 1.4.4 ATP-binding cassette transporter A9 (ABCA9)............................................................. 12 1.5 A possible mechanism for fatty acid transport from the plastid to the ER......................... 13 1.5.1 ER-localized LACS enzymes have access to the fatty acids synthesized in the plastid. .... 13 1.5.2 Membrane contact sites and their possible participation in fatty acid transfer between different cellular organelles................................................................................................... 14 1.5.3 TGD protein complex transfers glycerolipids between the ER and the plastid. .............. 16 1.6 Research objectives ...................................................................................................... 18 Chapter 2: Materials and methods. ................................................ 19 2.1 Nucleic acid preparation ............................................................................................... 19 2.1.1 Isolation of Arabidopsis genomic DNA ....................................................................... 19 vii 2.1.1.1 Quick genomic DNA preparation for genotyping ................................................. 19 2.1.1.2 Genomic DNA preparation for gene cloning ....................................................... 19 2.1.2 Isolation of Arabidopsis total RNA ............................................................................. 20 2.1.3 Plant cDNA preparation using RNA reverse transcription............................................. 21 2.2 Polymerase chain reaction (PCR) and vector construction ............................................... 22 2.2.1 Introduction of PCR .................................................................................................. 22 2.2.2 Primers used for PCR ................................................................................................ 24 2.2.3 Vector construction .................................................................................................. 25 2.2.3.1 Vector construction using digest-ligation ........................................................... 25 2.2.3.2 Vector construction using Gateway technology .................................................. 28 2.3 Bacterial transformation ............................................................................................... 30 2.3.1 Preparation and transformation of competent Escherichia coli and Agrobacterium tumefaciens cells .................................................................................................................. 30 2.3.2 Verification of bacterial colonies that contain desired vectors ..................................... 31 2.4 Plant transformation ..................................................................................................... 32 2.4.1 Plant growth conditions ............................................................................................ 32 2.4.2 Arabidopsis transformation using spray method ......................................................... 32 2.4.3 Screening for transgenic Arabidopsis in the T1 generation ........................................... 32 2.4.4 Crossing Arabidopsis plants ....................................................................................... 33 2.4.5 Nicotiana benthamiana infiltration ............................................................................ 33 2.5 Confocal microscopy..................................................................................................... 35 2.6 Analyses of seed oil and stem wax by gas chromatography (GC)...................................... 36 2.6.1 Single seed analysis .................................................................................................. 36 2.6.2 Analysis of seed batches ........................................................................................... 36 2.6.3 Arabidopsis stem wax analysis................................................................................... 37 2.7 Co-immunoprecipitation (Co-IP) analysis........................................................................ 38 Chapter 3: Retargeting of LACS9 from the chloroplast envelope to the ER results in complementation of the lacs4lacs9 double mutant ...... 40 3.1 Introduction ................................................................................................................. 40 3.2 Results ......................................................................................................................... 42 viii 3.2.1 Substitution of the N-terminal plastidial transit peptide (tp) with ER signal sequence (ss) results in re-targeting of LACS9 from plastid to the ER ............................................................ 42 3.2.2 The ER targeted LACS9 protein is able to rescue the mutant phenotype of lacs4lacs9 double mutant ..................................................................................................................... 45 3.3 Discussion .................................................................................................................... 49 3.3.1 Roles of plastid-localized LACS9 and ER-localized LACS4 in fatty acid transport from the plastid to the ER ................................................................................................................... 49 Chapter 4: Plastidial-LACS9, but not ER-LACS4, interacts with FAX1 in planta .......................................................................................... 52 4.1 Introduction ................................................................................................................. 52 4.2 Results ......................................................................................................................... 53 4.2.1 The iEM-localized FAX1 and oEM-localized LACS9 interact .............................................. 53 4.2.2 The iEM localized FAX1 and ER localized LACS4 do not interact ....................................... 55 4.3 Discussion .................................................................................................................... 57 Chapter 5: Conclusions and future directions .................................. 59 5.1 Conclusions from this research ...................................................................................... 59 5.1.1 ER-localized LACS enzymes have access to the fatty acids from the plastid envelope membranes .......................................................................................................................... 59 5.1.2 The iEM-localized FAX1 interacts with oEM-localized LACS9, but not ER-localized LACS4, to form a complex ................................................................................................................ 60 5.2 Future directions for this research ................................................................................. 61 References ................................................................................... 62 ix List of tables Table 2-1: PCR procedure ............................................................................................................. 22 Table 2-2: PCR mixture for genotyping using Taq polymerase..................................................... 22 Table 2-3: PCR mixture for cloning using Phusion polymerase .................................................... 23 Table 2-4: Primer sequences for cloning ...................................................................................... 24 Table 2-5: Primer sequences for sequencing and genotyping ..................................................... 25 Table 2-6: Reagents used for restriction digestion ....................................................................... 27 Table 2-7: Reagents used for ligation ........................................................................................... 27 Table 2-8: Reagents used for BP-reaction .................................................................................... 29 Table 2-9: Reagents used for LR-reaction ..................................................................................... 29 Table 3-1: Summary of characterized lacs mutants of Arabidopsis with detectable phenotypes 41 x List of figures Figure 1.1 Simplified schematic of the flow of carbon through the fatty acid and lipid metabolism in a generalized plant cell ............................................................................................................... 4 Figure 1.2 Schematic representation of fatty acid synthesis.......................................................... 5 Figure 1.3 Simplified schematic of the FAS and FAT functions ....................................................... 7 Figure 1.4 Schematic of lipid biosynthesis in WT, fax1, and FAX1-OX ........................................... 9 Figure 1.5 Model of the TGD complex .......................................................................................... 16 Figure 2.1 The construct used to complement the Arabidopsis lacs4-2lacs9-2 mutants ............ 26 Figure 2.2 Constructs used for Co-IP............................................................................................. 28 Figure 3.1: The subcellular localization of the native and ER-retargeted LACS9-GFPs ................ 44 Figure 3.2: Comple me ntation of the dwarf phe notype of the lacs4lacs9 double mutant by the LACS8ss-LACS9(-cTP)-GFP-KKXX.................................................................................................... 46 Figure 3.3: Comple me ntation of the lipid-related phenotypes of the lacs4lacs9 double mutant by the LACS8ss-LACS9(-cTP)-GFP-KKXX ............................................................................................. 47 Figure 3.4:Co-segregation analysis using the T2 generation of LACS8ss-LACS9(-cTP)-GFP-KKXX transformed lacs4lacs9 double mutants ...................................................................................... 48 Figure 3.5: Proposed diagram showing the distribution of LACS proteins between plastid and ER and their hypothetical topology. .................................................................................................. 50 Figure 4.1: Co-IP analysis of FAX1-GFP and LACS9-HA. ................................................................ 54 Figure 4.2: Co-IP analysis of FAX1-GFP and LACS4-HA. ................................................................ 56 Figure 4.3: Proposed diagram showing the Fatty acids made by the plastids transported by different complex.......................................................................................................................... 58 Figure 5.1: Proposed diagram showing the possible access of ER re-targetted LACS9 to the fatty acids from the plastid envelope membranes. .............................................................................. 60 xi List of abbreviations ACP acyl carrier protein DAG diacylglycerol DGAT diacylglycerol acyltransferase DGDG digalactosyldiacylglycerol DPA days post anthesis E.coli Escherichia coli EAR enoyl-ACP reductase ER endoplasmic reticulum FAD fatty acid desaturase FAE fatty acid elongation FAME fatty acid methyl ester FAS fatty acid synthase GC gas chromatograph GFP green fluorescent protein HAD hydroxyacyl-ACP dehydratase KAR 3-ketoacyl-ACP reductase KAS β-ketoacyl-ACP synthase KCS β-ketoacyl-CoA synthase LACS long-chain acyl-CoA synthetase MGDG monogalactosyldiacylglycerol PA phosphatidic acid PCR polymerase chain reaction RFP red fluorescent protein TAG triacylglycerol UTR untranslated region VLCFA very long chain fatty acid xii Acknowledgements First and foremost, I would like to thank my supervisors, Dr. George Haughn and Dr. Ljerka Kunst for giving me the space, time and advice needed to complete my research. Thank you as well to my committee members Dr. Abel Rosado and Dr. Yuelin Zhang for all the helpful feedback and comments during the committee meetings. Thank you to my external examiner Dr. Lacey Samuels, for the all the useful feedback and constructive criticism on my thesis during the defence. Lab work was the core of my experience as a graduate student here at UBC. Thank you to Dr. Gillian Dean, Dr. Tegan Haslam, Dr. Lifang Zhao, Dr. Yi-Chen Lee, Dr. Kresimir Sola, Dr. Robert McGee, Dr. Shuang Liu and Mr. Lewis Kurschner for their help and support during my project. My thanks also goes to Dr. Miranda Meents, and Dr. EunKyoung Lee for teaching me microscopy techniques and Dr. Di Wu for showing me co-immunoprecipitation techniques. All of this assistance during my research helped me to become a better scientist. Last but not least, thank you to my family members, especially my mom, who has been stuck here in Canada due to the pandemic. Your love and encouragements always keep me going without burdens. 1 Chapter 1: Introduction 1.1 Fatty acids and lipids Fatty acids are organic molecules that consist of a linear or branched aliphatic chain with usually even numbers of carbon atoms, and at least one carboxyl group on their end. They can be saturated and contain only single bonds between carbon atoms, or unsaturated, in which case they contain one or more double bonds between carbon atoms. Higher plants, like other organisms, use fatty acids as building blocks for a variety of complex lipids, including membrane glycerolipids and sphingolipids, storage triacylglycerols, and surface lipids (Somerville and Browse, 1991; Ohlrogge and Browse, 1995). Glycerolipids are generated by condensation of one, two or three fatty acids to the glycerol backbone. There are several types of glycerolipids distinguished by the type of molecule attached to the stereospecific numbering-3 (sn-3) position of the glycerol. If a phosphate group is present in this position, such molecule will be categorized as a phospholipid. Phospholipids are of utmost importance for membrane construction (Millar et al., 2001; Simons and Vaz, 2004; Furt et al., 2011), but also in signal transduction (Munnik 2001; Lim et al., 2017). If the sn-3 position is occupied by a sugar, then such a molecule will be categorized as a glycolipid. Glycolipids are abundant in plant photosynthetic membranes and essential for their function (Williams, 1998; Bahatyrova et al., 2004; LaBrant et al., 2018). They also play important roles in plant cell recognition and signal transduction (Hakomori and Igarashi 1995). Besides phospholipids and glycolipids, there are triacylglycerols with three acyl chains attached to their glycerol backbones. The most important role for plant triacylglycerols is energy storage, and as such they are commonly found in fruit and seeds (Stymne and Stobart 1987; Cox and Nelson, 2008; Fahy et al., 2011; Markham et al., 2006). However, triacylglycerols also play important roles in plant vegetative tissues in aspects of cellular energy balance, lipid homeostasis, growth, and maintenance (Xu and Shanklin 2016). Additionally, fatty acids are precursors of the plant surface lipids such as cutin and cuticular waxes, which form a barrier on the outer surfaces of epidermal cells of all aerial organs 2 known as the cuticle. The cuticle prevents excessive non-stomatal water loss and the entry of harmful substances and pathogens into plant cells. Similarly, fatty acids are also precursors of suberin, which forms a barrier in root cell walls, controll ing water or nutrient transport within the root as well as protecting roots from pathogen invasion (Kunst and Samuels, 2003; Franke and Schreiber, 2007; Watanabe et al., 2013). Given the diversity of fatty acid uses in plant cells, a comprehensive understanding of fatty acid metabolism and cellular trafficking is needed to assist with engineering of plants to produce higher agricultural yields and/or have better resistance to drought, pathogens, or insects. 1.2 Arabidopsis as a model organism to study lipid metabolism A small herbaceous plant, Arabidopsis thaliana (mouse-ear cress), which grows widely in moderate climate zones of the northern hemisphere, has not only become one of the most successfully established model organisms for plant research, but also one of the best characterized models for multicellular organisms in general (Koornneef and Meinke, 2010). There are several factors that contribute to the wide usage of Arabidopsis as a model for plant research. First, it reaches a maximal height of 30-35 cm; therefore hundreds of plants can be grown in a small amount of space. Second, it has a short life cycle of 6 - 8 weeks and at maturity produces more than 1000 seeds. Third, the breeding of Arabidopsis is very convenient because of its self-pollination. Hence, its fertilization is not reliant on exogenous factors like wind or insects. Last, as a species of the Brassicaceae family, it is closely related to Brassicaceae crops like Brassica napus (Oilseed rape) or Sinapis alba (White mustard) which have high economic values. Beside the biological aspects listed above, Arabidopsis also has many attributes which make it an excellent species for molecular genetics. It has a small genome of only 135 mega base pairs (Mbp) (Jiao and Schneeberger, 2020), which makes genetic studies much simpler compared to Brassica napus (2.4 giga base pairs, Gbp) or Triticum aestivum (16 Gbp). Its genome was completely sequenced in 2000 (Kaul et al, 2000) and to date, 27,000 genes have been annotated and 35,000 proteins (including splice variants) have been identified (Koornneef and Meinke, 2010; 3 Cheng et al., 2017). Furthermore, many useful mutants have been identified and studied in Arabidopsis and genetic transformation is well established, with the most commonly used technique being the floral-dip method with Agrobacterium tumefaciens (Clough and Bent, 1998). Arabidopsis has a high seed oil content (30%-35%) and has been used extensively for the genetic analysis of plant lipid metabolism and transport making it a useful tool for further studies in this area (Li et al., 2006). 1.3 Fatty acid synthesis in plant cells The synthesis of plant fatty acids, which happens primarily within plastids, and to a small extent in mitochondria (Hiltunen et al., 2010), provides building blocks for more complex lipids including glycerolipids, sphingolipids, as well as plant surface lipids (Fig 1.1). Fatty acid synthesis process is initiated by the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACCase) (Konishi et al., 1996). Malonyl-CoA:ACP transacylase then transfers the malonyl group from CoA-SH to ACP-SH to form malonyl-ACP. Malonyl-ACP is used as the carbon donor for fatty acid extension reactions. The extension of the fatty acid carbon chain is achieved by sequential addition of two-carbon units from the carbon donor to the acyl-ACP by the fatty acid synthase (FAS), a protein complex that consists of four subunits with distinct enzymatic activities (Fig 1.2) (Ohlrogge and Jaworski, 1997; Brown et al., 2006; Haslam and Kunst, 2013; Haslam, 2017). The key enzyme of FAS is 3-ketoacyl-ACP synthase (KAS), which catalyzes the condensation of an acyl-ACP with malonyl-ACP and results in irreversible C-C bond formation with release of CO2. Three types of KAS enzymes are known in the plant cell. KASIII catalyzes carbon chain elongation from C2 to C4, KAS I is responsible for the synthesis of acyl chains from C4 to C16, whereas KASII catalyzes elongation from C16 to C18 (Fig 1.3) (Shimakata and Stumpf, 1982; Clough et al., 1992). 4 Figure 1.1 Simplified schematic of the flow of carbon through the fatty acid and lipid metabolism in a generalized plant cell (ACCase: acetyl-CoA carboxylase; KAS: 3-ketoacyl-ACP synthase; VLCFA: very long chain fatty acid; PA: phosphatidic acid; ER: endoplasmic reticulum; PEX: peroxisome) (modified from Ohlrogge and Jaworski, 1997). Even though KAS enzymes play major roles in acyl chain extension, three additional enzymes are required for the formation of a fully reduced acyl-ACP. These include two reductases and one dehydratase. The first reductase is a 3-ketoacyl-ACP reductase (KAR) that converts 3-ketoacyl-ACP to 3-hydroxyacyl-ACP. The 3-hydroxyacyl-ACP is then dehydrated by a hydroxyacyl-ACP dehydratase (HAD), leading to the formation of an enoyl-ACP. The final step is the reduction of the enoyl-ACP, which is achieved by the second reductase, an enoyl-ACP reductase (EAR), using NADH or NADPH as a reductant to form the saturated fatty acid with two more carbons (Mou et 5 al., 2000; Li-Beisson et al., 2013). The four reactions mentioned above constitute a cycle of fatty acid elongation (Fig 1.2). Figure 1.2 Schematic representation of fatty acid synthesis (KAS: 3-ketoacyl-ACP synthase; KAR: 3-ketoacyl-ACP reductase; HAD: hydroxyacyl-ACP dehydratase; EAR: enoyl-ACP reductase; NADP+/NADPH: Oxidized/Reduced nicotinamide adenine dinucleotide phosphate) (Modified from Haslam, 2017). When the acyl-ACP chains reach 16 or 18-carbon-length, they can either be directly used by plastid localized acyltransferases for the synthesis of prokaryotic-like membrane glycerolipids within the plastid, characterized by a C16 acyl-chain on the sn-2 position of the glycerol backbone, or desaturated to C18:1 by stearoyl-ACP desaturase (SAD) (Shanklin and Cahoon, 1998) (Fig 1.2). They can also be removed from the ACP by enzymes called acyl-ACP thioesterases (FATs), discussed in more detail in the next section, and exported from the plastid to the ER. In the ER, they are used for the production of membrane and storage glycerolipids characterized by a C18 acyl-chain on the sn-2 position of the glycerol backbone, commonly referred to as the eukaryotic-lipid pathway. Some of these lipids are also returned to the plastid and used for the production of galactolipids for thylakoid membrane construction. The fatty acids which are exported to the 6 ER could also be elongated within the ER to make cuticular waxes in epidermal cells, or suberin (Li-Besson et al., 2013). 1.4 Proteins involved in fatty acid export from the plastid 1.4.1 Acyl-ACP thioesterases (FATs) As mentioned above, fatty acyl chains destined for export from the plastid will be hydrolyzed from the ACP by enzymes called fatty acyl-ACP thioesterases (FATs). Two types of FAT enzymes have been identified plants: FATA and FATB. In A. thaliana, they are encoded by two FATA genes (FATA1: AT3G25110 and FATA2: AT4G13050) and one FATB gene (AT1G08510), respectively (Salas and Ohlrogge, 2002). The FATA and FATB enzymes show different acyl-ACP substrate preferences. Both FATAs have the highest in vitro activity with C18:1-ACP as a substrate but much lower activity with either C16:0-ACP or C18:0-ACP as substrates (Fig 1.3) (Doermann et al., 1995). The FATB prefers saturated acyl groups such as C16:0-ACP or C18:0-ACP, but also exhibits activity with unsaturated acyl-ACPs, such as C18:1-ACP (Voelker et al., 1997; Bonaventure et al., 2003). While controlling production of either unsaturated or saturated fatty acids, FATA and FATB play different roles in the life of a plant. Their specific roles were investigated by analysis of mutants in corresponding genes (Bonaventure et al., 2003; Moreno-Pérez et al., 2012; Siebers 2016). Thus, the knock-down of Arabidopsis FATA1 and FATA2 genes generated by inserting T-DNA in the 5’ UTR region of these two genes resulted in a decrease of C18:1/C18:2 seed fatty acids and a proportional decrease in seed oil content (Moreno-Pérez et al., 2012). However, the essential role of FATA1 and FATA2 genes during Arabidopsis seed embryo development was not determined until the T-DNA insertional knock-out mutants, fata1 and fata2 had been identified. Whereas single fata1 or fata2 mutants were indistinguishable from the wild-type, a fata1fata2 double mutant resulted in the abortion of the embryo between late heart and early torpedo stage (Siebers 2016). Seed embryo abortion was also not observed in the knock-down mutants likely due to residual expression of the FATA1 and FATA2 genes (Moreno-Pérez et al., 2012). In contrast, the knock-out of FATB gene in Arabidopsis caused a dwarf phenotype as well as 7 compromised seed development, seed germination, and stem cuticular wax deficiency (Bonaventure et al., 2003). Figure 1.3 Simplified schematic of the FAS and FAT functions. Each FAS cycle contains four enzymes are shown on the left. The different KAS enzymes which recognize a specific chain length of acyl-ACPs are highlighted in red. 8 1.4.2 Fatty acid exporters (FAXs) After their release from the ACP by FATs in the plastid stroma, free fatty acids (FFAs) need to cross the two envelope membranes on their way from the plastid to the ER. Due to their hydrophobic nature, The C16 and C18 FFAs will not diffuse in the aqueous phase of a plant cell. In contrast, the FFAs will be dissolved in the lipid membrane due to the ‘like dissolves like’ principle. However, large amounts of FFAs in a membrane will act like a detergent that will destroy the membrane itself (Henriksen et al., 2010). Thus, protein-assisted transport has been proposed as a plausible option (Koo et al., 2004; Fan et al., 2015). Recent discovery of the FATTY ACID EXPORTER1 (FAX1) (Li et al., 2015a) suggest that this may indeed be the case. The FAX1 is one of the seven members of the TMEM-14C (Trans-membrane protein 14C) family in Arabidopsis (Li, 2012). It is an integral membrane protein with four predicted α-helixes and found to localize to the inner envelope membrane (iEM) of the chloroplast. A yeast complementation assay suggests that this protein is able to transport fatty acids into yeast cells (Li et al., 2015). Detailed analysis of the lipids in the leaves and flowers of the Arabidopsis T-DNA insertional fax1 mutants showed that a total of 190 lipid species were significantly changed compared to the wild-type. For example, glycerolipids generated by the eukaryotic pathway in the ER showed an obvious decrease in the fax1 mutant. Additionally, fax1 mutants also exhibited a dramatically reduced seed oil content (as much as 8-fold) (Li et al., 2015; Roberts, 2015). By contrast, FAX1-overexpression lines showed significant increases in both their membrane glycerolipid content as well as seed oil accumulation (Tian et al., 2018) This evidence suggests that FAX1 plays an important role in fatty acid export from the plastid (Fig 1.4). 9 Figure 1.4 Schematic of lipid biosynthesis and transport in WT, fax1, and FAX1-OX. Plastid membrane lipids generated by the prokaryotic pathway of glycerolipid synthesis within the plastid are indicated in red, whereas those generated by the eukaryotic pathway in the ER are indicated in blue (Adapted from Li et al., 2015). Developmentally, the fax1 mutant showed a dwarf phenotype (Li et al., 2015), as well as a decrease in the number of pollen grains released from its anthers compared to wild type (WT). An examination of the structural features of fax1 pollen indicated that the pollen exine failed to form. Pollen exine consists of lipid-rich sporopollenin and tryphine made by the tapetal cells of the anthers, using fatty acids as substrates (Li, 2012). Thus, such a phenotype is consistent with a defect in fatty acid delivery from the plastid to the ER. In addition, the stem wax ketone content is decreased in the fax1mutant while the total wax load remains unchanged. Besides FAX1, there are six additional members of the FAX family in Arabidopsis. Among them, the FAX1-FAX4 have predicted chloroplast transit peptides, while the FAX5-FAX7 do not. Experimental characterization of FAX2 and FAX4 demonstrated that they localize to the chloroplast envelope membranes. Compared to fax1, single fax2 or fax4 mutants do not show a decrease in seed oil content, whereas fax2fax4 double mutant does. Thus, there is a functional overlap between FAX2 and FAX4. Similar to FAX1 overexpressors, the FAX2 and FAX4 over-expressors showed an elevated seed oil content, suggesting that, in addition to FAX1, FAX2 and FAX4 also contribute to seed oil production. However, in contrast to FAX1, FAX2 and FAX4 are expressed exclusively in the seed embryo during seed development. (Tian et al., 2019; Li et al., 2020). 10 1.4.3 Long chain Acyl-CoA synthetases (LACSs) After FAX1-mediated transport across the iEM, fatty acids need to cross the aqueous chloroplast intermembrane space to be activated to fatty acyl-CoAs by long chain acyl-CoA synthetases (LACSs) on the plastid oEM. It is not clear how fatty acids reach the oEM, but it may be that FAXs serve as hydrophobic channels to deliver fatty acids to the oEM LACS for activation. Activation of fatty acids to fatty acyl-CoAs is necessary, because fatty acyl-CoAs are used as substrates for the synthesis of all complex lipids produced in the ER. The fatty acyl-CoAs are made by the following reaction catalyzed by a LACS enzyme: free fatty acid + CoA+ATPLACS→ fatty acyl − CoA+AMP Nine LACS enzymes have been identified in the Arabidopsis genome, and all of them use both saturated and unsaturated C16/18 fatty acids (Shockey et al., 2002). In addition, LACS6 and LACS7 showed high specificity for the seed-specific C20:1 fatty acid substrate (Fulda et al., 2004), whereas LACS1 showed a high specificity for C30 fatty acid substrates (Lü et al., 2009; Weng et al., 2010). The tissue expression profile of the nine LACS genes was also determined by RT-PCR and GUS staining. The result showed that the flower is the tissue that all LACSs are highly expressed, and it is the only organ where LACS5 is expressed. Seed is the tissue where LACS1/2/4/8/9 are expressed. Such pattern indicating functional importance of these five LACS enzymes in the seed during development and during germination. Stem, where the wax protection is needed, is the tissue that LACS1 is highly expressed. The substrate preference for C30 fatty acids of LACS1 has been demonstrated to be important for cuticular wax production as the Arabidopsis lacs1 mutant displays stem wax deficiency (Shockey et al., 2002; Schnurr et al., 2002; Lü et al., 2009). Of the nine LACSs in Arabidopsis, only LACS9 localizes to the plastid (Andrews and Keegstra, 1983; Shockey et al., 2002; Schnurr et al., 2002). Thus, it is reasonable to hypothesize that loss of function of LACS9 would severely compromise plastidial acyl–CoA production and 11 have a considerable effect on downstream plant lipid metabolism. Surprisingly, the lacs9-ko showed a wild-type phenotype, suggesting that other LACS enzymes may be able substitute for the loss of LASC9 activity (Jessen et al., 2015). Among the other eight LACS proteins, LACS1/2/4/8 have been shown to localize to the ER membrane (Weng et al., 2010; Zhao et al., 2010; Jessen et al., 2011), and LACS6/7 have been shown to localize to the peroxisome (Fulda et al., 2004). The localization of LACS3 and LACS5 have not been experimentally determined, but they are predicted to be ER-localized (Kalinger et al., 2020). Creation of double and triple lacs mutants revealed that many LACSs enzymes have overlapping functions in seed oil and cuticular wax biosynthesis, as well as in pollen coat formation. For example, analyses of lacs4 and lacs9 single mutants and lacs4lacs9 double mutant demonstrated that LACS4 and LACS9 are functionally equivalent (Jessen et al., 2015; Zhao et al., 2019). This result makes it reasonable to hypothesize that the ER-localized LACS4 has access to the fatty acids generated in the plastid and together contribute to the production of acyl-CoAs required for seed oil production. Similarly, examination of the lacs1lacs9 double mutant indicated functional redundancy of the plastidial LACS9 and ER-localized LACS1 in seed oil biosynthesis (Zhao et al. 2010). In addition, LACS4 and LACS9, as well as ER-localized LACS1, LACS2 and LACS4 have overlapping functions in Arabidopsis stem wax production (Zhao et al., 2019), whereas LACS1 and LACS4 enzymes also participate in the production of extracellular pollen coat lipids (Jessen et al., 2011). Research on peroxisomal LACS6/7 enzymes revealed that single lacs6 or lacs7 mutants were phenotypically indistinguishable from the wild type in various aspects of seed development, germination and growth. By contrast, the double lacs6lacs7 mutant showed a distinct seedling developmental phenotype after germination. The addition of exogenous sucrose was essential for lacs6lacs seedling establishment, similar to a mutant that lacks a peroxisomal ABC transporter and leads to β-oxidation deficiency (Fulda et al., 2004). 12 1.4.4 ATP-binding cassette transporter A9 (ABCA9) The plastid is the major site of the de novo synthesis fatty acids in plant cells. As mentioned above, some of these fatty acids are incorporated in the glycerolipids within the plastid, whereas depending on the plant species and tissue, 60-90% of the fatty acids synthesized by the plastid will be exported to the ER to be used for the production of glycerolipids, sphingolipids and surface lipids (Browse and Somerville, 1991). As discussed above, loss-of-function mutations in FAX and LACS proteins in Arabidopsis resulted in substantial reduction in seed TAG accumulation, an indication of their involvement in providing fatty acyl-CoAs for oil production. In addition, one of the Arabidopsis ATP-binding cassette (ABC) transporters, the ER-localized ABCA9, has been shown to be required in this process. (Kim et al., 2013). The abca9-ko lines showed, on average, a 35% seed oil content decrease, whereas ABCA9 overexpressors (OE) displayed a 40% increase in seed oil content. Seedlings of three independent abca9 alleles also showed growth retardation compared to the wild type on a medium without sucrose. Thus, ABCA9 plays a significant role in fatty acid transport to the ER necessary for TAG formation which supplies energy for Arabidopsis early seedling growth. Another noteworthy piece of information from AtABCA9 research is that even though the seed oil content of three OE lines was significantly increased, no change in fatty acid composition was observed. These data suggest that ABCA9 does not impact lipid assembly which happens downstream of fatty acids transport and that ABCA9 is not substrate specific when transporting different fatty acids. 13 1.5 A possible mechanism for fatty acid transport from the plastid to the ER Taking into consideration all the information discussed above, it is clear that an effic ient mechanism for the delivery of fatty acids between their site of synthesis in the plastid and the site of their utilization for the production of complex membrane and storage lipids and surface lipids in the ER must exist. It is also becoming increasingly obvious that this mechanism involves membrane proteins including the FAX family (FAX1/2/4) localized to iEM of the plastid, LACS9 localized to the oEM, ER localized LACS proteins (LACS1/2/4/8), as well as ABCA9 also localized to the ER membrane. However, even though proteins that participate in fatty acid transport from the plastid to the ER have been identified, it has not been determined if and how these proteins work together to accomplish this task. One possible mechanism is that they associate into complexes that result in membrane contact sites which provide a physical path for fatty acid movement between organelles. 1.5.1 ER-localized LACS enzymes have access to the fatty acids synthesized in the plastid. The notion that a contact site between the ER and the plastid may be involved in transorganellar movement of fatty acids is based on genetic evidence that ER-localized LACS4 can substitute for plastid-localized LACS9 and carry out fatty acid activation to acyl-CoAs required for their utilization in the ER in a loss-of-function mutant lacs9 (Jessen et al., 2015). Neither lacs4 nor lacs9 single mutants showed a detectable growth phenotype, reduced seed oil yield, or stem wax load, whereas lacs4lacs9 double mutants did. This result indicated that only one of these two LACS enzymes is necessary for fatty acid activation regardless of their different cellular localizations. Additional evidence for functional redundancy between the plastid and ER-localized LACS enzymes was provided by Zhao et al. (2011) who demonstrated that LACS1 found in the ER can also substitute for the deficiency of the plastidial enzyme LACS9. 14 The existence of transorganellar complementation has previously been documented in studies of tocopherol and carotenoid metabolism in Arabidopsis (Mehrshahi et al., 2013). In this study, wild-type enzymes for tocopherol and carotenoid biosynthesis, which are normally found in the plastid envelope membranes, could functionally complement the loss of function mutations in the endogenous genes even when they were re-targeted to the ER. Such a result suggests that the enzyme localized to the ER membrane must be able to access their corresponding substrates from the plastid. It is conceivable that similar to tocopherol and carotenoid metabolic enzymes, ER-resident fatty acid enzymes such as LACS1 or LACS4 can also access their fatty acid substrates in the plastid. 1.5.2 Membrane contact sites and their possible participation in fatty acid transfer between different cellular organelles. As suggested above, transient formation of membrane contact sites between the iEM and the oEM of the plastid and between the plastid oEM and the ER would create a physical path for transorganellar substrate exchange. Membrane contact sites are areas where two membranes are brought and held together in close proximity (in a range between 10~30 nm) by tethering or by protein complexes, but without any fusion between two membranes. Such contact sites have been shown to be involved in ion transport, signal transduction, or harbour a specific enzyme within the site (Prinz, 2014; Pérez-Sancho et al., 2016; Scorrano et al., 2019). Functional studies in yeast have highlighted the importance of contact sites between the plasma membrane (PM) and the ER for small molecule transfer, as well as signaling between the PM and the ER. For example, the yeast oxysterol-binding homology (Osh) proteins, that are localized to the PM-ER contact sites, are required to facilitate yeast Sac1 phosphoinositide (PI) phosphatases to adjust the PI4P levels in yeast PM (Stefan et al., 2011). In addition, the Osh3 protein, also regulates the synthesis of phosphatidylcholine (PC) by adjusting the activity of the phosphatidyl-ethanolamine N-methyltransferase enzyme, Opi3 (Tavasolli et al., 2013). Apparent membrane connections between the ER and other plant organelles have been captured by TEM studies (Whatley, 1993) and freeze-fracture SEM (McLean et al., 1988; Whatley, 15 1991). More recently, optical manipulation of plant organelles has added more evidence for the existence of stable, physical contact sites between the ER and plastidial envelopes (Andersson et al., 2007). It has also been reported that protein-protein interactions play a crucial role in maintaining plastid-associated-membranes (PLAMs) between plastids and the ER but to date, the function of PLAMs has not been elucidated (Andersson et al., 2007). Research in plants also demonstrated that proteins which facilitate lipid transfer between the plastidial iEM and oEM or between plastids and mitochondria act at membrane contact sites (Kelly et al., 2016; Michaud et al., 2016). The galactolipids monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG) are predominant structural constituents of the chloroplast thylakoid membranes. They are synthesized in the plastid envelope membranes by the galactolipid synthases MGD1 and DGD1. MGD1, localized to the iEM, is responsible for catalyzing galactosyl-transfer biosynthetic reactions that produce MGDG while DGD1 in the oEM, catalyzes the subsequent step that adds one more galactosyl group to MGDG to make DGDG. Thus, inter-membrane lipid transfer between plastid iEM and oEM is required for DGDG synthesis. Protein domain-switching experiments were conducted to show that the N-terminus of DGD1 is required for galactolipid translocation between plastid iEM and oEM possibly by mediating phosphatidic acid (PA)-dependent membrane fusion (Kelly et al., 2016). It has been shown that during phosphate starvation plastids are able to transfer DGDG to mitochondria (Jouhet et al., 2004). Clear-Native PAGE (CN-PAGE) based proteomics analysis suggested that a large lipid-enriched complex called the mitochondrial transmembrane lipoprotein (MTL) complex, which consists of proteins located in the two mitochondrial membranes as well as plastid envelope membranes, participates in lipid transfer between mitochondria and the plastids. A mitochondrial protein named AtMic60 played a key role as a member of the complex during the lipid transfer between plastids and mitochondria, probably through acting on membrane contact sites. Such a role was suggested by a significant increase of PE within mitochondrial lipids, correlated with a decrease of DGDG import into mitochondria in the absence of AtMic60. Plastid iEM proteins AtFAX1, AtFAX2, and the oEM protein AtLACS9 were also identified as members of the complex. Although this MTL complex participates in PE and 16 DGDG transfer between plastids and mitochondria, the authors suggest a possibility that proteins from both iEM and oEM could also form a complex with the ER (Michaud et al., 2016). 1.5.3 TGD protein complex transfers glycerolipids between the ER and the plastid. The best understood case of transorganellar lipid movement in plant cells involves the TGD complex-mediated transfer of glycerolipids, likely phosphatidic acid, from the ER, across the oEM and the inter-membrane space (IMS) to the stromal side of the plastid iEM (Fig 1.5). The TGD complex consists of five subunits, TGD1-5 (Xu et al., 2003; Awai et al., 2006; Lu et al., 2007; Xu et al., 2008, Fan et al., 2015). Experimental evidence suggests that the TGD5 subunit serves as the “bridge” between oEM-localized TGD4 and the iEM-localized TGD1, TGD2, and TGD3 through protein-protein interactions. Furthermore, a portion of the oEM-localized TGD4 is proposed to be associated with the ER-oEM contact site thus allowing lipid transfer from the ER to the plastid oEM (Wang et al., 2012). The energy required to transfer ER-derived lipid molecules across the membranes is presumably provided by TGD3-mediated ATP hydrolysis inside the plastid. Thus, this protein complex likely serves as a hydrophobic channel for the ER to plastid lipid transfer. Figure 1.5 Model of the TGD complex. Numbers correspond to TGD1-TGD5 proteins. The dimeric composition is shown for TGD1, TGD3, TGD4, and TGD5 and octameric for TGD2 (Adapted from Fan et al., 2015). 17 Although the TGD complex currently represents the best working model for explaining the mechanism of lipid transfer between the ER and the plastid, it is clear that the mechanism for fatty acid transfer from the plastid to the ER must be distinct from that of the TGD model for two reasons. First, the transported substrate are free fatty acids rather than a complex lipid like PA. Second, the fatty acid delivery is in the opposite direction from the TGD-complex, i.e. from the plastid to the ER. 18 1.6 Research objectives As explained above, fatty acids are made in the plastid by the FAS with the ACP as a carrier. When completed, they are removed from the ACP by a FAT and attached to CoA by the LACS9 on the plastid oEM before being moved to the ER where they are used for the biosynthesis of diverse cellular lipids. How fatty acids are transported between the plastid and the ER is not known. Because loss-of-function mutants of LACS9, the sole plastidial LACS, showed no detectable phenotype, it has been suggested that other members of the LACS family localized on the ER may have access to the fatty acids exiting the plastid and generate acyl-CoAs necessary for the downstream lipid metabolic reactions. The functional redundancy between plastidial -LACS9 and ER-LACS1 and LACS4, provided genetic evidence that ER-localized LACS enzymes’ have access to the fatty acids produced by plastids. The long-term objective of my research was to elucidate for the involvement of FAX1 and LACS enzymes in trafficking of fatty acids from the plastid to the ER. In specific, I hypothesize that the FAX1 and LACS9 are forming a complex in order to help fatty acids go across the plastid iEM and oEM. And the LACS enzymes in the ER have access to the fatty acids from the plastid envelope membranes. For my thesis research I addressed the following specific questions: 1. Do the ER-resident LACS enzymes have access to fatty acids in the plastid (Chapter 3)? 2. Do FAX1 and LACS9 form a complex in planta (Chapter 4)? 3. Is ER-localized LACS4 able to associate with FAX1 (Chapter 4)? The complementation of lacs4lacs9 by re-targeting the plastidial-LACS9 to the ER membrane would provide genetic evidence for question 1. While the co-immunoprecipitation would provide evidence for the protein interaction between FAX1/LACS9 and the association between FAX1/LACS4. 19 Chapter 2: Materials and methods 2.1 Nucleic acid preparation 2.1.1 Isolation of Arabidopsis genomic DNA 2.1.1.1 Quick genomic DNA preparation for genotyping In order to prepare DNA samples for genotyping, one cauline leaf from 2~3-week-old plants was pressed through a piece of parafilm using a glass tube to make a print onto the FTA card surface (GE Healthcare, 2010). One disc from each leaf sample was excised from the FTA card using a paper puncher and placed into a PCR tube. The paper puncher was cleaned between samples with a Kimi-wipe moistened with 70% EtOH. The excised disc was soaked in the 50 µL FTA reagent (10 mM Tris pH=7.5; 2 mM EDTA; 0.1% Tween-20) for 5 min, then rinsed with 150 µL of TE-1 (10 mM Tris, pH=8.0; 0.1 mM EDTA) for 5 min. The second rinse was accomplished using another 50 µL TE-1 and soaked for another 5 min. 2.1.1.2 Genomic DNA preparation for gene cloning To obtain high quality genomic DNA for gene cloning, young rosette leaves from 2~3-week-old plants were used for DNA extraction by the CTAB (cetyl-trimethyl-ammonium bromide) method. For each 100 mg of leaf tissue, 500 µl of CTAB Extraction Buffer (2% CTAB, 1% polyvinyl pyrrolidone, 100 mM, Tris-HCl; 1.4 M, NaCl; 20 mM, EDTA) was applied. The tissue was mixed and vortexed thoroughly, then heated at 65 °C for 20 min using a heat block. The mixture was then centrifuged at 12,000g for 5 min. The supernatant was transferred to a new tube and an equal volume of chloroform/isoamyl alcohol (24:1) was added to denature all the protein in the sample. The tube was vortexed for 5 sec then centrifuged at 12,000g for 1 min to separate the phases. The aqueous phase on the top was transferred to a new tube. The genomic DNA was then precipitated by adding 0.7 x volume of cold isopropanol and incubated at -20°C for 20 min, followed by centrifugation at 12,000 g for 10 min. The supernatant was discarded without disturbing the pellet, which was subsequently washed twice with 500 µL ice cold 70% EtOH and 20 air-dried long enough to remove all the residual alcohol, but without completely drying the DNA. DNA was dissolved in 50 µl of ddH2O and stored at -20℃ for further use. 2.1.2 Isolation of Arabidopsis total RNA The CTAB extraction method was used for RNA extraction from Arabidopsis tissues as follows. The CTAB extraction buffer was preheated to 65℃ using a heat block, then aliquoted into RNase-free tubes (500 µl per tube). Dithiothreitol (DTT) was added to a final concentration of 5mM and 5 µl of Spermidine trihydrochloride (SPD) (100X) were added. 2-3 dry ice blocks were ground in an empty mortar to chill it. 100 mg of Arabidopsis flower or root tissue were then placed into the mortar and ground into powder, followed by the addition of 500 µl of preheated CTAB buffer. After thorough mixing, the suspension was transferred to a new RNase-free tube, vigorously vortexed for 30s, and incubated at 65℃ for 30 min. An equal volume of chloroform/isoamyl alcohol (24:1) was added to denature all the protein in the sample, then centrifuged at 4℃ at 12,000g for 20 min. The top aqueous phase was transferred to a new tube, a 1/4 volume of 10M LiCl was added, vortexed vigorously and precipitated at -20℃ for at least 2 h (or overnight to yield more RNA). The precipitated sample was centrifuged at 12,000g for 30 min at 4℃. The supernatant was removed and the pellet was air-dried in the hood. An additional wash using the 70% EtOH could be applied to clean the pellet. Finally, the pellet was then dissolved in 50 µl DEPC-water. 21 2.1.3 Plant cDNA preparation using RNA reverse transcription Before using it for cDNA synthesis, the concentration and purity of the RNA was determined by measuring the sample absorbance values at 260 nm (A260) and 280 nm (A280) in a NanoDrop 8000 spectrophotometer (Thermo Scientific, CA). Samples with a A260/A280 ratio ranging from 1.8~2.0 were considered good quality samples. The quality of the Arabidopsis total RNA was further verified by agarose gel (1.0%) electrophoresis using GelRed as the gel stain. At least 500 ng of RNA were treated with DNase I (Amplification Grade; Invitrogen) prior to cDNA synthesis, according to the manufacturer’s protocol. First strand cDNA synthesis was then carried out using the iScript cDNA synthesis Kit (Bio-Rad, Hercules, CA) and oligo(dT)20 primers. 22 2.2 Polymerase chain reaction (PCR) and vector construction 2.2.1 Introduction of PCR PCR technology used for the amplification of a specific DNA fragment was first applied by (Mullis et al., 1989) and is widely used today. The technique nowadays has a very a broad range of applications for genotyping or gene cloning. A typical PCR protocol used for this research is outlined in Tables 2-1 and Table 2-2 below. Table 2-1: PCR procedure Table 2-2: PCR mixture for genotyping using Taq polymerase Step No Ingredients Notes 1 94 °C (Taq)/ 98 °C (Phusion) for 2 min. Denaturation 2 94 °C (Taq)/ 98 °C (Phusion) for 30s. Denaturation Cycles repeat for 30~35 times. 3 Specific Tm1 for 30s. Annealing 4 72 °C for a specific time2. Extension 5 72 °C for 5~10 min. More extension time to allow completion of the product synthesis. 6 12 °C For product storage 1. The Tm was calculated as Tm [°C] = 2*(sum of A + T) + 4*(sum of G + C). The Phusion requires a +3 °C based on the calculated value. 2. The Taq polymerase will generate 1kb/min of products, while Phusion polymerase will generate 2~4kb/min. Ingredients Volumes (µL) 10 X Taq Buffer 2.0 dNTPs mixture 1.6 Primer F 1.0 Primer R 1.0 ddH2O 13.3 Taq 0.1 DNA template ~1.0 1 Total ~20.0 1(i.e. plasmid DNA; colony DNA; DNA from FTA card). 23 Table 2-3: PCR mixture for cloning using Phusion polymerase Ingredients Volumes (µL) 5 X HF/GC Buffer 10.0 dNTPs mixture 4.0 Primer F 2.5 Primer R 2.5 ddH2O 25.5~29.5 Phusion 0.5 DNA template 1.0~5.0 1 Total 50.0 1 (i.e. plasmid DNA; gDNA; cDNA). 24 2.2.2 Primers used for PCR In order to amplify a specific region from the Arabidopsis genomic DNA or cDNA, primers listed in Table 2-4 were used with the procedure described in Table 2-1. All the restriction sites are highlighted in red. Table 2-4: Primer sequences for cloning Purpose Primer Name Sequence (5'-3') Amplification of the LACS9-promoter with desired restriction sites. The overlapping sequence with LACS8ss-F in order to fuse LACS9-Pro and LACS8ss is highlighted in yellow. AscI-LACS9-Pro-F AAAggcgccgcTCCTGTTTAACGACGGAGTTCCTC LACS9-Pro-R ttcactccagaatcttccatCTTTCACTAAACCCCCAAAC Amplification of the LACS8-signal sequence (ss) with desired restriction sites. The overlapping sequence with LACS9-Pro-R in order to fuse LACS9-Pro and LACS8ss is highlighted in yellow. LACS8ss-F GTTTGGGGGTTTAGTGAAAGatggaagattctggagtgaa PstI-KpnI-LACS8ss-R AAActgcagAAAggtaccTTCCTCGCCACCTACTTTGAT Amplification of the eGFP, and the KKXX retention signal is shown in green and the restriction site is shown in red. PstI-eGFP-LK-F AAActgcagATGAGCAAGGGCGAGGAGCTGTTC HpaI-eGFP-KKXX-R AAAgttaacGGCGGCGTCCTTCTTGTACAGCTCGTCCATGCC Amplification of the truncated LACS9 (without the predicted chloroplast transit peptide) coding sequence with desired restriction sites. The linker is highlighted in blue. KpnI-LACS9t-F AAAggtaccGGTTATGCTATTAGGAATCACAGGT PstI-LACS9t-R AAActgcagtgttccggcaccgggtgcgatcgcGGCATATAACTTGGTGAGATC Amplification of the whole LACS9 coding sequence for BP reaction. attB1-LACS9-F GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGAGATGGGTCGTCTGGTCTTCGGGTT attB2-LACS9-R GGGGACCACTTTGTACAAGAAAGCTGGGTAGGAGAATTGAGATTGAGCTTGACTCG Amplification of the whole FAX1 coding sequence for BP reaction. attB1-FAX1-F GGGGACAAGTTTGTACAAAAAAGCAGGCTATGAGAAAACATTCTCTTGATCG attB2-FAX1-R GGGGACCACTTTGTACAAGAAAGCTGGGTAGTTCTTCTTCCCACGACC Amplification of the whole LACS4 coding sequence with desired restriction sites. NotI-LACS4-F AAAgcggccgcAATGTCGCAGCAGAAGAAATACATC AscI-LACS4-R AAAggcgccgcTCCCTCTGGAAGCAAATTTTGCAT 25 Table 2-5: Primer sequences for sequencing and genotyping Purpose Primer Name Sequence (5'-3') Internal primers for LACS9 sequencing and genotyping. LACS9-1R TGCCGAAACTGTAGCTAGGAC LACS9-2R CTATTATCTCGAGGCAGCCATC LACS9-3F AACTAGTGATAAGCCGATGCC LACS9-4F TGATGTTCAGAAACTTGGCCGC Internal primers for LACS4 sequencing and genotyping. LACS4-1R AGATACACATCTTTCACAGTCAGAGC LACS4-2R GAACCTCTTTCGTGAGATCTTCAC LACS4-3F GGTGTCTCACGTGAACAAAAAGAAG LACS4-4F ATGTTCTCCAAGGATACGGTCTTAC Vector-specific primers for genotyping. M13-F GTAAAACGACGGCCAGT M13-R CAGGAAACAGCTATGAC Vector-specific primers for sequencing. pCAMBIA-1380-Rev CCTGTAATTCACACGTGGTGG Sequencing primer based on 35S promoter. 35S-B AGTGGAAAAGGAAGGTGGCT Sequencing primer based on eGFP. EGFP-N CGTCGCCGTCCAGCTCGACCAG Primers for testing L8-ss-L9-GFP-KKXX insert LACS8-ss-F ATGGAAGATTCTGGAGTGAA Primers for FAX1 genotyping FAX1-RP CCTCTACTGGCTCTGTGATGC For the genotyping of the transgenic Arabidopsis which contains L8-ss-L9-GFP-KKXX insert, LACS8-ss-F and LACS9-1R primers were used. For pDONR221-LACS9, M13-F and LACS9-1R primers were used, while for pDONR221-FAX1, M13-F and FAX1-RP primers were used. For pENTR-LACS4 M13-F and LACS4-1R primers were used for genotyping. 2.2.3 Vector construction 2.2.3.1 Vector construction using digest-ligation The plasmid DNA was prepared using Bio-Basic Plasmid Miniprep Kit (Bio-Basic, Toronto, ON) by following the manufacturer’s protocol. Plasmid DNA and PCR products of the LACS9 promoter, the LACS9 coding sequence, as well as GFP were then digested by specific restriction 26 enzymes, and FastAP (Thermo Scientific, CA) was applied to remove the 5’ phosphorous group of the digested plasmid or PCR product to prevent self-ligation. The plasmid DNA and PCR products were then separated on agarose gels, excised and purified using QIAquick Gel Extraction Kit (QIAGEN, Mississauga, ON) and ligated using T4 DNA ligase (Invitrogen, Carlsbad, CA). Figure 2.1 The construct used to complement the Arabidopsis lacs4-2lacs9-2 mutants (12803 bp in total). The 1108 bp upstream of the LACS9 coding region in the Arabidopsis genome was used for the promoter+5’UTR sequence; SS: LACS8-signal sequence (first 204 bp). L: Linker between LACS9 coding sequence (1959 bp) and eGFP (714 bp). KKXX: the ER-retention signal. The position of restriction-enzyme recognition sites is indicated. The construct pCAMBIA-LACS9-Pro-LACS8ss-LACS9 (-cTP)-eGFP-KKXX used for the transformation of the Arabidopsis lacs4-2lacs9-2 double mutants, was generated in the pCAMBIA1380 plasmid (Deblaere et al., 1987). The AscI-LACS9-Pro-LACS8-ss-KpnI-PstI fragment was inserted by restriction enzyme digestion and ligation, followed by the PstI-Linker-eGFP-KKXX-HpaI fragment. Finally, the KpnI-LACS9 (-cTP)-PstI fragment was added to the construct after the previous fragment was confirmed by sequencing (Macrogen Inc., MD). Because the sequence in the middle of the AtLACS4 is homologous to the attB2 recombination site, which would affect the BP-reaction and lead to a truncated LACS4 sequence in the pDONR221 or pDONR207 donor vectors, the digest-ligation was used to construct the pENTR-LACS4 donor vectors. The NotI-LACS4-AscI fragment was exercised by the corresponding restriction enzymes and was ligated to the pENTR vector which was cut with the same restriction enzymes. 27 All the reagents used for restriction digests and ligation are shown in Table 2-6 and Table 2-7 below. The restriction digestion was carried out at the 37°C for 4 h or overnight. The ligation was performed at RT overnight. All constructs were confirmed by DNA sequencing. Table 2-6: Reagents used for restriction digestion Table 2-7: Reagents used for ligation Ingredients Volumes (µL) Vector or PCR Fragment ~10.0 (~1.0-2.0 µg) 10 X Buffer 3.0 ddH2O Up to 30.0 Restriction Enzyme(s) 0.1~0.5 for each Total 30.0 Ingredients Volumes (µL) Digested vector ~1.0 (~50-100ng) Digested insert ~1.0 (~50-100ng) 10 X T4 Buffer 1.0 ddH2O Up to 10.0 T4 ligase 0.5 Total 10.0 28 2.2.3.2 Vector construction using Gateway technology The genomic DNA of FAX1 and the cDNA sequence of LACS9 were recombined into the Gateway pDONR221 (Invitrogen, Carlsbad, CA) donor vector using a BP-reaction. The LACS9 cDNA entry clone contained the LACS9 coding sequence (2,073 bp) starting from the ATG but without the TAA stop codon to keep the gene fusion in-frame with the C-terminal 3xHA tag in the destination vector pGWB14. Similarly, the FAX1 entry clone contained the FAX1 genomic sequence (1,585 bp) starting from the ATG and without the TGA stop codon to keep the gene fusion in-frame with the C-terminal GFP tag in the destination vector pGWB5. All the entry or donor vectors which contained the desired sequences were then used for LR-reaction to make the destination vectors with desired tags fused on the C-termini of the open reading frames. The regents used for BP and LR-reactions are shown in Table 2-8 and Table 2-9. All the reactions were carried out at RT overnight. Figure 2.2 Constructs used for Co-IP. The FAX1 genomic sequence was fused with eGFP, in the pGWB5 vector, while LACS9-CDS was fused with 3xHA tag, in the pGWB14 vector. Both genes are under the control of the 35S promoter. 29 Table 2-8: Reagents used for BP-reaction Table 2-9: Reagents used for LR-reaction Ingredients Volumes (µL) Fragments with –attB- overhangs ~1.0 (50-100ng) Empty Entry Vectors ~1.0 (~75ng) TE buffer (pH 8.0) Up to 5.0 BP Clonase II 1.0 Total 5.0 Ingredients Volumes (µL) Entry Vector ~1.0 (50-100µg) Destination Vector ~1.0 (50-75µg) LR Clonase II 1.0 ddH2O Up to 5.0 Total 5.0 30 2.3 Bacterial transformation 2.3.1 Preparation and transformation of competent Escherichia coli and Agrobacterium tumefaciens cells Competent E. coli cells (DH5α) were prepared by treatment with 0.1 M MgCl2 followed by 0.1 M CaCl2 for 10 min each, and were re-suspended in 0.1 M CaCl2 with 10% glycerol. The 100μL of aliquoted cell suspension was placed in a pre-cooled 2 ml tube and was instantly frozen on dry ice and stored at -80°C. Before transformation, cells were removed from -80°C and allowed to thaw on ice. Plasmid DNA or ligation product not exceeding 10% of the total volume was added to the cells, mixed gently, and left on ice for 25~30 min. Cells were then heat shocked at 42°C for 90 s, left on ice for another 3~5 min, and incubated with vigorous shaking at 37°C for 1 h after addition of 900 μL of fresh LB medium. Various amounts of the cell suspension were then spread on plates containing LB medium with 0.8~1.0% agar and the appropriate antibiotic for selection, and allowed to grow overnight at 37°C. The colonies appeared on plates after 14~16-h incubation if the transformation was done properly. In order to make Competent Agrobacterium tumefaciens (A. tumefaciens) cells (GV3101, pMP90) (Koncz and Schell, 1986), the cells were grown in LB medium containing 50 mg/L rifampicin and 50 mg/L gentamicin until the OD600 = 0.5~1.0 was reached. Then the cells were pelleted in a microfuge and re-suspended in ice-cold ddH2O three times, and frozen immediately in dry ice in 30~50 μL aliquots. Competent cells were stored at -80°C. For transformation with binary vectors, competent cells were thawed on ice, and 10~20 ng plasmid DNA was added and mixed gently. The mix was then placed into a pre-cooled cuvette, electroporated using the Bio-Rad Gene Pulser (Bio-Rad, Hercules, CA) and 1 ml fresh LB medium was added after the electroporation. Cells were incubated at 30°C for 2~3 h with gentle shaking, then spread on LB plates containing the appropriate antibiotics and allowed to grow for at least 48 h at 30°C. 31 2.3.2 Verification of bacterial colonies that contain desired vectors A number of independently transformed bacterial colonies were selected, their total DNAs isolated and used as templates for colony PCR to test whether the inserted DNA fragment is properly assembled in the vector. For this purpose, a set of primers which included one vector backbone-specific (often M13-F/R) and one insert-specific primer were used. The colonies containing the desired fragments were incubated at 37°C with 3~5 ml LB liquid media at 200 rpm using a shaker (New Brunswick Scientific, Edison, NJ). Plasmid DNAs was purified using the Bio-Basic Plasmid Miniprep Kit (Bio-Basic, Toronto, ON) from each small-scale culture, digested by the specific restriction enzyme(s) and run on an agarose gel stained with GelRed. Selected plasmids containing the desired inserts were sent out for sequencing (Macrogen, Maryland, USA. Restriction mapping and sequence analyses were carried out by SnapGene (https://www.snapgene.com/snapgene-viewer/) and ChromasPro (https://chromaspro.software.informer.com/2.0/) software. 32 2.4 Plant transformation 2.4.1 Plant growth conditions Arabidopsis seeds were sown on Arabidopsis thaliana (AT)-agar (0.8%) plates (Somerville and Ogren, 1982) containing the desired antibiotics for selection (for transgenic seeds only). The seeds were kept at 4°C for 2 d to break dormancy and then germinated at 20°C under continuous light (100~150 μE/m2/s of photosynthetically active radiation) in the Conviron growth chambers (Conviron, Winnipeg, MB). After 10-14 d, seedlings were transplanted into soil (Sunshine Mix 4, SunGro, Kelowna, BC) supplemented with AT liquid medium and grown at 20°C under continuous light conditions. 2.4.2 Arabidopsis transformation using spray method Spraying the plants with an Agrobacterium (A. tumefaciens) suspension was used for Arabidopsis transformation (Chung et al., 2000). For this purpose, Arabidopsis plants were grown at a density of 100~200 plants in a 12-inch pot. After Agrobacterium cells were grown to an appropriate concentration (~ OD600 = 1.0), the suspension was spun down at 5,000 g for 10 min at RT. Precipitated cell pellet was re-suspended in 100 mL of 5% sucrose, washed and then spun down again at 5,000 g for 10 min at RT. Finally, Agrobacterium pellet was re-suspended in 100 mL of 5% sucrose solution supplemented with 0.02% (v/v) Silwet L-77 (Lehle Seeds, Round Rock, TX) and used for spraying when the inflorescences were roughly 3-4 inches high. Transformed plants were covered with black plastic bags for 16-24 h before being returned to standard growth conditions. 2.4.3 Screening for transgenic Arabidopsis in the T1 generation Harvested seeds were sterilized using 5% bleach and 0.1% Triton X-100 and then germinated on the AT medium supplemented with 0.8% agar and hygromycin B (50 mg/L). Only seedlings that had acquired the transgene(s) could grow normally on the selective medium. The 33 hygromycin B- resistant seedlings were transferred into soil after 1~2 weeks. Along with transgenic plants, wild-type plants (most frequently Col-0 ecotype) were planted as controls for comparison. 2.4.4 Crossing Arabidopsis plants Arabidopsis is a self-fertilizing plant. To avoid self-fertilization, flowers used as the female parents must be manipulated before the anthers begin to shed pollen onto the stigma. Usually 2-3 of the oldest flowers on the main shoot that have not yet opened were selected for crossing. Other flowers and siliques present on the stem were removed by scissors. The intact carpels were kept following the excision of all the sepals, petals and stamens. An open flower from the transformation line containing the transgene LACS8ss-LACS9(-cTP)-GFP-KKXX that was visibly shedding pollen was chosen as a male parent. This flower was removed from the transformation line and was squeezed near the base with the tips of the forceps and used for brushing the stigmatic surface of the exposed carpels on the Arabidopsis RFP-HDEL line used as female parent. The pollinated carpels were then covered with plastic film to keep moist for 24 h. The crosses were checked the following day to determine if the pollination was successful using the elongation of the siliques as the sign. Siliques were collected when they began to turn yellow (2~3 weeks after crossing) and dried at RT for at least 2 weeks before planting. 2.4.5 Nicotiana benthamiana infiltration The Nicotiana benthamiana was grown at 22 °C under long day (16 h light/ 8 h dark) conditions until plants were 4-weeks-old. Plants were watered about 30 min before the infiltration to make the infiltration easier. The leaves about 3~5 cm across from the healthiest looking plants were selected for infiltration. The Agrobacterium strains harbouring binary vectors with the desired genes fused with the epitope tags were grown overnight at 30 °C at 200 rpm in 3~5 ml LB under an appropriate antibiotic selection. When they reached OD600 = 0.6~0.8, the cells were collected in 50 ml Falcon 34 tubes by centrifugation for 10 min at 3,500 g. The Agrobacterium pellet was re-suspended in 5~10 ml of infiltration solution (4.4mg/L MS powder; 50mM MES; 150μM Acetosyringone) to OD600 = 0.1~0.3. At least 1 ml of infiltration solution was used per leaf. The co-expression of two or more proteins was carried out by injecting tobacco with an equal volume mixture of two or more strains of Agrobacteria, each grown to the same desired OD. The infiltration was carried out with a 1 ml syringe on the underside of the leaf avoiding main veins, and applying counter-pressure with a finger on the other side of the leaf. The infiltrated area, easily seen as a dark water-soaked region, was marked with a black marker. The plants were kept from the light and harvested 2-3 days after infiltration. Usually, 1 g of tissue was enough for a single Co-IP analysis. Samples could be kept in Falcon tubes and stored at -80 °C for a month. 35 2.5 Confocal microscopy Leaves of Arabidopsis transformants containing the GFP fluorophores and chloroplast auto-fluorescence were examined with a Nikon C1 confocal laser scanning microscope (https:// www.nikoninstruments.com). A 488 nm excitation wavelength with the emission filter set at 509 nm was used for GFP. The co-localization analysis was carried out using Perkin-Elmer spinning-disk confocal microscope (www. perkinelmer.com) and the Volocity software (Improvision). A 488 nm laser line with the GFP emission filter (525/36 nm) or a 561nm laser with the RFP emission filter (595/50nm) were used. All confocal images obtained were processed with Fiji (https://fiji.sc/) and Adobe Photoshop (https://www.adobe.com/ca/products/photoshop.html) software. 36 2.6 Analyses of seed oil and stem wax by gas chromatography (GC) 2.6.1 Single seed analysis A single Col-0 wild type seed or a transgenic seed was placed into a screw top Pyrex tube containing 0.5 mL 1N methanolic-HCl (Supelco, College park, GA), as well as 10 μL of 1mg/ml THD (Triheptadecanion: C17:0 FA on glycerol backbone) in toluene as an internal standard (IS). Each tube was capped tightly and heated at 80°C overnight to convert fatty acids to fatty acid methyl esters (FAMEs) (Browse et al., 1986). The following day, samples were cooled down for 5 min at RT, and 0.5 mL of 0.9% NaCl was added to each tube together with 200 μL of hexane to extract the FAMEs. The tubes were then recapped and vortexed vigorously. The hexane top phase was carefully transferred from each tube to a clean GC vial. Samples were dried completely under a stream of nitrogen, re-suspended in 20 μL of hexane and transferred into the conical glass insert (Agilent Technologies, Mississauga, ON) for GC analysis. FAMEs were analyzed on an Agilent 6890A gas chromatography system with a 30 m × 0.25 mm DB-23 capillary column (J and W Scientific) using the method described (Kunst et al., 1992). Briefly, after heating the sample to the initial temperature of 180°C for 1 min, an increase at 4 °C/ min from 180°C to 240°C was applied, and was maintained for further 3 min. Injector and flame ionization detector (FID) temperatures were set to 250 °C. The gas flow rates applied were: H2 30 ml/min, air 300 ml/min, N2 makeup 30 ml/min. Retention times of reference standards were recorded and used for identifying the FAME components from each sample. 2.6.2 Analysis of seed batches About 30~50 dry Arabidopsis seeds from each line were weighed and transferred into a screw top Pyrex tube. 750 μL of freshly prepared 1N methanolic-HCl (Supelco, College park, GA) and 20 μL of toluene containing an internal standard (1mg/ml C17:0 FA on glycerol backbone) were added to each tube. All the tubes were then vortexed for 30 s and heated at 80 °C for at least 2 h. After cooling at RT for 5 min, 1.5 mL of 0.9% NaCl (w/v) were added to each tube and FAMEs were extracted using 500 μL hexane twice. The samples were then evaporated under 37 nitrogen stream, dissolved in 50 μL of hexane and transferred to clean GC vials. GC analysis was carried out under the same conditions as described in section 2.6.1. 2.6.3 Arabidopsis stem wax analysis The plant materials used for Arabidopsis stem wax analysis were the top 10 cm of 3-5-week-old primary Arabidopsis stems. Stem tissue was submerged for 30 sec in approximately 10 mL of chloroform with 10 µg tetracosane added as an internal standard. Chloroform was dried under nitrogen gas. Then the waxes were silylated by heating in 10 μL pyridine and 10 μL N,O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) in 1% tri-methylchlorosilane at 80°C for at least 1 hour. Samples were then dried under nitrogen gas and the waxes were resuspended in 50 μL of chloroform for GC-FID analysis. Waxes were analyzed by the Agilent 7890A gas chromatography system using the sequence described by Haslam and Kunst (2013). Tissue surface area determination was carried out by measuring two-dimensional area in Adobe Photoshop based on pixels, and for three-dimensional stems, the surface area value was calculated by multiplying the flat area value by π. 38 2.7 Co-immunoprecipitation (Co-IP) analysis In order to test the interactions between FAX1 and LACS9, -GFP and -3xHA tags were fused to the C-termini of these two proteins, respectively. The TGD4-HA and TGD5-GFP, two proteins that localized to the oEM and iEM of the plastid and already shown to interact with each other were used as a positive control for interaction (Xu et al., 2015). TGD4-HA and FAX1-GFP were used as a negative control because they are localized to the oEM and iEM of the plastid, but are not believed to belong to the same pathway. The Agrobacterium cells carrying FAX1/LACS9, TGD4-HA/TGD5-GFP and TGD4-HA/FAX1-GFP genes were co-infiltrated into N. benthamiana leaves as described in section 2.4.5. For protein extraction from the infiltrated N. benthamiana leaves for Co-IP analysis, the modified protocol initially described by Xu and Li (2015) was used. At least 1.0 g of infiltrated leaves was ground into fine powder using dry-ice pre-cooled mortar and pestle. The extraction buffer [10% glycerol; 25 mM Tris-HCl, pH=7.5, 1 mM EDTA; 150 mM NaCl; and 2% polyvinylpolypyrrolidone (PVPP), 10 mM DTT; 1x protease inhibitor (PI) and 1 mM PMSF added just before use] (2 ml extraction buffer was used per gram tissue) was added and placed in the 4°C cold room. When the powder started to thaw, the suspension was homogenized by grinding to break cells to completion. The suspension was then transferred into 1.5 mL tubes and centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was transferred into new tubes and centrifuged a second time. Clean supernatant was then moved into new tubes again and 10% NP40 (NEB) was added to a final concentration of 0.15% together with 30 μL EZview™ Red Anti -HA affinity beads (Sigma, St. Louis, MO). The sample was incubated for at least 4 h in the cold room to allow the tagged proteins (or protein complexes) to be captured by the beads. Finally, beads were washed with 800 μL extraction buffer 3 times, followed by release of the captured protein by elution buffer (0.1M Glycine-HCl, pH=2.5~3.0). For protein blot analysis, 10% acrylamide gels with 1% SDS were used. The resolving gels (15 mL set) were made by mixing together 5.7 mL H2O, 5 mL acrylamide (30%), 3.8 mL 1.5 M Tris-HCl (pH=8.8), 100 μL glycerol, 150 μL SDS (10%), 150 μL APS (10%), and 15 μL TEMED. The stacking gels (5 mL set) were made by mixing together 3.4 mL H2O, 830 μL acrylamide (30%), 630 μL 1.0 39 M Tris-HCl (pH=6.8), 50 μL SDS (10%), 50 μL APS (10%), and 5 μL TEMED. The 10-well combs were placed on the top and the gel was polymerized for 30 min. Protein samples mixed with 5x SDS loading buffer [250 mM Tris-HCl (pH= 6.8), 10% SDS, 50% glycerol, 0.05% bromophenol blue, 500 mM DTT], were boiled for 5 min and loaded together with the protein ladder on the gel. Gels were run under a constant voltage of 200 volts for 1h. The protein samples were then transferred from the gel to the nitrocellulose membrane using Bio-Rad semi-dry membrane transfer dock at 17 volts for 50 min. Membranes were washed by TBST [10 mM Tris-HCl, 150 mM NaCl (pH = 7.4), 0.1% tween-20] and blocked by TBST containing 5% non-fat milk for 45 min, then incubated with TBST containing anti-GFP (1:10,000 dilution, mouse) and anti-HA (1:5,000 dilution, rat) antibodies overnight at 4°C. Membranes were then washed 3 times by TBST and incubated with secondary antibodies (1:50,000 dilution) which contain horseradish peroxidase (HRP) for detection. Incubation was carried out at RT for 1 h followed by three washes with TBST. The chemiluminescent substrate (Thermo Scientific, CA) was then added to the membrane and images were captured using Bio-Rad Image Lab system, under chemiluminescence Hi-resolution mode. 30s and 90s exposure times were used to capture the membrane images incubated with anti-GFP and anti-HA, respectively. 40 Chapter 3: Retargeting of LACS9 from the chloroplast envelope to the ER results in complementation of the lacs4lacs9 double mutant 3.1 Introduction Among the nine LACS enzymes in A. thaliana, only LACS9 localizes to the plastid envelope and thus provides all the plastidial LACS activity (Andrews and Keegstra, 1983; Shockey et al., 2002; Schnurr et al., 2002). Since the plastid is the subcellular organelle where most of the plant de-novo FA synthesis takes place, it was expected that LACS9 would be essential for acyl-CoA supply to the rest of the cell. However, lacs9 knock-out mutants showed no obvious phenotype indicating that LACS9 is not necessary for cell viability, raising the possibility of functional redundancy among different LACS enzymes (Schnurr et al., 2002). It is well-established that LACS1, LACS2, LACS4, as well as LACS8 localize to the ER (Weng et al., 2010; Zhao et al., 2010), where the majority of complex lipid assembly and the biosynthesis of surface lipids take place. Similar to LACS9, mutants that have a mutation in a single LACS gene encoding ER-localized LACS do not show any visible phenotype. To determine if this is due to the functional overlap between different LACS enzymes, LACS double and triple mutants were generated (Table 3-1). Indeed, the lacs1lacs4 double mutant showed a decrease in stem wax content, illustrating the redundant roles of LACS1 and LACS4 during stem wax synthesis (Jessen et al., 2011). Double mutants deficient in the plastidial LACS9 activity and the activity of the ER-localized LACS4 were also produced. These double mutants showed severe growth phenotypes, as well as deficiency in seed oil accumulation, and stem wax load (Jessen et al., 2015; Zhao et al., 2019). The establishment of the triple LACS mutants, lacs4lacs8lacs9 resulted in embryo lethality demonstrating the crucial role of these LACS enzymes during plant embryo development (Jessen 2011; Kalinger et al., 2020). 41 Table 3-1: Summary of characterized lacs mutants of Arabidopsis with detectable phenotypes Previous research on tocopherol biosynthesis has shown that ER-localized enzymes have access to substrates produced by the plastids. The defects in genes encoding tocopherol biosynthetic enzymes could be rescued by genes encoding an ER-targeted version of the same enzyme, in a mechanism known as trans-organellar complementation (Mehrshahi et al., 2013). In order to demonstrate the existence of a mechanism allowing trans-organellar access of ER-localized LACS enzyme(s) to their fatty acid substrate in plastids, I conducted a complementation experiment where the LACS9 enzyme was retargeted to the ER. In this experiment, a chimeric gene encoding a LACS9 enzyme was targeted to the ER by replacing its N-terminal plastidial transit peptide with the ER signal sequence and an ER retention signal. This chimeric LACS9 gene was then introduced into the lacs4lacs9 double mutant which lacked both plastidial LACS9 and ER-LACS4 activity resulting in growth deficiency, a 30 % reduction in seed oil yield and a 25% stem wax decrease (Jessen et al., 2015; Zhao et al., 2019). The plant height, rosette leaf diameter, seed oil content, as well as stem wax accumulation of transgenic plants Mutant Ecotype Mutagen Phenotype References lacs1 Col T-DNA Reduced surface wax and fertility. Lü et al., 2009 lacs1lacs2 Col T-DNA More severe reduction in stem wax, increased cuticle permeability and male sterile in low humidity. Weng et al., 2010 lacs1lacs4 Col T-DNA Reduced surface wax and conditional sterility. Jessen et al., 2011 lacs4lacs9 Col T-DNA Dwarf phenotype, reduced seed oil and stem wax content. Jessen et al., 2015 lacs1lacs9 Col T-DNA Reduced seed oil content. Zhao et al., 2010 lacs1lacs8lacs9 Col T-DNA Reduced seed oil content. Zhao et al., 2010 lacs1lacs4lacs9 Col T-DNA Reduced growth rate, reduced seed oil and stem wax content. Jessen, 2011 lacs4lacs8lacs9 Col T-DNA Lethal. Jessen, 2011 42 were measured in order to determine if the retargeted enzyme can rescue the morphological and lipid-related phenotypes. 3.2 Results 3.2.1 Substitution of the N-terminal plastidial transit peptide (TP) with ER signal sequence (ss) results in re-targeting of LACS9 from plastid to the ER For most of the plastidial proteins encoded by the nuclear genome, the import into the organelle requires the presence of a targeting signal on the N-terminus known as the transit peptide (TP). Similarly, ER-localized proteins require a designated signal sequence (ss) on their N-terminus to be imported into the ER. Thus, substitution of the LACS9 N-terminal transit peptide with a proper ER signal sequence is required in order to re-target the protein to the ER. Using SignalP 4.1. (Nielsen, 2017), the first 25 amino acids of LACS9 were predicted to correspond to the transit peptide. To ensure removal of the complete transit peptide, a DNA fragment encoding the first 40 amino acids of LACS9 was replaced with a DNA fragment encoding the first 65 amino acids of the ER localized LACS8 (At2g04350) gene that included the first 50 amino acids, predicted to serve as an ER signal sequence. A GFP tag was added to the C-terminus of the recombinant protein, so that it would be possible to detect it by confocal microscopy, followed by a retention signal KKXX (X stands for any amino acids) to keep the protein in the ER, rather than have it transit through the secretory pathway (see Fig 2.1 for the scheme of the construct used for plant transformation). Thus, the final form of the engineered recombinant protein was LACS8ss-LACS9(-cTP)-GFP-KKXX. The gene encoding the recombinant protein was placed under control of the LACS9 native promoter and transformed into Arabidopsis. A total of 16 independent transgenic lines were obtained and, of them, three carrying a single copy of the LACS9pro::LACS8ss-LACS9(-cTP)-GFP-KKXX transgene were analyzed further. A 35Spro::LACS9-GFP was used as a negative control to show the plastid envelope membrane localization of the native LACS9. (Fig 3.1 A). The ER 43 localization of the recombinant protein was confirmed by confocal microscopy after crossing the LACS9pro::LACS8ss-LACS9(-cTP)-GFP-KKXX transgenic line 10 with a RFP-HDEL line that highlights the ER network. (Fig 3.1 B) 44 Figure 3.1 The subcellular localization of the native (plastidial) and ER-retargeted LACS9-GFP. A, LACS9-GFP with an intact N-terminal transit peptide localized to the plastid envelope of (Bar=5µm), in mesophyll cells of Arabidopsis leaves, harvested 3 days after infiltration; B, the recombinant LACS9-GFP with its N-terminal transit peptide replaced by the N-terminal LACS8 signal sequence and an ER retention sequence added to the C-terminus was observed in the reticular ER network of epidermal cells in 7-day cotyledons of transgenic Arabidopsis plants. The Arabidopsis RFP-HDEL line was used as an ER marker line for the co-localization analysis. The RFP channel, GFP channel, and the merged channel are A B RFP-HDEL erLACS9-GFP-KKXX Merge B 45 shown from left to the right, as indicated (Bar=10µm). Confocal microscopy was carried out and images were generated by Dr. Eun-Kyoung Lee. 3.2.2 The ER targeted LACS9 protein is able to rescue the mutant phenotype of lacs4lacs9 double mutant The plastid LACS9 and ER LACS4 are important for fatty acid activation to acyl-CoA, so that they can be used for the production of surface lipids or incorporated into complex lipids. The inhibition of acyl-CoA formation leads to inadequate acyl-CoA supply in the ER and consequently a dwarf plant phenotype. The fact that the lacs4 or lacs9 single mutants have no detectable phenotype while the lacs4lacs9 double mutants have a lipid-related and an obvious dwarf phenotype suggests that LACS4 and LACS9 are functionally redundant even though they are in different subcellular compartments. To determine if an ER-localized LACS9 enzyme can indeed substitute for the loss of plastidial LACS activity and complement both the biochemical and the dwarf phenotype of the lacs4lacs9 double mutants, I carried out detailed phenotypic analyses of the three independent transgenic lines that received the LACS8ss-LACS9(-cTP)-GFP-KKXX recombinant protein. First, the plant height and the rosette leaves diameter of the Arabidopsis Col-0 wild type, lacs4lacs9 double mutants, and lacs4lacs9 transformants were measured. The results show that the height and rosette diameters of the lacs4lacs9 LACS9pro::LACS8ss-LACS9(-cTP)-GFP-KKXX transformants were not significantly different from the wild type using a one-way analysis of variance (ANOVA) (Fig 3.2) indicating that the LACS8ss-LACS9(-cTP)-GFP-KKXX can complement the lacs4lacs9 mutant dwarf phenotype. 46 Col-0 lacs4-2 lacs9-2 Line10 Line14 Line20 Figure 3.2: Complementation of the dwarf phenotype of the lacs4lacs9 double mutant by the LACS8ss-LACS9(-cTP)-GFP-KKXX A, Photographs; B, heights; and C, rosette leaf diameters of 4-week-old Arabidopsis wild type plants (Col-0), lacs4lacs9 double mutant, and three independent lacs4lacs9 LACS9pro::LACS8ss-LACS9(-cTP)-GFP-KKXX transgenic lines. Columns show the average of three replicates (three longest stems for B and three measurements in different directions for C). Error bars on the top of each column represent standard error (SE). Statistical differences between samples are labeled with different non-capital letters at P < 0.01 using a one-way analysis of variance (ANOVA). A B C 47 Second, to determine if the ER-targeted LACS9 protein is able to complement lipid-related phenotypes of the lacs4lacs9 double mutant, the stem wax load and the seed oil content of three independent transgenic lines were measured by GC (Fig 3.3). The results show that the stem wax load, as well as the seed oil content of the three transgenic lines is similar to or greater than that of the wild type, demonstrating that the introduction of the ER-localized LACS8ss-LACS9(-cTP)-GFP-KKXX recombinant protein rescues the lipid mutant phenotypes of the lacs4lacs9 double mutant. Third, to ensure that the complementation of the lipid-related phenotypes was due to the introduction of ER-targeted LACS9 protein, I carried out co-segregation analysis (Fig 3.4). For this purpose, seed oil content and stem wax load were measured by GC and the presence of the LACS8ss-LACS9(-cTP)-GFP-KKXX transgene determined by PCR for 12 seedlings from the T2 generation of the transformed double mutant line 10. The results show that complementation of both seed oil content, as well as the stem wax load was correlated 100% with the presence of Figure 3.3: Complementation of the lipid-related phenotypes of the lacs4lacs9 double mutant by the LACS8ss-LACS9(-cTP)-GFP-KKXX A, Stem wax load of the Arabidopsis Col-0, lacs4lacs9 double mutant, and three independent lacs4lacs9 LACSpro::LACS8ss-LACS9(-cTP)-GFP-KKXX transgenic lines; B, Seed oil content of the three independent lacs4lacs9 LACS9pro::LACS8ss-LACS9(-cTP)-GFP-KKXX transformed lines; Columns showed the average of four (for wax) and three (for oil) biological replicates. Error bars on the top of each column represents standard error (SE). Statistical differences between samples are labeled with different non-capital letters at P < 0.01 and with capital letters at P < 0.05 using a one-way analysis of variance (ANOVA). A B 48 the transgene consistent with the hypothesis that the transgene is necessary for the complementation. Figure 3.4: Co-segregation analysis using the T2 generation of LACS8ss-LACS9 (-cTP)-GFP-KKXX transformed lacs4lacs9 double mutants A, Agarose-gel electrophoresis showing the genotyping result of PCR. Marker is presented on (lane 1), wild-type (WT) and double-mutant (DM) (lanes 2 and 3) and seedlings No.1~12 are presented; B, Seed oil content of the WT, DM and each seedlings from T2 generation; C, Stem wax load of the WT, DM and each seedlings from T2 generation; Columns showed the average of four (for wax) and three (for oil) biological replicates. Numbers on the top of each column represent the average value of the replicates. Error bars on the top of each column represents standard error (SE). WT DM 1 2 3 4 5 6 7 8 9 10 11 12 A 29.76 21.21 27.27 28.78 27.41 26.75 21.39 30.04 27.77 30.54 30.56 20.20 29.93 20.90 0.005.0010.0015.0020.0025.0030.0035.00WT DM 1 2 3 4 5 6 7 8 9 10 11 12Seed oil content (%)B 29.76 23.54 30.38 30.20 28.10 30.29 20.72 29.27 28.78 30.60 26.74 20.87 28.51 20.90 0.005.0010.0015.0020.0025.0030.0035.0040.00WT DM 1 2 3 4 5 6 7 8 9 10 11 12stem wax (ug/cm^2)C 49 3.3 Discussion 3.3.1 Roles of plastid-localized LACS9 and ER-localized LACS4 in fatty acid transport from the plastid to the ER Fatty acids (FA) are substrates for the synthesis of more complex lipids including glycerolipids, sphingolipids, as well as surface lipids in plants. The de novo synthesis of the fatty acids takes place in the plastids of plants but the production of complex lipids and surface lipids occurs in the ER (Bishop and Bell, 1988). Thus, transport of FAs from plastids to the ER is essential for normal plant growth and development. LACS enzymes play an important role in making fatty acids available for their downstream metabolic uses by catalyzing the conversion of FAs to acyl-CoAs (Shockey et al., 2002). Previous research has shown that loss of function of LACS9, the only LACS located in the plastid results in no obvious plant phenotype unless it occurs in combination with loss-of-function of one or more ER-localized LACS enzymes. (Zhao et al., 2010; Jessen et al., 2015; Zhao et al., 2019). Based on these data I hypothesized that LACS enzymes located in the plastid and ER can functionally substitute for each other. To test this hypothesis, I have re-targeted the plastid-localized LACS9 to the ER in a lacs9lacs4 double mutant background and examined the growth and lipid-related phenotypes of several independent transgenic lines. The LASC9 enzyme re-targeted to the ER was able to completely rescue the lacs4lacs9 double mutant growth phenotype, as well as its seed oil content and cuticular wax load deficiencies. My results demonstrate that in the case between LACS4 and LACS9, a LACS enzyme in either the plastid or the ER is sufficient to catalyze the formation of acyl-CoAs required for their utilization in the ER. Since the complementation occurred even after re-targeting of LACS9 to the ER compartment, LACS9 localized in the ER must have trans-organellar access to its FA substrate in the plastid. There are at least two different ways that such trans-organellar access could be explained. First, active sites of the plastidial-LACS9 and ER-LACS4 could both be localized in the cytosol, as shown in Figure 3.5A. If that is the case, in either the lacs4 (Fig 3.5 B) or lacs9 (Fig 3.5 C) single 50 mutant, the remaining functional LACS can access FAs exiting the plastid and convert them to FAacyl-CoAs to be used in the ER. Alternatively, there may be two distinct pathways for formation of acyl-CoAs: 1. the plastidial-LACS9 can convert the FAs to FAacyl-CoAs and these are transported to the ER and 2. FAs could also be transported to the ER first, and there they could be converted to acyl-CoAs by the ER-LACS enzymes. Figure 3.5: Proposed diagram showing the distribution of LACS proteins between plastid and ER and their hypothetical topology. A, diagram of plastid LACS9 and ER LACS4 in WT Arabidopsis; B, in lacs4 single mutant, only plastid LACS9 is functional during plastid-ER fatty acid transport; C, in lacs9 single mutant, only ER LACS4 is functional during plastid-ER fatty acid transport; D, in lacs4lacs9 double mutant, the ER-retargeted LACS9 is functional during plastid-ER fatty acid transport (purple cylinder indicates signal sequence and a predicted transmembrane domain from ER localized LACS8); A B D C 51 Whichever the case, it is not clear how FA or acyl-CoA physically make their way from the plastid to the ER. One possibility is that membrane contact sites are involved. Contact sites are often used for cellular communication or substrate delivery between organelles . For example, the ER-PM contact sites play a role in the regulations of intracellular Ca2+ level (Prinz, 2014; Pérez-Sancho et al., 2016; Scorrano et al., 2019), the proposed ER-plastid contact sites maintained by TGD complex play a role in lipid transportation from ER back to plastid, with a substrate preference of PA (Fan et al., 2015). So LACS9 and LACS4 may be associated with the plastid-ER membrane contact sites that participate in the delivery of FAs or acyl-CoAs to the ER. Further experiments are needed to identify additional proteins that may be involved in the formation of such potential contact sites, investigate their relationship with LACS9 and LACS4, and elucidate the mechanism of plastid to ER transport of FAs. 52 Chapter 4: Plastidial-LACS9, but not ER-LACS4, interacts with FAX1 in planta 4.1 Introduction In plant cells, the largest amount of lipid flux occurs between the plastids and the ER (Somerville and Browse, 1996; Benning et al., 2006). Almost all the fatty acids are produced in the plastid, so they need to be exported to the ER where they are used for the biosynthesis of a variety of complex lipids including glycerolipids, a portion of which is transferred back to the plastid (Li-Beisson et al., 2013). The mechanism by which glycerolipids are transported from the ER to the plastid has been relatively well-characterized thanks to the discovery of the TRIGALACTOSYL-DIACYLGLYCEROL (TGD) complex that contains five distinct polypeptide subunits (TGD1-5; Xu et al., 2003; Awai et al., 2006; Lu et al., 2007; Xu et al., 2008, Fan et al., 2015). Among these, TGD5 was found to interact with all the other four subunits including the iEM protein TDG4, and is believed to serve as a bridge between plastid iEM and oEM that facilitates lipid transfer. Relative to ER-plastid delivery of glycerolipids, relatively little is known about the export of newly synthesized fatty acids from plastid stroma across the envelope membranes to the ER. FAX1 is an iEM protein involved in the fatty acids movement across the iEM (Li et al., 2015). FAX2 and FAX4 are FAX1 homologs that, like FAX1, belong to the Transmembrane 14C family, localize to plastid envelope membranes and appear to promote fatty acid transport from the plastid to the ER. FAX2 is highly expressed in seeds and is required for seed oil biosynthesis. In addition, FAX2 has been shown to interact with LACS9 (Tian et al., 2019; Li et al., 2020), a chloroplast oEM protein that activates fatty acids to acyl-CoAs to make them available for incorporation into complex lipids. It is unclear whether such FAX-LACS interactions occur between LACS9 and other FAX proteins, including the better characterized FAX1, or what the role of such interactions might be in the movement of fatty acids out of the plastid. Since available data demonstrate that LACS9 can perform its function both the plastid oEM and when transferred to the ER, and that LACS9 and LACS4 are can complement each other (Jessen et al., 2015; this study) even though they are 53 present in different subcellular compartments. Based on that, I hypothesized that iEM FAX1 and oEM LACS9 are forming a complex, and even LACS4 which is in the ER may also be able to associate with FAX1. I tagged the FAX1 with GFP and LACS4/9 with HA (as described in section 2.7), as GFP and HA antibodies are commercially available, and used co-immunoprecipitation to test the interactions between FAX1/LACS9 and the association between FAX1/LACS4. 4.2 Results 4.2.1 The iEM-localized FAX1 and oEM-localized LACS9 interact In order to determine if FAX1 and LACS9 associate with one another in a complex, a Co-IP analysis was carried out. As I have mentioned previously, the TGD complex consists of 5 distinct polypeptides that localize to either the iEM (TGD1/2/3/5) or the oEM (TGD4) (Fan et al., 2015, Hölzl and Dörmann, 2019). Since the iEM protein TGD5 and oEM protein TGD4 have been shown to directly interact (Fan et al., 2015) they can be used as a positive control in my co-IP analysis. FAX1 and TGD4 transport distinct lipids in opposite directions. For this reason, they are not expected to interact and can serve as a negative control in my experiment. Two Agrobacterium strains carrying different binary vectors with either LACS9-HA, TGD4-HA, FAX1-GFP or TGD5-GFP were infiltrated into tobacco leaves and total protein was extracted from the injected tissue. The Co-IP analysis was done according to section 2.7, after 3 days of infiltration. The presence of each recombinant polypeptide was detected in the total leaf protein extract using antibodies to the relevant tags (HA or GFP; see Figure 4.1 Input, left panel). HA tagged polypeptides (LACS9 and TDG4) were immunoprecipitated using anti-HA affinity beads and the presence of GFP tagged polypeptides (TGD5, FAX1) in the precipitate was determined using GFP-specific antibodies (Fig 4.1, right panel). As expected, TGD5 but not FAX1 co-immunoprecipitated with TGD4. FAX1 co-immunoprecipitated with LACS9 demonstrating that these two plastid polypeptides are part of the same complex in planta. 54 Figure 4.1: Co-IP analysis of FAX1-GFP and LACS9-HA. Protein extracted from tobacco leaves injected with two Agrobacterium strains carrying binary vectors with one of LACS9-HA, TGD4-HA, FAX1-GFP and TGD5-GFP gene (see input samples in the left panel). The Co-IP pairs used in different lanes were labeled below each lane. Input LACS9-HA TGD4-HA FAX1-GFP TGD5-GFP HA-TGD4+FAX1-GFP HA-TGD4+TGD5-GFP LACS9-HA+FAX1-GFP IP-HA α-HA α-GFP LACS9-HA TGD4-HA FAX1-GFP TGD5-GFP HA-TGD4+FAX1-GFP HA-TGD4+TGD5-GFP LACS9-HA+FAX1-GFP 55 4.2.2 The iEM localized FAX1 and ER localized LACS4 do not interact Co-IP analysis was also carried out to determine whether FAX1 and LACS4 interact. The positive and negative controls are the same as section 4.2.1. Two Agrobacterium strains carrying different binary vectors with either LACS4-HA, TGD4-HA, FAX1-GFP or TGD5-GFP were injected into tobacco leaves and total protein was extracted from the injected tissue after 3 days of infiltration, using the protocol described at section 2.7. The presence of each recombinant polypeptide was detected using antibodies to the relevant tags (HA or GFP; see Figure 4.1 Input, left panel). HA tagged polypeptides (LACS4 and TDG4) were immunoprecipitated using anti-HA affinity beads and the presence of GFP tagged polypeptides (TGD5, FAX1) in the precipitate was determined using GFP-specific antibodies (Fig 4.1, right panel). As expected, TGD5 but not FAX1 co-immunoprecipitated with TGD4. FAX1 did not co-immunoprecipitate with ER-localized LACS4 suggesting that these two polypeptides are not part of the same complex in planta (Fig 4.2). 56 Figure 4.2: Co-IP analysis of FAX1-GFP and LACS4-HA. Protein extracted from tobacco leaves injected with two Agrobacterium strains carrying binary vectors with one of LACS4-HA, TGD4-HA, FAX1-GFP and TGD5-GFP gene (see input samples in the left panel). The Co-IP pairs used in different lanes were labeled below each lane. Input LACS4-HA TGD4-HA FAX1-GFP TGD5-GFP HA-TGD4+FAX1-GFP HA-TGD4+TGD5-GFP LACS4-HA+FAX1-GFP IP-HA α-HA α-GFP LACS4-HA TGD4-HA TGD5-GFP HA-TGD4+FAX1-GFP HA-TGD4+TGD5-GFP LACS4-HA+FAX1-GFP 57 4.3 Discussion The phenotypes of the fax1 single mutant and lacs4lacs9 double mutants indicate that FAX1 and LACS4/9 play roles in fatty acid transport from the plastid to the ER (Li et al., 2015). In addition, LACS9 and FAX2 have been shown to co-immunoprecipitate suggesting that LACS and FAX polypeptides can form a complex (Tian et al., 2019). Since FAX1-4 belongs to the Transmembrane-14C family and have the same plastidial localization, researchers also suggested they have functional redundancy (Li et al., 2015; 2020). So based on the result that FAX2 and LACS9 interact with each other and likely to form a complex, it is reasonable to hypothesize that FAX1 and LACS9 also form a complex that is responsible for the movement of FAs from the plastid stroma to the ER. In this chapter, I have provided evidence that LACS9 and FAX1 do indeed interact in vitro and likely to form a complex in planta. Currently, it is not clear whether this interaction is direct or if they are members of the same protein complex. I also cannot rule out the possibility that the FAX1-LACS complex includes additional proteins that are required for fatty acid transport. A mechanism called vectorial acylation first described in yeast (Zou et al. 2003), but also in Synechocystis (Von Berlepsch et al., 2012), may be involved, which allows an acyl activating enzyme to transport fatty acids across the membrane. Thus, it is possible that like the proteins of the TDG complex, FAX1 and LACS9 interact to transport lipids across both the iEM and oEM plastid membranes. Alternatively, LACS9 may be associated with a FAX1 fatty acid transport complex, but not directly involved in transport itself. The absence of a mutant phenotype in lacs4 and lacs9 single mutants and the ability of an ER targeted LACS9 to rescue the mutant phenotypes of the lacs4lacs9 double mutant (see Chapter 3) indicate that LACS4 and LACS9 are at least partially redundant. However, I have shown in this chapter that, unlike LACS9, LACS4 does not interact with FAX1. This result is not surprising considering that FAX1 and LACS4 are localized to different subcellular compartments, but it raises the question as to how LACS9 and LACS4 can functionally complement each other. ER- localized LACS4 must be able to access plastid fatty acids in the absence of LACS9. If LACS9 is solely responsible for fatty acid transport across the oEM, then the LACS4 would not be able to access 58 fatty acids in a lacs9 mutant background. Thus, either LACS9 is not essential for fatty acid transport across the oEM, or LACS4 is able to access plastid fatty acids via an alternate pathway (Possible mechanism shown by Fig 4.3). Further investigation into the LACS-FAX interactions may help answer such questions. Figure 4.3: Proposed diagram showing fatty acids made by the plastids transported by different complexes. A, diagram of plastid iEM-FAX1 and oEM-LACS9 forming a complex, which is able to transport fatty acids made by the plastids out of the plastids and made available to ER-LACS4; B, diagram of fatty acids made by the plastids are transported by an unknown complex (different from the FAX1-LACS9 complex) and made available to ER-LACS4; The flux of the FA from the plastid to the ER is marked by the grey arrow. A B 59 Chapter 5: Conclusions and future directions 5.1 Conclusions from this research 5.1.1 ER-localized LACS enzymes have access to the fatty acids from the plastid envelope membranes In plant cells, fatty acids are produced primarily by the plastid. They are used for the synthesis of complex lipids including phospholipids, sphingolipids, TAGs, and surface lipids, in the ER. The LACS enzymes are crucial for converting fatty acids to their corresponding acyl -CoAs, so that they can be used as substrates by the downstream biosynthetic enzymes in the ER. Because lacs9 single mutants showed no visible phenotype, it was obvious that other LACS enzymes must be able to compensate for the absence of the only plastidial LACS, LACS9. The lacs4 single mutants also did not exhibit a mutant phenotype, whereas the lacs4lacs9 double mutants displayed dwarfism and seed oil/stem wax decreases suggesting that LACS9 and LACS 4 have at least partially redundant functions (Jessen et al., 2015) even though they are localized in different cellular compartments. In this study, I have directly tested the hypothesis that ER-localized LACS enzyme can functionally substitute a plastid-localized LACS by re-targeting the LASC9 enzyme to the ER in the lacs4lacs9 double mutant. My data demonstrate that the ER-re-targetted LACS9 enzyme can complement the lacs4lacs9 double mutant supporting the conclusion that LACS enzymes localized to the ER must have access to the fatty acids from the plastid envelope membranes. The mechanism by which this access is achieved is not known, but it is independent of the activity of LACS9 (Fig 5.1). 60 Figure 5.1: Proposed diagram showing the possible access of ER re-targetted LACS9 to the fatty acids from the plastid envelope membranes. This diagram shows in lacs4lacs9 double mutant, where neither plastidial-LACS9 nor ER-LACS4 is available, the active sites (probably within the FA-binding domain of the protein) of the ER-retargeted LACS9 is able to get access to the fatty acids from the plastid envelope membrane maybe through the close proximity of the plastid envelope membrane (purple cylinder indicates signal sequence and a predicted transmembrane domain from ER localized LACS8). 5.1.2 The iEM-localized FAX1 interacts with oEM-localized LACS9, but not ER-localized LACS4, to form a complex The iEM-localized FAX1 and oEM localized-LACS9 coimmunoprecipitate suggesting that the two proteins cooperate in the transfer of fatty acids across the plastid envelope membranes. No such interaction was detected between iEM-localized FAX1 and ER-localized LACS4 further confirming that LACS4 functions independently from LACS9. 61 5.2 Future directions for this research The importance of FAX1 and LACS4/9 in fatty acids transport has been well documented. The ability of the ER-localized LACS enzymes to access fatty acids made in the plastid stroma and interactions between FAX1/LACS9 are presented in this thesis. Many questions about these processes remain. First, it is unclear how ER-localized LACS proteins access plastid fatty acids. Since access occurs in the absence of active LACS9, a LACS9 independent pathway for fatty acid transport must exist. One possibility is that the FAX1 complex transports fatty acids across both plastidial membranes even in the absence of LACS9, or a separate pathway for export of fatty acids is present in the plastid. Either way, additional proteins must be involved in this process. Such proteins could be identified by using an enhancer mutant screen in a lacs9 genetic background to identify mutants that have a lacs9lacs4-like mutant phenotype. Alternatively, co-immunoprecipitation of LACS9-FAX1 protein complex could be used to identify other proteins associated with the complex. Second, it is unknown how LACS9 and LACS4 can complement each other when they reside in membranes of different organelles. It is conceivable that the active site of both LACS proteins is facing the cytosol so that fatty acids exiting the plastid can be accessed by either one of these LACSs (Fig 3.5). This hypothesis can be tested in two ways. First, bi -molecular fluorescence complementation (BiFC) can be used to show that the active sites on the C-terminus of the LACS4 and LACS9 are in the same subcellular compartment – the cytosol. Another experiment that could be performed to test whether LACS4 and LACS9 are in close proximity (within 10 nm) is Turbo-ID-based proximity labelling. 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The mechanism for fatty acids trafficking from plastids to the endoplasmic reticulum
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