LIPID TRAFFICKING AND LIPID BREAKDOWN IN CHLAMYDOMONAS By Jaruswan Warakanont A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Biology ÑDoctor of Philosophy 2015 ABSTRACT LIPID TRAFFICKING AND LIPID BREAKDOWN IN CHLAMYDOMONAS By Jaruswan Warakanont With their high photosynthetic efficiency and the ability to synthesize triacylglycerol (TAG), unicellular microalgae have an important ecological role, as well as value for productio n of triacylglycerol lipids that can be converted to biodiesel. The recent state of knowledge about microalgal lipid metabolism had been deduced from that of a model seed plant, Arabidopsis. However, recent studies have revealed that aspects of lipid metab olism differ between microalgae and Arabidopsis. Investigating these differences was a cornerstone of this study, using Chlamydomonas, a representative of microalgae, and Arabidopsis. Two major approaches were undertaken: forward and reverse genetics. The forward genetic screening used insertional mutagenesis of Chlamydomonas and focused on a knockout mutation of a gene, which proved to be an orthologue of the Arabidopsis TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2). The tgd2 mutant exhibits increases in cellular concentrations of phosphatidic acid (PtdOH) and triacylglycerol (TAG); the latter contains signature fatty acids of monogalactosyldiacylglycerol (MGDG), pointing to its likely origin of synthesis. The mutant also experiences low viability in extended cult ure. Similar to AtTGD2, CrTGD2 is located in the chloroplast inner envelope membrane and binds PtdOH in vitro . Radioactive labeling experiments suggest that CrTGD2 functions in transferring a lipid precursor, presumably PtdOH, from the outer chloroplast en velope into the chloroplast. This study shows that, in contrast to prevailing assumptions , Chlamydomonas is able to import lipids from the endoplasmic reticulum ( ER) to the chloroplast, and utilizes the eukaryotic pathway to synthesize galactoglycerolipids . The reverse genetics investigation focused on CrLIP4, a putative TAG lipase. CrLIP4 is an orthologue of a major Arabidopsis TAG lipase. Reverse transcription PCR revealed that the CrLIP4 transcript is reduced in abundance during N deprivation when TAG a ccumulates. Down -regulation of this gene through an artificial microRNA construct resulted in delayed TAG degradation. Expression of CrLIP4 in Escherichia coli alters the pattern of neutral lipids. Recombinant CrLIP4 exhibited TAG lipase activity. These re sults show that CrLIP4 has TAG lipase activity both in in vitro and in vi vo. In summary, two Arabidopsis orthologues in Chlamydomonas were characterized through forward and reverse genetic approaches. The results elaborate and refine our understanding of Chlamydomonas lipid metabolism, and are likely relevant for other unicellular microalgae. Copyright by JARUSWAN WARAKANONT 2015 !!"!ACKNOWLEDGEMENTS In the Fall 2009, I was accepted to a PhD program in the Department of Crop and Soil Science at M ichigan State University by Prof. Mariam B. Sticklen. However, after a year had passed my classmate Paula Marquardt made me realized that I would be happier switching my research to different directions . I began look ing for a new lab and Benni ng lab was one of them. Through help and support from my Plant Biochemistry classmates, Cheng Peng, Benshen Liu and Chia -Hong Tsai, I had a chance to talk to Prof. Christoph Benning . In Decembe r 2010, t hrough a series of talking and discussion , I transfer red to the Plant Biology program and to the Benning lab with help from the director of graduate studies Prof. Alan Prather , my advisor at the time Prof. Mariam B. Sticklen, my current committee member Prof. Barbara B. Sears, my former advisor in Thailand D r. Yindee Chanviwattana, and my former SticklenÕs labmates Sang Hyuck Park and Thang Xuan Nguyen. The most important person is Prof. Christoph Benning who was kind enough to accept me into his lab without knowing me from a rotation system and with the fact that I had only a little experience in molecular biology ; zero in biochemistry. In the new lab, I have earned valuable experience , learned many techniques, and encountered many challenges including comprehensive exams and difficult experiments . I would not have gone through this unforgettable time without help, guidance and support from the following individuals to whom I express my profound gratitude. First and foremost , I would like to express my deepest appreciation to my advisor Prof. Christoph Benni ng for his e ndless support in many aspects. At the beginning of my research in his lab , I spent many months cloning a gene ( CrLIP4 ). At the same time , I also tried to identify a !!"#!mutation in the mutant ( tgd2 ) with two molecular biology techniques without a successful result. Christoph was very patient with me during that difficult time. At the end, he accepted my idea to sequence the genome of the tgd2 mutant, which then became a successful story in my thesis research. In general, h e has allowed me to think , tackle problems, and conduct research freely with my own pace. However, this does not stop him from giving me directions and advices when needed. He is a great scientist and advisor at the same time. Throughout his guidance and nurturing , he helps me de velop skill for being independent scientist in the future. Beside my advisor, I am immensely grateful to the rest of my committee, who always provide me knowledge and advice for my research . I sincerely appreciate Prof. Barbara B. Sears for her tremendous help on my writing and genetic analysis of the Chlamydomonas muta nt. I appreciate Prof. John B. Ohl rogge for his advice on radioactive labeling experiments and for hooding me in the graduation ceremony. I would like to thank Prof. Min -Hao Kuo for his sugg estions and comments on yeast experiments and for having his graduate stu dent Witawas Handee helped me in the yeast project . All of them are supportive and helpful. Their comments and questions always help me improv e my work. I would like to thank all of my labmates from the past and pre sent. I am very grateful to Rebecca Roston, Simone S−uner, and Yang Yang who spent hours discussing the results, giving me suggestions and knowledge. I am thankful to Astrid V ieler, Blair Bullard, Que Kong , Xiabo Li, and Bensheng Liu, who always willing to share their knowledge and exchanging nice conversation with me. My gratitude also goes to Elena J. S. Michel, an undergraduate who worked with me. Due to her talent and dedication, she help ed me complete the CrTGD2 project . I would like to thank other lab members : Chia -Hong Tsai, Agnieszka Zienkiewicz, Krzysztof Zienkiewicz, Wei Ma, Zheng Wang, Anna Hurlock, Kun Wang, Patric Horn, Zhi -Yan Du, Linda !!"##!Danhof, Rachel Miller, Sanjay, Bagya Muthan, Eric Poliner, Tomomi Takeuchi, Anastasiya Lavell, all the undergrad students and visiting scholars, for their enco uragement, support, and being great friends throughout my time here. I am thankful for financial support from a Royal Thai Government scholarship for the first five years a nd for the research assistantship through my advisor throughout my study. I am greatly indebted to my former advisor s, Dr. Yindee Chanviwattana and Assoc. Prof. Jarunya Narangajavana , as well as Dr. Oranuch Leelapon who encourage me to continue my graduat e study abroad. Without them I would never have a great academic and cultural experiences in the United States. In addition, I would like to thank all of my former teachers who educated me throughout my study from a kindergarten through a graduate school. I am grateful to my parents and my grandmother for their unconditional love, patience, and supportiveness. I fully appreciate my siblings who are always on my side and take care of our parents while I am away. All of my family m embers have shaped the way I am; no word can describe how important they are. I would like to thank my boyfriend Pawin Ittisamai warmly for helping me with everything, being with me here in the United States, and bein g so supportive and thoughtful. He always has a positive way of thinking. He is the only person who understands me in every aspect. He is a technical guy for me. He can help fixing and finding answers for computer, camera, telephone, television, bicycle, outdoor gears, etc. He helps me relieve the stress of graduate sc hool by entertaining me through different activitie s including watching movies, and traveling to several places in Europe and the US . Besides, he help ed me taking photos and finish all of my experimental dishes ; some of which I hesitated to try . !!"###!Another p erson who plays an important role in my life is Lisa Luchita . She is my first Italian friend. I knew her from Prof. Michael Morris class in Summer 2009. Lisa introduced me a different perspective of life . She emphasized how importance of travel toward the understand ing of the differences among people in different regions and backgrounds . She also in spired me about cooking which then became my passion through the GialloZafferano website. It is my pleasure to know her. I consider knowing her as a turning poin t in my life. I am thankful to my dearest friend Teeda Sasipreeyajan for being such a good friend since my first gr ade at the Chulalongkorn University Demonstration School or Satit Chula in short . She and her husband who is also my friend from Satit Chula moved to Ann Arbor in the fall of 2014. Since their arrival, we took turns visiting each other. We traveled to many places together. We exchange d storie s through many delicious meals. All of these activities keep me happy during the final stage of my PhD s tudy . Last but not least, my living in the US would not be completed without learn ing the American culture from the locals. I am thankful to Alan and Carol Prais who inviting Pawin and I to their house in the Pre -academic program in 2009. They continued in viting us to the American celebrations of Thanksgiving s and Christmas. Finally, I deep ly appreciate Pooh Stevenson and Richard Bowie from the Owosso Organics. They not only supplied me organic vegetables and eggs for five years but they are also ones of th e greatest persons I have ever met. Their way of thinking reflects how good and sustainable their farm is. I find that studying PhD in the US is not only giving me academic experience but also broadening my perspective toward many aspects. I hope my learn ing could help contribute improving our human society in the future. !!#$!TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... xiv LIST OF FIGURES .......................................................................................................................xv CHAPTER 1: Lipid metabolism in Chlamydomonas reinhardtii ....................................................1 WHY STUDY LIPID META BOLISM IN CHLAMYDOMO NAS? .................................2 LIPID METABOLISM IN CHLAMYDOMONAS VS. ARAB IDOPSIS ..........................4 Fatty acid composition .............................................................................................4 Lipid species ............................................................................................................5 Genes involved in lipid metabolism ........................................................................6 LIPOLYSIS AND LIPASE S ...............................................................................................7 Lipases are interfacial enzymes ...............................................................................7 Many TAG lipases contain a patatin domain ...........................................................7 Patatin-domain containing enzymes are unique l ipases ...........................................8 MUTANT SCREENING AND IDENTIFICATION OF A RESPONSIBLE GENE .........9 Forward genetic screening .......................................................................................9 Map-based cloning .................................................................................................10 PCR -based methods ...............................................................................................10 Whole genome resequencing .................................................................................12 Forward genetic screening in Chlamydomonas .....................................................13 AIM OF THE THESIS RE SEARCH ................................................................................14 REFERENCES ..................................................................................................................20 CHAPTER 2: Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue .........32 ABSTRACT .......................................................................................................................33 SIGNIFICANT STATEMEN T ..........................................................................................33 INTRODUCTION .............................................................................................................34 RESULTS ..........................................................................................................................37 The tgd2 mutant accumulates triacylglycerol enric hed in MGDG acyl groups ....37 Cultures of tgd2 show early senescence ................................................................38 Changes in the ultrastructure of tgd2 cells .............................................................38 Molecular and genetic analysis of the tgd2 mutant locus ......................................39 CrTGD2 is a presumed orthologue of AtTGD2 .....................................................40 Altered galactoglycerolipid labeling and impaired ER -to-plastid lipid trafficking in tgd2 ..................................................................................................40 Altered labeling of non -galactoglycerolipids in tgd2 ............................................42 Biosynthesis of MGDG is increased in the tgd2 mutant ........................................42 CrTGD2 is present in the chloroplast inner envelope membrane ..........................43 CrTGD2 binds PtdOH in vitro ...............................................................................44 DISCUSSION ....................................................................................................................45 Galactoglycerolipid metabolism is altered in tgd2 ................................................46 !!$!What happens to MGDG as it is metabolized in the tgd2 mutant ? ........................47 Does CrTGD2 play a role in the transfer of lipids from the ER to the chloroplast ? ............................................................................................................48 MATERIALS AND METHOD S .......................................................................................50 Alga l strains and growth conditions ......................................................................50 Generation of tgd2 mutant and genetic analyses ...................................................50 DNA isolation and Southern blot analysis .............................................................50 Whole genome resequencing .................................................................................50 Lipid analysis .........................................................................................................50 Viability assay ........................................................................................................51 Transmission electron microscopy ........................................................................51 Phylogenetic analysis .............................................................................................53 Heterologous complementation analysis ...............................................................52 [14C]-Acetate pulse -chase labeling ........................................................................52 DsRED-CrTGD2 pLW01 , DsRED -AtTGD2 pLW01 and DsRED pLW01 constructs, recombinant protein expression and purification ................................52 CrTGD2 antibody ..................................................................................................52 Immunoblotting ......................................................................................................52 Subcellular fractionat ion ........................................................................................53 Protease protection assay .......................................................................................54 MGDG synthase assay ...........................................................................................54 Lipid binding assay ................................................................................................54 ACCESSION NUMBERS .................................................................................................55 ACKNOWLEDGEMENT S ...............................................................................................56 APPENDICES ...................................................................................................................66 APPENDIX A. SUPPORTING FIGURES ...........................................................67 APPENDIX B. SUPPORTING TABLES .............................................................82 APPENDIX C. SUPPORTING DATA .................................................................85 APPENDIX D. SUPPORTIN G METHODS .........................................................87 REFERENCES ..................................................................................................................91 CHAPTER 3: Characterization of Chlamydomonas LIP4, a putative triacylglycerol lipase ......100 ABSTRACT .....................................................................................................................101 INTRODUCTION ...........................................................................................................102 RESULTS ........................................................................................................................104 CrLIP4 is down -regulated during N -deprivation .................................................104 CrLIP4 contains DUF3336, transmembrane, and pata tin domains and a large IDR at its C terminus ..................................................................................104 Phylogenetic analysis of CrLIP4 .........................................................................105 Down -regulation of CrLIP4 transcript resulted in slower TAG degradation ......106 CrLIP4 coding sequence cloned from Chlamydomonas dw15.1 contains four amino acid changes and a five amino acid insertion compared to the gene model ...........................................................................................................106 CrLIP4 was not able to rescue yeast tgl3 ! , tgl4 ! , or tgl3 ! tgl4 ! mutants ..........107 CrLIP4 could not complement Arabidopsis sdp1 mutants ..................................108 Recombinant CrLIP4 and SDP1 protein expression in Escherichia coli ............109 !!$#!Recombinant CrLIP4 showed TAG lipase activity in vitro .................................109 DISCUSSION ..................................................................................................................110 MATERIALS AND METHODS .....................................................................................112 Algal strain and growth condition ........................................................................112 Artificial microRNA (amiRNA) knockdown ......................................................112 RNA isolation and cDNA synthesis ....................................................................113 Quantitative reverse transcription PCR (qRT -PCR ) ............................................113 Bioinformatic analysis .........................................................................................113 Phylogenetic reconstructions ...............................................................................114 Lipid analysi s .......................................................................................................114 Cloning of CrLIP4 coding sequence ....................................................................114 Expression of CrLIP4 in yeast tgl3 ! tgl4 ! double, tgl3 ! , and tgl4 ! single mutants .................................................................................................................115 Western blot analysis ...........................................................................................115 Analysis of CrLIP4 overexpression in yeast tgl3 ! tgl4 ! double, and tgl3 ! and tgl4 ! single mutants .....................................................................................116 Plant materials and growth conditions .................................................................116 Genomic DNA isolation from Ara bidopsis leaves ..............................................116 Genotyping of sdp1 T-DNA insertion lines .........................................................116 Construction of pMDC32 -CrLIP4 plasmid .........................................................117 Preparation of Agrobacterium competent cells ....................................................117 Agrobacterium transformation .............................................................................117 Arabidopsis transformation ..................................................................................117 Construction of pET28 -AtSDP1 and pET 28-CrLIP4 plasmids ...........................118 Recombinant AtSDP1 and CrLIP4 protein production .......................................118 Recombinant protein purification ........................................................................118 Lipase assay .........................................................................................................119 ACCESSION NUMBERS ...............................................................................................119 REFERENCES ................................................................................................................141 CHAPTER 4: Conclusion and Perspective ..................................................................................148 SUMMARY OF PHYSIOLOG ICAL, GENETIC, AND B IOCHEMI CAL FEATURES OF CHLAMYDO MONAS TGD2 .............................................................149 Phenotype of the tgd2 mutant ..................................................................149!Identification of the mutation in tgd2 ......................................................149!Characterization of changes in lipid trafficking in the tgd2 mutant ........150!Synthesis of galactoglycerolipid in tgd2 is affected ................................150!Localizatio n and lipid binding property of CrTGD2 ...............................150!Function of CrTGD2 and consequence of its absence .............................150!New insights from this study ...............................................................................151 Remaining questions and future directions ..........................................................152 MGDG and DGDG syntheses ..................................................................152 Feedback inhibition of DGDG synthesis .....................................152 Inaccuracy in the measurement of DGDG synthesis ...................153 PtdOH inhibits DGDG synthase ..................................................153 Labeling of other lipids ................................................................153 !!$## !Metabolic flux analysis ................................................................153 Does CrTGD2 form a complex with CrTGD1 and CrTGD3 ? .................153 The absence of TGD4 and TGD5 in the Chlamydomonas genome .........154 TGD4 and TGD5 may be present in Chlamydomonas but may not be detectable ...................................................................154 Other potential function(s) of CrTGD2 ...................................................154 Which galactolipase and/or lipoxygenase is responsible for degradation of MGDG ? ...........................................................................155 Localization of MGDG and DGDG synthases ........................................155 CrLIP4, A PUTATI VE TAG LIPASE ............................................................................155 Conclusion ...........................................................................................................155 Future directions ..................................................................................................156 Heterologous expression in yeast and Arabidopsis experiments .............156 Heterologous expression of CrLIP4 in yeast double mutant .......156 Heterologous expression of CrLIP4 in single mutants of yeast ...156 Heterologo us expression of CrLIP4 in Arabidopsis mutant ........156 Substrate specificity and kinetics study of CrLIP4 ..................................157 Inhibitors of lipases ..................................................................................157 The function of the intrinsic disordered region (IDR) of CrLIP4 ............157 Target mutation to obtain a CrLIP4 loss -of-function mutan t ..................158 Targeting Induced Local Lesions in Genomes (TILLING ) ..........158 Homologous recombination .........................................................158 Zinc -finger nucleases ...................................................................158 Transcription activator -like effector nucleases ...........................159 CRISPR -Cas9 ...............................................................................159 SUMMARY .....................................................................................................................160 REFERENCES ................................................................................................................161 SUPPORTING CHAPTER: Critical Role of Chlamy domonas reinhardtii Ferredoxin -5 in Maintaining Membrane Structure and Dark Metabolism ..............167 ABSTRACT .........................................................................................................168 SIGNIFICANT STATEMEN T ............................................................................168 INTRODUCTION ...............................................................................................168 RESULTS ............................................................................................................170 fdx5 is a null mutant .................................................................................170 FDX5 localizes to chloroplasts ................................................................170 Disruption of FDX5 causes a dark growth deficiency .............................170 Photosynthesis and respiration decrease in dark -maintained fdx5 ...........171 Photosynthetic electron flow is altered in dark -maintained fdx5 .............171 Photosynthetic polypeptides are not altered in fdx5 ................................172 Dark -maintained fdx5 has altered membrane u ltrastructure ....................172 Membrane lipids are altered in dark -maintained fdx5 .............................173 FDX5 interacts with Cr!4FAD and CrFAD6 desaturases ......................174 Increased TAG in fdx5 mutant in the dark ...............................................174 DISCUSSION ......................................................................................................175 MATERIALS AND METHOD S .........................................................................178 !!$### !Strains, mutant isolation and growth conditions ......................................178 Complementation and transformation ......................................................178 Phenotyping and growth rates ..................................................................179 Photosyntheti c O 2 evolution and respiratory O 2 consumption ................179 Transmission electron microscopy (TEM ) ..............................................179 Analysis of total and membrane lipids .....................................................179 TAG analysis and staining of lipid droplets ............................................179 Mating -Based Split Ubiquitin assay ........................................................180 ACKNOWLEDGEMENTS .................................................................................180 APPENDICES .....................................................................................................185 APPENDIX A. SUPPORTING FIGURES .............................................186 APPENDIX B. SUPPORTING TABLES ...............................................206 APPENDIX C. SUPPORTING DATA ...................................................216 APPENDIX D. SUPPORTING METHODS ...........................................218 REFERENCES ....................................................................................................220 !!$#" !LIST OF TABLES Table 2.S1. Bacterial Artificial Chromosomes (BACs) used in the tgd2 complementation analysis ......................................................................................82 Table 2.S2. Sequences of primers for probing the deletion in chromosome 16 of the tgd2 mutant ....................................................................................................................83 Table 2.S3 . Sequences of primers used for constructing plasmids as indicated .......................84 Table 3.1 . Protein domains identified in CrLIP4 homologues .............................................136 Table 3.2 . Target and primer sequences for artificial microRNA of CrLIP4 .......................138 Table 3.3. Sequences of Primers used for various purposes as indicated .............................139 Table A.S1. Genes encoding ferredoxins in the Chlamydomonas genome .............................207 Table A. S2. Primers used in this st udy ....................................................................................208 Table A.S3 . Strains used in this study .....................................................................................210 Table A.S4 . Constructs used in this study ............................................................................... 211 Table A.S 5. Pull down assay using FDX5 to establish interacting proteins ............................212 !!$"!LIST OF FIGURES Figure 1.1. Chemical structures of fatty acids and lipids .........................................................15 Figure 1.2. Lipolysis of neutral lipids ......................................................................................16 Figure 1.3. Screening of Chlamydomonas mutants .................................................................17 Figure 1.4. Identification of the insertion locus in the Chlamydomonas tgd2 mutant using whole -genome resequencing ..................................................................................18 Figure 2.1. Lipid phenotypes of Chlamydomon as tgd2 mutant ...............................................56 Figure 2.2. Viability assay for the PL (dw15.1), tgd2 and complemented line TGD2 tgd2 grown in N -replete medium ...................................................................................58 Figure 2.3. Ultrastructural changes in the tgd2 mutant ............................................................59 Figure 2.4. [14C]-Acetate pulse -chase labeling of PL (dw15.1), tgd2 mutant, and TGD2 tgd2 complemented line ..............................................................................60 Figure 2.5. Galactoglycerolipid synthesis of PL (15.1) and tgd2 chloroplasts ........................61 Figure 2.6. Localization of CrTGD2 ........................................................................................62 Figure 2.7. Lipid binding assay ................................................................................................63 Figure 2.8. Proposed model of CrTGD2 function ....................................................................64 Figur e 2.S1. Separation of Phosphatidic acid (PtdOH) and oligogalactoglycerolipids by thin layer chromatography (TLC) ..........................................................................67 Figure 2.S2. Cell viability and acyl group composition of TAGs during extended culturing time ........................................................................................................................68 Figure 2.S3. Ultrastructure of chloropla st membranes ...............................................................69 Figure 2.S4. Southern blot analysis of the tgd2 mutant and the PL (dw15.1) ............................70 !!$"# !Figure 2.S5. Analysis of progeny from crosses between tgd2 and CC -198 ...............................71 Figure 2.S6. Mutant locus in the tgd2 genome and complementation .......................................73 Figure 2.S7. Amino ac id sequence alignment of AtTGD2 and CrTGD2 ...................................75 Figure 2.S8. Phylogenetic analysis of CrTGD2 homolog ues .....................................................76 Figure 2.S9. Lack of Arabidopsis TGD2 complementation in Chlamydomonas tgd2 mutant ..78 Figure 2.S10. Lack of Chlamydomonas TGD2 complementation in Arabi dopsis tgd2 mutant ..79 Figure 2.S11. Pulse chase [ 14C]-acetate labeling of MGDG in the PL (dw15.1) .........................81 Figure 3.1. Relative expression of CrLIP4 transcript compared to TAG concentration during N deprivation and N resupply ...................................................................120 Figure 3.2. Amino acid sequenc e analysis of CrLIP4 ............................................................121 Figure 3.3. Prediction of transmembrane helices (TMD) and alignment of intrinsic disorder regions (IDRs) of CrLIP4 homologues ..................................................122 Figure 3.4. Phylogenetic analysis of CrLIP4 homologues .....................................................124 Figure 3.5. Down regulation of CrLIP4 transcript with artificial microRNA (amiRNA) .....126 Figure 3.6. Sequence alignment of the CrLIP4 ......................................................................127 Figure 3.7. Heterologous expression of CrLIP4 in the yeast tgl3 ! tgl4 ! double mutant .......128 Figure 3.8. Heterologous expression of CrLIP4 in yeast tgl3 ! and tgl4 ! mutants ...............129 Figure 3.9. Heterologous expression of CrLIP4 in Arabidopsis sdp1 mutants ......................130 Figure 3.10. CrLIP4 recombinant protein expression in E. coli ...............................................132 Figure 3.11. In vitro lipase assay ..............................................................................................134 !!$"## !Figure A.1. The fdx5 mutant is unable to grow and has attentuated respiration and photosynthesis rates in the dark ...........................................................................182 Figure A.2. Altered membrane morphologies and lipid compositions in dark -maintained fdx5 ......................................................................................................................183 Figure A.3. C16:4 !4,7,10,13 fatty acid is decreased in dark -grown fdx5 and FDX5 interacts with fatty acid desaturases ...................................................................................184 Figure A.4. PGD1-mediated TAG accumulation in fdx5 in the dark ......................................185 Figure A.S1. Ferredoxin -mediated electron transfer reactions .................................................187 Figure A.S2. Gene ration and molecular analyses of fdx5 mutant .............................................188 Figure A.S3. Localization of FDX5 ..........................................................................................190 Figure A.S4. fdx5 growth at different light intensities ..............................................................192 Figure A.S5. The fdx5 dark growth deficiency is linked to paromomycin resistance ...............193 Figure A.S6. Elevated li ght-induced O 2 consumption occurs in fdx5 maintained in the dark ..194 Figure A.S7. Fluorescence and spectroscropic analyses show impairment of photosynthetic electron transport .................................................................................................195 Figure A.S8. Immunoblot of representative photosynthetic proteins ........................................197 Figur e A.S9. Profiles of fatty acids in MGDG after growth in the light and dark ....................198 Figure A.S10. Profiles of fatty acids in DGDG after growth in the light and dark .....................199 Figure A.S11. Localization of Cr!4FAD and CrFAD6 ..............................................................200 Figure A.S12. Separation of neutral lip ids in different cell types under different light conditions .............................................................................................................201 Figure A.S13. Genotyping of the fdx5pgd1 double mutants .......................................................203 !!$"### !Figure A.S14. Fatty acid profiles in fdx5 and the fdx5#13 strains in the dark at 48 h . ...............203 Figure A.S15. Model for FDX5 -mediated regulation of thylakoid membrane structure in the dark ............................................................................................................204 Figure A.S16. The impact of PGD1 on lipid production in the various strains in the dark ........205 !!%! CHAPTER 1 Lipid metabolism in Chlamydomonas reinhardtii !!&! Photosynthetic organism s play an important role in utilizing solar energy to produce chemical energy in the form of carbon -based molecules. This energy makes life on earth possible. In todayÕs world, microalgae contribute to almost half of the total carbon assimilation on earth (Moroney & Ynalvez, 2001) . Microalgae serve as primary producers in different ecological systems. They synthesize energy -rich compounds such as starch, triacylglycerol (TAG), and hydrogen. Many species of microalgae can produce polyunsaturated fatty acids (PUFAs) that are valuable for nutrition and provide the basis of healthy Òfish oilsÓ (Riediger et al. , 2009) . Due to their short life cycle, minimal space requirements, and the ability of many species to grow in salt or wastewater, microalgae are a good op tion for the production of biofuels and bio -products (Demirbas & Fatih Demirbas, 2011; Dismukes et al., 2008; Koller et al., 2014; Spolaore et al. , 2006) . Both biodiesel and PUFAs are derived from the metabolism of lipids. Most of what we know about lipid metabolism of microalgae has been deduced by analogy to either plants or fungi. However, microalgae appear to have some unique characteristics indicating that their metabolic processes may differ from those of plants. In this chapter , I will emphasize (i) the importance of studying lipid metabolism in a model algal species, Chlamydomonas reinhardtii , (ii) comparison of lipid metabolism between Arabidopsis and C. reinhardtii , (iii) the action of triacylglycerol (TAG) lipases for lipolysis, and (iv) mutant screening and identification. WHY STUDY LIPID METABOLISM IN CHLAMYDOMONAS ? Chlamydomonas sp. is a unicellular eukaryotic microalga in the division Chlorophyta of the kingdom Viridiplantae. Early research on various species of genus Chlamydomonas focused on genetic mechanisms, flagella, and photosynthesis (Goodenough, 2015) for many reasons. First, Chlamydomonas sp. harbors a haploid genome that allows genetic analysis to be carried out easily (Harris, 1989) . Second, mutants occur spontaneously or can be induc ed through the application of chemicals, irradiation or procedures for gene disruption (Harris, 2001) . These two reasons make Chlamydomonas a good model organism for studying gene function since mutants can be selected that immediately show a phenotype. Th ird, C. reinhardtii in particular can grow under heterotrophic conditions, which allows photosynthesis -deficient mutants to survive in the presence of acetate. Finally, the C. reinhardtii genome has been sequenced (Merchant et al. , 2007). For these reasons , much research has been focused on C. reinhardtii, which I will refer to as Chlamydomonas in this dissertation. !!'!Chlamydomonas was not considered to be an oleaginous alga and was reported to be unable to synthesize very long chain PUFA according to the US Department of EnergyÕs survey (Sheehan et al. , 1998) . However, recent research has shown that the alga can accumulate TAG under stress conditions especially in starchless mutants (James et al. , 2011; Y. Li et al. , 2010; Wang et al. , 2009; Work et al. , 201 0). The attempt to find alternative renewable energy to replace fossil fuels has stimulated research on algal lipid metabolism using Chlamydomonas as a model organism. This is due to the fact that its genetics and physiology are well -characterized, and mol ecular genetics and genomics tools are more developed than for other algae (Day & Goldschmidt -Clermont, 2011; Michelet et al. , 2011; Rochaix, 2002) . Moreover, as mentioned previously, its genome has been sequenced (Merchant et al. , 2007) . Therefore, using Chlamydomonas as a model organism to study lipid metabolism in microalga has advantages over other microalgae that accumulate more oil but are not as well characterized. Initial research on lipid metabolism of Chlamydomonas focused on identification of the specific lipid molecules, sites of lipid biosynthesis and fatty acid composition (Li-Beisson et al., 2015). These biochemical analyses revealed differences in lipid metabolism of Chlamydomonas and plants, as I will discuss in the following section. More t han 10 mutants defective in lipid metabolism have been identified and characterized since 1989 as reviewed by Li-Beisson et al. (2015). Among these mutants, novel genes were identified that are involved in biosynthesis and degradation of different lipid sp ecies; i.e. fatty acid desaturases, TAG and polar lipid synthases, and a lipase. These investigations have illustrated the value of mutant screening for identifying new genes involved in the metabolism of lipids. Although 48 orthologues of plant and yeast lipid proteins were annotated in the Chlamydomonas genome (Riekhof et al. , 2005) , many of the proteins have not been characterized. One example of these is a TAG lipase as I will discuss later in this Chapter and in Chapter 3. Conceivably, gene orthologue s present in the genome of the alga have a different role than they do in plant metabolism. This issue will be addressed in Chapter 2. !!(!LIPID METABOLISM IN CHLAMYDOMONAS VS. AR ABIDOPSIS Our current knowledge of algal lipid metabolism is based on studies in Arabidopsis. Regarding in silico analysis of lipid genes in Chlamydomonas, t he overall picture of lipid metabolism in this alga is similar to and simpler than that of Arabidopsis (Riekhof et al. , 2005) . In general, we believe that major pathways for fatty acid synthesis, diacylglycerol (DAG) assembly and glycerolipid biosynthesis are conserved between Chlamydomonas and Arabidopsis. This assumption is based on the presence of homologues of genes encoding enzymes involved in the lipid common pathways (Li -Bei sson et al. , 2015) . In most cases, Chlamydomonas lipid genes are present in fewer copies than those in Arabidopsis. However, analyses of Chlamydomonas fatty acid components, lipid species, and lipid biosynthesis enzymes have revealed that evolutionary dive rgence of microalgae and seed plants has resulted in differences in synthetic pathways. These differences will be addressed in the following three subsections. Fatty acid composition . Chlamydomonas fatty acid composition is similar to that of Arabidopsis i n that glycerolipids contain fatty acids with 16 or 18 carbons (C16 or C18). As described in more detail in Chapter 2, in plants, the presence of C16 or C18 acyl groups in the sn-2 position of the glycerol backbone is reflective of the synthetic pathway of the corresponding lipid. Lipids containing C16 acyl groups at their sn-2 position are derived from prokaryotic pathway and are synthesized within the chloroplast, while those with C18 are derived from the endoplasmic reticulum -based eukaryotic pathway (He inz & Roughan, 1983; Roughan & Slack, 1982). The discrimination between these two pathways is based on substrate preferences of acyltransferases located either in the chloroplast or the endoplasmic reticulum (ER) (Frentzen et al. , 1983; Kim et al. , 2005; K unst et al. , 1988; Roughan & Slack, 1982) . Positional analysis by Giroud et al. (1988) concluded that the plastidic and extraplastidic lipids of Chlamydomonas are almost exclusively synthesized through the prokaryotic and eukaryotic pathways, respectively. However, substrate preferences of acyltransferases in Chlamydomonas still remain to be tested. One difference from Arabidopsis is that Chlamydomonas synthesizes unique fatty acids containing front -end ! 4 or ! 5 double bonds (Figure 1.1A). The ! 4 double bo nd is present in 16:4!4,7,10,13 (number of carbons : number of double bonds with !Number indicating the position of the double bond counted from the carboxyl end) of monogalatosyldiacylglycerol (MGDG) (Giroud et al. , 1988; Z−uner et al. , 2012) , while the !5 double bond can be found in 18:3 !5,9,12 !!)!and 18:4 !5,9,12,15 of diacylglyceryl -N,N,N-trimethylhomoserine (DGTS) and phosphatidylethanolamine (PtdEtn) (Giroud et al. , 1988; Kajikawa et al. , 2006). The 16:4 !4,7,10,13 in MGDG is catalyzed by the chloroplas t-located ! 4 desaturase named Cr!4FAD from 16:3 !7,10,13 (Z−uner et al. , 2012) . Down-regulation of Cr!4FAD leads to a lower abundance of 16:4 !4,7,10,13 and also MGDG. This indicates that a tight relationship may exist between the acyl group and the correspo nding lipid, as reflected by the stable ratio of 16:4!4,7,10,13 and 18:3 !9,12,15 within MGDG. Thus, the altered ratio of these two acyl groups leads to less MGDG being made. The importance of 16:4 !4,7,10,13 to the synthesis of MGDG and to the photosynthe tic membranes of Chlamydomonas is suggested in the Appendix of this dissertation. In that study, it is shown that desaturation of 16:3 !7,10,13 to produce MGDG with 16:4 !4,7,10,13 contributes to the ability to grow of the alga in the dark. Synthesis of 18 :3!5,9,12 and 18:4 !5,9,12,15 take s place in the ER by the action of the ! 5 desaturase (CrDES) from 18:2 !9,12 and 18:3 !9,12,15 , respectively (Kajikawa et al. , 2006) . Similar to Cr ! 4FAD, CrDES contains a cytochrome b5 domain, which is commonly found in fron t-end desaturases in the ER (Petra Sperling & Heinz, 2001; P. Sperling et al. , 1995) . In addition to their presence in Chlamydomonas, the 18:3 !5,9,12 and 18:4 !5,9,12,15 fatty acids are widely distributed in gymnosperms, but not in angiosperms, suggesting t hat the ability to desaturate the !5 position was lost from the angiosperm lineage. Lipid species . Chlamydomonas and Arabidopsis contain similar lipid species, with three exceptions. Biochemical analysis by Giroud et al. (1988) revealed that phosphatidylc holine (PtdCho) and phosphatidylserine (PtdSer) were absent from the alga. Instead, Chlamydomonas contains DGTS that is not present in Arabidopsis. DGTS is thought to be a substitute for PtdCho due to their similarity in structural (Fig. 1.1 B & C) and bio physical properties (B. Liu & Benning, 2013; N. Sato & Murata, 1991) . Although not found in seed plants, DGTS has been identified in other algae, bacteria, and non -seed plants (Moellering et al. , 2010). Although DGTS has a structure and chemical propertie s similar to PtdCho, it might not be able to substitute for the biochemical function of PtdCho. Because DGTS contains an ether bond instead of an ester bond as in PtdCho, it is an inferior substrate for DAG synthesis (B. Liu & Benning, 2013) . Hence, DGTS i s not as versatile as PtdCho as a central metabolite for biosynthesis of different lipids. !!*! The lack of PtdSer in Chlamydomonas is in accordance with an absence of the gene for its biosynthesis (Riekhof et al., 2005) . Since synthesis of PtdEtn relies on Pt dSer as a substrate, the authors suggested that PtdEtn in Chlamydomonas is carried out through a transfer of phosphoethanolamine to a DAG moiety instead of through the decarboxylation of PtdSer as in yeasts, bacteria, and plants (Voelker, 1997) . Genes invo lved in lipid metabolism . In general, Chlamydomonas harbors fewer copies of lipid genes compared to Arabidopsis (Riekhof et al. , 2005) . For example, Arabidopsis contains three and two copies of MGDG and digalactosyldiacylglycerol (DGDG) synthases, respecti vely; while Chlamydomonas has only one copy of each gene. In Arabidopsis, as summarized by Petroutsos et al. (2014) the major MGDG and DGDG synthase are MGD1 and DGD1, respectively; they are located in the inner and outer envelope membrane of the chloropla st, respectively. These two enzymes synthesize galactolipids for the thylakoid membrane in photosynthetic tissue. In contrast, two other MGDG synthases (MGD2 and MGD3) and DGDG synthase (DGD2) are located in the outer envelope membrane (Awai et al. , 2001) . They are expressed in non -photosynthetic tissues and in response to phosphate limitation (Botte et al., 2011a; Kobayashi et al. , 2004) . A broad phylogenetic analysis of MGDG synthases indicates that the MGDG synthase in Chlamydomonas is a pre -angiosperm M GD1 orthologue (Botte et al., 2011b; Petroutsos et al., 2014; Yuzawa et al. , 2012) . This implies that MGDG and DGDG synthases in Chlamydomonas are mainly responsible for synthesizing galactoglycerolipids of photosynthetic membranes. The absence of MGD2, MG D3, and DGD2 orthologues in the alga suggests the existence of a different response to phosphate limitation in the alga (Moellering et al. , 2010). In contrast to the biosynthesis pathway for membrane lipids for which plants have a higher number of gene co pies, algae have a higher number of genes for the pathway of TAG synthesis. In Chlamydomonas, six copies of diacylglycerol acyltransferases (DAGATs) were identified and categorized into two types; one DGAT, and five DGTTs (Deng et al. , 2012; Merchant et al ., 2012). A more extreme case is seen in Nannochloropsis sp., an olegenous marine alga that has 13 copies of predicted DAGAT encoding genes (Vieler et al., 2012) . This higher number of gene copies for putative DAGATs in the algae compar ed to Arabidopsis co uld suggest gene loss during evolution due to the fact that vegetative tissues of higher plants do not accumulate TAG in high quantity. !!+!LIPOLYSIS AND LIPASES Lipolysis is a crucial reaction that results in the generation of lipid messengers, membrane rem odeling and lipid homeostasis. Lipases hydrolyze either an ester or amide bond of an acyl chain from various types of lipids, resulting in a free fatty acid and the corresponding lysolipid. Lipases are diverse enzymes including TAG lipases, phospholipases, galactolipases, ceraminidases, cholesterol ester hydrolases and retinol ester hydrolases (M. Li & Wang, 2014) . Figure 1.2 shows lipolysis of neutral lipids by TAG, DAG, or monoacylglycerol (MAG) lipase. For the purpose of TAG homeostasis in Chlamydomonas that is discussed in Chapter 3, I will focus on TAG lipase in this section. Lipases are interfacial enzyme s. This is due to the fact that the enzyme is hydrophilic whereas the substrate is lipophilic. This phenomenon was originally observed by Sch¿nheyder and Volqvartz (1945) and then reinvestigated by Sarda and Desnuelle (1958) . In a low concentration of substrate, lipase activity was almost undetectable. However, when the concentration of the hydrophobic substrate exceeded the solubility limit , thus allow ing the substrate to be present in the form of micelles or emulsion drops, the lipase activity was increased sharply. The enzyme is activated by its interaction with the aqueous -hydrophobic interface. For this reason, kinetics of the TAG lipase does not fo llow normal Michaelis -Menten kinetics as reviewed by Gill and Parish (1997); Verger (1976) ; Reis et al. (2009). The mechanism of interfacial activation has been explained through a three dimensional structure of a Mucor miehei TAG lipase (Brady et al., 199 0) and of human pancreatic lipase (Winkler et al., 1990) . In the inactive stage, the active site of the lipase, containing serine, histidine, and aspartate residues, is protected from the hydrophilic environment by a lid structure. Upon interaction with aq ueous -lipid interface, the enzyme undergoes a conformational change exposing the active site to the hydrophobic substrate. Many TAG lipases contain a patatin domain. Patatins (Pfam01734) are potato tuber pro teins that carry an evolutionarily conserved este rase box GXSXG (where G is glycine, X is any amino acid, and S is serine) and exhibit acyl -hydrolyzing activity (Andrews et al., 1988) . This group of proteins was classified into the patatin -related phospholipase A family (pPLA) , which is a member of the p hospholipase A 2 (PLA 2) superfamily (Scherer et al. , 2010) . This PLA 2 superfamily is comprised of the cytosolic or Ca 2+-activated cPLA 2, the Ca 2+-independent iPLA 2 !!,!and the secreted sPLA 2 as summarized in (Scherer et al. , 2010) . The pPLAs can be further classified into 4 groups; pPLAI, pPLAII ( ", #, $, %, &), pPLAIII ( ", #, $, %) (Scherer et al., 2010) , and group 4 subclass (M. Li & Wang, 2014) . Group 4 contains the only enzymes that catalyze hydrolysis of TAG while the other members of pPLAs hydrolyze phosph olipids and/or galactoglycerolipids. Members of group 4 pPLAs are Arabidopsis SDP1, SDP1 -like, and adipose triglyceride lipase -like (ATGL -L) (Eastmond, 2006) . As discussed in detail in Chapter 3, SDP1 is responsible for TAG mobilization during seed germina tion and the establishment of photosynthesis. Patatin -domain containing enzymes are unique lipases. As mentioned previously, patatin domain containing lipases contain an esterase motif and exhibit acylhydrolase activity as do typical lipases. However, crys tallography of Pat17, an isozyme of potato patatin, revealed that patatin did not possess a cannonical catalytic triad (Serine -Aspartate -Histidine) and did not adopt an " / # hydrolase fold structure as do other lipases (Rydel et al. , 2003) . Instead Pat17 ca rries a catalytic dyad (Serine -Aspartate) with an " / # / " structure. In addition, the study showed that Pat17 lacked a lid structure that was proposed to protect the catalytic site of a lipase. Therefore, the authors concluded that patatin did not likely int eract interfacially, as opposed to other lipases. The activation of patatin was proposed to occur through translocation of the enzyme from the storage vacuole to the cytosol (H. Sato & Frank, 2004) . In the same study of Pat17 by Rydel et al. (2003), an am phipathic helical structure was identified within its topology that is equivalent to the lid structure of human cytosolic phospholipase A2 (cPLA 2) (Dessen et al., 1999) . Furthermore, low -resolution homology models of human patatin -like phospholipases (PNPL As) suggested a loop structure that could function as a lid but with minimal flexibility (Wilson et al., 2006) . Since mammalian ATGL is a member of PNPLAs (Kienesberger et al., 2009) and also a homologue of SDP1 (Eastmond, 2006) in group 4 pPLA, it is stil l unclear whether members of group 4 pPLA possesses a lid structure that allows the enzyme to be activated at the water -lipid interface. Biochemical and structural analyses are necessary to unravel this problem. !!-!MUTANT SCREENING AND IDENTIFICATION OF A RESPONSIBLE GENE Fundamental questions in biology have been studied through model organisms such as Caenorhabditis elegans , fruit fly, Arabidopsis, zebrafish and mouse. Knowledge from these studies can be further applied to other organisms including economic ally important ones, e.g. food crops and livestock as well as humans. For this purpose, genetic screening serves as a great tool to study gene function in model organisms. This approach can be classified into two types; forward and reverse genetics. Forwar d genetics starts with a particular phenotype and seeks to identify the responsible gene. In reverse genetics, the site of mutation is known, and the phenotype is characterized. While reverse genetics specifically studies the function of a particular gene, forward genetics allows one to discover a novel gene. In this section, I will focus on mutagenesis and gene identification through forward genetic screening. Forward genetic screening Forward genetic screens utilize chemical, physical or biological agent s to mutate DNA of an organism. Chemical mutagens, such as ethylmethan ol sulphonate (EMS) and nitrosomethylurea (NMU) , yield a high mutation rate and depending on the mutagen, they generate various types of mutation including base substitutions and small i nsertions and deletions (Alonso & Ecker, 2006) . The major disadvantage of using chemical mutagens is the unknown location of the mutated genes. This is due to the fact that point mutations are difficult to locate, especially in a large genome. Physical age nts such as fast neutrons, X -rays or accelerated ions can be utilized to introduce large insertions/deletions and rearrangement s in the genome (Alonso & Ecker, 2006) . Finally, certain biological agents such as T -DNA and transposons can also be used to indu ce mutations. Selectable markers can also be used for mutagenesis by random insertional gene disruption. These include antibiotic or herbicide resistance, and genes that confer the ability to grow under specific conditions (prototrophy). Forward genetic sc reens using biological agents are widely used in Chlamydomonas because identification of the mutation is relatively easy. Using a specific DNA sequence that is carried by the biological agent, PCR -based techniques can be used to identify the mutation withi n adjacent DNA. Once a population of transformants is generated, screening for mutants with a diagnostic phenotype is performed. For instance, Chlamydomonas mutants accumulating high amounts of TAG can be screened by Nile Red fluorescence staining which i s specific for neutral lipids (Kimura et al. , 2004) . The signal is detected with a plate reader (X. Li et al. , 2012) or a flow !!%.!cytometry (Cagnon et al. , 2013) . In my study, the identification of the Chlamydomonas tgd2 mutant in Chapter 2 was carried out fo llowing a random insertional mutagenesis with a DNA fragment carrying a hygromycin resistance gene ( AphVII ). The mutant population was screened by Western blot using an antibody against the major lipid droplet protein (MLDP). It abundance is tightly correl ated with the amount of TAG accumulating in the cells. A diagram of the screening procedure is depicted in Figure 1.3. After the mutants have been identified, the responsible genes that cause the phenotype need to be identified. Different methods are discu ssed below. Map -based cloning . Before DNA sequencing and/or PCR became available, the method to identify mutation s relied on map -based cloning (also called positional cloning) which was first introduced in 1913 (Sturtevant, 1913) . The principle of this app roach is that the distance between the gene of interest and the marker correlates to the recombination frequency. In general, the more infrequent the recombination events, the closer the gene of interest is to the marker. Therefore, this technique relies o n the availability of markers, which were limited until the invention of ways to detect DNA differences between isolates or ecotypes including restriction fragment length polymorphisms (RFLPs) (Botstein et al. , 1980) , random amplified polymorphic DNAs (RAP Ds) (Williams et al. , 1990) , simple sequence repeats (SSRs) (Bell & Ecker, 1994) , single -nucleotide amplified polymorphisms (SNAP) (Drenkard et al. , 2000) , and amplified fragment length polymorphisms (AFLP) (Vos et al. , 1995) . The advantage of mapping with DNA-based markers is that no prior knowledge about a specific gene is required. The time for mapping a specific gene in Arabidopsis has been shortened from 3 -5 years to a single year (Jander et al. , 2002) , and most recently to months using combined mappin g and sequencing strategies involving whole genome -resequencing techniques, which are discussed in the following sections. PCR-based methods . PCR -based techniques exploit the known DNA sequence that was used for creating an insertional mutation as a key t o identify the unknown flanking region. Only one defined primer -binding site is known, and it lies within the DNA insertion. Several different strategies are used to enable a second oligonucleotide to prime the reaction from the adjacent unknown DNA as dis cussed in three examples below. !!%%!The first example is inverse PCR, which is based on the inversion of an insert and flanking sequence to generate two primer -binding sites at both ends of the fragment (Triglia et al., 1988) . The technique employs one restri ction enzyme, which cuts the genomic DNA outside the insertion. The digested product is ligated to itself to create a circular molecule. This molecule is then digested with a second restriction enzyme to produce a linearized DNA fragment having unknown seq uence flanked by known DNA sequence at both 5Õ and 3Õ ends. With the known DNA sequence at both ends, PCR amplification can be carried out and the specific product can be amplified. The second approach is to attach another known DNA fragment to the sequenc e that flanks the insertion. An example of this approach is called SiteFinding -PCR (Tan et al. , 2005) . In this approach, the first round of PCR is carried out with a primer specific for the insertion and another primer attached to a SiteFinder that contain s a restriction site and a semi -random sequence. The random sequence in the SiteFinder of the second primer allows semi non -specific amplification. More specific amplicons can be obtained through nested PCRs. The PCR product can then be digested with a res triction enzyme specific to the site at the SiteFinder and cloned into a vector for determining the sequence of a gene of interest. The third method is thermal asymmetric interlaced (TAIL -) PCR (Y. G. Liu et al. , 1995; Y. G. Liu & Whittier, 1995) . This me thod does not require any modification of DNA molecule. A series of PCRs are carried out with one specific primer that can bind to an insert fragment and an arbitrary degenerate primer. The alternating PCR cycles between high and low stringency are used to increase specificity of the amplification, followed by another two sets of PCR with nested primers to enhance specificity. PCR -based methods are relatively easy and require less time compared to map -based cloning. However these approaches can be applied o nly to the mutants generated through biological mutagens with known sequence. In addition, high false positive rates can occur through the amplification of non -specific sequences due to the fact that only one primer -binding site is known. Finally, since th is method relies on amplification at the insertion site, if the insertion is located in a high GC region, the amplification can be difficult. An example of the last case can be seen in the identification of the tgd2 mutant in Chapter 2 in which inverse PCR and SiteFinding PCR both failed to identify the insertion site. !!%&!Whole genome resequencing . Both conventional and next generation DNA sequencing technologies have enabled many genome projects from diverse organisms starting with bacteriophage MS2 in 1976 (Fiers et al., 1976) . Twenty years later the first eukaryote to have its genome sequence was Saccharomyces cerevisiae (Goffeau et al. , 1996) . From that point on, many genome projects of model organisms have been completed. These include Caenorhabditis elegans (Consortium, 1998) , Arabidopsis (Arabidopsis Genome, 2000) , fruit fly (Adams et al. , 2000), human (Venter et al. , 2001) , and Chlamydomonas (Merchant et al. , 2007) , etc. The availability of next generation sequencing, which offers high -throughput results at an affordable price, and an extensive genome database provided new tools for identifying all kinds of mutations by directly comparing genomes of mutants to that of the reference genome. Resequencing allows direct comparison of the genome sequence of t he mutant with the reference genome from the database. Sequencing reads of the mutant can be aligned with those of the reference genome through a computer program that uses algorithms based on the Burrows -Wheeler Transform such as Bowtie (Langmead et al. , 2009) and BWA (H. Li & Durbin, 2009) . For example, 46 somatic mutations in four types of chronic lymphocytic leukemia were identified through an alignment between tumor DNA sequences and the human reference genome (Puente et al. , 2011) . The major advantage of this method is that it can be applied to mutations with single nucleotide changes or short insertions/deletions depending on the parameter settings and software. However, the application of this approach is limited to a mutant generated in the same bac kground as the reference genome. In addition, detection of large insertions/deletions or rearrangements can be challenging. As a variation, the site of insertion of a specific DNA sequence can be identified based on construction of contigs that contain the alien DNA. This approach can overcome the drawbacks of the read alignment approach because it can be applied to a mutant that is not generated from a reference genome background. However, this method is also limited to insertional mutagenesis. An example of this approach is the identification of the Chlamydomonas tgd2 mutant in Chapter 2. This mutant was generated in Chlamydomonas strain dw15.1 (CC -4619 cw15 nit1 mi +), which is not the same strain as the reference genome (CC -503 cw92 mt +). In this case, th e huge number of polymorphisms between the two Chlamydomonas strains can overwhelm the real mutation. However, alignment of the insert sequence to the contigs of assembled reads allowed its site of insertion to be established. Figure 1.4 illustrates this process. !!%'!A point mutation that is generated in a non -reference background can be detected through a method called mapping by sequencing. The principle of this approach is to sequence a pooled group of the F 2 generation that carry the mutant phenotype produ ced from a cross between the mutant in the non -reference background to the reference genome. The read alignment between the mutants and the reference genome can identify both polymorphisms and mutations. These two types of reads can be distinguished based on the principle of genetic recombination as in the conventional mapping. More information about this technique can be found in a number of articles (Candela et al. , 2015; Ossowski et al. , 2008; Schneeberger et al. , 2009) . An essential component of this me thod is the availability of a reference genome. Recently a new technique has been invented to eliminate this requirement. Based on the differences in frequency of k-mer (subsequence of length k from a sequencing read) between two closely related genomes, e .g. wild type and mutant, a single nucleotide change can be detected even in a mutant generated from non -reference background (Nordstrom et al. , 2013). Forward genetic screening in Chlamydomonas A number of forward genetic screen s in Chlamydomonas ha ve bee n carried out over the past 60 years. These screens led to identification of genes involved in photosynthesis, mobility, mating, nitrogen assimilation, and biosynthetic pathways, as summarized by Jinkerson and Jonikas (2015) . Some of these genes were scree ned based on their lipid phenotype, i.e. sqd1 (Riekhof et al., 2003) , crfad7 (Nguyen et al., 2013) , and pgd1 (X. Li et al., 2012) and cht7 (Tsai et al. , 2014) . Mutations have been created by several mutagens, including chemicals (EMS and N-methyl -NÕ-nitro -N-nitrosoguanidine, MNNG), physical insult (UV, X -ray and gamma irradiation), and biological agents (e.g., using hygromycin and paromomycin resistance genes on a transforming DNA ). The mutant alleles were mapped with various methods including classical gen etic mapping and PCR -based techniques. In 2012, whole genome sequencing was first reported to identify mutations and polymorphisms in Chlamydomonas (Dutcher et al. , 2012) , followed quickly by others (Lin et al., 2013a; Lin et al., 2013b; Tulin & Cross, 201 4). Recently, two UV -induced Chlamydomonas mutants exhibiting high light tolerance were also identified through this approach (Schierenbeck et al., 2015) . These examples show that discovery of novel genes in Chlamydomonas can be accomplished through differ ent types of forward genetic screening. In addition, many methods can be employed to identify a causative gene. Details about genetic screening in Chlamydomonas to discover genes in lipid metabolism can be found !!%(!in several recent articles (Cagnon et al. , 2 013; Jinkerson & Jonikas, 2015; Terashima et al. , 2015). AIM OF THE THESIS RESEARCH As previously mentioned , unicellular microalgae are crucial for a well -balanced ecological system as primary producers and they have a great potential for alternative ener gy and bio -product production. Our current state of knowledge indicates that not all biological and biochemical processes in microalgae are identical to those of land plants or other kingdoms of life. With respect to lipids, microalgae produce unique fatty acids and lipids that are absent from plants. On the other hand, some lipids found in plants are missing in algae. Moreover, enzymes for some important metabolic functions have not been characterized. This thesis research attempts to resolve these discrep ancies at least in part. Chlamydomonas reinhardtii, was selected as a model organism to represent unicellular microalgae and their lipid metabolism. The study is composed of two major parts. First, a forward genetic screen was performed to identify novel genes involved in lipid metabolism. The function of one gene in lipid metabolism of Chlamydomonas was then characterized. The details of this project can be found in Chapter 2. Second, a Chlamydomonas orthologue of a central triacylglycerol lipase in Arabid opsis seeds was characterized in Chapter 3. In addition, a collaborative project was carried out to study a gene important for synthesizing a unique fatty acid found in the chloroplast of Chlamydomonas. This topic will be addressed in the Appendix. !!%)! Figure 1.1. Chemical structures of fatty acids and lipids (A) 16:4 !4,7,10,13 , 18:3 !5,9,12 , and 18:4 !5,9,12,15 fatty acids (B) phosphatidylcholine (PtdCho) (C) diacylglyceryl -N,N,N-trimethylhomoserine (DGTS) !!!!%*!!!Figure 1.2. Lipolysis of neutral lipids (A) Degradation of triacylglycerol (TAG) by TAG lipase yields 1,2- or 2,3 -diacylglycerol (DAG) and free fatty acid (FFA). ( B) Degradation of DAG by DAG lipase yields monoacylglycerol (MAG) and FFA. ( C) Degradation of MAG by MAG lipase yields FFA and glycero l backbone. !!%+! Figure 1.3. Screening of Chlamydomonas mutants A random insertional mutagenesis was performed with the AphVII gene from the pHyg3 plasmid in the Chlamydomonas parental line (PL) strain dw15.1. The transformed colonies were selected on Tris -Acetate-Pho sphate (TAP) medium with hygromy cin. The transformants were picked and grown in liquid TAP medium (under nitrogen replete condition, N+) in 96 -well culture plates. To induce TAG accumulation, the cultures were transferred to TAP medium without n itrogen (N -) and incubated for 48 hours. The cultures were then reintroduced into TAP medium with nitrogen (NR), in order to observe TAG degradation. A mutant defective in this process is expected to have a high level of TAG compared to the PL. The amount of TAG can be determined by immun o-dot blot against the major lipid droplet protein (MLDP). The MLDP signal was normalized with the density of the cell s measured by a plate reader. Colonies showing a strong signal for MLDP were then tested for TAG content. During this screen the tgd2 mutant was discovered and shown to have increased TAG levels. Further detail s of this screening procedure can be found in Chapter 2. !!%,! Figure 1.4. Identification of the insertion locus in the Chlamydomonas tgd2 mutant using whole -genome resequencing . !!%-!Figure 1.4. (Continued) The genome of the Chlamydomonas tgd2 mutant was sequenced using the Illumina Hi -Seq paired -end method ( A). Each DNA fragment was sequenced at both ends yielding 2 sequencing reads. Paired sequencing reads were used for de novo genome assembly ( B) based on a known distance between the two reads illustrated as broken lines. This assembly yielded a number of contigs of the tgd2 genome. Identification of the insertion site was carried out in two major steps (C). First, the AphVII sequence was located in the tgd2 contigs by a BLAST search. Second, the tgd2 sequence flanking the AphVII was used as a query for another BLAST search in the reference genome. Once the insertion was located, alignment between the tgd2 contig containing the insertion and the reference genome in the corresponding location was carried out to determine the nature of the insertion site and possible deletions close by. The structure of the locus in the mutant was then further probed and confir med with PCR. Further details of this process can be found in Chapter 2. !!&.! REFERENCES !!&%!REFERENCES Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R . F., George, R. A., Lewis, S. E., Richards, S., Ashburner, M., Henderson, S. N., Sutton, G. G., Wortman, J. R., Yandell, M. D., Zhang, Q., Chen, L. 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CHAPTER 2 Chloroplast lipid transfer processes in Chlam ydomonas reinhardtii involving a TRIGALACTOSYL DIACYLGLYCEROL 2 (TGD2) orthologue * !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!/!This project was carried out in the collaboration with sever al colleagues and has been published in t he Plant Jour nal in Jaruswan Warakanont, Chia -Hong Tsai, Elena J. S. Michel, George R. Murphy III, Peter Y. Hsueh, Rebecca L. Roston, Barbara B. Sear s, and Christoph Benning (2015) Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTO SYLDIACYLGLYCEROL 2 (TGD2) orthologue. Plant J. 84: 1005 -10200!doi: 10.1111/tpj.13060. In collaboration with me, Chia -Hong Tsai screened the mutants. Elena J. S. Michel was an undergraduate student under my supervision. She contributed to Figure 2.1B, 2.S1 B, 2.S9, and 2.S10. George R. Murphy III and Peter Y. Hsueh were undergraduate students under the supervision of Rebecca L. Roston. They contributed in liposome binding assay in Figure 2.7, and made DsRED -CrTGD2 pLW01 construct used in Figure 2.7 and in th e production of antiserum against CrTGD2. Barbara B. Sears performed crossing between the tgd2 mutant and CC -198 wild type, providing progenies for genetic analysis in Figure 2.S5 and for electron microscopy in Figure 2.3. ! !!''!ABSTRACT In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl -constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas does not use the ER pathway. However, the Chlamydo monas genome encodes presumed plant orthologues of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER -to-chloroplast lipid trafficking in plants. To resolve this conundrum, we identified a muta nt of Chlamydomonas deleted in the TGD2 gene and characterized the respective protein, CrTGD2. Notably, the mutantÕs viability was reduced showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldi acylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conve rsion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impair ed in the tgd2 mutant. SIGNIFICANCE STATEMENT In plants, lipids in the chloroplast membrane are synthesized both in the ER and in the chloroplast envelope membranes. However, in Chlamydomonas, the chloroplast lipid composition has suggested that the ER pat hway was not involved. Here, analyzing a mutant in a chloroplast lipid transporter, we show that the C hlamydomonas chloroplast does likely import precursors from the ER for thylakoid lipid biosynthesis . !!'(!INTRODUCTION Microalgae play an important role as pr imary biomass producers in diverse ecosystems due to their ability to efficiently convert solar into chemical energy. In fact, estimates based on radioactive bicarbonate labeling of sea water samples from the Atlantic ocean suggest that marine algae accoun t for nearly a quarter of total global carbon fixation (Jardillier et al. , 2010) , which is in part due to their high photosynthetic efficiency (Melis, 2009; Weyer et al., 2010) . In addition to their importance in natural ecosystems, microalgae have receive d special attention as biofuel feedstocks due to their rapid life cycle and their potential to accumulate high levels of biomass in limited space. Chlamydomonas reinhardtii (Chlamydomonas) is a unicellular green alga, which has served for many decades as a n instructive model in studies of photosynthesis, flagella development and function, and more recently to explore the regulation of metabolism in response to different growth conditions. Chlamydomonas cells are haploid during vegetative growth and the Chla mydomonas genome often has a single copy of many plant orthologous genes facilitating genotype -phenotype relationship studies. Finally, availability of multiple resources including a sequenced genome (Merchant et al. , 2007) facilitates research on this mic roalga. Photosynthesis takes place in thylakoid membranes in bacterial cells or inside chloroplasts present in algae and land plants. Photosynthetic membranes from cyanobacteria, the presumed predecessor of chloroplasts, algae, and plants analyzed to date contain four lipids: the two galactoglycerolipids mono - (MGDG) and digalactosyldiacylglycerol (DGDG), the sulfoglycerolipid sulfoquinovosyldiacylglycerol (SQDG), and the only phosphoglycerolipid phosphatidylglycerol (PtdGro) (Boudi‘re et al. , 2014) . Due to their different properties, each lipid class plays distinct roles in photosynthetic membranes. Therefore, these four lipids are present in a conserved ratio in order to maintain the structure and function of photosynthetic membranes (Boudi‘re et al. , 2014). Among these conserved lipids, the galactoglycerolipids MGDG and DGDG are the most abundant throughout Viridiplantae (Boudi‘re et al., 2014) . In plants, the galactoglycerolipids are synthesized either through the eukaryotic or the prokaryotic pathways (Roughan & Slack, 1982) . In the case of the eukaryotic pathway, fatty acids synthesized in the chloroplast are exporte d, converted to acyl -CoAs and incorporated at the endoplasmic reticulum (ER) into membrane !!')!lipids, e.g. phosphatidic acid (P tdOH ) or phospha tidyl choline (PtdCho) . ER-assembled lipids then return to the chloroplast and are converted to diacylglycerol (DAG), which is then galactosylated in the two envelope membranes to form MGDG and subsequently DGDG . In contrast, the prokaryotic pathway in plan ts and algae occurs entirely in the chloroplast envelope membranes . Fatty acyl groups attached to acyl carrier proteins (ACPs) synthesized in the chloroplast are incorporated directly into lipid precursors to give rise to PtdOH, DAG and then galacto glycero lipids. In plants, the bulk of MGDG is synthesized in the inner envelope while DGDG is synthesized at the outer envelope requiring exchange of lipids between the two envelope membranes (C Benning, 2009) . In plants, lipids derived from either pathway can be distinguished by the number of carbon s at the sn-2 position of the glyceryl backbone ; 16 -carbon fatty acids (C16) for the prokaryotic and 18-carbon fatty acids (C18) for the eukaryotic pathway -derived lipids, respectively (Heinz & Roughan, 1983) . This is thought to be due to the substrate specificity of lyso -PtdOH acyltransferases located in the chloroplast that prefer C16, while those at the ER prefer C18 (Frentzen et al. , 1983; H. U. Kim et al. , 2005; Kunst et al. , 1988; Roughan & Slack, 1982) . Because C hlamydomonas has exclusively C16 fatty acids at the sn-2 position of its galactoglycerolipids , it is thought to not utilize the eukaryotic pathway for galacto glycero lipid synthesis and therefore should not require ER-to-chloroplast lipid trafficking (Girou d & Eichenberger, 1988) . However, this conclusion is based on the untested assumption that the lyso -PtdOH acyltransferases at the ER and the chloroplast membranes in plants and Chlamydomonas have the same distinct substrate specificity. In Arabidopsis, the ER-to-chloroplast lipid trafficking of the eukaryotic pathway is mediated through TRIGALACTOSYLDIACYLGLYCEROL (TGD1, -2, -3 and -4) proteins (Koichiro Awai et al. , 2006; Lu et al. , 2007; Xu et al. , 2008; Xu et al. , 2003) and a recently discovered TGD5 pro tein (J. Fan et al., 2015) . Mutations in any of the respective Arabidopsis genes cause a secondary phenotype resulting in the accumulation of oligogalactolipids, e. g. tri galactosyldiacylglycerol (TGDG), after which the mutants were named. Th ese oligogalact olipids are synthesized by activation of a galactolipid : galactolipid galactosyl -transferase encoded by SENSITIVE TO FREEZING2 (SFR2 ), a gene absent from Chlamydomonas, which, therefore, presumably does not synthesize oligogalactolipids . In Arabidopsis, T GD1, -2 and -3 form an ABC (ATP -binding cassette) transporter complex in the !!'*!inner envelope membrane of the chloroplast and function as a permease, a substrate binding protein, and an ATPase, respectively (C Benning, 2009; R. L. Roston et al. , 2012) . TGD4 is localized in the outer envelope membrane of the chloroplast as a homodimer (Wang et al., 2012) . It has been suggested that TGD4 transports the ER lipid precursor from ER to the TGD1, -2, -3 complex (C Benning, 2009; Hurlock et al., 2014) and that TGD5 l inks this complex to TGD4 (J. Fan et al. , 2015) . The TGD1, 2, 3 complex then transfers the ER lipid precursor to the inner envelope membrane of the chloroplast. It should be noted that the exact nature of the transported lipid species for the TGD1, 2, 3 co mplex or TGD4 is not known at this time. Bacterial orthologues of TGD proteins have been suggested to be involved in resistance to organic solvents or toxic chemicals. In Pseudomonas putida , TtgA, -B, -C (toluene tolerance genes) and SrpA, -B, -C (solvent -resistant pump) have been proposed to act as toluene efflux pumps , although their direct biochemical function has yet to be demonstrated (Kieboom et al. , 1998a; Kieboom et al. , 1998b; K. Kim et al. , 1998; Ramos et al. , 1998) . Loss of function of these proteins leads to toluene sensitiv ity. In Escherichia coli , MlaD, -E, -F proteins are proposed to function in maintaining an asymmetric lipid distribution in the outer membrane (Malinverni & Silhavy, 2009) . Loss of function of any of these proteins result s in increased outer membrane permeability , possibly due to an inability to remodel the outer membrane lipid composition in response to chemical stress. The implication is that the bacterial orthologues of the plant TGD proteins transport lipids between the cel l membrane and the outer membrane to allow remodeling in response to chemical insults. The genome of Chlamydomonas harbors genes encoding putative TGD1, -2 and -3 orthologues (Merchant et al., 2007) . This raises two possibilities: 1. If Chlamydomonas does not require the import of lipids from the ER for thylakoid lipid assembly as suggested by the molecular species composition of its thylakoid lipids, these proteins may be primarily involved in transferring lipids between the two envelope membranes as in ba cteria. 2. Alternatively, these proteins are involved in lipid trafficking from the ER to the chloroplast, in which case the plant paradigm for distinguishing thylakoid lipid species derived from the ER versus the chloroplast species does not apply to Chla mydomonas. To test these possibilities, we identified and studied a Chlamydomonas mutant deleted in TGD2. !!'+!RESULTS The tgd2 mutant accumulates triacylglycerol enriched in MGDG acyl groups. Triacylglycerols (TAGs) typically accumulate to high levels in Chl amydomonas only following nutrient deprivation and are degraded following refeeding. The tgd2 mutant was generated by insertional mutagenesis and identified during a screen for mutants delayed in TAG degradation following nitrogen (N) deprivation and refee ding (Tsai et al. , 2014) . When TAG levels were monitored during N -replete, N -deprived and N -resupplied growth conditions, the tgd2 mutant showed elevated levels under all conditions tested (Figure 2.1A). Therefore, we focused the subsequent analysis on cel ls grown in N -replete medium. When the steady -state levels of all glycerolipids and free fatty acids (FFA) were analyzed during N -replete growth, only TAG and PtdOH were increased with statistical significance in tgd2 compared to the parental line (PL, Figure 2.1B). The central lipid intermediate PtdOH was the least abundant lipid included in the analysis and was identified based on its relative co -chromatography with standards (Figure 2.S1A) and its distinct acyl composition. While the ratios of different acyl groups in the total lipid fraction did not obviously change (Figure 2.1C), the TAG fraction of tgd2 was enriched in 16:4 !4,7,10,13 and 18:3 !9,12,15 acyl groups (number of carbons : number of double bonds with !Number indicating the position of the double bond counted from the carboxyl end), with a concomitant reduction in 16:0 acyl groups (Figure 2.1D) normally found in TAG synthesized following nutrient deprivation. These highly unsaturated acyl groups are found typically only in MGDG (Figure 2.1E) and their abundance in TAG of tgd2 suggests that its DAG moiety or its acyl groups are derived from MGDG. Subtle changes in MGD G and DGDG molecular species were notable as well. In MGDG of tgd2 higher levels of 16:0, 16:1 and 18:1, and lower levels of 16:4 !4,7,10,13 and 18:3 !9,12,15 were observed (Figure 2.1E). In the case of DGDG of tgd2 , the levels of 16:0 and 16:3 were reduced, while those of the 16:1 and 16:4 were increased (Figure 2.1F). We specifically tested whether the Chlamydomonas tgd2 mutant accu mulates TGDG, as observed for Arabidopsis tgd mutants . However, none of the Chlamydomonas samples showed TGDG detectable by thin -layer chromatography (Fig ure 2.S1B), consistent with the absence of an SFR2 -like activity from Chlamydomonas. !!',!Cultures of tgd2 show early senescence . During routine maintenance of long -term cultures on agar -solidified medium , w e observed that t he tgd2 mutant had a shorter culture lifespan than the PL. Subsequently, viability assays were carried out to further investigate this phen otype. Liquid cultures of PL, tgd2 , and TGD2 tgd2 cells (a complemented line expressing TGD2 in the tgd2 mutant background , see below ) were inoculated at 0.5 x 10 6 cells/mL. Cultures were examined after 3, 7, 14, 21 and 28 days. After 21 days, the tgd2 cul ture beg an to turn yellow which was even more obvious on day 28 (Figure 2.2A). This observation correlated with a continuously increasing fraction of dead cells over time observed in the tgd2 culture using live cell stains (methylene blue and phenosafranin ) (Figure S2a). The tgd2 culture accumulated more TAG throughout the culturing time (Figure 2.2B), and at day 3 substantial levels of MGDG derived acyl groups were detected in the TAG fraction (Figure 2.S2B). It seems likely that initially TAG is derived f rom turnover of fully desaturated MGDG peaking at day 3, while later during prolonged culturing, as nutrients are depleted, de novo TAG synthesis is induced. This is indicated by a steady increase of 16:0 and 18:1 !9 acyl groups characteristic of de novo synthesized TAG. The prolonged culturing of tgd2 also led to an accumulation of high levels of malondialdehyde, which is a product of the reaction between polyunsaturated acyl groups and reactive oxygen species (ROS ) (Figure 2.2C). As cells die, ROS typically accumulates consistent with the observed decrease in viability of the tgd2 mutant. Changes in the ultrastructure of tgd2 cells. The original tgd2 mutant was isolated in the cell wall mutant background dw15 .1. Ho wever, strains with cell walls (cw +) are more amenable to ultra -thin sectioning for transmission electron microscopy (TEM) than are cw - lines, such as dw15.1. Hence, we moved the tgd2 mutation into a cw + line, by crossing it with wild -type strain CC-198. F or the TEM studies, the tgd2 mutant was compared with CC -198, to determine ultrastructural changes caused by the tgd2 muta tion at mid -log, stationary and late stationary phases. In contrast to CC -198 (Figure 2.3A -C), which contained lipid droplets during l ate stationary phase (black arrows), tgd2 showed lipid droplets at every time point (Figure 2.3D -F, black arrows). The tgd2 mutant had more lipid droplets, which were larger in size over time. These lipid droplets were observed in the cytoplasm of tgd2 also harboring mitochondria and were often adjacent to the chloroplast outer envelope membrane (Figure 2.3G). In addition, lipid droplets from the tgd2 mutant stained darker (Figure 2.3D -G compared to 2.3C, black arrows), which is consistent with a higher des aturation level of TAGs as osmium tetroxide stains !!'-!unsaturated lipids more intensely. During late stationary phase at day 17, membranes of tgd2 became disorganized (Figure 2.3H, white arrows) compared to PL and earlier stages of tgd2 (Figure 2.S3). About h alf of the population appeared to be dead ghost cells, lighter in color and with less distinct internal structures (Figure 2.3I). Although a cw + complemented line was not available for this experiment, these ultrastructural changes corroborate observations made on the tgd2 mutant and complemented lines in the dw15.1 background described above and are likely due to the ablation of the TGD2 gene. Molecular and genetic analysis of the tgd2 mutant locus . The tgd2 mutant was generated by random genomic insertion of a plasmid carrying the Hygromycin B resistant gene ( AphVII ). Southern blot analysis of genomic DNA of tgd2 cut with Bam HI and using a probe covering a region of AphVII that does not contain the Bam HI restriction site indicated the presence of a single insertion (Figure 2.S4). As a first step to identify the mutant locus, a g enetic analysis was carried out to determine linkage of the primary lipid phenotype and the Hygromycin B resistance marker. For this purpose, CC -198 as already mentioned above was cr ossed with tgd2 in the dw15 .1 background . The progenies of this cross were tested for TAG content, TAG acyl profile and sensitivity to Hygromycin B. Without exception, all Hygromycin B resistant progenies tested (11 resistant and 12 susceptible progenies f rom 6 zygotes) showed high TAG levels and TAGs with highly unsaturated acyl groups (Figure 2.S5A and B). This result suggested close linkage of the Hygromycin B resistant marker and the mutation that caused the TAG phenotype of the tgd2 mutant. Because PCR -based methods were not successful in identifying DNA flanking the Hygromycin B marker, whole genome resequencing was used to determine the location of the mutation responsible for the primary phenotype in the genome of the tgd2 mutant. Towards this end, g enomic DNA of the tgd2 mutant was subjected to Illumina -HiSeq paired -end sequencing. The reads were assembled de novo with velvet 1.2.07 (Zerbino & Birney, 2008) using the k-mer length of 21 . Contigs containing AphVII were searched against the Chlamydomona s reference genome (v5.3) to identify flanking sequences revealing a possible insertion site in chromosome 16. When the respective section of the reference genome of chromosome 16 was used as a template for the assembled contigs from the tgd2 genome, a 31 kb deletion in chromosome 16 of tgd2 became apparent . This deletion, which was confirmed by PCR probing, affected six genes either fully or partially as shown in Figure 2.S6A. !!(.!We introduced into tgd2 genomic DNA fragments containing each affected gene (fro m approximately 1 kb 5Õ of the start codon to about 0.5 kb 3Õ of the stop codon) derived from a bacterial artificial chromosome covering the region. Each genomic DNA fragment was co -introduced into tgd2 along with a linearized Paromomycin resistance gene ( AphVIII), which was under selection. Of the six genes disrupted or missing from the tgd2 genome, only introduction of CrTGD2 was able to restore TAG content, TAG acyl group profile, cell viability, and PtdOH content close to PL levels (Figure 2.S6). Theref ore, deletion of TGD2 is the cause of at least the four phenotypes of the tgd2 mutant discussed. Because the phenotype of compl ement ed line C3 was nearly fully restored to that of the PL (Figure 2.S6C and C), this line was included in all subsequent analys es, except where indicated otherwise, and is designated TGD2 tgd2 . CrTGD2 is a presumed ortholog ue of AtTGD2 . The translated sequence of the Chlamydomonas CrTGD2 gene has 40% amino acid identity with the Arabidopsis protein AtTGD2. Both are similar to sub strate binding protein components of bacterial ABC transporters (Casali & Riley, 2007) . Both also contain a Mammalian Cell Entry (MCE) domain (Figure 2.S7), which in the respective Mycobacterium tuberculosis protein for which this domain was named, is requ ired for pathogenesis. In addition phylogenetic analysis of the MCE domain of predicted CrTGD2 orthologues across plants, green algae, and bacteria revealed that CrTGD2 falls into the same clade as the respective plant proteins and is divergent from bacter ial orthologues ( Figure 2.S8 and APPENDIX C. for the alignment). Using TMHMM (Krogh et al. , 2001) , CrTGD2 is predicted to contain one transmembrane domain (Figure 2.S7), similar to the Arabidopsis orthologue AtTGD2 (Koichiro Awai et al. , 2006) . Based o n these similarities, we hypothesized that CrTGD2 may have similar functions as AtTGD2 and, hence, those two proteins may be true orthologues. To more directly test their functional equivalence, we introduced (i) codon optimized Arabidopsis TGD2 in the Chl amydomonas tgd2 mutant, and (ii) Chlamydomonas TGD2 in Arabidopsis tgd2 -1 mutant. However, despite the presence of the recombinant proteins, lipid phenotypes were not restored (Figure 2.S9 and 2.S10). Thus, the two TGD2 proteins seem to be sufficiently div ergent to not substitute for each other in a heterologous protein complex. Altered galactoglycerolipid labeling and impaired ER -to-plastid lipid trafficking in tgd2 . Pulse chase analysis provides a proven in vivo method to examine general substrate -product relationships in metabolic pathways and lipid trafficking in plants in particular, and it was used in the original analysis of the tgd1 -1 mutant of Arabidopsis, e.g. (Xu et al. , 2003) . Here, we !!(%!carried out pulse -chase labeling experiments with [ 14C]-acetate using mid -log phase cultures of PL, tgd2 and TGD2 tgd2. The cells were incubated in the presence of labeled substrate until 20 -40% incorporation of label was observed before the labeled medium was replaced for the chase. Total lipids were extracted at d ifferent times during the chase phase and individual lipids were separated by thin -layer chromatography followed by liquid scintillation counting to determine the fraction of incorporation of radiolabel into all lipids analyzed. Multiple individual repetit ions of this experiment were carried out and all of these replicates showed similar trends, but the absolute values differed making it difficult to average results from the different experiments. Therefore, a representative experiment is shown in Figure 2. 4. The most noticeable differences in labeling between the different cell lines were observed for MGDG and DGDG. MGDG in tgd2 was labeled to much higher levels with a subsequent rapid decrease in label during the chase phase (Figure 2.4, top panels). In co ntrast, MGDG labeling in PL and in the TGD2 tgd2 line was lower, and less of a decrease in label was observed during the chase. As the bulk -steady -state levels of MGDG in tgd2 did not significantly change compared to the PL (Figure 2.1b), the result sugges ts that MGDG was more rapidly synthesized and metabolized in tgd2 . Since DGDG is derived from MGDG, one would expect that its labeling would follow that of MGDG at least in the initial chase phase (within the first five hours) and that label would appear t o move from MGDG to DGDG as was the case for the PL and TGD2 tgd2 lines during the first five hours of the chase. However, DGDG labeling was severely delayed and not as high in tgd2 (Figure 2.4, top panels) suggesting a disruption in the conversion of MGDG to DGDG in tgd2 or the activation of pathways competing for MGDG as a substrate. The detailed shape of the time course for MGDG labeling also was different in the mutant. In the PL and TGD2 tgd2 lines the MGDG labeling time course showed a ÒdipÓ and rebou nd during the first five hours of the initial stage of the chase, but in the tgd2 mutant, MGDG labeling declined steadily (Figure 2.4, top panel). This result was not a chance observation of this particular experiment, because independent repeats showed th is phenomenon reproducibly (Figure 2.S11). The equivalent experiment with wild -type Arabidopsis leaves shows a comparable MGDG labeling time course which is interpreted as initial rapid labeling of MGDG by the chloroplast pathway, followed by label dilutio n during the initial chase phase decreasing the label followed by a gradual increase in labeling of MGDG over time as lipids move back from the ER as part of the ER pathway of galactoglycerolipid biosynthesis (Xu et al. , 2003) . In !!(&!the Arabidopsis tgd1 -1 mutant MGDG labeling is also very high and steadily declines during the chase phase, interpreted as a reduction in lipid species returning from the ER to the chloroplast. The observation of the typical ÒdipÓ in MGDG labeling in the Chlamydomonas PL is curren tly the most direct indicator for ER -to-chloroplast lipid trafficking in this alga. The lack of the ÒdipÓ in the tgd2 mutant suggests that TGD2 is involved in this process. Altered labeling of non -galactoglycerolipids in tgd2 . In addition to the difference s in MGDG and DGDG labeling, the labeling time course of the betaine lipid diacylglyceryl -trimethylhomoserine (DGTS) and P tdGro also showed changes in the tgd2 mutant . In contrast to the PL , DGTS labeling during the chase in tgd2 started at a lower level a nd continue d to increase without leveling off (Fig ure 2.4, middle panels). This result would be consistent with a decreased rate of precursor conversion into DGTS in the tgd2 mutant. Less label was also found in PtdGro in tgd2 (Fig ure 2.4, top panels) , alt hough the shape of the labeling time course looked similar to that of the PL and the TGD2 tgd2 complemented line . It should be noted that DGTS and P tdGro steady state levels were not altered in the mutant (Fig ure 2.1B). Therefore the relative rate of synth esis of these two lipids was decreased (or turnover increased) compared to that of MGDG in tgd2 . While incorporation of label into the TAG fraction in the PL and the complemented line TGD2 tgd2 was minimal, label in TAG of the tgd2 mutant steadily increase d during the chase phase in parallel with a decrease in label in MGDG (Fig ure 2.4 lower panel). Given also the observation that MGDG specific acyl groups were found in TAG under steady -state conditions in tgd2 (Figure 2. 1A, B, D and E), the labeling result was consistent with a conversion of mature MGDG to TAG in the tgd2 mutant. The labeling of other lipids did not show much difference in the tgd2 mutant (Fig ure 2.4). Biosynthesis of MGDG is increased in the tgd2 mutant. The increase in acetate labeling of MGDG prompted us to investigate MGDG synthesis directly by using a more specific substrate, UDP-galactose. Intact chloroplasts from PL and tgd2 were fed with 300 mCi/mmol UDP -[14C]-galactose. Total lipids were extracted and incorporation of labeled galact ose into MGDG and DGDG was monitored and normalized based on an equal number of chloroplasts. We observed higher levels of labeled MGDG and DGDG in chloroplast s from tgd2 than from the PL (Fig ure 2.5A) consistent with a higher galactolipid synthesis activi ty in the tgd2 mutant. It is important to !!('!note that MGDG is synthesized by addition of one galactose from UDP -galactose to DAG and DGDG is synthesized by transfer of one galactose from UDP -galactose to MGDG. Thus in the case of DGDG, four different molecul ar species can be obtained during this experiment, depending on whether only the distal or the proximal galactose, or both or none of the galactoses are labeled . This makes it challenging to directly determine the MGDG -to-DGDG precursor product relationshi p. However, i n order to compare rate s of conversion of MGDG to DGDG, ratios of labeled DGDG/labeled MGDG were calculated. This ratio is significantly lower in tgd2 (Fig ure 2.5B), which indicates a lower rate of conversion of MGDG to DGDG in tgd2 . In Arabid opsis, MGDG synthases can be localized in either of the two chloroplast envelope membranes (K. Awai et al., 2001) , but the bulk of MGDG synthesis involves MGD1 at the inner envelope membrane (Jarvis et al. , 2000) . To test whether the observed MGDG synthase activity of isolated chloroplasts might be associated with the outer envelope membrane in Chlamydomonas, we treated the chloroplasts with Thermolysin, a large protease that cannot penetrate the outer envelope membrane (see also below). As shown in Figure 2.5A, MGDG and DGDG syntheses were sensitive to Thermolysin suggesting that both, the MGDG and DGDG synthases are located in the outer envelope membrane in Chlamydomonas. CrTGD2 is present in the chloroplast inner envelope membrane. Based on amino acid sequence analysis with different online prediction tools including PredAlgo (Tardif et al., 2012) , the loca tion of CrTGD2 was initially ambiguous , but likely in the chloroplast. Fractions containing c hloroplasts, mitochondria and microsomal membranes were iso lated from whole cell lysates and CrTGD2 was detected using immunoblotting with antiserum raised against the recombinant protein . Antisera against m arker proteins for each cell compartment were tested as well. Whole cell lysate of the tgd2 mutant lacking C rTGD2 was used as a negative control to confirm the presence of the CrTGD2 signal in the PL extracts . As shown in Figure 2.6A each subfraction was enriched with its respective marker protein, while CrTGD2 was clearly detected in the chloroplast fraction an d whole cell lysate. This suggests that CrTGD2 is localized in the chloroplast. Because CrTGD2 was predicted to contain one transmembrane domain (Fig ure 2.S7 ), a protease protection assay was carried out to determine the possible insertion of CrTGD2 in to one of the chloroplast envelope membranes . This assay takes advantage of size difference between !!((!Thermolysin and Trypsin (Cline et al., 1984) . In general, Thermolysin is too big to penetrate the outer envelope membrane of the chloroplast. In contrast, Trypsin is sufficiently small to gain access to the intermembrane space and proteins in the inner envelope membrane. Different concentrations of either Thermolysin or Trypsin were used to treat isolated intact chloroplasts of the PL . Antisera against t hree mark ers for specific compartments were: anti -Toc34 detecting an outer envelope membrane protein , anti -ARC6 detecting an inner envelope membrane protein facing the intermembrane space, and anti -Tic40 detecting an inner envelope membrane protein facing the strom a, respectively. As a result of Thermolysin treatment at increasing concentration s, Toc34 decreased in abundance as predicted for an outer envelope membrane protein , but the other markers and CrTGD2 did not (Fig ure 2.6B, left panel). Following treatment with Trypsin, all proteins were susceptible at increasing concentration s of the protease (Fig ure 2.6B, right panel). This result suggests that CrTGD2 is localized in the inner envelope membrane as was previously observed for the AtTGD2 orthologue (Koichiro A wai et al. , 2006). CrTGD2 binds P tdOH in vitro . The Arabidopsis orthologue AtTGD2 binds P tdOH in vitro (Koichiro Awai et al. , 2006; Lu & Benning, 2009) . Therefore, a liposome binding assay was used to test for lipid binding by CrTGD2. As done for AtTDG2 , a recombinant CrTGD2 protein truncated from the N -terminus to just beyond the membrane -spanning domain was fused to the C-terminus of DsRED to improve solubility (Lu & Benning, 2009) and was produced in E. coli. This fusion protein also contained a His -tag at its C -terminus and was designated DsRED -CrTGD2 -His-tag. In parallel, DsRED -AtTGD2-His-tag was used as a positive control. As an internal negative control, DsRED by itself, which does not bind lipids, was included with the samples. Both AtTGD2 and CrTGD2 showed strongest binding to P tdOH of all lipids tested (Fig ure 2.7A and B). The double band is due to an internal autocatalytic cleavage of DsRED as previously reported (Gross et al. , 2000). !!()!DISCUSSION Lipid metabolism of Chlamydomonas was thought to dif fer from that of Arabidopsis in at least three aspects: 1. Chlamydomonas does not synthesize phosphatidylcholine (PtdCho); 2. It has the betaine lipid DGTS; and 3. It is thought to not utilize the ER -pathway for precursors of thylakoid lipid assembly (Giro ud & Eichenberger, 1988) . In fact, because the lipid precursor transported from the ER to the chloroplast is still unknown (Hurlock et al., 2014) , but might involve PtdCho, these three differences could be related. However, the genome of Chlamydomonas enco des TGD1, 2, and 3 proteins, which have been shown to be involved in lipid trafficking from the ER to the chloroplasts in Arabidopsis (C Benning, 2009; Hurlock et al. , 2014) . Thus, if Chlamydomonas were truly lacking ER -to-plastid lipid transport, the pres ence of these proteins in Chlamydomonas presents an interesting conundrum. The analysis of the tgd mutants of Arabidopsis is complicated by the fact that loss of function of TGD genes is lethal and that in the available leaky mutants a galactoglycerolipid synthesizing enzyme, SFR2, is induced. Its activity leads to the formation of di - and higher order oligogalactoglycerolipids from MGDG. Therefore, the absence of SFR2 activity from Chlamydomonas allows us to reevaluate more directly the function of TGD pro teins in the biosynthesis of the thylakoid lipids MGDG and DGDG in this organism without having to consider the competing SFR2 -based pathway present in Arabidopsis. Thus, studying the role of TGD2 in Chlamydomonas with a loss -of-function tgd2 mutant that i s not lethal, but has a rapid senescence phenotype, provides a unique opportunity to more fully understand the role(s) of TGD proteins in the chloroplast envelope membranes . Moreover, only single genes for MGDG and DGDG synthases respectively have been ide ntified in the Chlamydomonas genome further simplifying the analysis of its galactoglycerolipid metabolism. One caveat is that we do not yet know the exact location of the two galactoglycerolipid synthases in Chlamydomonas, but our analysis based on the se nsitivity of the two activities in isolated chloroplasts to Thermolysin (Figure 2.5A) suggests that they are both associated with the outer envelope membrane. In Arabidopsis, the main MGDG synthase activity encoded by MGD1 is associated with the inner enve lope membrane facing the intermembrane space, while the DGDG synthase, DGD1, is associated with the outer envelope membrane facing the cytosol (C. Benning & Ohta, 2005) . Additional MGDG synthases MGD2 and MGD3 are present in the outer envelope membrane in Arabidopsis, but are thought to be conditionally involved in synthesis of galactoglycerolipids !!(*!during phosphate starvation or in specific tissues (Kobayashi et al. , 2009) . How the products of the two enzymes move between the envelope membranes in Arabidops is or any other organism is currently unknown. Galactoglycerolipid metabolism is altered in tgd2. A first indication that MGDG synthesis and turnover are affected in the tgd2 mutant arises from the fact that tgd2 accumulates TAG with 16:4 and 18:3 acyl gro ups typically found only in MGDG (Fig ure 2.1D and E). Moreover , osmium tetroxide -stained lipid droplets of tgd2 (Fig ure 2.3D-F) appeared darker compared to those of the PL (Figure 2.3C) consistent with an increased desaturation of acyl groups associated with lipid droplets (Bahr, 1954; Korn, 1967) . Second, altered MGDG metabolism in the tgd2 mutant became obvious during acetate pulse -chase labeling studies. While the steady state bulk levels of MGDG and DGDG in tgd2 were similar to those of the PL (Fig ure 2.1B), acetate-labeling experiments and subsequently direct measurements of MGDG synthase activity in isolated chloroplasts show ed that MGDG is more actively synthesized in tgd2 (Fig ure 2.4 and 2.5A). Interestingly, this higher level of MGDG labeling did no t translate into higher or more rapid labeling of DGDG presumably formed by galactosylation of MGDG. In fact, this result would be consistent with an impairment in the conversion of MGDG into DGDG in the tgd2 mutant. Similarly, UDP-galactose labeling of isolated chloroplasts showed a decrease in the conversion of MGDG into DGDG in the tgd2 mutant as the ratio of labeled DGDG to MGDG strongly decreased (Fig ure 2.5B). It should be noted that in this assay DGDG is more highly labeled than MGDG, which is opposi te to what is observed with isolated Arabidopsis chloroplasts (Xu et al. , 2005) . One reason could be that in Chlamydomonas substrate channeling occurs between the MGDG and DGDG synthases, which is decreased in the tgd2 mutant indicated by the strongly decr eased ratio of labeled DGDG to MGDG. Another reason might be the absence of SFR2 from Chlamydomonas, such that there is no further redistribution of label from MGDG into higher order galactoglycerolipids. How TGD2 might be affecting galactoglycerolipid me tabolism in Chlamydomonas is outlined in Figure 2.8. Formally based on the labeling data alone, TGD2 could provide MGDG substrate to the DGDG synthase. However, TGD2 is likely a component of a lipid transporter shuttling lipids between the outer and the in ner envelope membranes while the two galactoglycerolipid synthases appear to be both localized in the outer envelope membrane in !!(+!Chlamydomonas. Because we do not know the lipid substrate of this transporter, it might well be that the TGD1, 2, 3 complex tra nsfers galactoglycerolipids from the outer to the inner envelope membrane. An alternative possibility is that TGD2 as proposed for Arabidopsis is involved in transferring PtdOH from the outer to the inner envelope membrane. After all, it is similar to subs trate binding proteins associated with ABC transporters and binds primarily PtdOH just like the TGD2 orthologue from Arabidopsis (Figure 2.7). Its absence could lead to an increase in PtdOH in the outer envelope membrane. We indeed observed an increase in cellular PtdOH content in the tgd2 mutant (Figure 2.1 B), although we could not determine the membrane association of this additional PtdOH. As is known for the Arabidopsis MGDG synthase (Dubots et al., 2010) , this increased PtdOH content if associated with the outer envelope membrane could stimulate MGDG synthase activity leading to increased MGDG biosynthesis as observed in the tgd2 mutant (Figure 2.8). In fact , there is some resemblance in the labeling results obtained for Arabidopsis tgd mutants (Xu et a l., 2005; Xu et al. , 2003) . First, Arabidopsis tgd1 -1 showed initially a strongly increased incorporation of label into MGDG during acetate pulse -chase labeling (Xu et al. , 2003) . Second, differences in the labeling of MGDG and DGDG are increased as well in the Arabidopsis tgd1 -1 mutant (Xu et al. , 2003) . Thirdly , during UDP-galactose labeling of isolated chloroplasts , MGD1 activity of tgd1 -1 was increased while DGDG labeling did not increase (Xu et al. , 2005) . Thus disruption of the TGD complex in both organisms seems to stimulate MGDG synthesis without stimulating DGDG synthesis and formally could appear as a disruption of transfer of precursors to the DGDG synthase in labeling experiments. What happens to MGD G as it is metabolized in the tgd2 mutant? It i s critical for the cell to maintain a set ratio of the non -bilayer forming lipid, MGDG, and the bilayer forming lipid, DGDG, in its photosynthetic membrane (Dırmann & Benning, 2002) . Because MGDG synthesis is increased over that of DGDG in the tgd2 mutant, to maintain lipid homeostasis and prevent accumulation of bulk MGDG, its turnover rate must also be increased as suggested by the acetate pulse -chase labeling experiment (Fig ure 2.4). As acyl groups normally specifically found in MGDG of the PL are presen t in TAG accumulating in the tgd2 mutant (Fig ure 2.1D and E), it appears that some of the DAG moieties of MGDG or its acyl groups are converted to TAG and are sequestered in lipid droplets (Fig ure 2.3D-F). In plants, MGDG can be hydrolyzed and its DAG or a cyl groups are converted to TAG that is stored in plastoglobuli inside the plastid during !!(,!photosynthetic stress (Youssef et al. , 2010) or during leaf senescence (Kaup et al. , 2002) . Thus lipid droplets may serve as buffer for storing otherwise toxic acyl g roups derived from membrane lipids. In addition, TAG synthesis in Chlamydomonas may involve the release of acyl groups from newly formed MGDG by the activity of the lipase PGD1 (Figure 2.8), as has been shown to occur during N deprivation (Li et al. , 2012). In case of the tgd2 mutant of Chlamydomonas , lipid droplets were observed in the cytosol in contact with the outer envelope membrane of the chloroplast (Fig ure 2.3G). This observation supports the hypothesis that Chlamydomonas can synthesize TAG in its chloroplast membranes (Jilian Fan et al. , 2011; Liu & Benning, 2013) . The Arabidopsis tgd mutants, e.g. tgd1 -1, also accumulate TAG in the cytosol of lea ves (Xu et al. , 2005) . However, the TAG accumulat ing in the Arabidopsis tgd1 -1 mutant has an acyl profil e more similar to that of PtdCho , but not MGDG as in the case of Chlamydomonas tgd2. It was concluded that accumulation of TAG in the tgd1 -1 mutant was the result of increased conversion of lipid precursors accumulating at the ER due to impaired ER to chlo roplast lipid trafficking. TAG accumulation in Arabidopsis tgd1 -1 involves the conversion of PtdCho by phospholipid : DAG acyltransferase (PDAT, Jilian Fan et al. , 2013). In addition, studies on the Arabidopsis tgd1 -1 sfr2 double mutant revealed that the D AG moiety of TAG accumulating in the Arabidopsis tgd1 -1 mutant is derived from DAG generated by SFR2 activity (Jilian Fan et al., 2014) . However, as Chlamydomonas lacks SFR2 activity, the mechanism of TAG biosynthesis in the Chlamydomonas tgd2 mutant must be different. It seems also likely that acyl groups derived from MGDG undergo a different fate as they can become oxidized as seen in the higher level of malondialdehyde derived from fatty acid peroxidation in the tgd2 mutant (Fig ure 2.2C). It is largely accepted that polyunsaturated fatty acids are targets for oxidation by autooxidation or lipoxygenases (Feussner & Wasternack, 2002). Products of lipid peroxidation are mainly aldehydes, which are toxic to nucleic acids and proteins (Esterbauer et al. , 1991) . Thus, it seems possible that increased oxidization of MGDG derived acyl groups in tgd2 leads to lower viability of tgd2 in prolonged cultures (Fig ure 2.2A and 2.3I). Does CrTGD2 play a role in the trans fer of lipids from the ER to the chloroplast ? In the Arabidopsis tgd1 ,2,3,4 mutants, increase in the ratio of C 16/C18 acyl groups of thylakoid lipids indicates lack of lipid trafficking from the ER to the chloroplast (Koichiro Awai et al., 2006; Lu !!(-!et al., 2007; Xu et al., 2008; Xu et al., 2003). In addition, changes in acetate pulse -chase labeling of MGDG in tgd1-1 seedlings (a lack of a transient decrease in MGDG labeling in the mutant as lipid precursors move from the chloroplast to the ER and then return) also indicates the lack of lipi d tran sfer between the two compartments (Xu et al. , 2003) . A s imilar, albeit more subtle change in the labeling time course for MGDG was observed for the Chlamydomonas tgd2 mutant ( Figure 2.4, top panel , Figure 2.S11 ). Th us these labeling data suggest that lipid precursors could be transferred from the ER to the chloroplast envelope membranes for the synthesis of thylakoid lipids. If this conclusion is correct, the ER located lyso -PtdOH acyl transferase must have a different substrate specificity in Chlamyd omonas compared to Arabidopsis to explain the absence of 18 carbon fatty acids at the sn-2 position of thylakoid lipids in Chlamydomonas. Thus, a thorough analysis of the acyltransferases in Chlamydomonas will be required to ultimately solve this conundrum . Like in Arabidopsis, s ubcellular localization and protease protection assay s suggested that CrTGD2 is localized in the inner envelope membrane of the chloroplast (Fig ure 2.6A and B). Furthermore, similar to AtTGD2 (Koichiro Awai et al., 2006; Lu & Benni ng, 2009; R. Roston et al. , 2011) , in vitro lipid binding assay s showed that CrTGD2 bind s primarily to P tdOH (Fig ure 2.7A and B), which is a candidate for transferred lipid species. Phylogenetic analysis showed that CrTGD2 is in the same clade as those of land plants but not bacteria (Fig ure 2.S8 ), but TGD2 proteins from either Arabidopsis or Chlamydomonas were not functional in the opposite host in our hands, respectively (Figure 2.S9 and 2.S10), perhaps because both proteins are too divergent to function in a heterologous complex. However, based on all other data presented here , it is likely that the TGD2 protein of Chlamydomonas is also a component of a lipid transporter transferring lipid precursors between the envelope membranes as proposed for the homo logous system in Arabidopsis. However, because TGD4 and TGD5 are seemingly absent from Chlamydomonas, it remains to be seen what proteins might be involved in lipid transfer between the ER and the outer envelope membrane in Chlamydomonas. Furthermore, beca use the actual lipid species transported is not yet known, a possibility remains that the TGD1, 2, 3 complex of Chlamydomonas also plays a role in the transfer of the galactoglycerolipids synthesized at the outer chloroplast envelope membrane to the inner envelope membrane in Chlamydomonas. !!).!MATERIALS AND METHODS Algal strains and growth conditions . Chlamydomonas reinhardtii cell wall -less strain dw15 .1 (cw15, nit1, mt +) provided by Arthur Grossman (Carnegie Institute for Science, Department of Plant Biolog y, Stanford Universit y) was used as wild-type PL with regard to TGD2 to generate the tgd2 mutant. A c ell-walled strain CC -198 (er -u-37, str -u-2-60, mt -) obtained from the Chlamydomonas Resource Center ( http://www.chlamycollection.org ) was used to generate tgd2 cell-walled progenies for linkage analysis and for TEM. Unless specified, the algal cultures were grown in Tris -acetate-phosphate (TAP) medium (Gorman & Levine, 1965) . For a ll experiments except for chloroplast preparation, algal cultures were grown u nder continuous light at 80 µmol m-2 s-1 and at 22¡C. The c ell concentration was monitored with a Z2 Coulter Counter (Beckman Coulter). Generation of tgd2 mutant and genetic analyses. The tgd2 mutant was generated by insertional mutagenesis in the same exp eriment as described previously for the cht7 mutant (Tsai et al. , 2014). The details of the genetic analysis are described under APPENDIX D. Details about the Bacterial Artificial Chromosomes used for genetic complementation can be found in Table 2 .S1. Sequences of primers used for testing genetic complementation are listed in Table 2.S2. DNA isolation and Southern blot analysis. DNA isolation was carried out as previously described (Keb -Llanes et al. , 2002) with some modifications as detailed in t he APPENDIX D. Southern blot analysis was done with the same probe as described in (Li et al. , 2012). Whole genome resequencing . The genome of the tgd2 mutant was sequenced by Illumina Hi -Seq using the paired -end method at the MSU -Research Technolo gy Support Facility . Details of the analysis are described under APPENDIX D. Sequences of primers used for identifying the deletion can be found in Table 2.S2. Lipid analysis . Total lipid was extracted as previously described (Bligh & Dyer, 1959) from pellets of either freshly harvested cultures or from pellets stored at -80¡C. In general, pellets from 15 mL algal culture were resuspended in 3 mL extraction solvent. The extracted lipid s were dried under an N2 stream and stored at -20¡C. Individual l ipids were separated on thin layer chromatography (TLC) plates (TLC Silica gel 60, EMD). For PtdOH separation, ammonium sulfate treated TLC plates (C Benning & Somerville, 1992) were employed. Different solvents were used for different lipid classes: for neutral lipids petroleum ether, diethyl ether, acetic acid !!)%!(80:20:1 v/v ); for polar lipids chloroform, methanol, acetic acid and water (75: 13: 9: 3 v/v ); for PtdOH chloroform, methanol, ammonium hydroxide (65: 25: 5); and for oligogalactolipids chloroform, methanol, 0.9% sodium chloride and water (60: 35: 4: 4 v/v ). Lipids on TLC plates were visualized by briefly staining with iodine vapor. Alternatively, g alactoglycero lipids wer e stained with "-nap hthol as described in (Wang & Benning, 2011) . Lipids were isolated and processed for generation of fatty acid methyl esters (FAMEs) as described in (C Benning & Somerville, 1992) . Quantifications of FAMEs were performed by gas liquid chromatography using an HP6890 instrument equipped with a DB -23 column (both Agilent Technologies, Santa Clara, CA) with a temperature profile and running condition s as described in (Z−uner et al. , 2012). Viability assay . The PL (dw15 .1), tgd2 mutant and TGD2 tgd2 inoculated at 0.5 million cells/mL were grown in TAP medium. Cells were harvested at day 3, 7, 14, 21 and 28 for viability staining, lipid analysis, and thiobarbituric acid-reactive -substances (TBARS) assay. Viability stain ing was performed as descr ibed (Chang et al. , 2005) with minor modifications. The cell samples were mixed with an equal volume of staining solution (0.0252% methylene blue, 0.0252% phenosafranin and 5% ethanol) and incubated for 5 min. The two dyes are excluded from living cells, w hich remain green, while dead cells taking up the dyes stain purple. The two cell types were counted with a hemocytometer. For lipid analysis, cell were harvested as described above, flash frozen in liquid N 2, and stored at -80¡C for later lipid analysis (see above) . Lipid peroxidation was estimated with a TBARS assay. Two aliquots of 5 to 10 ml of algal culture were harvested by centrifugation as described and the algal pellets were resuspended in 1 mL of 20% trichloroacetic acid either with or without 0. 5% thiobarbituric acid. The mixtures were heated at 95¡C for 15 min. Absorbance was measured at 440, 532 and 600 nm. The concentration of malondialdehyde was calculated as described in (Hodges et al. , 1999). Transmission Electron Microscopy . Walled strains were fixed as previously described (Harris, 1989). Images were taken with a JEOL100 CXII instrument (Japan Electron Optics Laboratories, Tokyo, Japan). Phyloge netic analysis . The analysis was done as detailed under APPENDIX D. !!)&!Heterologous comple mentation analysis. The heterologous complementation analysis of the tgd2 mutant was performed as described in detail under APPENDIX D. Sequences of primers used for generating constructs are listed in Table 2.S3. [14C]-Acetate pulse -chase labeling . The different lines were grown in 200 m L TAP medium to mid-log phase. After 10 -fold concentration into 20 m L TAP medium , 10 µ L of 1 mCi/m L [14C] sodium acetate (55 mCi/mmo l) w ere added to the culture. The cultures were incubated at room temperature for 1 -2 h until the incorporation of label reached 20 -40%. To initiate the chase, t he cultures were centrifuged at 3,000 X g for 3 min. The cell pellets were then resuspended in 200 mL unlabeled TAP medium and the cultures were incubated at room temperature. At given intervals, 20 m L of the cultures w ere harvested. Individual lipids were separated on TLC plates as describe d above . Silica powder containing each lipid was i solated from the TLC plates and subjected to liquid scintillation count ing in 10 m L complete counting cocktail 4a20 TM (Research Products International Corp.). Radioactivity was measured with a PerkinElmer Liquid Scintillation Analyzer Tri -Carb 2800TR. DsRED -CrTGD2 pLW01 , DsRED -AtTGD2 pLW01 and DsRED pLW01 constructs, recombinant protein expressio n and purification . Details of the construction of the plasmids and recombinant protein production are as described under APPENDIX D. Primers used in this experiment are listed in Table 2.S3. CrTGD2 antibody. DsRED -CrTGD2 fusion protein was used as antigen to raise antiserum in rabbits (Cocalico Biologicals ). The antigen was prepared with FreundÕs adjuvant and inoculated in rabbits with 3 additional boosts. Two test bleeds were tested for immunore action against pre -bleed with Chlamydomonas PL and tgd2 mutant proteins. Final bleed antiserum was used as primary antibody for detect ion of immunoreactio ns. Immunoblotting . Protein samples for localization of CrTGD2 and for heterologous tgd2 complementation analysis were resuspended in protein extraction bu ffer (0.1 M Tris -HCl pH 6.8, 1% SDS, 15% glycerol and 5% #-mercaptoethanol). The mixtures were incubated at 95¡C for 5 min. The protein w as cooled down on ice and centrifuged at 20,000 X g for 10 min at 4¡C. Pellets were discarded. Protein concentrations w ere determined with bovine serum albumin (BSA) as a standard according to (Bradford, 1976) . In general, equivalents of 10 µg protein (2 µg chlorophyll equivalents for experiments described in Figure 2.6 B and 40 µg of protein in Figure !!)'!2.S9 B) were separated by SDS -PAGE. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated in blocking solution (5% non -fat dry milk in TBST containing 20 mM Tris -HCl pH 7.5, 150 mM NaCl and 0.05% Tween 20 (v/v)) for 30 mi n. Following the addition of primary antiserum in blocking solution , t he membranes were incubated at 4¡C overnight. The membranes were washed 6 times with TBST at 5 min interval s. Secondary antisera conjugated with horseradish peroxidase were incubated with the membranes for 1 -2 h at room temperature. The membranes were washed again as described previously prior to detection of immunoreaction with Clarity TM Western ECL Substrate (BIO -RAD) as a substrate using a ChemiDoc TM MP Imaging System (BIO -RAD). Antibo dies against CrTGD2, Tic40 ( provided by John Froehlich , Michigan State University ), BIP (Santa Cruz Biotechnology) , cytochrome C (BD Pharmingen TM), Toc34 (Agrisera) , ARC6 (provided by Katherine W. Osteryoung , Michigan State University ) and AtTGD2 (Koichiro Awai et al. , 2006) were used at 1:500, 1:2000, 1:1000, 1:250, 1: 10,000 , 1:2500 and 1:2000 dilutions , respectively. Secondary anti -rabbit antibodies were used for each primary antibody, except for anti Ðcytochrome C, for which an anti -mouse antibody (SIGMA ) was used. In case of the lipid binding assay described below , the protein samples were resuspended in 2x SDS sample buffer (0.12 M Tris -HCl pH 6.8, 4% SDS, 20% glycerol, 10% #-Mercaptoethanol and 0.05% bromophenol blue) and processed as described above. The protein samples were separated by SDS -PAGE and transferred to PVDF membranes. The se membranes were processed in the same manner as described above. Primary an d secondary antibodies were the His-tag antibody (GenScript) and anti -mouse conjugated with horseradish peroxidase, respectively. The immunoreaction was detected as described above. Subcellular fractionation . Chlamydomonas PL dw15.1 was grown under 12 h li ght and 12 h dark cycles to mid -log phase. A 500 m L culture was harvested by centrifugation at 3000 X g for 10 min at 4¡C. Isolation of chloroplast, mitochondria l and microsomal membrane s was based on a procedure described in (Klein et al. , 1983) with modi fications. In brief, cells were broken by digitonin. The pellet from an 800 X g centrifu gation was considered the chloroplast fraction . The chloroplast s were further purified on a 20-40-65% Percoll step gradient made with isotonic solution. Intact chloropl asts were obtained at the transition between 40% and 65% Percoll layers following centrifugation at 4,000 X g for 15 min at 4¡C. The s upernatant of the 800 X g centrifugation contained mitochondria and microsomal membrane s. This supernatant was !!)(!centrifuged at 5,000 X g for 10 min at 4¡C. The p ellet from this centrifugation contained a mitochondrial crude fraction which w as further purified as previously described in (Eriksson et al. , 1995) using a 20% Percoll gradient made from isotonic solution compatible with the chloroplast isolation buffer. The s upernatant from the 5,000 X g centrifugation was further centrifuged at 100,000 X g for 90 min at 4¡C. The pellet from this centrifugation was considered the microsomal membrane fraction . All fractions were proce ssed by immunoblotting as described above. Protease protection assay . Isolated chloroplasts were treated with Thermolysin or Trypsin. For Thermolysin treatment, 50 µg/m L chlorophyll equivalent of chloroplasts were incubated with 0 -200 µg/m L Thermolysin in buffer containing 20 mM Tricine -NaOH pH 7.7, 150 mM mannitol, 1 mM MgCl 2, 1 mM MnCl 2, 2 mM EDTA and 0.5 mM CaCl 2. The treatment was carried out on ice for 15 min. The reaction was stopped with the above buffer by adding 10 mM EDTA. The treated chloroplasts were overlaid on a 40% Percoll gradient made from treatment buffer with the addition of 5 mM EDTA. The chloroplasts were centrifuged at 1,500 X g for 5 min. The pellet was washed with treatment buffer containing 5 mM EDTA. Trypsin treatment was carried ou t in a similar manner with a few modifications. The Trypsin treatment buffer was the same as the Thermolysin buffer without 0.5 mM CaCl 2. The reaction was stopped with buffer containing 200 µg/m L T rypsin inhibitor. Instead of 5 mM EDTA in the 40% Percoll g radient and in the wash buffer 100 µg/m L Trypsin inhibitor were added . Protease -treated chloroplasts were processed for immunoblotting as described above or assayed for MGDG synthase activity . MGDG synthase assay . Intact chloroplasts either treated with 10 0 µg/mL Thermolysin or left untreated as described above were resuspended at 125 µg chlorophyll equivalent in 100 µ L assay buffer containing 20 mM Tricine -NaOH pH 7.7, 150 mM mannitol, 5 mM MgCl 2, 2.5 mM EDTA. The reaction was started by adding 0.3 µCi of UDP-[14C] galactose (300 mCi/mmol) and incubated at room temperature under light for 2 min. Lipid was extracted as a mean s to stop the reaction. MGDG and DGDG were separated by TLC and radioactive lipids analyzed as described above. Lip id binding assay . Liposomes consisting of different test lipids were prepared with dioleoyl PtdCho at a 40:60% molar ratio. The mixtures of tested lipid and P tdCho were dried under a stream of N2. Dried lipids were resuspended in 200 µ L of TBS (50 mM Tris -HCl pH 7.0 and 0.1 !!))!M NaCl). The mixtures were incubated in a water bath at the highest lipid transition temperature (37¡C for dioleoyl lipids) for 1 h. The liposomes w ere washed one time and resu spended in 95 µL TBS. Five µ L of 3.7 µg DsRED -AtTGD2-6xHi s and 2 µg of DsRED , or 3.6 µg of DsRED -CrTGD2 -6xHis and 2 µg DsRED were added to the liposomes . DsRED served as internal negative control. The mixtures were incubated at room temperature for 30 min. Liposome -protein complexes were recovered by centrifugation at 13,000 X g for 10 min at 4¡C. The pellets were washed twice in TBS. The liposome -protein pellets were pr ocessed to detect the DsRED proteins by immunoblotting as described above. ACCESSION NUM BERS Read sequences of the tgd2 mutant obtained from whole genome resequencing can be found in the Sequence Read Archive of the National Center for Biotechnology Information under accession number SRP061379. Chlamydomonas TGD2 sequence accession number Cre16.g694400.t1.2 was obtained from the Joint Genome Institute. Arabidopsis TGD2 sequence accession num ber AT3G20320 was obtained from The Arabidopsis Information Resource (www.arabidopsis.org) . Accession numbers used for phylogenetic tree reconstruction are shown in Figure 2.S8 following the species names . ACKNOWLEDG MENT S We are grateful to Dr. Simone Z−uner and Dr. Yang Yang for valuable discussions. We would like to thank Dr. Likit Preeyanon for helping with de novo genome assembly and Alicia Pastor for he lping with electron microscopy. We thank Tomomi Takeuchi and Dr. Shin -Han Shiu for helping with phylogenetic tree construction. We thank Dr. Setsuko Wakao and Dr. John Froehlich for valuable suggestions for subcellular fractionation and the protease protec tion assay, respectively. We appreciate Dr. Pawin Ittisamai of photographs in Figure 2.2A. J. W. has been supported by a Royal Thai Government Scholarship . This work was supported in parts by grants to C .B. from the US NSF (MCB 1157231), the US AFOSR (FA95 50-11-1-0264), by a Strategic Partnership grant from the MSU Foundation, and by MSU AgBioResearch. !!)*! Figure 2.1. Lipid phenotypes of Chlamydomonas tgd2 mutant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igure 2.1. (contÕd ) (A) Triacylglycerol (TAG) concentration in fmol/cell of parental line (d w15.1) PL (solid bars) and tgd2 (open bars) grown in N -replete medium (N+) until mid -log phase , followed by 48 h of N-depriv ation (N-), followed by 24 h of N-resupp ly (NR). (B) Cellular concentration of lipids of the PL and tgd2 during mid -log phase grown in N -replete medium in order of presentation: monogalactosyldiacylglycerol, MGDG; digalactosyldiacylglycerol, DGDG; sulfoquinovosyldiacylglycerol, SQDG; phosphatidylglycerol, PtdGro; digalactosyl -N,N,N-trimethylhomoserine; DGTS; phosphatidylethanolamine, P tdEtn; phosphatidylinositol, PtdIns; phosphatidic acid, PtdOH; triacylglycerol, TAG; diacylglycerol, DAG; and free fatty acids, FFA. (C) Acyl group profile of total lipids of the PL and tgd2 during mid -log phase grown in N -replete medium. Standard nomencla ture for fatty acids is used and indicated at the bottom axis in (F): number of carbons : number of double bonds with position of double bounds indicated counting from the carboxyl end. (D) Acyl group profile of TAG of the PL and tgd2 during mid -log phase grown in N -replete medium. (E) Acyl group profile of MGDG of the PL and tgd2 during mid -log phase grown in N -replete medium. (F) Acyl group profile of DGDG of the PL and tgd2 during mid -log phase grown in N -replete medium. In all cases, three biological replicates were averaged and standard deviations are shown. Differences in means of PL and tgd2 were compared with a paired -sample student t-test (* p -value ' 0.05, ** p-value ' 0.01). !!),! Figure 2.2. Viability assay for the PL (dw15.1), tgd2 and complemented line TGD2 tgd2 grown in N -replete medium. (A) Images of c ultures of PL, tgd2 and TGD2 tgd2 at day s 3, 7, 14, 21 and 28 of cultivation. The culture was inoculated at 0.5 x 10 6 cells/ml at day 1. (B) Cellular concentration of triacylglycerol (TAG, fmol/cell ) of the PL, tgd2 and TGD2 tgd2 at days 3, 7, 14, 21 and 28 of cultivation. (C) Cellular concentration of malondialdehyde (fmol/cell) of the PL, tgd2 and TGD2 tgd2 at day 3, 7, 14, 21 and 28. In all cases error bars indicate standard deviations based on th ree replicates. Analysis of variance was performed with Origin Pro 8.0 (** p -value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igure 2.3. Ultrastructural changes in the tgd2 mutant. Electron micrographs of the wild -type cw + strain (CC -198) and tgd2 grown in N -replete medium are shown du ring mid -log phase (day 3), stationery phase (day 10) and late stationary phase (day 17). Black and white arrows indicate lipid droplets and other organelles, respectively. Bars represent size as indicated. (A) Cells of CC-198 during mid -log growth with subcellular structures abbreviated: eyespots, E; Golgi apparatus, G; mitochondria, M; nucleus , N; pyrenoid, P; and thylakoid membrane, T. (B) Cells of CC-198 during stationery phase with starch granules (S). (C) Cells of CC -198 during late stationary phase. (D) Cells of tgd2 during mid -log phase. (E) Cells of tgd2 during stationary phase. (F) Cells of tgd2 during late stationary phase. (G) Representative tgd2 cell during mid -log phase showing the thylakoid membrane (T), inner envelope membrane (iEM), outer en velope membrane (oEM), mitochondria (M) and electron -dense lipid droplets (black arrows). (H) Representative tgd2 cell during late stationary phase showing compromised membrane structures (white arrows). (I) Population of tgd2 cells during late stationary phase showing dead and living cells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igure 2.4. [14C]-Acetate pulse -chase labeling of PL (dw15.1), tgd2 mutant, and TGD2 tgd2 complemented line. Cells were labeled with [ 14C]-acetate until 20 -40% of label was incorporated (pulse). The chase shown here b eginning at time 0 (h) was initiated by changing to unlabeled medium. The fraction of label found in each lipid (%) relative to the total label incorporated in the lipid fraction is shown during the 24 h chase time course. Abbreviations of lipid are as def ined in the legend to Figure 2.1. Results from one representative experiment of four are shown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igure 2.5. Galactoglycerolipid synthesis of PL (15.1) and tgd2 chloroplasts. (A) UDP-[14C]-galactose incorporation into monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) of PL and tgd2 chloroplasts. All samples were either not treated with protease ( -) or treated (+) with Thermolysin (T -lysin) as indicated. (B) Ratio of labeled DGDG/labeled MGDG from non -protease treated chloroplasts f or the PL and the tgd2 mutant. In each case n ine replicates from different experiments were statistically analyzed for each treatment and the results displayed as box plots (solid for PL, open for tgd2 ). A central square within each box plot represents the mean, the line the median. The extreme values are indicated by *. Statistical test s of different means were performed using a paired -sample student t-test (p-value ' 0.05). The following pairs indicated by numbers were significantly different: 1MGDG PL ( -) was significantly higher than MGDG PL (+) . 2MGDG PL (-) was significantly lower than MGDG tgd2 (-). 3MGDG tgd2 (-) was significantly higher than MGDG tgd2 (+). 4DGDG PL (-) was significantly higher than DGDG PL (+). 5DGDG/MGDG PL ( -) was significantly higher than DGDG/MGDG tgd2 (-). !0.050.100.150.20PLtgd201234T-lysin - 0+-+-+-+ PL tgd2PL tgd2MGDGDGDGUDP-[14C]galactose incorporation(amol/chloroplast/min)DGDG/MGDGAB12, 33451, 245Figure 5. Galactoglycerolipid synthesis of the PL (15.1) and the tgd2 chloroplasts. (A) UDP-[ 14C]-galactose incorporation into monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) of PL and tgd2 chloroplasts. All samples were either not treated with protease (-) or treated (+) with Thermolysin (T-lysin) as indicated. (B) Ratio of labeled DGDG/labeled MGDG from non-protease treated chloroplasts for the PL and the tgd2 mutant. In each case nine replicates from different experiments were statistically analyzed for each treatment and the results displayed as box plots (solid for PL, open for tgd2). A central square within each box plot represents the mean, the line the median. The extreme values are indicated by *. Statistical tests of different means were performed using a paired-sample student t indicated by numbers were significantly different: 1MGDG PL (-) was significantly higher than MGDG PL (+). 2MGDG PL (-) was significantly lower than MGDG tgd2 (-). 3MGDG tgd2 (-) was significantly higher than MGDG tgd2 (+). 4DGDG PL (-) was significantly higher than DGDG PL (+). 5DGDG/MGDG PL (-) was significantly higher than DGDG/MGDG tgd2 (-). !!*&! Figure 2.6. Localization of CrTGD2. (A) Subcellular fractionations of the PL (dw15.1) were compared to whole cell lysates o f tgd2 . Fractions of the PL were as follows: whole cell lysate, wcl; chloroplasts, Chl; mitochondria , Mit; and microsomal membrane, mm . A Western blot is shown. Total protein of each fraction was used to detect Tic40, a chloroplast marker (Chl), cytochrome C (Cyt C), a mitochondrial marker (Mit), BIP, an endoplasmic reticulum (ER) marker, and CrTGD2 using the respective antisera. (B) Protease treatment of PL chloroplasts to determine envelope membrane association of TGD2. Chloroplasts were treated with vari ous concentrations of Thermolysin or Trypsin as indicated. A western blot is shown detecting Toc34, a chloroplast outer envelope membrane (oEM) marker, ARC6, a chloroplast inner envelope membrane marker facing the intermembrane space (iEM -int), Tic40, a ch loroplast inner envelope membrane marker facing the stroma (iEM -str), and CrTGD2. Toc34 (oEM) ARC6 (iEM-int)Tic40 (iEM-str) CrTGD2Tic40 (Chl) Cyt C (Mit) BIP (ER) CrTGD2wcl Chl Mit mm wclPLtgd2AB0 20 50 100 200 0 20 50 100 200 Thermolysin (g/ml) Trypsin ( g/ml)Figure 6. Localization of CrTGD2.(A) Subcellular fractionations of the PL (dw15.1) were compared to whole cell lysates of tgd2. Fractions of the PL were as follows: whole cell lysate, wcl; chloroplasts, Chl; mitochondria, Mit; and microsomal membrane, mm. A Western blot is shown. Total protein of each fraction was used to detect Tic40, a chloroplast marker (Chl), cytochrome C (Cyt C), a mitochondrial marker (Mit), BIP, an endoplasmic reticulum (ER) marker, and CrTGD2 using the respective antisera. (B) Protease treatment of PL chloroplasts to determine envelope membrane association of TGD2. Chloroplasts were treated with various concentration of Thermolysin or Trypsin as indicated. A western blot is shown detecting Toc34, a chloroplast outer envelope membrane (oEM) marker, ARC6, a chloroplast inner envelope membrane marker facing the intermembrane space (iEM-int), Tic40, a chloroplast inner envelope membrane marker facing the stroma (iEM-str), and CrTGD2. !!*'! Figure 2.7. Lipid binding assay. Liposomes made from different lipids as indicated (see legend of Figures 2.1 for lipid abbreviations) were incubated with DsRED -TGD2 recom binant proteins from Arabidopsis (A) or Chlamydomonas (B). Proteins associated with the liposomes were detected by Western blotting using a His -tag antibody. Each reaction contained DsRED protein as an internal negative control. A control lane containing 1 0% of the proteins without liposomes used in each assay is shown in the middle of each blot. PtdChoPtdOHPtdEtnPtdGroPtdInscontrolMGDGDGDGSQDGDGTSPtdChoPtdOHPtdEtnPtdGroPtdInsMGDGDGDGSQDGDGTSABcontrolDsRED-AtTGD2DsREDDsRED-CrTGD2DsREDFigure 7. Lipid binding assay. Liposomes made from different lipids as indicated (see legends of Figures 1 for lipid abbreviations) were incubated with DsRED-TGD2 recombinant proteins from Arabidopsis (A) or Chlamydomonas (B). Proteins associated with the liposomes were detected by Western blotting using a His-tag antibody. Each reaction contained DsRED protein as an internal negative control. A control lane containing 10% of the proteins without liposomes used in each assay is shown in the middle of each blot.!!*(! Figure 2.8. Proposed model of CrTGD2 function. Different steps (as numbered in solid circles) involved in galactoglycerolipid synthesis are shown in the PL (lef t panel) and in the tgd2 mutant (right panel). Lipid molecular species symbols are explained in a box below the scheme. Fatty acid synthesis (FAS) takes place in the chloroplast generating acyl groups bound to acyl carrier protein (acyl -ACP) used for the s ynthesis of chloroplast -derived phosphatidic acid (Chl -PtdOH) (1). Free fatty acids (FFA) are also exported to endoplasmic reticulum (ER) and activated to acyl -CoAs for the synthesis of ER -derived PtdOH (ER -PtdOH), which is then returned to the chloroplast outer envelope membrane (oEM) (2). It is postulated that the two PtdOHs derived from the two different pathways in Chlamydomonas cannot be distinguished unlike those produced in plants. TGD2 situated in the !!*)!Figure 2.8. (contÕd ) chloroplast inner envelope membrane (iEM) transports ER -PtdOH from the oEM to the stroma side of the iEM (Loria et al. ). PtdOH from both sources can give rise to diacylglycerol (4). On the oEM, PtdOH stimulates MGDG synthase (MGD) (5) to produce MGDG from diacylglycerol and UDP -gala ctose (6). This newly synthesized MGDG composed of 18:1 and 16:0 acyl chains is a common substrate for three reactions. First, desaturation by MGDG specific desaturases (7) produces mature MGDG with 18:3 and 16:4 acyl chains. Second, DGDG synthesis (8) yie lds newly synthesized DGDG with 18:1 and 16:0 acyl chains, which then undergoes desaturation (9) resulting in mature DGDG with mainly 18:3 and 16:0 acyl chains. Third, under N deprivation (Villena et al. ), newly synthesized MGDG is degraded (10) by the act ion of PLASTID GALACTOGLYCEROLIPID DEGRADATION1 (PGD1). Fatty acids derived from PGD1 degradation is then used for the synthesis of triacylglycerol (11) containing 18:1 and 16:0 acyl chains which is stored in lipid droplets (LD). In the tgd2 mutant lackin g TGD2, ER -PtdOH accumulates in the oEM and hyper -stimulates MGDG synthase (5) indicated by the star. This results in higher MGDG synthesis (6) indicated as thick arrows. Note that MGDG in the tgd2 mutant is primarily synthesized from Chl -PtdOH. Under norm al growth conditions, desaturation of MGDG is more efficient than degradation by PGD1 or synthesis of DGDG leading to an increased formation of mature MGDG. To avoid an accumulation of mature MGDG it is then degraded by galactolipase(s) (12) yielding 16:4 and 18:3 fatty acids or diacylglycerol with these two acyl groups. These lipid precursors are exported to the cytosol to give rise to triacylglycerol unique to the tgd2 mutant (13). The additional MGDG molecules produced or fatty acids derived from them ca n also be substrates for lipid peroxidation (14) resulting in the accumulation of reactive oxygen species (ROS), presumably causing lower viability of the tgd2 mutant. !!**!!!!!!!!!!!!!!!!!!!!!APPENDI CES !*+!!APPENDIX A . SUPPORTING FIGURES Figure 2.S 1. Separ ation of Phosphatidic acid (PtdOH) and oligogalactoglycerolipids by thin layer chromatography (TLC). (A) Lipids from the PL (dw15.1), the tgd2 mutant, and the TGD2 tgd2 complemented line were separated by TLC and stained with i odine vapor. PtdOH and phosph atidylinositol (PtdIns) standards were loaded on the left of the plate to indicate positions of the lipids. Brackets indicate the areas from which silica containing PtdOH was isolated and used for quantification of PtdOH by gas chromatography in Figure 2.1 B. The number of cells used for lipid loading of each lane was not equal. (B) Lipids from the same cell lines as in (A) were sep arated by TLC and stained with "-naphthol. A lipid extract from E. coli producing galactoglycerolipids was included as standard (Std) containing MGDG, DGDG, trigalactosyldiacylglycerol (TGDG), tetragalactosyldiacylglycerol (TeGDG), and pentagalactosyldiacylglycerol (PGDG). !*,!! Figure 2.S 2. Cell viability and acyl group composition of TAGs during extended culturing time. (A) Number s of living and dead cells of the PL (dw15.1) , the tgd2 mutant , and the TGD2 tgd2 complemented line kept in N -replete medium for 3, 7, 14, 21 and 28 days . The cel ls were stained with methylene blue and phenosafranin to distinguish living and dead cells. Cel ls were counted with a hemo cytometer. (B) TAG acyl group profile of the PL (dw15.1) , the tgd2 mutant , and the TGD2 tgd2 complemented line kept in N -replete medium for 3, 7, 14, 21 and 28 days . Three biological replicates were average d and standard deviatio ns are shown. !*-!! Figure 2.S3. Ultrastructure of chloroplast membranes Electron micrographs of chloroplast membranes of the wild -type cw + strain (CC -198) and tgd2 grown in N -replete medium are shown during mid -log phase (day 3), stationery phase (day 10) and late stationary phase (day 17). ( A) Chloroplast membranes of a representative CC -198 cell during mid -log phase. ( B) Chloroplast membranes of a representative CC -198 cell during stationery phase. ( C) Chloroplast membranes of a representative CC -198 cell during late stationery phase. ( D) Chloroplast membranes of a representative tgd2 during mid -log phase. ( E) Chloroplast membranes of a representative tgd2 cell during stationery phase. ( F) Chloroplast membranes of a representative tgd2 cell during late stat ionery phase. !+.!! Figure 2.S4. Southern blot analysis of the tgd2 mutant and the PL (dw15.1). Genomic DNA was restriction digested with Bam HI. Linearized pHyg3 plasmid was used as a control. The p robe was complementary to a fragment from the pHyg3 plasmid (see Methods) . A line between tgd2 and pHyg3 plasmid indicates different exposure time s of the same blot. !+%!! Figure 2.S5. Analysis of progeny from crosses between tgd2 and CC -198. !+&!!Figure 2.S5. (contÕd ) (A) TAG content of the PLs (dw15.1 ) and (CC-198), the original tgd2 mutant in the dw15.1 background, and progenies from 6 zygotes with HS indicating Hygromycin B -sensitive and HR Hygromycin B -resistant progenies . (B) TAG acyl group profile s of the same lines as described under (A). !+'!! Figure 2.S6. Mutant locus in the tgd2 genome and complementation. !+(!!Figure 2.S6. ( contÕd ) (A) Alignment of tgd2 assembled contigs with chromosome 16 of the Chlamydomonas reference genome (v5.3). The AphVII gene insertion, affected genes and their annotation are shown. ( B) Cel lular TAG concentrations of parental line (PL, dw15.1), tgd2 mutant, CrTGD2 complemented lines (C1 -C3) and empty vector control (EV). (c) TAG acyl groups (16:0, 16:4 and 18:3) of the same lines as described under ( B). (B, C) Three replicates were averaged and standard deviations are shown. ( D) Cultures (30 day -old) of the PL (dw15.1), the tgd2 mutant and transgenic lines into which the six deleted genes were individually introduced into the tgd2 mutant. Three independent lines per gene introduced are shown. (E) Cellular PtdOH concentrations of the PL, the tgd2 mutant and CrTGD2 complemented lines ( TGD2 tgd2 , C3). !+)!! Figure 2.S7. Amino acid sequence alignment of AtTGD2 and CrTGD2. The alignment shows transmembrane and Mammalian Cell Entry (MCE) domains. The alignment was carried out with MUSCLE (3.8). !"#$%&'&("#$')*+&#,$ -#&&#.,#$)/'..)0$1"2)*+&#,$!"#$%&''''()$*+,)-,+'../(+...()'!0+1,.+*$,'+2/++3+1#1'4/,,1!!.*.05#$%&''''6666666666'6666666666'6666666666'6666666666'6(,)4!.!.-!"#$%&''''%!!4$-+..%'$$3*+/#,,/'%,+1*)71-#'/3+/.%8$8$'31.)79$$,$05#$%&''''$%!9.-+$83'-$66666666'66666/8$.)'!3./.%2$)$'33.)79$$,$!"#$%&''''/8),.$!#//'!/.7!7/1$8'-(1.38132-'#,89/.4!.$')0#$#+,1)105#$%&''''/8,/!$$$!!',!/,!7!1$*'!/1#$6#+2-'!#)98+/!0$')-)$#+,1)1!"#$%&''''$,#,$#))1,'*+./3*)9!,'!9)9%%3)))'+1*./,9,*-'.$//(9#()%05#$%&''''$,-,$-,/!,'3+./91,%,/',9,*%,.#,)'+1*.,)9!*-'.$/)!9+/,%!"#$%&'''')(+1*+)+9+'.,$+/4+90$'39$/),0%1-'#)3$,-$,./'%9/,$)8#1)05#$%&'''')#+-,+,+%2'1!/+49+10-'%9./),0.*$'4)-$1-$,!/'%%/,2)(#1/!"#$%&''''$19,9!)$,!'*#2./!91!!'.,)99!1+//'33)-!(!9%!'-+//.981%.05#$%&''''!1-!9*%$,%'3,8!!!9.!#'-/(93!!+/,'..!!9/,9-/'#+/(!9/1$$!"#$%&''''$//39,90/#'1./#-!.%%/'13,*..)(#+'9*#9/)-3.)'2#/,2#/3*,05#$%&''''$/,$*)9!/#'1#!!%!!!%)'11/-$.,/#9'%*,1!/1-!,'/#/03#/%4,!"#$%&''''9.)..%)/$8'#$%9!#13*/'3//)3./.1/'/6605#$%&''''9.).!%,.)/'!1%.$,-1*/'3#/,-!/.1/'/%%!+*!! Figure 2.S8. Phylogenetic analysis of CrTGD2 homologues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igure 2.S8. ( contÕd ) The evolutionary history was inferred based on Maximum Likelihood . The tree with the highest log likelihood ( -4302.1023) is sh own. The percentage of trees in which the associated taxa clustered together (1000 repeats) is shown above the branches. The tree is drawn to scale, with branch lengths corresponding to the number of substitutions per site. The accession number for each pr otein is given in the parenthesis following the scientific name. Accession numbers for Chlamydomonas, Arabidopsis and other organisms are from the Joint Genome Institute (JGI), the Arabidopsis Information Resource (TAIR), and National Center for Biotechnol ogy Information (NCBI), respectively. Groups of organisms are shown as colored bars. !+,!! Figure 2.S9 . Lack of Arabidopsis TGD2 complementation in Chlamydomonas tgd2 mutant. (A) CrAtTGD2 -pSI103 construct. Genomic DNA of Chlamydomonas TGD2 (CrTGD2 ) containing promoter region, intron, transit peptide and transmembrane domain (TMD) was assembled with codon optimized Arabidopsis TGD2 (AtTGD2) membrane extrinsic portion, followed by Chlamydomonas genomic DNA containing the terminator of CrTGD2 . The assembled seque nce was cloned into pSI103 -AphVIII containing the Aph VIII Paromomycin resistance gene. (B) Western blot analysis of CrAtTGD2 protein expression in the tgd2 mutant background ( AtTGD2 tgd2 ). Arabidopsis TGD2 recombinant protein (AtTGD2) was used as a positiv e control. White lines separate irrelevant lanes removed from the blot. (C) TAG concentration (fmol/cell) of the PL, the tgd2 mutant, the empty vector control in tgd2 mutant background (EV), and AtTGD2 tgd2 line 1 -4. !+-!! Figure S10 . Lack of Chlamydomonas T GD2 complementation in Arabidopsis tgd2 mutant. !,.!!Figure S10 . (contÕd ) (A) AtCrTGD2 -pMDC32 construct. Genomic DNA of Arabidopsis TGD2 (AtTGD2) containing transit peptide and transmembrane domain (TMD) was assembled with Chlamydomonas TGD2 (CrTGD2 ) transmemb rane extrinsic portion. The assembled sequence was cloned into pMDC32 containing 2x35S promoter and nos terminator. ( B) Western blot analysis of AtCrTGD2 fusion protein expression in the Arabidopsis tgd2 mutant background ( AtCrTGD2 tgd2) line 2, 6, 10, 15 and 20 compared to Col -2, tgd2-1 and the AtTGD2 cDNA complemented line ( AtTGD2 tgd2 -1). (C) TLC plate separating oligogalactoglycerolipids shows trigalactosyldiacylglycerol (TGDG) and tetragalactosyldiacylglycerol (TeGDG) of Arabidopsis lines as shown in ( B). ( D) Acyl group composition of monogalactosyldiacylglycerol (MGDG) of lines in ( B). ( E) Acyl group composition of digalactosyldiacylglycerol (DGDG) of lines in ( B). !,%!! Figure 2.S11. Pulse chase [ 14C]-acetate labeling of MGDG in the PL (dw15.1). Replic ates from four independent experiments during a 0 -9 h chase are shown. The highest radioactivity incorporations were set as 1, while the lowest were set as 0. !! !,&!!APPENDIX B. SUPPORTING TABLES Table 2.S 1. Bacterial Artificial Chromosomes (BACs) used in th e tgd2 complementation analysis. Corresponding BAC identifiers from the Joint Genome Institute (JGI) and from the Clemson University Genomics Institute (CUGI) are shown. Restriction endonucleases used for isolating genomic DNA fragments containing individ ual genes and corresponding product sizes are shown. The amount of genomic DNA and that of the AphVIII gene used for transformation as well as names of primers used for testing the insertion are listed. The primer sequences are given in Table 2.S2. Phytozo me, (V5.3) Number Restriction endonuclease Product size (kb) Amount of DNA used Testing primers JGI number CUGI number DNA from BAC (µg) AphVIII from pSI103 (ng) Cre16.g694550 PTQ2066 6C14 Dra I and Xho I 9.5 1.6 34.8 ch16 -1, ch16 -4, ch16 -8 and ch16 -14 Cre16.g694500 PTQ2066 6C14 SnaBI, Rsr II and Eco RI 9.9 1.5 30.0 ch16 -16, ch16 -20 and ch16 -21 Cre16.g694450 PTQ12548 33H17 Dra III and Kpn I 8.0 0.5 11.0 ch16 -21, ch16 -22, ch16 -25 and ch16 -26 Cre16.g694400 PTQ12548 33H17 Apo I, Msc I and Mlu I 8.1 1.0 25.0 ch16 -29, ch16 -32, TGD2_5Õ and TGD2_3Õ g15584 PTQ12548 33H17 Apa I, Bsu 36I and Sfi I 7.5 1.0 25.0 ch16 -32 and ch16 -33 g15585 PTQ12548 33H17 Dra III, Zra I and Nco I 6.5 0.5 25.0 ch16 -37, ch16 -38 and ch16 -40 !,'!!Table 2.S 2. Sequences of primers for probing the deletion in chromosome 16 of the tgd2 mutant. Gene/ primer names Forward primer sequence (5Õ -3Õ) Reverse primer sequence (5Õ -3Õ) Covered region on chromosome 16 From To Hygromycin ACTTCGAGGTGTTCGAGGAG AC GCTGCAAGGCGATTAAGTTGGG N/A N/A ch16 -1 GATTAACGGTGCCTGATG GGAACTAGCGTGATGATG 230406 231550 ch16 -2 CGACACATGGCGCTTGAT CGATTGGCCAGTATTCGT 231416 232578 ch16 -3 CCGCACCTTGCCAAT CT AGCGATGGCCGATGTCA 232058 233209 ch16 -4 ATAGCAGTCCTGCGAGTG ACGTGTGCTTGGTAATGG 233056 234297 ch16 -5 GCGTGCAACCAACTAGTA CAACATCACCACCACAAC 234095 235303 ch16 -7 ACACCTACCTGGAATTCG CGTACACGCGCAATAA 236170 237339 ch16 -8 GCACCTATCCGCACCTTATT TGTCCGGTGTTGTC AGTC 237309 238693 ch16 -9 GCTGAGTATGCAGCTTCCA GTGCGAACCGTGAAGACA 225080 226389 ch16 -14 GGCTCAGCTGTTCTTGTC CCATTGGTCACGTGTCTA 238609 239330 ch16 -15 CGGCTGCTTAACTCGTTG GCAGGTGGAGGTGTTCA 239168 240594 ch16 -16 GTTGTAGCTCAGGCTGTA CTGGAGCGACTTCAACT 240227 241327 ch16 -18 GCTGCTGCTGCTACTTCT GCAGCATGTGTCGTTATCG 242251 243390 ch16 -20 GTGTCGTACGCCTCCTTGA CAGCAGCCTCGGTTGACTA 244128 245414 ch16 -21 GTTACGCTCAGCTTGTCGTT AATGCTCATGCGGCCTTG 245224 246297 ch16 -22 CGACCACGACTACCACTTA GCAGGAGGAGGTTGATGT 246223 247360 ch16 -23 GCAGCTGTTGCAGTTCCT CGATTGGAGCACGAGCTA 247377 248719 ch16 -24 TAGCTCGTGCTCCAATCGT TGTCGCATCGTGTCTTGA 248702 249668 ch16 -25 CTCGCTGCCAAGTACCAT GCGTCTGAGTCAGCTTGTAG 249183 250444 ch16 -26 GCGTTACCGCTGTGTAAGG GCAGGCGGTTAAGCGAA 250253 251488 ch16 -29 TCAAGCAGTGGTGGTAGTG ACGGTGAAGTCCGTGAT 253446 254368 ch16 -32 CCTTGCCGTCTCTGTTGTT CAGTGCCTGGTACTTGCTA 256655 257724 ch16 -33 GGTGCCATGTGAGGAACGAT AGGTGCGTCACCGACTTGT 258158 259348 ch16 -34 GCATCGCATCGCATACAC CCAGGTCCTCACTCCACTT 259233 260470 ch16 -37 GTGGAGA ACGGAGTGAGTA GTGGACCGACATCGCATTA 263345 264599 ch16 -38 CGCGTCCTGTCCTTAAGAT TGCGGTAGGTGGCCTAATA 264324 265679 ch16 -40 GGCGTTGCCAACCTCTT CTCCGCCTGCGATGTTA 267314 268421 ch16 -43 CGCGCAGTATGTACGGTT CCATCAGGCACCGTTAATCC 229674 230424 ch16 -44 CGGCATTCGGCCTTA CAAGA TGCGGAGTGTGGACATAGTG 230638 231246 ch16 -45 GCGGCGGCTTATTATT ACATGGCGGTCTATCT 265814 266436 ch16 -46 CGGCATTCGGCCTTACAAGA ATGCGGCTTAGGCGGAATAC 230638 231091 ch16 -47 CGACCACCGTCATATAGT CGGTAGTCAGCGCATTA 230779 231072 ch16 -48 GGCATTCGGCCTTACAAGA TGCT GTGCCAGTATGCTTC 230639 230975 ch16 -49 GCTGCCGTGGCTGAGTATT GACCTCGGCTGTGTCTGTAA 230237 230554 TGD2_3' CATGCCTGACGGTGAATCCT GTAGCACGGTGAAGCTCTGT 251097 251804 TGD2_5' GTGCCATGTGAGGAACGATT TGTCGAGGACGGAGTTACAA 258159 258814 !,(!!Table 2.S 3. Sequences of prime rs used for constructing plasmids as indicated. Plasmids Purpose Forward (5Õ -3Õ) Reverse (5Õ -3Õ) DsRED -CrTGD2 pLW01 CrTGD2 cloning ATGGTCATCCACGCCAGCG TCAGTCGTCCAGCAGCCTG DsRED -CrTGD2 pLW01 CrTGD2 amplification for DsRED -CrTGD2 pLW01 CAGGATCCGCTCGCGG CAAC GCGCT TGGTCGACGAGTCGTCCAGCA GCCTGCTG CrAtTGD2 pSI103 AtTGD2 amplification TGGTGGCGTGGGCTCGCGAAT TCGGCTTCCAGATGCGGAG CAAGTTCCGCAAGTACCAAA AAGCTTTCAGAGCAGGCGCGA CAGGCTCTTGATCAGCAGCTTC AGGTTCTTGCGGGTGGC CrAtTGD2 pSI103 Amplification of CrTGD2 containing promoter and transmembrane domain TCAGGCTGCGCAACTGTTGGG AAGGGCGATCGGTGCGGGCG TTCAGGGGTTCGCAGGGTT CTCCGCATCTGGAAGCCGAAT TCGCGAGCCCACGCCACCAGC GCCACCGCCGCGCCGCCG CrAtTGD2 pSI103 pSI103 amplification GCCCGCACCGATCGCCCTTCC CAACAGTTGCGCAGCCTGAAT GGCGAATGGGACGCGCC C TGGCGGCCGCTCTAGAACTAG TGGATCCCCCGGGCTGCAGGA CGGCGGGGAGCTCGCTGA CrAtTGD2 pSI103 Amplification of CrTGD2 containing terminator GCTGCTGATCAAGAGCCTGTC GCGCCTGCTCTGAAAGCTT GGCGCGCGAGGGGCGAGGAG CTGCAGCCCGGGGGATCCACT AGTTCTAGAGCGGCCGCCAAG TGCGCCACGCCGCCACGC AtCrTGD 2 pDONR221 Amplifying pDONR221 GATGCTGCCAACTTAGTC GCTGGATACGACGATTCC AtCrTGD2 pDONR221 Amplifying AtTGD2 Annealing synthesis portion of CrTGD2 and pDONR GCAGCGCGTTACCTCGCAAC CAAGCCCA ACGGAATCGTCGTATCCAGC ATGATTGGGAATCCAGTAATT CAAGTTCC AtCrTGD2 pDONR221 Amplifying CrTGD2 synthetic, overlapping AtTGD2 and CrTGD2 from DsRED GTTGCGAGGTAACGCGCTGC GCACCGGC CCGTGGACACGTCGTTCACCT CCACCAGCACGTC AtCrTGD2 pDONR221 Amplifying CrTGD2 from DsRED , overlapping CrTGD2 synthetic and pDONR GGTGAACGACGTGTCCACGG TCATCCCG TCGACTAAGTTGGCAGCATCT CAGTGGTGGTGGTGGTG !,)!!APPENDIX C. APPENDIX C. Amino acid sequence alignment of Mammalian Cell Entry domains of CrTGD2 homologues used for building the phylogenetic tree in Figure 2.S7. MUSCLE (3.8) multiple sequence alignment Ignatzschineria larvae IEYKVITN-ESVAGLSINSPIDYRGVNVGKVAAIELNNNDPRYVTILLNI--DVGTPIKR Xylella fastidiosa --YRVVFR-EAVTGLSVGSPVQYNGIAIGSITQLTLAPNDPRQVIAHLRV--NATTPIKK Escherichia coli MS 115-1 --FN-----EPVSGLSQGSTVQYSGIRVGEVTQLRLDRDNPNKVWARIRV--SASTPIRE Pseudomonas putida --YEVVFN-EAVSGLSRGSSVQYSGIKVGDVTSLRLDPNDPRRVLAQVRLSAD--TPVKE Caenispirillum salinarum --YTI---YDNVGGVKFGTPVLYEGYTVGQVEDVEPQMTD-EGTRFRVEMSVQEGWPIPE Actinomadura flavalba -PYNISVEFASSPGLHPGFEVDYLGLRIGKIDSVRLAGDK---VVVKLDI--DKDVEVPR Mycobacterium tuberculosis --NTVVAYFTQANALYVGDKVQIMGLPVGSIDKIEPAGDK---MKVTFHY--QNKYKVPA Gordonia otitidis -TKTITAYFPSVNGLYTGDTVRVLGVKVGKVAAITPRSGD---VKVTLDV--DRSTPIPA Streptomyces sp. Tu_6176 -GTRVTAYFDRAVGIYAGSDLRILGVRAGAVKSVRPQGTQ---VRVELEL--DDGIQVPR Intrasporangium calvum -PTTISADFTRAVGLYPGSDVRILGVKVGQVDVVEPQGRH---VRVTFSV--DSRHRIPA Synechococcus sp. PCC 6312 -TYEVTITLADAPGLVVGTPVRYRGVRVGSISDVQVGPMG---IIAKAKLR-D--VIIPR Selenomonas artemidis -EYTLYVGFGRAVGLNPEAQVLLSGVPVGHVEKVGSDGTG---VTVAISVS-DD-VKIPR Phascolarctobacterium succinatutens -GYELRINYPQVSGLMPGHVVRYAGVQVGTVKKINVAHDK---VEVITEIN-DD-IKIPQ Stanieria cyanosphaera PCC 7437 -SYQVIAQFPNVNGIQVGDSVRYRGLKVGKITDIMPGTNG---VDVMMEIS-SSDLLIPK Chlamydomonas reinhardtii -PYQATIEFPLACGIQIGTPVRIRGVQVGQVLAVKPSLER---VDVLVEVN-DVSTVIPR Volvox carteri f. nagariensis -PYKATIEFPLACGITIGTPVRVRGVQVGQVLAVKPSLER---VDVLVEVN-DVSTVIPR Coccomyxa subellipsoidea C-169 -GYQCVLEFPLACGITVGTPVRIRGVPIGSVLNLNASLEK---VEVLTEVK-KSTTVIPR Chlorella variabilis -GYQAILEFPVACGITVGTPVRIRGVPVGGVLSVQPSLEK---VDVLVEMK-DSTTVIPR Auxenochlorella protothecoides -SYQAILEFPVACGISVGTPVRIRGVPVGGVLGVQPSLEK---VEVLVEIR-DSTTVIPR Micromonas pusilla CCMP1545 --YQAFVEFPFACGIQVGTQVRVRGVKVGNVLSVRPNLER---VEVLVEMD-DDGIVIPR Ostreococcus tauri --YQAFIEFPVACGIQVGTNVRVRGVKAGTVLSVQPSLEK---VDVLVEMD-DKNVPIPR Tarenaya hassleriana -KYQTVFEFPQASGICTGTPVRIRGVNVGNVIRVNPSLKN---IEAVTEID-DDKIIIPR Arabidopsis thaliana -KYQTVFELSHASGICTGTPVRIRGVTVGTIIRVNPSLKN---IEAVAEIE-DDKIIIPR Brassica rapa -KYQTVFELSQASGICTGTPVRIRGVTVGTVIRVNPSLKN---IEAVAEIE-DDKIIIPK Solanum tuberosum --YLAVLEFEQACGICTGTPVRIRGVSIGNVIRVNPSLRN---VEAVVEVE-DDKIIIPR Nicotiana tomentosiformis -KYLAVLQFEQACGICTGTPVRIRGVNIGNVIRVNPSLRN---VEAVVEVE-DDKIIIPR Cicer arietinum -KYTATIEFSQACGICTGTPVRIRGVTVGDVIRVNPSLRS---IEAVVEIE-DDKTIIPR Brachypodium distachyon -KYQAVLEFGQACGICVGTPVRIRGVTVGNVVRVDSSLSR---IDAVVEVD-DEKIVVPR Musa acuminata subsp. malaccensis -KYQVVFEFSQACGICVGTPVRIRGVNVGNVVRVDSTLRS---IDAIAEVD-DDKIIVPR Phoenix dactylifera -KYQAVFEFSQACGICVGTPVRIRGVTVGSVVQVNSSLKS---IDATVEVE-DDKIIIPQ Aegilops tauschii -KYNAVFEFSQACGICVGTPLRIRGVTIGSVVRVDSSLRS---IDAYVEVE-DDKIIVPR Oryza sativa Indica Group -KYQAVFEFTQACGICVGTPVRIRGVTVGNVVRVDSSLKS---IDAYVEVE-DDKIIVPR Sorghum bicolor -KYNTVFEFTQACGICVGTPVRIRGVTVGSVVRVDSSLRS---IDATVEVE-DDKIIIPR Zea mays -KYNTVFEFTQACGICVGTPVRIRGVTVGSVVRVDSSLRS---IDATVEVE-DDKIIIPR Setaria italica -KYNTVFEFTQACGICVGTPVRIRGVTVGSVVRVDSSLRS---IDALVEVE-DDKIIIPR Prunus mume --YFAVFEFTQACGISTGTPVRIRGVNVGSVVRVNSSLES---IEAVVEVE-DDKTVIPR Fragaria vesca subsp. vesca --YFAVFEFTQACGIATGTPVRIRGVTVGNVIRVNSSLQS---IEAVVEVE-DDKTVIPR Coffea canephora --YLAVFEFEQACGICTGTPVRIRGVNVGSVIRVNPSLNS---IEAVVEVD-DDKVIIPR Theobroma cacao --YLAVFEFAQASGICTGTPVRIRGVTVGNVVRVNPSLKS---IEAVVEVE-DDKIFIPR Jatropha curcas --YLAVFEFAQAGGICTGTPVRIRGVTVGNVIKVNPSLRC---IEAVVEVE-DDKIIIPR Nelumbo nucifera -KYQAVFEFAQACGICMGTPVRIRGVTVGNVIRINPSLKS---IEAVVEVE-DDKVIIPR Hevea brasiliensis --YVAVFEFAQACGICTGTPVRIRGVTVGNVIQVNPSLRS---IEAVVEVE-DDKIIIPR Ricinus communis -KYTAVLEFAQACGICTGTPVRIRGVTVGNVIQVNPSLKS---IEAVVEVE-DDKIIIPR Vitis vinifera --YLAVFEFTQACGICKGTPVRIRGVTVGNVIQVNPSLKS---IEAVVEVE-DDKIIIPQ Selaginella moellendorffii -KYFATFEFAKAWGITVGTPVRIRGVDVGTVIRVKPTLEK---LDVEVQIV-DANLVIPR Physcomitrella patens -KYEAVFEFQLAQGITVGTPVRIRGVDVGNVVQVRPSLEK---IDVVVELS-DAGIVVPR Microcoleus sp. PCC 7113 -SYQFIVKFANVAGMKTGAMVRYRGVKVGRITEVTPETNG---VNATVEIS-DPDLLIPK Oscillatoria nigro-viridis -SYKFAVEFASAQGMQIGTPIRYRGVAVGKITALKPGSNG---VDVTLEIA-PGTLVIPR Anabaena sp. 90 -SYQATIEFANAGGMQKGSAVRFRGVKVGTITNVKPGSNA---IDVEIQIN-SPDLIIPS Gloeocapsa sp. PCC 7428 --YSAIIEFANVGGMQEGGVVRYRGVNVGNIAAIRPGPNG---VEVDVEIA-PANLIIPR Calothrix sp. PCC 7103 -NYKIFVDFSNAGGMQKGAPVRFRGVKVGRIAAIRPGPNN---VEVELEIS-QRDLIIPR Nostoc sp. PCC 7120 --YKAVVEFANAGGMQRGATVRYRGVKVGRISQIQPGPNA---VEVEIEFA-QSNLIIPR Richelia intracellularis -TYKVIVEFTNAGGMQKGAVVRYRGVKVGRVNSIQPGPNT---VEVEIEIS-QSELIIPK Megasphaera sp. NP3 ----IHTEFNDANGLQKGNSVRYVGVHVGKVEKVTPSRNG---VDVTMKI--DKGTEIPR Anaeromusa acidaminophila -GYPIQAVFSQVGGLKDGAIVRYAGVDVGRVQSVEMTATG---VTVNLRIF-DH-VRIPR .: : * * : : : Ignatzschineria larvae DTEAVLMSRGITGIVNVSLTG- Xylella fastidiosa DTRAKLAITSLTGPSIIQLSG- Escherichia coli MS_115-1 DTQARLTVAGITGTSNIQFSS- !,*!!Pseudomonas putida DTQAKLTLTGITGTSFIQLSG- Caenispirillum salinarum DSSADVAASGFLGGMMINITGG Actinomadura flavalba GVHAAAARKSAVGEPVVELTP- Mycobacterium tuberculosis NASAVILNPTLVASRNIQLEP- Gordonia otitidis DARAVVVAQSLVSGRFVQLTP- Streptomyces sp. Tu_6176 GAHAVIVAPSVVADRFVQLTP- Intrasporangium calvum DARAAIVAPSLVSDRYVQLLP- Synechococcus sp. PCC_6312 DAIPEVRQSGFVGSSFLDF--- Selenomonas artemidis GSSVTIAQPGIMGDKFVIITP- Phascolarctobacterium succinatutens GATFTISSDGIMGEKFVSVIP- Stanieria cyanosphaera PCC 7437 NALIQASSSGLIGETFVAIIP- Chlamydomonas reinhardtii NSVIEANQSGLIAEPLVDITP- Volvox carteri f. nagariensis NSVIEANQSGLIAEPLVDITP- Coccomyxa subellipsoidea C-169 NSHIEANQSGLIAEPLIDITP- Chlorella variabilis NSLIEANQSGLIAEPLIDITP- Auxenochlorella protothecoides NSLIEANQSGLIAEPLIDITP- Micromonas pusilla CCMP1545 NSLVEANQSGLIAETIIDITP- Ostreococcus tauri NSVIEANQSGLIAETIIDITP- Tarenaya hassleriana NSLVEVNQSGLLMETMIDVTP- Arabidopsis thaliana NSLVEVNQSGLLMETMIDIMP- Brassica rapa NSLVEVNQSGLLMETMIDITP- Solanum tuberosum NSLVEVNQSGLIMETMIDITP- Nicotiana tomentosiformis NSLVEVNQSGLIMETMIDITP- Cicer arietinum NSSVEVNQSGLLMETVIDITP- Brachypodium distachyon NSVVEVNQSGLLMDTLIDITP- Musa acuminata subsp. malaccensis NSLVEVNQSGLLMETLIDITP- Phoenix dactylifera NSLVEVNQSGLLMETLIDITP- Aegilops tauschii NSLVEVNQSGLLMETMIDITP- Oryza sativa Indica Group NSVVEVNQSGLLMETLIDITP- Sorghum bicolor NSVVEVNQSGLLMETLIDITP- Zea mays NSMVEVNQSGLLMETLIDITP- Setaria italica NSLVEVNQSGLLMETLIDITP- Prunus mume NSLIEVNQSGLLMETRIDVTP- Fragaria vesca subsp. vesca NSLIEVNQSGLLMETRIDITP- Coffea canephora NSLVEVNQSGLLMETLIDITP- Theobroma cacao NSLIEVNQSGLLMETLIDITP- Jatropha curcas NSLIELNQSGLLMETIIDITP- Nelumbo nucifera NSLIEVNQSGLLMETLIDITP- Hevea brasiliensis NSLIEVNQSGLLMETLIDITP- Ricinus communis NSLIEVNQSGLLMETLIDITP- Vitis vinifera NSLIEVNQSGLLMETLIDITP- Selaginella moellendorffii NALVEVNQSGLVSETLIDITP- Physcomitrella patens NALVEVNQSGLISETLIDVTP- Microcoleus sp. PCC 7113 DVVIEANQAGLVGETSIDITP- Oscillatoria nigro-viridis DVTIEANKSGLIGESSIDITP- Anabaena sp. 90 NSIIEANQSGLISENIIDITP- Gloeocapsa sp. PCC 7428 DVQIAANQSGLISEVSIDITP- Calothrix sp. PCC 7103 DVKVEANQSGLIAESLIDITP- Nostoc sp. PCC 7120 DVVIEANQTGLISESIIDITP- Richelia intracellularis NIVVEANQSGLIGESVIDITP- Megasphaera sp. NP3 DSKIVITTDGLLGEKIVSISPG Anaeromusa acidaminophila GSVFTIASEGLLGEKYITILP- . : . !!,+!APPENDIX D. SUPPORTING METHODS Generation of tgd2 mutant and genetic analyses. The tgd2 mutant was generated by insertional mutagenesis in the same experiment as described previously for the cht7 mutant (Tsai et al. , 2014). For genetic analysis, t he orig inal tgd2 mutant ( in dw15 .1) was crossed with the cell -walled strain CC -198 as previously described (Li et al. , 2012) . The progenies were test ed for cosegregation of Hygromycin B resistance and TAG lipid phenotype. Genetic complementation analysis was init iated by generating tgd2 lines into which fragments of genomic DNA were introduced that were derived from bacterial artificial chromosome s ( Clemson University Genomics Institute ). Genomic DNA fragments covering individually each affected gene with ~ 1 kb 5Õ of the start codon and 0.5 kb 3Õ of the stop codon w ere cut from BAC s with restriction endonucleases (for details refer to Table 2.S1). Approximately 0.5-2.0 µg of purified genomic DNA was co-introduced into the tgd2 mutant with the P aramomycin resistan ce gene AphVIII at a 1/10 molar ratio . AphVIII was prepared by KpnI/PstI digestion from plasmid pSI103 (Chlamydomonas Resource Center ; http://chlamycollection.org/ ). The a mount s of DNA used for the transformation s are listed in Table 2.S1. The transformants were selected on TAP agar contai ning 20 µg/m L Paromomycin. Single colonies were picked and grown in 200 µ L TAP medi um in 96 -well culture plates. Of the mid-log phase culture 150 µ L were transferred into 96-well PCR plates and centrifuged at 3000 X g for 5 min. The supernatant was removed . The pellet was used to extract DNA with Chelex -100 (SIGMA) as previously described (Cao et al. , 2009) . Presence of the introduced DNA was confirmed by PCR using p rimer s and sequences listed in Table s 2.S1 and 2.S2, respectively. Colonies positive for the introduced gene w ere then gr own in TAP liquid medium for lipid analysis. DNA isolation and Southern blot analysis. DNA isolation was carried out as previously described (Keb -Llanes et al. , 2002) with some modifications. A m id-log phase culture (15 mL) was harvested by centrifugation at 3,000 X g for 5 min. The pellet was resuspended in 400 µL extraction buffer A without polyvinylpyrrolidone, ascorbic acid and #-mercaptoethanol. The mixture was then incubated at 60¡C for 1 h. 400 µ L of phenol/chloroform (1: 1 v/v) w as added. The mixture was then mixed by repeated inverting and centrifuged at 13,000 X g for 1 min. The upper phase was transferred to a new tube. The process was repeated with chloroform. DNA was precipitated by adding 1 volume of isopropanol, fol lowed by centrifugation at 13,000 X g for 5 min. The supernatant was discarded. Th e pellet was washed with 70% ethanol, dried and !!,,!resuspended in 200 µ L of 10 mM Tris -HCl pH 8.0. The isolated DNA was treated with 10 µ L of 0.5 mg/m L DNase-free RNase (Roche). The treated DNA was purified, precipitated , and resuspended again as mentioned ab ove. Southern blot analysis was done with the same probe as described in (Li et al. , 2012). Whole genome resequencing . The genome of the tgd2 mutant was sequenced by Illumina Hi -Seq using the paired -end method at the MSU -Research Technology Support Facilit y. Reads were quality checked and trimmed with the FASTX toolkit 0.0.13 (Patel & Jain, 2012) . Read assembl y was performed with velvet 1.2.07 (Zerbino & Birney, 2008) using the 21 k-mer length of 21 . The presence of the AphVII gene was detected in a ssembled reads with BLASTN 2.2.26+ (Park et al., 2012) using the default setting. Reads containing the AphVII gene were analyzed for flanking genomic sequences against the Chlamydomonas reinhardtii V5.3 reference genome using BLAST (Altschul et al. , 1997) . The ide ntified deletion was confirmed by PCR using primers a s listed in Table 2.S2. Phyloge netic analysis . Amino acid sequences were obtained from blast p searches with CrTGD2 as query against the amino acid sequence database through BLASTP 2.2.31+ at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.giv/, Altschul et al. , 1997; Altschul et al., 2005) . Protein domain searches w ere carried out against the Pfam protein families database (Finn et al. , 2014) with hmmscan 3.1b1 (Eddy, 2009) . Ma mmalian Cell Entry (MCE) domain (PF02470.15) was a common domain for every amino acid sequence and was used for alignment with MUSCLE (Edgar, 2004) . The gap penalty was set to -9 for gap open and to -3 for gap extension. The aligned amino acid sequences we re then used for phylogenetic tree reconstruction with MEGA6 (Tamura et al., 2013) using Maximum Likelihood method based on the JTT matrix -based model (Jones et al. , 1992) . Test of phylogeny was carried out with the Bootstrap method (Felsenstein, 1985) set for 1000 replicates. Heterologous complementation analysis. In order to test the activity of AtTGD2 in the Chlamydomonas tgd2 mutant, a fusion protein containing the CrTGD2 transmembrane domain and the AtTGD2 extrinsic portion was expressed from a constru ct assembled into the pSI103 vector (Sizova et al. , 2001) . The CrTGD2 transmembrane domain encoding portion also contained 1 kb 5Õ of the start codon including an intron followed by the extra membrane portion encoded by codon -optimized AtTGD2. In addition, 1 kb CrTGD2 sequence 3Õ of the stop codon !!,-!was included (Figure 2.S9). This construct was produced using a G ibsonÕs assembly kit (New England Biolabs). Primers used are listed in Table 2.S3. Two µg of this construct was used to transform the Chlamydomonas tgd2 mutant. For testing CrTGD2 complementation of the Arabidopsis tgd2 mutant, a fusion protein consisting of the transmembrane domain encoded by AtTGD2 and the membrane extrinsic portion encoded by CrTGD2 was expressed from a construct assembled into the pMDC32 vector (Curtis & Grossniklaus, 2003) (Figure 2.S10) . The CrTGD2 fragment was obtained by gene synthesis (165 bp) and DsRED-CrTGD2 pLW01 (672 bp). AtTGD2 and CrTGD2 fragments were introduced by Gibson assembly (New England Biolabs) into pDONR ª221 (Invitrogen TM). This construct was then used as a template for assembly into pENTR ª/D-TOPO¨ (Invitrogen TM) eliminat ing the His-tag. At-CrTGD2 pENTR was used as a donor vector to construct At-CrTGD2 in pMDC32 using Gateway¨ LR Clonaseª II enzyme mix (Invitrogen TM). Primers used to make this construct are listed in Table 2.S3. At-CrTGD2 pMDC32 was i ntroduced into Agrobacterium strain G3101. Agrobacterium containing At-CrTGD2 pMDC32 was used for Arabidopsis tgd2 mutant transformation using the floral dip method as previously described (Clough & Bent, 1998). DsRED -CrTGD2 pLW01 , DsRED -AtTGD2 pLW01 and DsRED pLW01 constructs, recombinant protein expression and purification . DsRED-AtTGD2 pLW01and DsRED pLW01 were obtained from Binbin Lu (Lu & Benning, 2009) . DsRED-CrTGD2 pLW01 was constructed in order to test the function of CrTGD2. Based on s equence alignment between CrTGD2 and AtTGD2 and hydrophobicity analysis the C -terminal membrane external porti on of CrTGD2 was identified (Fig ure 2.S7 ). The corresponding DNA fragment was amplified with Phusion polymerase using p rimers listed in Table 2.S3. The CrTGD2 fragment and pLW01 (a provided by Dr. Michael Garavito , Michigan State University ) containing DsRED and a His-tag were digested with Bam HI and SalI. The products were ligated together to produce DsRED-CrTGD2 pLW01. Protein expression was carried out according to (Lu & Benning, 2009) with minor modifications. P rotein expression was induced with 100 µM instead of 50 µM IPTG. For protein purification, the supernatant from the cell lysate was incubated with HisPur Ni -NTA resin (Thermo SCIENTIFIC). The eluted protein was dialyzed in dialysis buffer containing 125 mM !!-.!NaCl in addition to 10 mM KH 2PO4. The pro tein was stored in 50% (v/v) glycerol at -20¡C until used. !!-%! REFERENCES !!-&!REFERENCES Altschul, S. F., Madden, T. L., Sch−ffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). 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A Cytochrome b5 -containing plastid -located fatty acid desaturase from Chlamydomonas reinhardtii . Euk Cell, 11 (7), 856-863. doi: 10.1128/ec.00079 -12 Zerbino, D. R., & Birney, E. (2008). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res, 18 (5), 821-829. ! !!%..! CHAPTER 3 Characterization of Chlamydomonas LIP4, a putative triacylglycerol lipase € !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!€ This project was conducted in collaboration with Witawas Handee who contributed to Figure 3.8. !!%.%!ABSTRACT The metabolism of triacylglycerol (TAG) is no t only important for maintaining an organismÕs homeostasis, but it is also key for coping with environmental stress. In microalgae, TAG accumulates in lipid droplets during stress conditions such as nutrient deprivation. When normal growth conditions resum e, TAG is degraded to free fatty acids, which can be used for membrane synthesis or further metabolism to supply carbon in the form of acetyl -CoA and energy in the form of ATP and NADH to support growth and development. TAG lipase is the first enzyme of TAG degradation. Whole genome sequencing of Chlamydomonas has identified 130 genes for putative lipases; however none of these candidates has been directly shown to possess TAG lipase activity. A transcriptomic study revealed nine putative lipases, CrLIP1 th rough CrLIP9, which are differentially expressed during nitrogen deprivation. Based on its amino acid sequence, one of these, CrLIP4, is a homologue of a major Arabidopsis seed TAG lipase, SDP1. The goal of this study was to assess the TAG lipase activity of the CrLIP4 protein. Down -regulation of CrLIP4 through artificial microRNA showed reduced TAG degradation in Chlamydomonas. The coding sequence of the gene was cloned and used for heterologous expression in both Arabidopsis and yeast and in vitro lipase assays. Introduction of CrLIP4 cannot rescue the Arabidopsis sdp1 mutant nor yeast TAG lipase mutants. However, the CrLIP4 recombinant protein showed TAG lipase activity toward triolein and Chlamydomonas TAG substrates in vitro . In summary, CrLIP4 showed T AG lipase activity in both Chlamydomonas and in vitro assays, but not in heterologous systems. !!%.&!INTRODUCTION All e ukaryotic and some prokaryotic cells (such as Mycobacterium, Streptomyces, Rhodococcus, and Norcardia, Alvarez & Steinbuchel, 2002) sequest er triacylglycerol (TAG) inside lipid droplets (LDs) in the cytosol as a way to store both energy and substrates for membrane lipid synthesis . During normal growth and development, TAG will be synthesized, stored, and broken down in response to the needs o f the cell. In animals imbalanced LD homeostasis and TAG metabolism can lead to diseases such as lipodystrophies, obesity, insulin resistance and type 2 diabetes as summarized by Gross and Silver (2014) . In angiosperms, TAG accumulates mainly in seeds. Du ring germination, TAG is degraded to support growth of seedlings prior to the establishment of photosynthesis. In leaves, TAG is synthesized from membrane lipids in response to stress, e.g. freezing (Moellering et al. , 2010) and ozone fumigation (Sakaki et al. , 1990a; Sakaki et al. , 1990b) . Senescence can also stimulate TAG accumulation in vegetative tissue as discussed in Troncoso -Ponce et al. (2013). In microalgae, TAG accumulation is observed in many stress responses such as high light and hypoxia as sum marized by Goold et al. (2015), and nutrient deprivation, including nitrogen (N), iron (Fe), zinc (Zn), sulfur (S) and phosphorus (P) (Boyle et al. , 2012; Kropat et al. , 2011; Matthew et al. , 2009). TAG breakdown is mediated through TAG lipase yielding di acylglycerol (DAG) and free fatty acid. In some cases, TAG lipase can further hydrolyze DAG or monoacylglycerol (MAG) yielding free fatty acids and the gly cerol backbone. TAG lipase is a patatin -related phospholipase A (pPLA) (Scherer et al. , 2010) , which has an esterase box G XSXG containing a serine in it a s an active site. Lipases hydrolyze ester bonds at the interface of organic and aqueous phases as originally monitored by Sch¿nheyder and Volqvartz (1945) . This is due to the difference in solubility of the enzyme and its substrate. In addition, conformational changes are required for the enzyme to function properly (reviewed in Gill & Parish, 1997; Verger, 1976) . In the inactive conformation, the catalytic motif is hidden. Upon exposure to an interface, the catalytic motif is exposed for substrate interaction. This process is controlled by a short helical fragment that acts as a lid covering the catalytic domain (Brady et al. , 1990; Brzozowski et al. , 1991; Kim et al. , 1997). Many lipases have been discov ered in several organisms; examples of some of these are described below. In yeast, Tgl3p, Tgl4p and Tgl5p were identified as having the conserved lipase motif and patatin domain (Athenstaedt & Daum, 2003, 2005; Kurat et al. , 2006) . These three TAG lipase s !!%.'!are associated with LDs and contain hydrophobic domains but not transmembrane domains. An in vitro study revealed that Tgl3p acts on both TAG and DAG. Deletion of TGL3, the gene that encod es Tgl3p , resulted in increased amounts of TAG and hypersensitivit y to cerulenin, a fatty acid synthase inhibitor (Athenstaedt et al. , 1999) . Lipase assays showed that Tgl4p degrades TAG (preferentially TAG -containing myristic and palmitic acids) but not DAG (Kurat et al. , 2006). In contrast to Tgl3p and Tgl4p, which bot h showed lipase activity in vitro and in vivo , Tgl5p only showed activity in vitro on TAG that contains 26:0 fatty acid (Athenstaedt & Daum, 2005). The tgl3 ! tgl4 ! tgl5 ! triple mutant accumulated high levels of TAG; furthermore, no TAG degradation was observed in the presence of cerulenin (Athenstaedt & Daum, 2005) . Adipose Triglyceride Lipase (ATGL), a major intracellular TAG lipase in mammals, was discove red for its TAG lipase activity by three different groups (Jenkins et al. , 2004; Villena et al. , 2004; Zimmermann et al. , 2004) . The transcript level of ATGL in mice and humans is highest in adipose tissue, for which the enzyme was named. ATGL contains a l ipase motif and patatin domain and is localized in LDs. Mice deficient in ATGL exhibited increased adipose mass that led to accumulation of TAG in several tissues (Haemmerle et al., 2006) . High levels of TAG in the heart of these mice led to cardiac dysfun ction and premature death. ATGL is regulated through hormonal stimulation by the protein Comparative Gene Identification -58 (CGI -58). During inactivation, CGI -58 binds to perilipin A; once stimulated, perilipin A is phosphorylated, and CGI -58 is free to bi nd ATGL and induce TAG lipase activity (Zechner et al. , 2009). The major TAG lipase in seeds of Arabidopsis is named SUGAR -DEPENDENT 1 (SDP1) (Eastmond, 2006) . SDP1 was discovered through ethyl methanesulfonate (EMS) mutagenesis. The sdp1 -1 point mutant i s impaired in TAG degradation in the seed, which is important for seedling growth. SDP1 encodes a protein containing a patatin -like acyl -hydrolase domain, which is also found in yeast Tgl3p and human ATGL. Recombinant SDP1 hydrolyzed TAG and DAG in vitro. Fusion of SDP1 with the green fluorescent protein revealed that SDP1 is associated with the surface of LDs. An attempt to identify other TAG lipases in Arabidopsis was carried out in double, triple and quadruple mutants of SDP1 -LIKE (SDP1L ), ATGL-LIKE and CGI -58-LIKE in the sdp1 -5 (a T -DNA insertion line) background (Kelly et al. , 2011) . Only SDP1 and SDP1L were found to be required for seedling growth. !!%.(!Although more than one hundred putative lipases are encoded in the Chlamydomonas genome (Merchant et al., 2007), none of the TAG lipases has been characterized. Transcriptomic analysis in Chlamydomonas revealed nine putative lipases, named CrLIP1-CrLIP 9, that are either up or down regulated during N -replete and N -deprived conditions (Miller et al. , 2010) . Bas ed on in vitro analyses, CrLIP1 has been shown to possess DAG lipase activity and it can hydrolyze polar lipids in vitro (Li et al. , 2012) . Down -regulation of CrLIP1 resulted in slower degradation of TAG in the mutant relative to wild type when N is resupp lied to starved cells, in which TAG has accumulated in LDs. In addition, expression of CrLIP 1 in a yeast tgl3 ! tgl4 ! double mutant can rescue the TAG phenotype, which is similar to that of the tgl3 ! tgl4 ! tgl5 ! triple mutant. Among the nine lipases, CrLIP4 shares the most sequence identity to the well -characterized Arabidopsis SDP1. The current study sought to d etermine if CrLIP4 has TAG lipase activity. RESULTS CrLIP4 is down -regulated during N -deprivation . A t ranscriptomic study of Chlamydomonas showed that expression of CrLIP4 was down -regulated during N -deprivation, during which TAG accumulates (Miller et al., 2010) . In order to monitor the expression pattern of this gene in detail, quantitative reverse transcription PCR (qRT -PCR) was used to analyze CrLIP4 transcript levels at different time points. The culture of the parental line Chlamydomonas strain dw15.1 (PL) was grown in N -replete Tris -Acetate-Phosphate (TAP) medium until mid -log phase (0 h) was achieved. The culture was N -deprived for 48 h and then resupplied with N and grown for another 48 h. RNA was isolated from samples taken at 24 h intervals; qRT -PCR of CrLIP4 was performed and normalized to an internal standard. The level of CrLIP4 transcript dropped sharply after 24 h of N deprivation and remained low throughout this condition (Figure 3.1). After N -resupply, the CrLIP4 transcript increased to abou t 30% of the initial level. The level of CrLIP4 transcript was inversely related to the TAG level (Figure 3.1), pointing to its potential role as a TAG lipase. CrLIP4 contains DUF3336 , transmembrane, and patatin domains and a large IDR at its C terminus . Structure and domains of CrLIP4 were predicted based on the amino acid sequence translated from the coding sequence through hmmscan 3.1b1 (Eddy, 2009) . Apparently, CrLIP4 contains a !!%.)!DUF3336 (domain of unknown function) and a patatin domain (Figure 3.2A). T hese two domains are also present in Arabidopsis SDP1 and yeast Tgl3p Ð 5p (Figure 3.3). The domain homology allowed us to hypothesize that CrLIP4 may function as a TAG lipase. In addition, the carboxyl end of CrLIP4 contains a large intrinsically disorder ed region (IDR) as predicted by four different programs (Figure 3.2B -3.2E). Furthermore, more phosphorylation sites were predicted to be localized in the IDR compared to the rest of the protein by the Disorder Enhanced Phosphorylation Predictor (DEPP) (Iak oucheva et al. , 2004) (Figure 3.2F). A transmembrane domain search through the transmembrane hidden Markov model (TMHMM) (Krogh et al. , 2001; Sonnhammer et al. , 1998) revealed that CrLIP4 contains one transmembrane domain (TMD) (Figure 3.3A). Although the gene sequences are divergent (Fig 3B), the G XSXG motif of the patatin domain is consistently preceded by a TMD in CrLIP4 , Tgl3p, Tgl5p and ATGL, and by hydrophobic regions in SDP1 and Tgl4p (Figure 3.3A), suggesting that the patatin domain needs to be anc hored and/or situated close to a membrane. Note that SDP1, Tgl4p and Tgl5p also contain IDRs (Figure 3.3A), however, sequence alignments show no similarity among these regions (Figure 3.3B). In addition, secondary structure prediction revealed that CrLIP4 , SDP1, Tgl3p, Tgl4p, Tgl5p, and ATGL contain multiple helices ( "-, (-, and 3 10-helix) upstream of IDR regions (Figure 3.3A). Phylogenetic analysis of CrLIP4. Based on a homology search using the CrLIP4 sequence against sequences deposited at the National Center for Biotechnology Information (NCBI), 45 orthologues were selected for further analysis. A domain search through hmmscan 3.1b1 (Eddy, 2009) revealed that all of the homologues contain a patatin domain (Table 3.1). The DUF3336 domain is also common t o all presumed homologues except ATGL. For this reason, human ATGL was excluded from the alignment and phylogenetic tree reconstruction. Structurally, the DUF3336 domain is always present at the N terminus followed by a few amino acids and the patatin doma in. Prot ein alignment was performed for the regions containing DUF3336 and the patatin domains with MUSCLE (Edgar, 2004) using the same criteria as described in Chapter 2. Phylogenetic tree reconstruction was undertaken from the protein alignment with the same setting as described in Chapter 2. This analysis revealed that CrLIP4 is grouped with presumed orthologues from plants but not with those from fungi or bacteria (Figure 3.4). !!%.*!Down -regulation of CrLIP4 transcript resulted in slower TAG degradation . In vivo analysis of CrLIP4 was conducted through artificial microRNA (amiRNA) knockdown in Chlamydomonas. Two constructs of amiRNA vectors targeting CrLIP4 at either the 5Õ or 3Õ region were designed based on WMD3 - Web MicroRNA Designer (http://wmd3.weigelw orld.org/cgi -bin/webapp.cgi?page= Home ;project=stdwmd ). The linearized plasmid containing AphVIII , a paromomycin resistance gene, was introduced with the glass bead method as described in the Materials and Methods section into the Chlamydomonas PL genome. The mRNA levels of the transformants were first tested with qRT -PCR. From 15 tested transformants of each construct, amiRNA targeting the 3Õ region of CrLIP4 provided more clones with reduced CrLIP4 transcript level than did the amiRNA targeting the 5Õ regi on (Figure 3.5A). Eleven amiRNA knockdown lines showed highly reduced levels (less than 75% of PL level) of the CrLIP4 transcript from both constructs (Figure 3.5A). Among these eleven lines, seven of them were screened for TAG degradation during N -resupp ly. From the seven, four lines accumulated more TAG than did the empty vector control. TAG levels in these four lines were then tested at different time points: 48 h after removal of N (time 0); 10 and 24 h after N -resupply. Three of the four lines showed higher TAG levels at 24 h after N -resupply. Another experiment was carried out to include additional time points at 12, 16 and 20 h after N -resupply. The result confirmed that these three lines showed higher TAG levels at 24 h after N -resupply (Figure 3.5B ). This result suggested that lowering the expression level of CrLIP4 resulted in a delay in TAG degradation. Note that the three lines contain amiRNA targeting the 3Õ region of CrLIP4 . This suggested that knocking down CrLIP4 was more effective when the 3 Õ region of the gene was targeted. CrLIP4 coding sequence cloned from Chlamydomonas dw15.1 contains four amino acid changes and a five amino acid insertion compared to the gene model . The CrLIP4 nucleotide sequence contains a region with extremely high GC content (79%). The overall GC content of the coding sequence is 72%. This high GC content made cloning of the full CrLIP4 coding sequence very challenging. Thus, the coding sequence of CrLIP4 was cloned in five overlapping fragments from Chlamydomonas PL c DNA. These five segments were joined two fragments at a time by PCR using the forward primer from the 5Õ piece and the reverse primer from the 3Õ piece. The sequence of the cloned CrLIP4 differs from the gene model 319691 available in the !!%.+!JGI v.4 database, with differences in the identity of four amino acids and with a five amino acid insertion in the IDR region (Figure 3.6). These differences are likely to be due to the unusually high GC content of the gene, which causes technical difficulties for standard sequencing. Indeed, I was unable to sequence this region with the normal Sanger procedures. To eliminate DNA secondary structure during the sequencing reactions, dimethyl sulfoxide, betaine and 7 -deaza -dGTP were added to the sequencing reaction mix (Musso et al. , 2006) . The cloned coding sequence was used for all subsequent experiments. CrLIP4 was not able to rescue yeast tgl3 !, tgl4 !, or tgl3 !tgl4 ! mutants. CrLIP4 shares 26, 45 and 32% amino acid identity with yeast Tgl3p, Tgl4p and Tgl5p, respectively. Tgl5p was reported to exhibit only in vitro TAG lipase activity toward TAG with 26 -carbon acyl groups (Athenstaedt & Daum, 2005) . In addition, the phenotype of the tgl3 ! tgl4 !tgl5 ! triple mutant and tgl3 ! tgl4 ! double mutant are the same. Therefore, function al analysis was conducted by overexpressing CrLIP4 with an HA tag in the yeast tgl3 ! tgl4 ! double mutant. Yeast WT strain yMK839 (Kuo et al. , 1998) and/or tgl3 ! tgl4 ! were transformed with either pMK595 empty vector or pMK595 -CrLIP4 . Expression of CrLIP4 was detected with HA antibodies in tgl3 ! tgl4 ! transformed with pMK595 -CrLIP4 (CrLIP4 -HA tgl3 ! tgl4 !) but not the WT or tgl3 ! tgl4 ! transformed with the pMK595 empty vector (Figure 3.7A). Note that multiple bands were detected with the HA antibody in the CrLIP4 -HA tgl3 ! tgl4 ! lines. These multiple bands could be a result of protein degradation in the yeast cell, multiple start codons or early termination of mRNA translation . The yeast cultures were then grown until stationary phase was achieved. Cerulenin (an inhi bitor of fatty acid synthesis) or the same volume of ethanol was added to the cultures. Cell density (cells/ml) and TAG concentration (fmol/ml) were monitored at 0, 3 and 7 h after addition of cerulenin or ethanol. In the presence of cerulenin, all cell t ypes remained in stationary phase (Figure 3.7B, upper left), however TAG concentrations in all cell lines dropped as the cells metabolized their storage lipids. The TAG concentration of CrLIP4 tgl3 ! tgl4 ! was essentially identical to the tgl3 ! tgl4 ! double mutant (Figure 3.7B, lower left), and both were higher than TAG levels of wild -type cells. At 7 h after cerulenin addition, TAG concentrations of CrLIP4 tgl3 ! tgl4 ! and tgl3 ! tgl4 ! were reduced to less than half of the original value. Without cerulenin, cell numbers of all cell types were constant during the first 3 h and then increased to almost double (Figure 3.7B, upper right). The TAG concentration of CrLIP4 !!%.,!tgl3 ! tgl4 ! was similar to that of t he double mutant and higher than that of WT (Figure 3.7B, lower right) . At 3 h, the TAG concentration of all three lines dropped, while at 0 and 7 h, TAG concentration of all was relatively unchanged. This drop in TAG concentration at 3 h could be due to s maller cell size of newly budded cells as mentioned above. It is crucial to note that this TAG analysis in CrLIP4 tgl3 ! tgl4 ! experiment was carried out only once. Without more replicates, a solid conclusion cannot be drawn from this result. These experiments showed that CrLIP4 was unable to rescue the TAG phenotype of the yeast tgl3!tgl4! double mutant. However, it w as possible that the impact of the loss of lipase activity in the tgl3!tgl4! double mutant is greater than any compensation CrLIP4 activity could provide. Therefore, introduction of pMK595 -CrLIP4 into each single mutant ( tgl3! or tgl4!) was carried out. St ationary phase cultures of tgl3 ! and tgl4 ! with pMK595 empty vector (EV) or pMK595 -CrLIP4 were used to determine TAG concentration. The HA antibody detected a smaller than expected size of CrLIP4 -HA in both CrLIP4 tgl 3! and CrLIP4 tgl 4! mutants (Figure 3.8 A). TAG concentrations in both CrLIP4 tgl3 ! and CrLIP4 tgl4 ! were higher than the mutants with empty vector controls (Figure 3.8B). Despite the large error bars, these results were similar to that of CrLIP4 overexpression in the yeast double mutant. CrLIP4 could not complement Arabidopsis sdp1 mutants . Since the CrLIP4 amino acid sequence shares 41% identity to SDP1 and carries patatin and DUF3336 domains similar to SDP1, I hypothesized that CrLIP4 would rescue the Arabidopsis sdp1 mutant phenotype. In order to test this hypothesis, CrLIP4 cDNA under the control of the CaMV 35S promoter was introduced into sdp1 -4 or -5 null mutants, which are T -DNA insertion lines. Expression of the CrLIP4 transcript was measured in the first generation of transgenic plants (Figure 3.9A). Transgenic plants were selected and self -crossed. Only the transgenic lines that contain a single insertion and are homozygous were selected for further analysis. Since the original sdp1 -1 mutant was reported to have a post -germinati on growth phenotype, root length of four -day old seedlings grown in Murashige and Skoog (MS) medium without sucrose was measured (Figure 3.9B). The sdp1 mutants and the sdp1 mutants expressing CrLIP4 (CrLIP4 sdp1 ) had shorter roots than did Col -0 WT when grown i n no sucrose medium (Figure 3.9B and 3.9C). This difference in root length was not observed in seedlings grown in regular medium (Figure 3.9D). I conclude that CrLIP4 was unable to rescue the sdp1 mutant phenotype. !!%.-!Recombinant CrLIP4 and SDP1 protein prod uction in Escherichia coli . Despite the unsuccessful attempts to complement phenotypes of yeast and Arabidopsis TAG lipase mutants, the amiRNA experiment pointed to the role of CrLIP4 as a TAG lipase. To test for lipase activity in vitro , a recombinant His-tagged CrLIP4 was expressed in E. coli strain BL21 -CodonPlus(DE3) -RP (Stratagene), which contains extra tRNAs corresponding to rare codons usually present in GC -rich genomes. To prevent leaky expression before induction, glucose was added to the medium. R ecombinant CrLIP4 was mainly in the insoluble fraction after both lysis buffer extraction and re -extraction with 0.25% Tween 20 (Figure 3.10A, middle and right panels). This co uld be due to either high level of expression or to the insertion of CrLIP4 into membranes. The latter case is possible since CrLIP4 is predicted to contain one transmembrane domain at the N -terminus (Figure 3.3A). A minute amount of recombinant CrLIP4 was detected with anti -His antibody in the soluble fraction (s1 in Figure 3.10A). I n order to avoid protein denaturation, protein purification with an Ni -NTA column was carried out from this soluble fraction. In addition, neutral lipids from E. coli expressing CrLIP4 were separated by thin layer chromatography (TLC). CrLIP4 expression ca used changes in the pattern of neutral lipids and free fatty acids on the TLC plate (Figure 3.10B). SDP1 recombinant protein was also produced to use as a positive control in the lipase assay. Recombinant His -tagged SDP1 was expressed in E. coli strain BL2 1-CodonPlus(DE3) -RIPL (Stratagene), containing extra tRNAs for AT -rich genomes. The SDP1 protein was also mostly insoluble (Figure 3.10C). Note that the protein was degraded over time (Figure 3.10C). Since SDP1 is not predicted to contain a transmembrane d omain (Figure 3.3A), lack of protein solubility could be due to its high level of expression. Despite the very low amounts of recombinant protein in the soluble fraction, SDP1 was detected and purified (Figure 3.10C). Recombinant CrLIP4 showed TAG lipase a ctivity in vitro . Different TAG substrates (Chlamydomonas TAG or triolein) were used to assay for lipase activity of various amounts of recombinant CrLIP4, with recombinant SDP1 as a positive control. The reactions were incubated at room temperature for 6 h. Total lipids were extracted and separated on a TLC plate. In the presence of recombinant SDP1 or CrLIP4, DAG and free fatty acid (FFA) were present at higher levels compared to the negative control (no protein) for both types of substrate (Figure 3.11A) . DAG and FFA bands were stronger for 2 µg of protein than 1 µg of protein and stronger TAG lipase activity was observed when triolein was used as a substrate (Figure 3.11A). Therefore, !!%%.!further characterizations were conducted with 2 µg of triolein as a su bstrate and 2 µg of recombinant protein. Longer incubation times yielded more FFA (Figure 3.11B). The pH optimum was tested from 5.0 to 11.0. CrLIP4 showed TAG lipase activity from pH 5.0 to 9.0 (Figure 3.11C), with the highest activity detected at pH 8.0. DISCUSSION Metabolism of neutral lipid s, e.g. triacylglycerol (TAG), is not only essential to cellular homeostasis, but it is also a key part of survival for an organism undergoing stress . TAG mobilization serves as the initial step of energy utilization for growth and development. TAG lipases are responsible for the first step of TAG catabolism. This study has been focused on characterization of a putative TAG lipase in Chlamydomonas. Despite the fact that Chlamydomonas has been studied intensely, TAG li pase has not been identified in Chlamydomonas (Li -Beisson et al. , 2015). A transcriptomic study of Chlamydomonas growing under N -deprived compared to N -replete conditions revealed 9 differentially -expressed putative lipases named CrLIP1 through CrLIP9 (Mil ler et al. , 2010) . Eight of these were cloned and tested for TAG lipase activity in a yeast tgl3 !tgl4 ! double mutant (Li et al., 2012) . One of the putative lipases CrLIP1 not only rescued the yeast mutant but showed lipase activity toward diacylglycerol. Only one of the eight putative lipases, CrLIP4 , was not amenable to clone at the time. Since CrLIP4 is a homologue of the Arabidopsis major seed TAG lipase, SDP1, it deserved further attention, and the gene was ultimately cloned and characterized in this study. As mentioned earlier, two transcriptomic experiments showed that CrLIP4 transcript is reduced during N -deprivation (Miller et al. , 2010; Tsai et al. , 2014) . This observation was confirmed by gene -specific amplification through quantitative reverse transcription PCR (Figure 3.1). In addition, gene -expression is reduced when the cell enters s tationa ry and later declining phase and during the period of TAG accumulation (Lv et al. , 2013). Furthermore, dark anoxia can induce transcription of CrLIP4 within 30 minutes of treatment (Hemschemeier et al. , 2013). Since the transcript of CrLIP4 is less abundant during N -deprivation, it was expected that when N was resupplied, the transcript of CrLIP4 would increase to the same level as during the N-replete condition. However, while TAG levels recovered to original amounts, the transcript level of CrLIP4 did not fully recover (Figure 3.1). Conceivably, transcription of CrLIP4 requires more time than allowed in this experiment. In addition to transcriptional regulation, post -!!%%%!translational regulation could also play a role in CrLIP4 activity. By analogy, the Arabidopsis homologue SDP1 has transcript levels that do not correlate with enzyme activity during seed maturation (Eastmond, 2006) . The author suggested that SDP1 is regulated posttranscriptionally. Finally, it is possible that one or more additional TAG lipases could be (fully or partially) responsible for TAG degradation during N -resupply. In order to investigate the contribution of CrLIP4 to TAG metabolism, a combination of Western blot analysis of CrLIP4 and lipase activity assays was used. Knocking d own CrLIP4 transcript through artificial miRNA resulted in slower degradation of TAG during N -resupply following N -starvation (Figure 3.5). Therefore, I concluded that CrLIP4 likely acts as a TAG lipase during N -resupply. CrLIP4 contains patatin and DUF333 6 domains, which typify TAG lipases in other biological systems, including Arabidopsis SDP1 and yeast Tgl3p, Tgl4p and Tgl5p (Figure 3.3A and Table 3.1). The patatin domain is well established for its esterase/hydrolase activity (Scherer et al. , 2010) , whi le the DUF3336 domain is of unknown function. CrLIP4 shares 41% amino acid identity with Arabidopsis SDP1. Based on TMHMM, CrLIP4 contains one hydrophobic region, which could be a transmembrane domain (Figure 3.3A). This hydrophobic region is located at th e beginning of the patatin domain before the G XSXG motif. The presence of a hydrophobic region and its position and "-helices are conserved in CrLIP4 homologues including SDP1, Tgl3p, Tgl4p, Tgl5p, and ATGL (Figure 3.3A). As summarized by Thiam et al. (2013), three types of proteins can be targeted to lipid droplets; amphiphathic helix -containing proteins, unfolded helix -containing proteins, and hairpin -containing proteins. Since the Arabidopsis, yeast, and human lipases associate with lipid droplets (Athen staedt & Daum, 2003, 2005; Eastmond, 2006; Gronke et al. , 2005; Zimmermann et al. , 2004) , their hydrophobic regions and "-helices are likely to be responsible for their targeting to lipid droplets. However, the mechanisms for protein targeting to lipid droplets are largely unknown and more conclusive evidence is needed to test this hypothesis . Since the catalytic site of TAG lipase is in the soluble portion of the protein, it is possible that the enzyme undergoes conformational changes upon interaction with the hydrophobic region in lipid droplets, which would allow the hydrophobic substrate ( TAG) to enter the active site of the enzyme. A lipase assay showed that recombinant CrLIP4 exhibited TAG lipase activity toward triolein and Chlamydomonas TAG substrates (Figure 3.11). This in vitro activity supports the !!%%&!conclusions of in vivo activity in the artificial miRNA experiment (Figure 3.5B). For future experiments, the kinetics of CrLIP4 activity should be tested. Despite these similarities to the Arabidopsis and yeast proteins, introduction of CrLIP4 into the Arabidopsis sdp1 mutant and the yea st tgl3 ! , tgl4 ! , tgl3 !tgl4 ! mutants failed to rescue the phenotypes. These unsuccessful attempts to complement the phenotypes could be due to the fact that these proteins are too evolutionarily divergent. The failure to obtain heterologous complementation using CrLIP4 is similar to our results from attempts to introduce CrTGD2 into different organisms as shown in Chapter 2. In the cases of both CrLIP4 and CrTGD2, amino acid similarity between the Arabidopsis and the Chlamydomonas proteins are about 40%. The diverg ence of intrinsic disorder regions (IDRs) in CrLIP4 (Figure 3.2 and 3.3A) could also contribute to the failure of the attempts at heterologous complementation. Although IDRs were also predicted in SDP1, Tgl4p and Tgl5p, sequence alignments showed lack of s imilarity in these regions (Figure 3.3B). IDRs can function in protein -protein interaction or serve as a regulatory or signaling region (Dunker et al. , 2002; Uversky, 2013a, 2013b) . This is also indicated by the presence of many predicted phosphorylation s ites (Figure 3.2F). Therefore, function of CrLIP4 could be dependent on a protein partner, which may not be present in divergent organisms. Another possible explanation is the high GC content of the Chlamydomonas gene, which can cause poor protein expressi on especially in the case of the Arabidopsis sdp1 mutant, since the Arabidopsis genome is AT rich. Although I could monitor transcription of the recombinant gene, in the absence of an epitope tag or antiserum against CrLIP4, I could not determine if the pr otein was expressed. MATERIALS AND METHOD S Algal strain and growth condition . Chlamydomonas cell wall -less strain dw15.1 (cw15, nit1, mt+) was used in this study. Growth conditions were the same as previously described in Chapter 2. Artificial microRNA (a miRNA) knockdown . Target sequences of amiRNA were determined through the Target function of WMD3 - Web MicroRNA Designer (http://wmd3.weigelworld.org/cgi -bin/webapp.cgi?page= Home ;project=stdwmd ). Primers for amiRNA of CrLIP4 were designed at either the 5Õ o r 3Õ ends of the transcript through the Designer page of WMD3. Sequences of amiRNA and primers can be found in Table 3.2. !!%%'!Oligonucleotides were then cloned into the pChlamiRNA3int vector according to (Molnar et al., 2009). The resulting amiRNA constructs w ere linearized with KpnI restriction enzyme. The digested products were separated via agarose gel electrophoresis. The bands were cut and purified. The linearized plasmids were then used to transform a Chlamydomonas culture by the glass bead method based o n (Kindle, 1990) with m inor modifications; the cells were grown in Tris -acetate-phosphate (TAP) liquid medium, and the use of polyethyleneglycol (PEG) was omitted. The transformants were selected on TAP medium with 10 µg/m L Paromomycin. Selected colonies were grown on TAP liquid medium and used to test transcript level by real -time PCR and TAG level by thin layer chromatography (TLC) and gas chromatography (GC) as described below. RNA isolation and cDNA synthesis . RNA was prepared from 5 -10 ml mid -log phase culture of Chlamydomon as with RNeasy Plant Mini Kit (QIAGEN) following the manualÕs instruction. The isolated RNA was treated with Rnase -Free DNase (QIAGEN) to eliminate genomic DNA contamination. Two µg of RNA were then used for cDNA synthesis with either RETROscript ¨ Reverse Transcription Kit (Ambion) or Quantiscript Reverse Transcription (QIAGEN). Quantitative reverse transcription PCR (qRT -PCR). The transcript level of CrLIP4 was monitored with qRT -PCR on either the Applied Biosystems 7500 Fast real -time PCR system or Eppend orf realplex 2. Primers Lip4 -jw2 F and Lip4 -jw2 R were used to amplify the CrLIP4 transcript. Sequences of these primers are given in Table 3.3. The reference gene used for Chlamydomonas samples was Receptor of activated protein kinase C (RACK1 ), while Isopentenyl-diphosphate Delta-isomerase II (IPP2) was used as a reference gene for Arabidopsis samples. Sequences of primers used in these experiments are shown in the Table 3.2. The qRT -PCR data were calculated based on the 2 -!!CT method as previously describ ed (Livak & Schmittgen, 2001) . Bioinformatic analysis . A homology search of CrLIP4 was carried out by BLASTP at the web site of the National Center for Biotechnology Information (NCBI) (Altschul et al., 1997) . Amino acid sequences that had more than 60% co verage and had an e -value lower than e -10 were selected for further analysis. Protein domains were searched against amino acid sequences from the blast search result with hmmscan 3.1b1 (Eddy, 2009) . Disorder prediction was carried out with 4 different prog rams; PONDR -VSL2, PONDR -VLXT, PONDR -VL3 !!%%(!(http://www.pondr.com/cgi -bin/PONDR/pondr.cgi ), and IUPred ( http://iupred.enzim.hu/ ) (Dosztanyi et al. , 2005) . Phosphorylation sites associated with intrinsically disordered regions were predicted by DEPP (http://www .pondr.com/cgi -bin/PONDR/depp.cgi). Transmembrane helices were predicted with TMHMM ( http://www.cbs.dtu.dk/services/TMHMM/ ). Protein secondary structure was analyzed with PredictProtein ( https://www.predictprotein.org/home ) (Yachdav et al. , 2014). Phyloge netic reconstructions Amino acid sequence alignment of CrLIP4 homologues in the region encompassing the patatin domains through DUF3336 was performed with MUSCLE3.8 as described in Chapter 2. The phylogenetic reconstruction was carried out through MEGA6 as described in Chapter 2. Lipid analysis . Lipid analysis of TAG for Chlamydomonas was carried out as described in Chapter 2. For the lipase assays, the solvent for neutral lipid separation consisted of chloroform, acetone and acetic acid (96: 4: 1 v/v/v). L ipid extraction from yeast was carried out in a similar manner to lipid extraction from Chlamydomonas except glass beads (425 -600 µm, Sigma -Aldrich) were added to the cells resuspended in extraction solvent. Cloning of the CrLIP4 coding sequence . Because CrLIP4 has a region with high GC content (about 70%), cloning the entire cDNA in one experiment was not successful. Therefore, the cDNA was cloned as five overlapping fragments. PCR reactions were carried out with either GoTaq ¨ DNA polymerase (Promega) or P husion ¨ High -Fidelity DNA Polymerase (New England Biolab). In addition to dNTP and MgCl 2, 0 -8% dimethyl sulfoxide (DMSO) was added to the PCR reactions. Sequences of primers used for amplifying each fragment, joining fragments and sequencing are provided i n Table 3.3. Each fragment, including joined fragments, was ligated with pGEM ¨-T Easy Vector (Promega) and transformed into E. coli strain DH5 " . Transformed bacteria were selected on Luria Bertani (LB) medium with 100 µg/ml ampicillin, 5 mmol of Isopropyl #-D-1-thiogalactopyranoside (IPTG) and 1 mg of 5 -bromo -4-chloro -3-indolyl -#-D-galactopyranoside (X -Gal). Plasmids were prepared from E. coli transformants and sent for sequencing. Clones containing the correct DNA sequence were used for fragment joining. Fragments of CrLIP4 were cut out from the pGEM -T Easy plasmid with EcoRI. Fragment joining was performed sequentially two fragments at a time with PCR. Fragments 1 and 2, and !!%%)!fragments 3 and 4 were joined with primers F -1 and R2, and primers F3 and F4.3, respectively. The resulting fragment, 3 -4, was joined to fragment 5 with primers F3 and R6. The 2 large fragments, 1 -2 and 3 -4-5, were joined using primers F -1 and R6. The full CrLIP4 fragment was cloned into the pGEM ¨-T Easy vector for sequence determinat ion. The clone containing the correct sequence was then used as a template for PCR amplification for pENTR TM/D-TOPO (Invitrogen TM) cloning with Lip4 -F1 kozak and Lip4 -R6 primers. Expression of CrLIP4 in yeast tgl3 !tgl4 ! double , tgl3 !, and tgl4 ! single mut ant s. CrLIP4 from pENTR/D -CrLIP4 was amplified with Phusion polymerase using 595Lip4 F and R primers. 10% DMSO was added to the reaction. The sequence was determined with MHK98 and MHK99 primers. Sequences of cloning and sequencing primers are provided in Table 3.3. A restriction digest of the pMK595 vector (Luo et al. , 2010) was performed with NotI. Linearized pMK595 and CrLIP4 DNA from PCR amplification were co -transformed into the yeast WT strain yMK839 following the method from (Gietz et al., 1992) . The linearized vector and CrLIP4 were then joined by endogenous homologous recombination of the yeast cell (Ma et al. , 1987) . The yeast transformants were selected on synthetic complete medium without uracil (SC -U). A few single colonies were picked and grown on liquid SC -U medium for testing protein expression. Colonies showing expression of the CrLIP4 cDNA were used for plasmid isolation. Plasmids from the WT yeast were then transformed into E. coli DH5 " for sequencing the plasmid pMK595 -CrLIP4 . The pMK595 plasmid and the pMK595 -CrLIP4 plasmid of correct DNA sequence were then used to transform the yeast tgl3 ! tgl4 ! double mutant , strain yXL005 (Li et al. , 2012) and the yeast tgl3 ! and tgl4 ! single mu tants. The transformants were tested for CrLIP4 expression by western blot analysis against HA -tag. Western blot analysis . Total protein extracts for yeast were prepared from either freshly harvested or frozen yeast cell pellets as previously described in Chapter 2 with some modifications. The cell pellets were resuspended in 2X sample buffer, and acid washed glass beads were added. The mixture was vortexed vigorously, followed the same remaining steps as described in Chapter 2. Total protein preparation fr om E. coli was carried out in a similar manner without addition of glass beads. Protein quantification, gel electrophoresis, blotting and immuno detection were performed similarly to that described in Chapter 2. Primary antibodies for yeast and E. coli samples were anti -HA and anti -His mouse antibodies, respectively. The secondary antibody was anti -mouse, conjugated with horseradish peroxidase. !!%%*!Analysis of CrLIP4 overexpression in yeast tgl3 !tgl4 ! double , and tgl3 ! and tgl4 ! single mutant s. Single colonies of yeast WT or double mutant containing pMK595 empty vector or pMK595 -CrLIP4 were inoculated in SC -U liquid medium. The cultures were grown at 30¡C for 20 h (stationary phase). The cult ures were then diluted with fresh SC -U liquid medium at OD 600 = 3 for a total volume of 25 ml. Either 25 µl of ethanol or 10 mg/ml cerulenin was added to the culture. Cells were harvested at 0, 1, 3, 5 and 7 h after addition of cerulenin for TAG separation , and direct FAME and cell concentration monitoring with Z2 Coulter Counter (Beckman Coulter) were performed. In the case of tgl3! and tgl4! single mutants, 4 -day-old cells were harvested for lipid analysis after growth in casamino acid medium lacking urac il (CAA -U). Plant materials and growth conditions . Arabidopsis sdp1 -4 (SALK_102887) and sdp1 -5 (SALK_076697) mutants were obtained from the Salk Institute for Genomic Analysis Laboratory. Arabidopsis Col -0 was used as a wild -type control in all Arabidopsis experiments throughout this study. The plants were germinated on Murashige and Skoog (MS) solid medium either with or without 1% (w/v) sucrose. The seeds were stratified for at least 48 h prior to incubation in a growth chamber to induce germination. One - to two -week old seedlings were transferred to soil. The plants were maintained in a 16/8 h light/dark cycle with temperatures of 22/20¡C and light intensity at 120/0 µmol.m -2.sec -1. Genomic DNA isolation from Arabidopsis leaves . A young leaf of Arabidopsi s was used to isolate genomic DNA. The leaf was ground in 200 µl of extraction buffer (0.2 M Tris -HCl, pH 7.5, 0.25 M NaCl, 25 mM EDTA and 0.5% SDS) followed by addition of 400 µl of absolute ethanol. The mixture was then shaken gently and centrifuged at 1 3,000 X g for 5 min. The supernatant was discarded. The pellet was washed with 200 µl of 70% ethanol, centrifuged at 13,000 X g for 1 min and air dried. The dried pellet was resuspended in TE buffer (10 mM Tris -HCl, pH7.5 and 1 mM EDTA) and stored at 4¡C u ntil further use. Genotyping of sdp1 T-DNA insertion line s. Determination of homozygosity for the sdp1 -4 and sdp1 -5 mutants was carried out based on http://signal.salk.edu/tdnaprimers.2.html . Primers used in this analysis are listed in Table 3.3. PCR ampli fication of the WT gene was carried out with primers LP -sdp1 -4 or LP -sdp1 -5 and RP -sdp1 -4 or RB -sdp1 -5. The T -DNA inserted gene was amplified with primers LBb1.3 and RP -sdp1 -4 or RP -sdp1 -5. Two separate PCR reactions were carried out for each plant tested; one for amplifying the WT locus and another for amplifying the !!%%+!T-DNA insertion. The WT plants show one band for LP and RP primers. Heterozygous plants show a band for LBb1.3 and RP primers in addition to another band for LP and RP primers. Homozygous plan ts show only one band for primers LBb1.3 and RP. Construction of pMDC32 -CrLIP4 plasmid . CrLIP4 was cloned into pMDC32 vector through a clonase reaction from pENTR/D -CrLIP4 following instructions from Invitrogen. Preparation of Agrobacterium competent cell s. A single colony of Agrobacterium tumefacien s strain GV3101 was grown in 5 ml of LB medium with 25 µg/ml gentamycin and 34 µg/ml rifampicin overnight at 28¡C. This culture was then inoculated into a larger volume of fresh medium and grown until OD 600 rea ched 0.3 -0.6. One ml of this culture was then aliquoted into a 1.5 ml centrifuge tube and chilled on ice for 2 -3 min before centrifugation at 13,000 X g for 3 min at 4¡C. The supernatant was removed, and the pellet was resuspended into 1 ml cold 10 mM Tris -HCl, pH 7.5. The cells were then centrifuged at 13,000 X g for 3 min at 4¡C to remove the sup ernatant. The pellet was then resuspended in 100 µL of cold LB medium. The cells were flash frozen with liquid N 2 and stored at -80¡C. Agrobacterium transformation . Agrobacterium competent cells were thawed on ice. One to two µg of pMDC32 -CrLIP4 plasmid DN A was added to the cells. The mixture was frozen in liquid N 2 for 5 min and quickly transferred to a 37¡C water bath for 5 min. One ml of LB medium was added to the cell mixture which was then incubated at 28¡C with shaking for 2.5 h. After incubation, the cells were centrifuged at 5,000 X g for 5 min to remove the supernatant. The cell pellet was then resuspended in 100 µl LB medium and spread onto LB medium containing Bacto agar. The cells were incubated at 28¡C for 2 days until colonies formed. Arabidopsis transformation . Transformation o f Agrobacterium containing pMDC32 -CrLIP4 plasmid into Arabidopsis was carried out with the floral dip method as described previously (Clough & Bent, 1998) . Seeds obtained from transformed plants were screened for transformants with Hygromycin B resistance as described previously (Harrison et al. , 2006) . The number of insertions and homozygosity were tested in the second and third generations by determining the ratio of seedlings resistant and susceptible to Hygromycin B. Transgenic plants were grown and self-crossed until homozygosity was obtained in the third generation. Transgenic plants with a single insertion were selected for further analysis. !!%%,!Construction of pET28 -AtSDP1 and pET28 -CrLIP4 plasmids. AtSDP1 coding sequence was cloned into pENTR/D -TOPO wi th SDP1 -pENTR F and SDP1 -pENTR R primers using Q5 ¨ High -Fidelity DNA Polymerase (New England Biolabs). Sequences of primers are provided in Table 3.3. The AtSDP1 -pENTR/D -TOPO plasmid was transformed into E. coli strain DH5 " . Single colonies were picked and grown in liquid LB medium for plasmid preparation. Sequences of the plasmids were determined with primers listed in Table 3.3. A clone containing the correct sequence was then used as a template for pET28 -AtSDP1 construction with primers SDP1 -pET28_NheI F and SDP1 -pET28_NotI R. pENTR/D -CrLIP4 was used as a template to construct pET28 -CrLIP4 with Lip4 -pET28 -F4 and Lip4 -pET28 -R2 primers. AtSDP1 and CrLIP4 were cloned into pET28b+ (Novagen), an E. coli expression ve ctor, with a restriction digestion and ligation method. For AtSDP1 , the pET28b+ vector and the PCR product of SDP1 were digested with NheI and NotI. In the case of CrLIP4 , the PCR product and pET28b+ vector were digested with HindIII and Xho I. After digest ion, ligations of the two sets of genes and vectors were carried out. The ligation products were transformed into E. coli DH5 " for recovery of plasmids. Recombinant AtSDP1 and CrLIP4 protein production. Since AtSDP1 could not be produced in the BL21(DE3) s train of E. coli, it was produced in the E. coli strain BL21 -CodonPlus(DE3)-RIPL (Stratagene), which can express proteins from AT -rich genomes. After transformation, a single colony was inoculated into 100 mL LB medium with 50 µg/mL kanamycin at 37¡C for 8 h. Eight mL of the seed culture was then inoculated into 50 mL fresh medium and incubated for another 75 min. Protein expression was induced by adding 1 mM IPTG into the culture. The cells were harvested for protein purification after 30 min of induction at 37¡C. CrLIP4 was also unable to be produced in the E. coli strain BL21(DE3). As an alternative, E. coli strain BL21 -CodonPlus(DE3) -RP (Stratagene), which is able to express proteins from GC -rich genomes, was used. The culture of E. coli containing pET28 -CrLIP4 was grown in LB medium containing 50 µg/mL kanamycin and 1% glucose at 37¡C for 8 h. The seed culture was used to inoculate into fresh medium with 1% glucose and was grown overnight. Expression of the cloned cDNA was induced in fresh medium without glucose with addition of 0.1 mM IPTG. The cells were incubated at 37¡C for 6 h and harvested by centrifugation for protein purification. Recombinant protein purification . The cell pellet was resuspended in an equal volume of lysis buffer (50 mM NaH 2PO4, 3 00 mM NaCl and 10 mM imidazole) to the original culture volume. Cells were lysed by addition of 1 mg/ml lysozyme and shaken for 30 min on ice. The cells were !!%%-!then sonicated on ice at power level 4.0 for 10 sec a total of 6 times (MISONIX Sonicator 3000). The soluble and non -soluble fractions were separated by centrifugation at 10,000 X g for 30 min at 4¡C. The supernatant was then collected and mixed with 1 volume of Ni -NTA slurry to 4 volumes of supernatant. The mixture was incubated with shaking at 4¡C fo r 30 min and loaded into a column. The flow -through was allowed to drain. The column was washed with 4 volumes of wash buffer (50 mM NaH 2PO4, 300 mM NaCl and 20 mM imidazole) to the initial lysate -Ni slurry volume. Finally, the protein was eluted from the column with elution buffer (50 mM NaH2PO4, 300 mM NaCl and 250 mM imidazole) 6 times. A small volume from each step was collected for further analysis by western blot. Lipase assay . TAG substrates were prepared from either Chlamydomonas total lipids or com mercial triolein. Chlamydomonas TAG was separated from total lipids as described in Chapter 2. Triolein was separated on a TLC plate to remove free fatty acids and other products of TAG degradation. The silica powder containing the TAG band from triolein s eparation was scraped and used for lipid extraction. Both Chlamydomonas TAG and triolein were dried and resuspended in chloroform. The concentration of TAG was determined by GC. Dry TAG was resuspended at 100 mM with 5% (w/v) gum arabic in water. The mixtu re was sonicated at 20 W for 30 sec. A lipase assay was carried out according to (Eastmond, 2006) . TAG substrate in gum arabic was mixed with assay buffer (50 mM Bis -Tris propane -HCl pH 8.0) at 1 -2 µM final concentration. In addition, 2 mM dithiothreitol (DTT) and 2 mM CaCl 2 were added to the reaction. The mixture was incubated at room temperature wit h shaking for 6 h. The reaction was stopped by adding lipid extraction solvent. The lipid was separated on a TLC plate with a neutral lipid solvent system as described above. The resulting TLC plate was stained with iodine vapor. ACCESSION NUMBERS CrLIP4 a ccession number, 319691, was obtained from Joint Genome Institute (JGI) v4. Arabidopsis SDP1 accession number, AT5G04040.1, was obtained from The Arabidopsis Information Resource (TAIR, www.arabidopsis.org). Accession numbers of amino acid sequences used f or the phylogenetic tree reconstruction in Figure 3.4 were given in the parenthesis following the scientific name . !!%&.! !!!!Figure 3.1. Relative expression of CrLIP4 transcript compared to TAG concentration during N deprivation and N resupply. Relative expression of CrLIP4 transcript compared to the Receptor of ac tivated protein kinase C (RACK1 ) (left y -axis), and relative TAG level (right y -axis) compared to total lipid fatty acids of Chlamydomonas PL. Arrows indicate the times when N was deprived (-N) and resupplied ( NR). Three biological replicates were averaged and standard deviations are shown. !!!%&%!!Figure 3.2. Amino acid sequence analysis of CrLIP4. (A) Protein domains present in CrLIP4. Disorder prediction by (B) PONDR -VSL2, ( C) PONDR -VLXT, ( D) PONDR -VL3, and ( E) IUPred . (F) Phosphory lation prediction sites predicted by DEPP. The broken line at value 0.5 indicates the threshold of disorder or putative phosphorylation sites. !!!%&&! Figure 3.3. Prediction of transmembrane helices (TMD) and alignment of intrinsic disorder regions (IDRs) of CrLIP4 homologues. !!%&'!Figure 3.3. (contÕd ) (A) Prediction of TMD of amino acid sequences through TMHMM Topology, secondary structures (helix and #-strand), domains and conserved motives of each protein are shown above the plots. ( B) Amino acid sequence alignment of IDRs as shown in ( A) from CrLIP4, Arabidopsis SDP1, yeast Tgl4p and Tgl5p and human? ATGL. The alignment was carried out through MUSCLE3.8. !!%&(! Figure 3 .4. Phylogenetic analysis of CrLIP4 homologues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igure 3 .4. (contÕd ) The evolutionary history was inferred based on Maximum Likelihood . The t ree with the highest log likelihood ( -12809.1964) is shown. The percentage of trees in which the associated taxa clustered together (1000 repeats) is shown above the branches. The tree is drawn to scale with branch lengths corresponding to the number of su bstitutions. The accession number for each protein is given in the parenthesis following the scientific name. Accession numbers for Chlamydomonas, Arabidopsis and other organisms are from the Joint Genome Institute (JGI), the Arabidopsis Information Resour ce (TAIR), and National Center for Biotechnology Information (NCBI), respectively. Groups of organisms are indicated by colored bars. !!%&*! !Figure 3.5. Down regulation of CrLIP4 transcript with artificial microRNA (amiRNA) . (A) Relative expression of CrLIP4 transcript compared to the RACK1 of the PL and amiRNA knockdown lines. The amiRNA knockdown lines targeting the 5Õ or 3Õ regions were named with N or C followed by number, respectively. ( B) TAG concentration (TAG/total fatty acids) of selected amiRNA knockdown lines from ( A) at various time points after N -resupply following N-deprivation. Three technical ( A) or biological ( B) replicates were averaged and standard deviations are shown. Differences in means of PL and amiRNA lines in ( B) were compared wi th a paired -sample student t-test (* p -value ' 0.05). !!%&+!! Figure 3.6. Sequence alignment of the CrLIP4 . Sequence alignment of the CrLIP4 coding sequence cloned from C hlamydomonas strain dw15.1 and the CrLIP4 gene model (accession number 319691 from JGI v4). Boxes show DUF3336 and patatin domains and the intrinsically disordered region (IDR). A conserved lipase domain (G XSXG) is underlined. !!!%&,! !Figure 3.7. Heterologous expression of CrLIP4 in the yeast tgl3 ! tgl4 ! double mutant . (A) Western blot analysis of pMK595 or pMK595 -CrLIP4 introduced into WT or the tgl3 ! tgl4 ! yeast double mutant. An arrow indicates the size of the expected CrLIP4 protein. ( B) Growth (million cells /ml) and TAG concentration (fmol/cell) of yeast WT, tgl3 ! tgl4 ! double mutant, and the double mutant expressing CrLIP4 (CrLIP4 tgl3 ! tgl4 !). Cerulenin (10 mg/ml, left panel) or equal volume of ethanol (mock, right panel) was added to the stationary phase cul tures at time 0. !!%&-! Figure 3.8. Heterologous expression of CrLIP4 in yeast tgl3 ! and tgl4 ! mutants . (A) Western blot analysis against HA tag of pMK595 or pMK595 -CrLIP4 introduced into the yeast tgl3 ! and tgl4 ! mutants. Two biological replicates were shown. Numbers on the left indicate molecular weight distribution in kDa. An arrow indicates expected size of CrLIP4 at 115 kDa. ( B) TAG concentration of tgl3 ! and tgl4 ! with either pMK595 or pMK595 -CrLIP4 introduced into their genomes. Two biological replicates were averaged and standard deviations are shown. !!%'.!! Figure 3.9. Heterologous expression of CrLIP4 in Arabidopsis sdp1 mutants. !!%'%!Figure 3.9. (contÕd ) Seeds were germinated in the absence (A, B, C) or presence ( D) of 1% sucrose. (A) Relative expression of CrLIP4 transcript compared to Isopentenyl -diphosphate Delta -isomerase II (IPP2 ) in the T1 generation by RT -PCR . Three technical replicates were averaged and standard deviations are shown. ( B) Root length (mm) o f seedlings of WT (Col -0), the sdp 1-4 and sdp1 -5 mutants, and the T2 or T3 generations of CrLIP4 expressed in either the sdp1 -4 or sdp1 -5 background ( CrLIP4 sdp1 -4 or CrLIP4 sdp1 -5). In all cases, one hundred biological replicates were averaged and standar d deviations are shown. (C) Photographs of seedlings of lines in ( B) grown in ) MS medium without sucrose. ( D) Photographs of seedlings of lines in (B) grown in ) MS medium with 1% sucrose. !!%'&!!Figure 3.10. CrLIP4 recombinant protein expression in E. coli . !!%''! Figure 3.10. (contÕd ) (A) Western blot analysis of CrLIP4 recombinant protein expression compared to an empty vector ( EV, pET28b+) with addition of IPTG and/or 1% glucose at different time points (left and middle panels), and recombinant protein pu rification (middle and right panels) showing supernatant (s) and pellet (p) at different steps. White lines separate different membranes or indicate removal of irrelevant lanes. ( B) Separation of neutral lipids on TLC plates isolated from E. coli cells exp ressing CrLIP4 or EV. The solvent system wa s petroleum ether, diethyl ether, acetic acid (80:20:1 v/v ). The lipid bands were visualized by staining with iodine vapor . Arrows indicate changes in abundance of lipids or free fatty acids due to the presence of the recombinant CrLIP4 at various concentration s of IPTG, with or without 1% glucose inhibition at different time points . The white line indicates removal of irrelevant lane s. (C) Western blot analysis of AtSDP1 recombinant protein expression compared to an empty vector ( EV, pET28b+) with addition of IPTG at different time points (left and right panels) and recombinant protein purification (right panel) showing supernatant (s) and pellet (p). The white line indicates two different membranes. !!%'(! Figure 3.11. Lipase assay. !!%')! Figure 3.11. (contÕd ) Separation of neutral lipids with chloroform, acetone and acetic acid (96:4:1 v/v/v) on a TLC plate. Lipids were stained with iodine vapor. Olive oil, diacylglycerol (DAG) and monoacylglycerol (MAG) were used as st andards. Each assay had a negative control ( -ve contÕd ) containing no protein. Arrows indicate DAG and free fatty acid (FFA) bands. (A) Chlamydomonas TAG or triolein was used as a substrate for 1 -2 µg of either recombinant SDP1 or CrLIP4. The reactions wer e carried out for six hours. White lines indicate reordering of Ðve contÕd and 1 µg SDP1 lanes from the same plate. ( B) TAG lipase activity of CrLIP4 was tested at different time points. Triolein was used as a substrate. ( C) TAG lipase activity of CrLIP4 w as tested against different pH levels from 5.0 to 11.0. No dithiothreitol (DTT) control (contÕd ) was included. !!%'*!Table 3.1. Protein domains identified in CrLIP4 homologues . Query name Domain Patatin DUF3336 Overall Position Position From To From To From To Acinetobacter sp. (gi514963557) 4 151 157 343 4 343 Alcanivorax sp. (gi551599646) 1 139 145 331 1 331 Arabidopsis thaliana SDP1 (AT5G04040.1) 86 226 232 436 86 436 Arabidopsis thaliana SDP1 -like (AT3G57140.1) 87 227 233 435 87 435 Aspergillu s oryzae (gi391866040) 1 129 138 334 1 334 Auxenochlorella protothecoides (gi760448007) 94 235 241 424 94 424 Brassica napus (gi674952444) 88 234 240 444 88 444 Camelina sativa (gi727560138) 86 226 232 435 86 435 Candida albicans (gi712885502) 59 205 211 416 59 416 Capsella rubella (gi565458145) 86 226 232 435 86 435 Chlamydomonas reinhardtii CrLIP4 (Cre17.g699100.t1.1) 1 145 151 348 1 348 Coccomyxa subellipsoidea (gi545354428) 1 68 74 270 1 270 Congregibacter litoralis (gi495571297) 1 140 146 333 1 333 Debaryomyces hansenii (gi294657254) 62 205 211 416 62 416 Elaeis guineensis (gi743786505) 86 226 232 435 86 435 Eutrema salsugineum (gi567170164) 86 226 232 435 86 435 Fusarium graminearum (gi758210332) 63 207 218 414 63 414 Genlisea aurea (gi527 206944) 89 229 235 437 89 437 Gossypium raimondii (gi823261503) 86 226 232 435 86 435 Helicosporidium sp. (gi633903867) 23 120 126 306 23 306 Homo sapiens (gi58759051) - - 10 179 10 179 Hordeum vulgare (gi326512766) 85 225 231 433 85 433 Lodderomyces elongisporus (gi149246010) 77 220 226 432 77 432 Marinobacter nanhaiticus (gi750601676) 3 141 147 333 3 333 Metarhizium robertsii (gi594722286) 69 211 225 421 69 421 Mortierella verticillata (gi672823045) 194 340 346 540 194 540 Musa acuminata (gi69501 1186) 87 228 234 437 87 437 Nevskia ramosa (gi703385018) 3 140 146 332 3 332 Nevskia soli (gi659872973) 3 140 146 332 3 332 Oryza sativa (gi125588353) 83 223 229 432 83 432 Paraglaciecola arctica (gi494890748) 2 140 146 331 2 331 Prunus mume (gi645271 062) 86 226 232 434 86 434 Pseudogymnoascus pannorum (gi682355077) 62 208 221 418 62 418 Ricinus communis (gi255578433) 86 226 232 435 86 435 Saccharomyces cerevisiae Tgl3p (gi323346999) 56 198 204 364 56 364 Saccharomyces cerevisiae Tgl4p (gi767174926 ) 132 276 282 482 132 482 Saccharomyces cerevisiae Tgl5p (gi768490581) 32 177 183 387 32 387 !!%'+!!Table 3.1. (contÕ d) !Query name Domain Patatin DUF3336 Overall Position Position From To From To From To Scheffersomyces stipitis (gi150864168) 14 155 161 366 14 366 Setaria italica (gi514782261) 85 226 232 434 85 434 Solanum tuberosum SDP1 -like (gi565369529) 90 230 236 440 90 440 Solimonas flava (gi654541222) 3 146 152 338 3 338 Sorghum bicolor (gi242058771) 85 226 232 434 85 434 Spathaspora passal idarum (gi598070323) 45 187 193 398 45 398 Verticillium dahliae (gi697089701) 59 206 218 414 59 414 Volvox carteri (gi302836077) 124 274 280 477 124 477 Zea mays SDP1 -like (gi670433428) 85 226 232 434 85 434 !!%',!Table 3.2. Target and primer sequences for artificial microRNA of CrLIP4 . !Target position on the CrLIP4 transcript Target sequence/Primer name Sequence from 5Õ to 3Õ 5Õ Target sequence TGAAACCCACGTCTAACTCGA Lip4AmiRNA_b_Fwd CTAGTTCGAGTTAGACGTGG GATTCATCTCGCTG ATCGGCACCATGGGGGTGGT GGTGATCAGCGCT ATGAAACCCACGTCTAACTCGA G Lip4AmiRNA_b_Rev CTAGCTCGAGTTAGACGTGG GTTTCATAGCGCTG ATCACCACCACCCCCATGGT GCCGATCAGCGAG ATGAATCCCACGTCTAACTC GAA 3Õ Target sequence TTTCACAATGGTCTTCCTCAA Lip4AmiRNA_e_Fwd CTAGTTTGAGGAAGACCATT GAGAAATCTCGCTG ATCGGCACCATGGGGGTGGT GGTGATCAG CGCT ATTTCACAATGGTCTTCCTC AAG Lip4AmiRNA_e_Rev CTAGCTTGAGGAAGACCATT GTGAAATAGCGCT GATCACCACCACCCCCATGG TGCCGATCAGCGA GATTTCTCAATGGTCTTCCT CAAA !!! !!%'-!Table 3.3. Sequences of Primers used for various purposes as indicated. "#$%&'( !)*+( !,(-#(./(!012!3&!42!56$(/36& .7!KLMN!8J! !"#$%& !Lip4 -jw2 -fwd CTTCTCCAACAGCCGCGC Lip4 -jw2 -rev CGCTCGTACGCCTCCAGG KLMN!8J! '(!)* !BEN3433 GTCATCCACTGCCTGTGCTT BEN3434 CCTTCTTGCTGGTGATGTTG KLMN!8J! $%%+ !QK194 GTATGAGTTGCTTCTCCAGCAAAG QK195 GAGGATGGCTGCAACAAGTGT MI8C#C:!8J ,!"#$%&, fragment 1 Lip4 -F0 TGGAGGAGGCCGATATG Lip4 -R0!GGCTGCCCTTGTTGATG !MI8C#C:!8J ,!"#$%&, fragment 2 Lip4 -F2 GGTTTCTTCTCCAACAGC Lip4 -R2!CACCAGGAAGTGGTTGC !MI8C#C:!8J ,!"#$%&, fragment 3 Lip4 -F3 CCACCAATGACTCGCTG Lip4 -R3!AATGGTCTTGCTCAGGTG !MI8C#C:!8J ,!"#$%&, fragment 4 Lip4 -F4.3 GTGGCTCATGCTGTTCAC Lip4 -R4.3 !TCTGAAGCGCTGTCCTC !MI8C#C:!8J ,!"#$%&, fragment 5 Lip4 -F5 TGAGGACAGCGCTTCAGAC Lip4 -R5!CCTCCTCAGTACGCAAACAC !Joining !"#$%&, fragment 1 and 2 and 3 -4-5 Lip4_F -1 ATGGACGCCCGCCAC Joining !"#$%&, fragment 5 w ith 3 -4 and 1 -2 Lip4 -R6 GTACGCAAACACGTCCAGAGCA CrLIP4 sequencing Lip4 -1424 CCAACATCGCCAACCAGGTC Lip4 -1817 TGGCCCTTCTGAACCTG Lip4 -1924 ATGCCGGCCTCTTACAG pENTR TM/D-TOPO cloning Lip4 -F kozak GAAATGGACGCCCGC pMK595 cloning 595Lip4 F TATCCATATGACGTTCCAGA TTACGCTGC TCAGTGCGGCCGCATGGACGCCCGCCACA GA 595Lip4 R !GAATTTCGACGGTATCGGGGGGATCCACT AGTTCTAGCTAGATCAGTACGCAAACACG TC !!%(.!Table 3.3. (contÕ d) "#$%&'( !)*+( !,(-#(./(!012!3&!42!56$(/36&.7 !pMK595 sequencing !MHK98 CAAGTATAAATAGACCTG !MHK99 CAAGTATAAATAGACCTG OEC82P 3#C:!8J!Q