L3! {3: it w. .3! .r x l a: . {to ‘93.. .522.» I #33537 . .35.. I; s. sham. nah. I... 563.0 an. a». .1... an "HESS /”\... '\ I; . ~, N, - 5 [09 095/ :1 ,7 i This is to certify that the dissertation entitled CHLOROPLAST ANIONIC LIPID BIOSYNTHESIS AND PhD FUNCTION presented by Bin Yu has been accepted towards fulfillment of the requirements for the degree in Genetics Program W Major Professor’s Signature‘J 2/61/13 Ll Date MSU is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE a 2012 6/01 c:/ClFiC/DateDuap65-p.15 CHLOROPLAST ANIONIC LIPID BIOSYNTHESIS AND FUNCTION Bin Yu A DISSERTATION Submitted to Michigan Sate University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program 2004 ABSTRACT CHLOROPLAST ANIONIC LIPID BIOSYNTHESIS AND FUNCTION By Bin Yu The thylakoid membranes of photosynthetic organisms contain four major glycerolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG), phosphatidylglycerol (PG), and the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). These lipids are critical for maintaining chloroplast structure and function. This dissertation focuses on dissecting the biosynthesis and function of the two anionic lipids. SQDG is anionic at physiological pH because of its 6-deoxy-6-sulfonate-glucose (sulfoquinovose) head group. Using a reverse genetic approach, I identified the sulfoquinovosyltransferase, designated as SQD2, which catalyzes the transfer of the sulfoquinovose moiety from UDP-sulfoquinovose to diacylglycerol. Insertion of a transfer DNA into this gene in Arabidopsis led to complete lack of sulfolipid in the respective .9qu mutant. This mutant showed reduced growth under phosphate-limited growth conditions. These results support the hypothesis that sulfolipid can function as a substitute for anionic phospholipids in order to maintain constant amounts of anionic lipids under phosphate-limited growth conditions. PG is the only major phospholipid in the thylakoid membranes. Using a forward genetic approach, we isolated a PG-deficient mutant of Arabidopsis, pgp] -1 , in which the overall content of PG was reduced by 30%. The pgp] -1 mutant is a pale green plant with impaired photosynthetic activity. Further analysis suggested that this mutant carries a point mutation in the CDP-alcohol phosphotransferase motif of the phosphatidylglycerol- phosphate synthase (EC 2.7.8.5) isoforrn. The mutant showed an 80% reduction in plastidic phosphatidylglycerolphosphate synthase activity consistent with the plastidic location of this particular isoforrn. To test the SQDG/PG substitution hypothesis, a double mutant, sqd2 pgpi-I was constructed. The double mutant exhibited an approximately one-third reduction in total anionic lipids, which resulted in pale yellow cotyledons and leaves with reduced chlorophyll content. The double mutant showed compromised photoautotrophic growth and impaired photosynthetic activity. In particular, the photosynthetic electron transfer at the level of photosystem II (PSII) was affected. Furthermore, it was observed that the double mutant had a reduced number of mesophyll cells and chloroplasts with altered ultrastructure. Observations of the sqd2pgp1—1 mutant led to the conclusion that the total content of anionic thylakoid lipids is limiting for chloroplast structure and function, and is critical for overall photoautrophic growth and plant development. Phosphatidic acid is a key intermediate for chloroplast membrane lipid biosynthesis. Using the combination of a reverse genetic approach and heterozygous complementation, I identified a plastidic acylglycerolphosphate acyltransferase, ATSZ, catalyzing the acylation of the sn—2 position of lysophosphatidic acid. A green— fluorescent protein fusion with ATSZ localized to the chloroplast. Disruption of the AT SZ gene of Arabidopsis by T-DNA insertion caused embryo lethality. Development of the embryos was arrested at the globular stage. Therefore, plastidic LPAAT appears to be essential for embryo development in Arabidopsis. Furthermore, the mutant shed light on the origin and function of phosphatidic acid in plastids. ACKNOWLEDGMENTS First, I would like to thank my advisor and mentor, Christoph Benning for taking me as his Ph.D student. I thank him for the inspiration, challenge and great help. None of this work could have been accomplished without his support. I also thank members of my guidance committee, Dr. Douglas Gage, Dr. John Ohlrogge and Dr. Jack Preiss for their thought-provoking discussion and insightful suggestions. My positive graduate experience would be impossible without their expertise, encouragement, and support. I would like to thank all members of the Benning Lab, past and present, for their companionship. I thank Jilian Fan, Sherrie Sanda, Changcheng Xu, Setsuko Waka for collaboration in several experiments presented in this thesis; Alex Cemac, Mie Shimojima, Carl Andre, and Heiko Hartel for their helpful discussion and entertainment. Wayne R. Riekhof and Todd A. Lydic are specially acknowledged for their critical reading of my thesis and correcting my English among many other things. I especially acknowledge the Genetics program, the program director, Helmut Bertrand, andithe secretary, Jeannine Lee for their support and help. I also like to thank Hobao Gao, and Alicia Pastor for their help with confocal and transmission electron microscopy. Finally, I owe many thanks to my family, especially to my wife, Lahong Sheng, for years of love, support, and understanding. This dissertation research was funded in parts by grants from the US Department of Energy Bioscience Program and the MSU Center for Novel Plant Products. TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ............................................................................................................. x ABBREVIATIONS ........................................................................................................... xii CHAPTERI: Introduction: Lipid Biosynthesis, Function and Regulation ......................... 1 1.1. The two pathway hypothesis for chloroplast lipids biosynthesis ............................. 7 1.1.1. The Prokaryotic Pathway .................................................................................. 8 1.1.2. The eukaryotic pathway .................................................................................. 1 1 1.2. The biosynthesis and functions of PG .................................................................... 12 1.2.1. The biosynthesis of PG ................................................................................... 13 1.2.2. The function of PG in photosynthetic organisms ........................................... 15 1.3. The biosynthesis and function of sulfoquinovsyldiacylglycerol (SQDG) ............. 20 1.3.1. The biosynthesis of SQDG .............................................................................. 23 1.3.1.1. Genes involved in SQDG biosynthesis .................................................... 24 1.3.1.2. The biosynthesis of UDP-sulfoquinovose ................................................ 25 1.3.1.3.The assembly of SQDG ............................................................................. 28 1.3.1.4. The biosynthesis of acyl-SQDG ............................................................... 30 1.3.2. The function of SQDG .................................................................................... 3O 1.3.2.1.The function of SQDG in photosynthetic organisms ................................ 31 1.3.2.2. Function of SQDG in non-photosynthetic organisms .............................. 33 1.3.3. Other potential applications for SQDG ........................................................... 34 1.4. Phosphate regulation of lipid biosynthesis. ............................................................ 35 1.5. Overview ................................................................................................................ 38 References ..................................................................................................................... 40 CHAPTER 2: Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth ............................................................................................. 51 Abstract ......................................................................................................................... 52 Introduction ................................................................................................................... 53 Materials and Methods .................................................................................................. 57 Results ........................................................................................................................... 62 Identification of an Arabidopsis SQD2 Candidate Gene .......................................... 62 Isolation of an .9qu T-DNA Insertion Mutant .......................................................... 65 The sqd2 Mutant is Completely Devoid of Sulfolipid .............................................. 67 Expression of Arabidopsis SQDI and SQD2 in E. coli ............................................. 71 The effect of sulfolipid deficiency on the sqd2 mutant ............................................. 74 Discussion ..................................................................................................................... 79 References ..................................................................................................................... 83 CHAPTER 3: The pgp] Mutant Locus of Arabidopsis Encodes a Phosphatidylglycerol- phosphate Synthase with Impaired Activity ................................................................. 86 Abstract ......................................................................................................................... 87 Introduction ................................................................................................................... 88 Materials and Methods .................................................................................................. 90 Results ........................................................................................................................... 96 Isolation of a new phosphatidylglycerol-deficient mutant of Arabidopsis ............... 96 vi The pgp] locus maps in the proximity of a putative PGP synthase gene on chromosome 2 .................................................................................................... 100 The pgp] mutant is recognizable by a distinct RFLP in BAC locus T168247 ..... 104 Expression of a cDNA derived from wild-type locus T16B24.7 restores PG biosynthesis in the pgp] mutant background ..................................................... 105 BAC locus T16B24.7 encodes a fully functional PGP synthase in the wild-type but not in the pgp] mutant ........................................................................................ 106 Plastidic PGP synthase activity is strongly reduced in the pgpl mutant ................ 1 1 1 Photosynthesis is impaired in the pgp] mutant ....................................................... l 15 Discussion ................................................................................................................... 1 17 Referrenc es .................................................................................................................. 120 CHAPTER 4: Anionic lipids are required for chloroplast structure and function in Arabidopsis ................................................................................................................. 1 25 Abstract ....................................................................................................................... 126 Introduction ................................................................................................................. 127 Materials and methods ................................................................................................ 129 Results ......................................................................................................................... 132 Growth of the .9qu pgp] -1 double mutant is reduced ............................................ 132 The proportion of total anionic lipids in the double mutant is decreased ............... 137 Photosynthetic activity is compromised in the double mutant ................................ 140 Leaf structure and chloroplast ultrastructure are altered in the double mutant ....... 147 Discussion ................................................................................................................... 153 References ................................................................................................................... l 59 CHAPTER 5: Loss of plastidic lysophosphatidic acid acyltransferase causes embryo- lethality in Arabidopisis ............................................................................................. 162 vii Abstract ....................................................................................................................... 1 63 Introduction ................................................................................................................. 164 Materials and methods ................................................................................................ 167 Results ......................................................................................................................... 171 Identification of an Arabidopsis A T S2 (A C T 2) candidate gene .............................. 171 Expression of A T 52 cDNA complement an E. coli LPAAT mutant ....................... 174 Localization of the ATS2 protein ............................................................................ 176 Isolation of mutants with T-DNA insertion into AT 52 ........................................... 176 A transient increase in AT SZ expression in developing siliques in the wild type corresponds to the arrest of embryo development in the mutant ........................ 182 Discussion ................................................................................................................... 188 References ................................................................................................................... l 91 CHAPTER 6: Conclusion and future direction ............................................................... 194 References ................................................................................................................... 201 viii LIST OF TABLES Table 2.1. Quantitative analysis of leaf lipid extracts ....................................................... 70 Table 2.2. Pigment content and chlorophyll fluorescence of wild type and sqd2 mutant. ...................................................................................................................................... 77 Table 3.1. Leaf Lipid Composition (mol%) of Arabidopsis Wild Type and Mutants ...... 99 Table 3.2. Pigment content chlorophyll fluorescence of wild-type and mutant fully expanded leaves .......................................................................................................... 1 14 Table 4.1. Pigment content of wild-type and mutant lines ............................................. 141 Table 5.1. Segregation and complementation analysis of the two allelic heterozygous ats/A T S2 lines ............................................................................................................. l 80 ix LIST OF FIGURES Figure 1.1. Structure of the major fatty acids and glycerolipid referred to plant cell membranes ...................................................................................................................... 4 Figure 1.2. Schematic representation of the pathways of glycerolipids biosynthesis ......... 5 Figure 1.3. Organization of sulfolipid genes in purple bacteria and cyanobacteria .......... 21 Figure 1.4. The pathways for sulfolipid biosynthesis cyanobacteria ............................... 22 Figure 2.1. The pathway for sulfolipid biosynthesis in Arabidopsis ................................ 54 Figure 2.2. Structure of the SQD2 gene carrying the T-DNA insertion and induction of SQD2 expression following phosphate deprivation ..................................................... 63 Figure 2.3. The sqd2 mutant is SQDG dificent ................................................................. 68 Figure 2.4. Expression of Arabidopsis SQD] and SQD2 in E. coli .................................. 72 Figure 2.5. Growth curves of wild type and the .9qu mutant .......................................... 75 Figure 3.1. Growth and morphology of Arabidopsis wild type and pgp] mutant ............ 97 Figure 3.2. Lipid phenotype of pgp] and molecular defect. ........................................... 102 Figure 3.3. The PGP] locus on chromosome 2 of Arabidopsis ...................................... 107 Figure 3.4. Active site mutation of PGP synthase in the pgp] mutant ........................... 109 Figure 3.5. Chloroplastic PGP synthase and galactolipid biosynthetic activities in wild type and pgp] mutant ................................................................................................. 112 Figure 4.1. Appearance and growth of wild-type and mutant lines ................................ 133 Figure 4.2. Growth curves of wild type and mutant lines ............................................... 135 Figure 4.3. Quantitative analysis of polar lipids ............................................................. 138 Figure 4.4. Light dependence of steady-state chlorophyll fluorescence in fully expanded leaves. ......................................................................................................................... 143 Figure 4.5. Sensitivity to DCMU of wild-type and mutant lines .................................... 145 Figure 4.6. Light microscopy of the leaf sections ........................................................... 148 Figure 4.7. Ultrastructure of chloroplasts from mesophyll cells ..................................... 150 Figure 5.1. Evolutionary relationship between LPAATs ................................................ 172 Figure 5.2. Heterozygous complementation of E. coli J C201 by expression of A T S2 175 Figure 5.3. Subcelluar localization of the ATSZ protein ................................................. 177 Figure 5.4. Isolation and characterization of AT S2 T-DNA insertion lines .................... 178 Figure 5.5. Expression of AT S2 in various tissues and during silique development ...... 183 Figure 5.6. Arrest of embryo development in AT S2/ats2 ................................................ 185 xi ASQD CDS DAG DCMU DGDG DHA ER F AB-MS F ADS GGGT GPAT LPAAT LTP MGDG ORF PA PAPS PC PE PG ABBREVIATIONS adenosine 5'-phosphosulfate 2'-O-acyl-sulfoquinovosyldiacyl glycerol cytdine triphosphate: diaylglycerol cytidylyltransferase diaclyglycerol 3(3,4-dichlorophenyl)-l,1—dimethyurea di galactosyldiacyl glycerol dihydroxyactone endoplasmic reticulum fast atom bombardment mass spectroscopy fatty acid desaturases processive galactolipid: galactolipid galactosyltransferase glycerol-3-phosphate acyltransferase lyso-PA acyltransferase lipid transfer proteins monogalactosyldiacylglycerol open reading frame phosphatidic acid 3'-phosphoadenosine-5'-phosphosulfate phosphatidylcholine phosphatidylethanolamine phosphatidyl glycerol xii PGPS pLPAAT PSII PSI SQDG SQ-DHA UDP-SQ phosphatidylglycerol phosphate synthase plastidic lyso-PA acyltransferase photosystem II photosystem I sulfolipid sulfoquinovosyldiacylglycerol sulfoquinvose- 1 -O-dihydroxyactone sulfoquinovose 6’-deoxy- a -D-glucosyl—6’-sulfonic acid xiii CHAPTER 1 Introduction: Lipid Biosynthesis, Function and Regulation Lipids are, loosely defined, structurally divergent compounds that are preferentially soluble in non-polar organic solvents such as chloroform. The most abundant lipids in cells are fatty acid derived compounds (Ohlrogge and Browse, 1995). Other lipids include many pigments and some secondary metabolites that are not related to fatty acids. These lipids are essential for living organisms. A sheet-like bilayer of polar lipids and associated proteins are the main components of the biological membranes critical to life. The hydrophobic nature of the lipid bilayer ensures that the membrane is a barrier to water-soluble compounds. They delineate the cell boundary and therefore facilitate the creation and maintenance of distinct enviromnents between the inside and outside of cells. Lipid membranes also establish the eukaryotic organelles such as nucleus, mitochondria, and chloroplast. In addition, membranes are the sites where the two most important energy conversion processes occur: Photosynthesis in which light energy is converted into the energy of chemical-bonds is carried out in the chloroplast. Oxidative phosphorylation, in which adenine triphosphate is formed by oxidation of fuel molecules, takes place at the inner mitochondria membrane. Lipids themselves, such as triacyl- glyerol, also serve as a substantial chemical reserve of energy. In plants, the major membrane lipids are glycerolipids, in which fatty acids are esterified to the 511-1 and 372-2 position of the glycerol backbone and a polar head group attached to the sn-3 positions (Figure 1.1, below). Due to the presence of the hydrophobic fatty acids and the polar head group, glycerolipids have amphipathic physical properties essential for forming the bilayer structure of membranes. Like in membranes of animal cells, phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) are the most abundant membrane lipids in extraplastidic plant membranes. In the chloroplast where oxygenic photosynthesis occurs, PC is only found in the cytosolic leaflet of the outer envelope of chloroplasts (Dome et al., 1985; Joyard et al., 1998) and PG is the only major phospholipid found in thylakoids (Marechal et al., 1997). Beyond PG, chloroplast membranes chiefly consist of glycolipids, the galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) and the sulfolipid sulfoquinovosyldiacyglycerol (SQDG) (J oyard et al., 1998; Slabas, 1997). MGDG and DGDG represent up to 80% of the thylakoid membrane glycerolipids, of which MGDG constitutes 50% (J oyard et al., 1998b). PG and SQDG are acidic anionic lipids. Beyond their role as membrane building blocks, anionic lipids have specific roles in chloroplast development and function. It is hypothesized that under severe phosphate limitation, SQDG substitutes for PG ensuring a constant amount of anionic lipids even under adverse conditions (Benning et al., 1993; Giller et al., 1996). Biochemical and genetic approaches have been used to understand the biosynthesis of glycerolipids in plants, focusing on the biosynthesis of the diaclyglycerol (DAG) backbone, the head group, and the assembly of backbone and head group (Browse and Somerville, 1991; Ohlrogge and Browse, 1995; Joyard et al., 1998; Dorrnann and Benning, 2002). Phospholipids Glycolipids O NH2 0 _1, 0 32—1—35 I—l—I RZOC—O OH R 100—0 . . phosphatidylethanolamine (PE) 0 II T T it” P c r: CH3 Rzoc—O (LH 1L IL H3 R1OC—O _ , phosphatidylcholine (PC) i T T C P C i l NH2 Rzoc—o (LH H COOH R 1oc—o , _ phophatldylsenne (PS) 7:000” O'COR1 sulfoquinovosyldiacylglycerol (SQDG) RT and R2 =Fatty acid phosphatidylinositol (Pl) 18:3 Linolenic H00 W 16:3 Roughanoic HOOW/m . - . H3 H3 18-2 L'nOIG'C HOOWCW 16:1-transA3 HOOC 18.1 Oleic H00 18:0 Stearic HOOK/\Ms CH3 W 16:0 Palmltic HOOCWWW H3 H3 Figure 1.1. Structure of the major fatty acids and glycerolipid in plant cell membranes. Note that the fatty acids are referred to by the number of carbon atoms, colon, followed by the number of double bonds. Figure 1.2 Schematic representation of the pathways of glycerolipids biosynthesis discussed in the text. The glycerol backbones with the typical carbon length of fatty acid in the C—1 and C-2 positions are indicated to illustrate the main molecular species derived from each pathway. The two pathways for chloroplast lipid biosynthesis are indicated, (1): the prokaryotic pathway; (II): the eukaryotic pathway. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol. Endoplasmic 18 16 PG,H reticulum PE P OH PCho PC 16 18 16 18 16 18 P OH """* PCho TH l—l—l 18 18 18 18 1a 1a Chloro last p DGDG MGDG Gm 0“ SQDG 50 Gal — Gal 1 I I I 0601 16 18 MGD1 16 1a 7 16 18 16 18 <--- ____—’ Gal — Gal Gal OH so 18 18 18 18 18 18 18 18 isnsAcp ATS? P OH MGDG Gal Gal —Gal DGDG ATS1 M601 0601 —————> |? 7 18 16 18 13 15 GW PG 18 16 18 5° SQDG 16 1.1. The “eukaryotic” and “prokaryotic” pathway hypothesis for chloroplast lipids biosynthesis One of the most intriguing hypotheses in plant lipid metabolism is the two pathway hypothesis for chloroplast lipid biosynthesis (Roughan and Slack, 1982). According to this model (Figure 1.2), after 16:0 and 18:1 fatty acids are synthesized de novo in the chloroplast (which is the major site for fatty acid biosynthesis in seed plants, see review by Browse and Somerville, 1991; Ohlrogge and Browse, 1995), they may be used directly for lipid biosynthesis via the “prokaryotic” pathway in the chlor0plast envelope, or exported to the endoplasmic reticulum (ER) where the “eukaryotic” pathway of lipid biosynthesis occurs. Subsequently, lipid linked fatty acid desaturases (FADs) introduce double bonds into lipids to produce the desaturated forms typical for plant membranes (Browse and Somerville, 1991; Ohlrogge and Browse, 1995, Joyard et al., 1998 a, b). A fraction of the diacylglycerol moieties from PC synthesized via the “eukaryotic” pathway is returned to the chloroplast and used for thylakoid lipid biosynthesis (Roughan and Slack, 1982). Due to the substrate specificity of the acyltransferases in some plants, phosphatidic acid (PA) produced via the “prokaryotic” pathway has 16:0 at the sn-2 position and 18:1 at the sn-1 position and PA from the “eukaryotic” pathway has 18:1 at the sn-2 position and 16:0 when present, is at the sn-1 position. In some species, the “prokaryotic” pathway only contributes to PG biosynthesis, and the “eukaryotic” pathway is responsible for the rest of membrane lipid biosynthesis. In some other species, both pathways contribute to MGDG, DGDG and SQDG biosynthesis. Because MGDG synthesized from the “prokaryotic” pathway contains a substantial amount of 16:3 at the sn-2 position, e. g. in Arabidopsis and spinach, these plant species are called “16:3" plants to distinguish from other species (18:3 plants) whose galactolipids are synthesized completely by the eukaryotic pathway. 1.1.1. The Prokaryotic Pathway In the plastid of all plants, an acyl-ACP: glycerol-3-P acyltransferase (ACT) catalyzes the biosynthesis of lyso-PA, the first step of the “prokaryotic” pathway. cDNAs encoding this enzyme have been cloned from many plant species (Ishizaki et al., 1988; Bhella and Mackenzie 1994; Liu et al., 1999; Manaf and Harwood 2000; Weber et al., 1991; Nishida et al., 1993;1shizaki-Nishizawa et al., 1995). In some species, this enzyme is highly specific for 18: l-ACP. Inactivation of plastid glycerol-3-P acyltransferase in the Arabidopsis act] mutant leads to the loss of the “prokaryotic” pathway for glycolipid biosynthesis (Kunst et al., 1988). This deficiency is compensated by increased flux from the eukaryotic pathway and therefore the act] mutant is changed from a 16:3 to an 18:3 plant. In all seed plants, the prokaryotic pathway is responsible for chloroplast PG biosynthesis. However, the act] mutant still contains 70% of the wild type level of PG suggesting the existence of an alternative pathway or altered regulation of that pathway (Kunst et al., 1988). Based on this fact, one may hypothesize that the putative alternative pathway or a lyso-PA pool from the “eukaryotic” pathway is used to synthesize “prokaryotic” PA molecular species utilized specifically for PG biosynthesis. How plants control PA partitioning between PG and glycolipids is an intriguing question. Following the first step of lyso-PA formation, a lyso-PA acyltransferase (LPAAT), highly specific for 16:0-ACP, completes the “prokaryotic” PA biosynthesis. A plastidic LPAAT, BA T2, from Brassica napus, has been isolated by functional complementation of an E. coli LPAAT deficient mutant plsC. This enzyme is chloroplast-localized and shows 16:0-ACP preference to 18:1-ACP (Bourgis et al., 1999). However, the in vivo role and the specific sub-organelle localization of this protein are still in need of further investigation. PA synthesized via the prokaryotic pathway enters PG biosynthesis in all seed plants. In 16:3 plants, it is converted to DAG by a PA-phosphatase located at the inner membrane. This DAG pool is used to synthesize glycolipids such as MGDG, DGDG and SQDG (Joyard, 1993). The DAG pool can’t be formed in 18:3 plants due to the absence of a PA-phosphatase (Heinz and Roughan, 1983) and thus DAG precursor entirely derived from the eukaryotic pathway is used for glycolipid biosynthesis. The biosynthesis of MGDG is catalyzed by UDP-galactose:DAG galactosyl- transferase (EC 2.4.1.46). MGDG synthase was first cloned from cucumber (Shimojima et al., 1997) and subsequently from Arabidopsis. Arabidopsis contains three MGDG synthases, MGDl, MGD2 and MGD3 belonging to the glycosyltransferase gene family GT28 ( http://afmb.cnrs-mrs.fr/CAZY/) (Miege et al., 1999; Jorasch et al., 2000; Jarvis et al., 2000; Awai et al., 2001). MGDl is located at the inner chloroplast envelope and proposed to be the major enzyme for MGDG synthesis, because a 75% reduction in MGDG synthase activity resulted in the 42% reduction of MGDG content (Jarvis et al., 2000). Among these three genes, MGD2 and MGD3 are more closely related to each other based on sequence similarity. They are located at the outer envelope of the chloroplast and are suggested to be involved in phosphate-stress regulated glycolipid formation (Awai et al., 2001). In Arabidopsis, the DGDG synthase gene DGD] was isolated by map-based cloning in the DGDG-deficient mutant dgd] , in which the amount of DGDG is reduced by 90% (Dormann et al., 1999). In agreement with previous biochemical studies, the DGDl protein is associated with the outer chloroplast envelope. The protein lacks a cleavable-chloroplast targeting sequence, and is inserted into the outer enveIOpe by a mechanism independent of the general import pathway (Froehlich et al., 2001). The C—tenninal end of DGDl is proposed to contain the enzymatic domain required for galactosylation based on its similarity to the plant and bacterial glycosyltransferase family GT4. The function of the N-terminal domain of DGDl is unknown (Dormann and Benning 2002). Utilizing a reverse genetic approach, a second DGDG synthase, DGD2, was isolated in Arabidopsis and other plant species (Kelly and Donnann, 2002). It was proposed that this enzyme activity is responsible for the synthesis of DGDG under specific conditions such as phosphate-limitation (Kelly and Dormann, 2002). It was found that DGDl and DGD2 use the UDP-Galactose as galactose donor to synthesize DGDG and oligogalactosyldiacylglycerol trigalactosyl- diacylglycerol and tetragalacosyldiaylglycerol (Kelly et al., 2003; Kelly and Dormann, 2002). The Galactosyltransferase activity reponsible for biosynthesis of DGDG and oligogalactosyldiacylglycerol is still present in the dgd] dgd 2 double null mutant, suggesting the existence of a progressive galactolipid synthase independent from DGDl and DGD2 in Arabidopsis. This enzyme activity has been proposed to be induced in the tgd] mutant carrying a mutation in a permease-like gene presumably involved. in lipid trafficking between the ER and chloroplast (Xu et al., 2003). This galactolipid synthase was thought to be related to a processive galactolipid:galactolipid galactosyltransferase (GGGT) catalyzing the galactosylation of MGDG using a second MGDG molecule as the galactose donor ( Kelly et al., 2003, Xu et al., 2003). This GGGT was previously proposed to be the DGDG synthase that synthesizes both DGDG and the unusual 10 oligogalactosyldiacylglycerol (Dome et al., 1982; Cline and Keegstra. 1983; van Besouw and Wintermans, 1978). The biosynthesis of SQDG and PG will be addressed below in detail. 1.1.2. The eukaryotic pathway After the fatty acids are exported to the ER as acyl—CoA from the chloroplast, they are incorporated into PA to give rises to molecular species with 18:1 at the sn-2 position and18:1 or 16:0 at the sn—1 positon by two ER acyltransferases. This PA then is incorporated into PC, PE, PI and extraplastidic PG (review by Browse and Somerville, 1991; Ohlrogge and Browse, 1995, Joyard et al., 1998 a, b). Experiments show that PC synthesized in the ER gives rise to PC in the chloroplast, which lacks the enzymes for de novo PC biosynthesis (J oyard et al., 1991), thus the intact diacylglycerol moiety from PC can be returned to the chloroplast envelope and contribute to chloroplast lipids (Slack et al., 1977). For instance, pulse-chase labeling experiments using ‘4 [C] acetate on intact leaves showed that PC was labeled first and subsequently the label from PC is incorporated into MGDG and later into DGDG (Slack et al., 1977). However when the incorporation of label into fatty acids at each position of the DAG moiety is analyzed, differences between the 811-1 and the sn-2 position of PC and MGDG is revealed (Mongrand et al., 1997, 2000). Based on these results, it was postulated that lyso-PC rather than PC is returned into the chloroplast (Mongrand et al., 1997, 2000). Similar experiments also indicated that an extensive modification of the DAG moiety of PC occurs and that only a specific metabolic pool of PC is used to supply chloroplast lipid biosynthesis (Williams et al., 2000). Thus, the molecule transported and the transport 11 mechanism itself remains unknown. Lipid transfer proteins (LTP) have been postulated to be involved in the lipid transporting process between ER and chloroplast. However, the expected ER or secretory pathway localization of LTP from maize, spinach and barley are inconsistent with a role in intracellular lipid trafficking (Browse and Somerville, 1991). Recently, a mutant of Arabidopsis, tgd], was isolated by Xu et al (2003). Several pieces of evidences suggest tgd] is deficient in ER to plastid lipid trafficking. First, in contrast to the act] mutant, the proportion of chloroplast lipids derived from the ER pathway in tgd] is reduced to below 20%. Second, pulse chase experiments showed that the transfer of ER-derived PC to the plastid or the incorporation of diacylglycerol backbones derived from PC into the thylakoid lipids MGDG and DGDG is impaired in the mutant. Furthermore, the TGDl protein is similar to the permease subunit of some bacterial ATP binding cassette (ABC) transporters. Understanding the biochemical function of TGDl and the identification of the components of the TGDl complex will provide important insights in understanding the lipid trafficking between ER and chloroplast. 1.2. The biosynthesis and functions of PG Phosphatidylglycerol (PG) is a common membrane lipid present in animals, plants and microorganisms. In all organisms, the biosynthesis of PG starts with the biosynthesis of phophatidylglycerol phosphate by a reaction of CDP-diacylglycerol with glycerol- phosphate, followed by cleavage of phosphate by a phosphatase to produce PG (Moore, 1982; Andrews and Mudd, 1986; Kinney, 1993). Genetic disruption of the PG biosynthesis pathway and therefore depletion of PG is detrimental to E. coli (Kikuchi et 12 al., 2000), in which PG is the major anionic lipid and Saccharomydes cerevz’siae (Ostrander et al., 2001), where PG is predominantly associated with mitochondria, suggesting essential roles for PG in non—photosynthetic organisms. In photosynthetic organisms, PG is mainly associated with the photosynthetic membrane, and it is the predominant phospholipid present in thylakoid membrane, although it also exists in extraplastidic membranes. PG is the only phospholipid present in cyanobacteria, and PG typically constitutes 8—10% of chloroplast lipids (Browse and Somerville, 1993). Thus the biosynthesis and function of PG in photosynthetic organism is an interesting question. 1.2.1. The biosynthesis of PG Three enzymes are involved in PG biosynthesis. The first is cytdine triphosphate: diaylglycerol cytidylyltransferase (CDS) catalyzing the biosynthesis of CDP- diacylglycerol, the precursor of the minor lipids PI, PG, cardiolipin, and possibly phosphatidylserine (Moore, 1982) and therefore is not specific for PG biosynthesis. The next two enzymes in the pathway are phosphatidylglycerol phosphate synthase (PGP) and phosphatidyl glycerol phosphate phosphatase (Joyard et al., 1998b). In the anoxgenic photosynthetic bacterium, Rhodobacteria sphaeroides, CDS enzyme activity has been shown to co-localize with the phosphatdiylglycerol phosphate synthase activity at the cytoplasmic membrane consistent with the idea that phospholipids are synthesized there and then transferred to the intracytoplasmic membrane system (Cain et al., 1984; Radcliffe et al., 1985). In cyanobacteria, the open reading frame (ORF) encoding this enzyme has been identified based on a similarity search with CDS rom other organisms. The mutant defective in this gene requires the supplementation of 13 PG to the medium for growth, suggesting that this gene indeed encodes CDP-DAG synthase and is essential for PG biosynthesis (Sato N et al., 2000). In higher plants, CDS activity is localized in microsomal membranes which might account for PI biosynthesis (Moore, 1982), the inner membrane of mitochondria where CDP-DAG is the substrate for PG and cardiolipin biosynthesis (Frentzen and Griebau, 1994), and the inner membrane of the chloroplast , which is the major site for PG biosynthesis in leaves (Andrews and Mudd, 1985). The CDS gene has been cloned from Arabidopsis and Potato (Solanum tuberosum) and has been characterized in some detail (Kopka et al., 1997). However, the localization and in vivo roles of the enzyme still need further investigation. The committed step for the PG biosynthesis pathway is the biosynthesis of phosphatidylglycerol phosphate (PGP) catalyzed by the phosphatidylglycerol phosphate synthase (PGPS). There are two phylogeneticially distinct groups of PGP synthases, the bacterial PGP synthase of about 20 KDa containing the typical CDP-alchohol binding motif (Muller and Frentzen, 2001; Hagio et al., 2000; Dryden and Dowhan., 1996; J iang et al., 1997; Tuller et al., 1998; Ohta et al., 1981), and the mitochondrial PGP synthase of about 60 KDa which, like the bacterial cardiolipin synthases, belongs to a conserved protein family defined by an invariant motif, HXK(X)4D, denoted as HKD (Ponting and Kerr, 1996; Mtiller and Frentzen, 2001; Kawasaki et al., 1999; Chang et al., 1998; Tropp, 1997). In plants, labeling studies indicate that enzymatic activity involved in PG biosynthesis is associated with the inner mitochondrial membrane, the endoplasmic reticulum, and the chloroplast (Moore, 1982; Kinney, 1993), suggesting that at least three isoforrns of PGP synthases and PGP phosphatases might exist in plant cells. However, little is known about plant PGP synthases other than their cellular compartmentation. Recently, two isofonns of PGP synthases, PGPl and PGP2, have been identified and characterized from Arabidopsis (Miiller and Frentzen, 2001). Both PGPl and PGP2 belong to the bacterial PGP synthases group. PGPl contains a cleavable transit peptide for import into plastids and with a lower probability for import into mitochondria. Consistent with this hypothesis, PGPl, which is not active in E. coli, can be imported into the yeast mitochondria and processed to a catalytically active protein (Muller and Frentzen, 2001). PGP2 has lO-times higher activity in E. coli than in yeast where the PGP2 activity is predominantly located in the microsomal fraction. Thus, PGP2 is possibly the microsomal enzyme (M'Liller and F rentzen, 2001 ). The R. sphaeroides PGP synthase, pgsA, has been identified. Overexpression of this gene in both R. spaeroides and E. coli do not have dramatic effects on the phospholipid composition of either organism indicating that this enzyme activity is tightly regulated, although the mechamism is unknown (Dryden and Dowhan 1996). 1.2. 2. T he function of PG in photosynthetic organisms As the major anionic phospholipid, PG has critical roles for the proper function of photosynthetic membranes. It is also thought to play a critical role in cold acclimation of plants. Based on a survey of lipids present in the chloroplast membranes from a variety of plant species representing both chilling-sensitive and chilling—resistant types, chilling sensitivity is proposed to closely correlate with the proportion of specific molecular species of PG, specifically disaturated PG, containing only 16:0, 18:0 or 16:1 trans, which has physical properties similar to that of a saturated fatty acid (Murata, 1983). The identification and analysis of genetic mutants that are impaired in PG biosynthesis, their 15 biochemical and physiological analysis, and the analysis of transgenic plants with an altered firaction of PG has began to offer new insights into the functions of PG in photosynthetic organism (Murata, 1983; Murata and Nishida, 1990; Murata and Yamaya,1984; Sato et al., 2000). Several biochemical studies have demonstrated the essential roles of PG in photo- synthesis. In vitro experiments showed that PG is enriched in the light-harvesting pigment-protein complexes of photosystem II (PSII) (Murata et al., 1990; Tremolieres et al., 1994) and is essential for the dimerization of the P811 reaction center core pigment- protein complex (Kruse et al., 2000). Furthermore, the recently published crystal structure of photosystem I (PSI) contains three molecules of PG in tight coordination with the core and the antenna complex (Jordan et al., 2001) and PG is required for the in vitro reconstitution of the light-harvesting pigment-protein complexes of PSI (Schmid et al., 1997). Thylakoid membranes treated with phospholipase A2 were PG depleted and were inhibited in their photosynthetic electron transport activities (Jordan et al., 1983; Siegenthaler et al., 1987). The function of PG in photosynthesis has been studied with a mutant of the cyanobacterium Synechocystis sp. PCC6803, which is defective in the biosynthesis of PG. The growth of this mutant requires supplementation with PG and the photosynthetic activity of the mutant has been shown to decrease with a concomitant decrease in the PG content in thylakoid membranes primarily due to a decrease in the PSII activity. These results provided the first in vivo evidence for an essential function of PG in PSII. Further analysis of this mutant indicated that PG is indispensable for the PSII reaction center complex, and that PG is required to maintain the structural integrity of the QB-binding site (Gombos et al., 2002). In Arabidopsis, it has been known that the 16 relative proportion of PG is reduced in the pho] and act] mutants (Hartel et al., 1998; Kunst et al., 1988; Poirier et al., 1991). However because both mutants are affected in multiple aspects of lipid metabolism, they are of limited usefulness for the investigation of specific functions of PG in the photosynthetic membranes of seed plants. Therefore, identification of mutants deficient in the PG biosynthetic pathway was expected to provide new insights into the specific functions of PG. It has been proposed that one primary event during chilling injury is a La-to-LB lipid phase transition (liquid crystalline to gel phase) resulting in alterations in the metabolism of chilled cells that leads to injury and death of the chilling-sensitive plants (Lyons, 1973; Raison, 1973). It has been observed that PG is the only glycerolipid exhibiting a significant phase transition and PG prepared from chilling-sensitive plants has been shown to undergo this phase transition at 30 0C while PG purified from chilling—resistant plants underwent phase transition at 15 OC. Based on these data and the correlation between disaturated PG and chilling—sensitivity, it has been proposed that disaturated chloroplast phosphatidylglycerol (PG) confers chilling sensitivity to plants (Murata, 1983; Murata and Yamaya, 1984; Murata and Nishida, 1990). The substrate specificity of the glycerol-3-phosphate acyltransferase of chloroplasts (GPAT) is proposed to be the dominant factor in determining the proportion of disaturated PG, because chloroplasts always contain 16:0 or trans 16:1 at the sn-2 position (Roughan, 1985). It has been observed that GPAT from chilling-resistant plants prefers 18:1-ACP to 16:0-ACP as a substrate, resulting in low level of disaturated PG in chloroplast membranes, while the enzyme from chilling-sensitive plants hardly distinguishes between 18:1-ACP and 16:0-ACP, resulting in a high proportion of 17 disaturated PG. To directly test the effect of disaturated PG on chilling sensitivity, the GPAT cDNAs from chilling resistant and chilling sensitive plants have been used in transgenic experiments. In transgenic tobacco (a chilling sensitive plant) expressing a GPAT cDNA from squash (a chilling sensitive plant) increased the proportion of disaturated PG (76%). In the converse experiment, disaturated PG is decreased (28%) in transgenic tobacco expressing GPAT cDNA from Arabidopsis, a chilling resistant plant. The increase of disaturated PG was positively correlated with low-temperature induced photoinhibition, an indication of chilling sensitivity, and the decreased amount of disaturated PG was correlated with decreased chilling sensitivity (Murata et al., 1992). These results showed the first direct evidence for the causal correlation between the amount of disaturated PG and low-temprature induced injury. Further analysis of transgenic tobacco with increased amount of PG suggests that the variation of disaturated PG amount has no effect on the rate of low—temperature induced photoinhibition. Instead, the rate at which damaged PSII can be repaired is affected in the transgenic plants. It has been hypothesized that the replacement of damaged D1 protein at the photosystem II reaction center with newly synthesized D1 protein is effected by the amount of disaturated PG (Moon et al., 1995). However, the possibility that reduced recovery from photoinhibition contributes to plant chilling sensitivity has been debated (Moon et al., 1995; Somerville, 1995). For example, the analysis oftransgenic Arabidopsis plants expressing the E. coli plsB gene suggests that an increase of disaturated PG has no effect on their ability to recover from photoinhibition (Wolter et al., 1992, Bruggemann and Wolter, 1995). In agreement with this result, Vijayan and Browse (2002) found that there is no difference between wild-type and the fab] mutant in terms of the speed of recovery from photoinhibition temperatures between 7°C and 27°C. The Arabidopsis fab] mutant is deficient in the elongation of palmitic acid which leads to leaf PG containing 70% high-melting-point fatty acids (Wu and Browse, 1995). The fab] mutant shows no obvious difference with wild-type in low temperature induced injury. This result is not necessarily in conflict with those of Moon et al., (1995). Instead, it suggests that a critical threshold for disaturated PG is required to cause rapid chilling injury. Recently, transgenetic experiments with cDNAs of GPAT have been successfully used to improve the chilling sensitivity of rice (Yokoi et al., 1998; Ariizumi et al., 2002) suggesting a practical application of this research in the improvement of the chilling sensitivity of crops. 19 1.3 The biosynthesis and function of sulfoquinovsyldiacylglycerol (SQDG) The sulfolipid sulfoquinovosyl diacylglycerol (SQDG) is an abundant sulfur containing bio-organic compound in nature. Its structure was elucidated by Benson and coworkers (1959) over 40 years ago, as 1’-O-(6’-deoxy-q-D-glucosyl—6’-sulfonic acid)-3- O-diacylglycerol. The head group of SQDG, referred to as sulfoquinovose (6’-deoxy- or - D-glucosyl-6’-sulfonic acid) is hydrophilic and negatively charged at biological pH value. As the diacylglycerol backbone is also hydrophobic, SQDG has strong amphipathic characteristics (Benning, 1998). SQDG has been found in most photo- synthetic organisms, including all seed plants, mosses, ferns, algae and photosynthetic bacteria (Haines 1973; Araki et al., 1991; Dembitsky et al., 1990, 1991). SQDG was also identified from the nonphotosynthetic bacteria Rhizobium mclitoti (Cedergreen et al., 1994) and Bacillus acidocaldarius (Langworthy et al., 1976) showing that SQDG is not restricted to photosynthetic organisms. In seed plants, SQDG is exclusively associated with chloroplast membranes. In other photosynthetic organisms, SQDG is likewise found associated with photosynthetic membranes. Thus, SQDG has long been thought to play a role in photosynthesis (Barber and Gounaris, 1986). Recently, the identification of genes essential for SQDG biosynthesis (Benning, 1998) (Figure 1.3) has provided strong evidence in support of the proposed sugar- nucleotide pathway (Barber, 1963; Pugh et al., 1995; Benning, 1998) (Figure 1.4). Furthermore, the analyses of SQDG deficient mutants have provided new insight into the 20 Bacteria (alpha Group) fir w squ .9qu squ squ Cyanobacteria """_>“-—"- squ squ 313:1. Figure 1.3 Organization of sulfolipid genes in purple bacteria and cyanobacteria. bp, base pairs. Adapted from (Benning C, 1998) 21 HO CH20HO SQDB HO CH2$O§O SQDX Ho CH2$O§O H H H H H—FH H H H H ——> H H H H H HO OH OH Ho OH o\ Ho Ho OH 0 UDP (I? \Cl) —>——\o 0H 0“ .I, t. UDP—GIC UDP-SQ DAG SQDG Figure 1.4. The pathways for sulfolipid biosynthesis in cyanobacteria. SQDB is specific to the first reaction. SQDX from cyanobacteria is the SQDG synthase. DAG, diaylglycerol; R, fatty acyl groups; UDP-Glc, UDP-glucose; SQDG, sulfoquinovosyldiacyl glycerol; UDP-SQ, UDP-sulfoquinovose 22 functions of SQDG (Benning, 1998). However, in higher plants, the pathway for SQDG biosynthesis was still incomplete, because the gene, encoding SQDG synthase catalyzing the final assembly of SQDG, was not isolated. Also, the function of SQDG in higher plants was unclear due to the absence of an SQDG deficient mutant. The identification of SQDG synthase in Arabidopsis was necessary to verify the SQDG biosynthetic pathway in plants. In addition, by construction of an SQDG-deficient mutant, the function of SQDG could be tested. 1.3.1. The biosynthesis of SQDG It has become clear that SQDG is synthesized via the sugar nucleotide pathway which is a two step reaction, initially utilizing UDP—glucose as the direct precursor of UDP-sulfoquinovose (UDP-SQ), followed by the transfer of sulfoquinovose to diacylglycerol (DAG) to complete the final assembly of SQDG (Pugh, 1995; Benning, 1998). The most elusive aspect in this pathway has been the origin of UDP-SQ, i.e. the nature of the sulfur-donor. How SQDG is assembled posed yet another question. In the past, progress toward answers to these questions has been hampered because of the lack of a suitable in vitro system for SQDG biosynthesis and a large number of sulfurous water-soluble compounds present in extracts of higher plants (Benning, 1998). But recently it became possible to solve these problems with the cloning of genes involved in SQDG biosynthesis and the isolation of SQDG deficient mutants in different organisms. 23 1.3.1.1. Genes encoding enzymes involved in SQDG biosynthesis More than 12 years ago, a combination of biochemical and genetic approaches was used to identify genes essential for the biosynthesis of SQDG in various organisms. Initially, four genes involved in SQDG biosynthesis (squ, squ, squ, and squ)(Benning, 1992a, b; Rossak 1995), were isolated from the purple bacterium R. sphaeroia’es. The squ, squ, and squ genes were isolated by screening SQDG- deficient mutants and followed by complementation. The genes are organized in two transcriptional units as shown in Figure 1.3. Subsequently an SQDD gene was identified by insertional inactivation of an open reading frame flanked by squ and .9qu (Rossak at al., 1995). The sequence of the squ product shows similarity to sugar nucleotide- modifying enzymes, UDP-glucose-4-epimerases and nucleotide-hexose-4, 6- dehydratases, suggesting that the gene product may be responsible for the conversion of UDP-Glc to UDP-SQ (Benning, 1992). The similarity of the .9qu sequence to glycosyltransferases suggests that it may be a SQDG synthase (UDP-sufquinovose: diacylglycerol sulfoquinovsyltransferase), an enzyme catalyzing the final assembly of SQDG (Rossak, 1995). It has been suggested that the squ protein provides substrate specificity to the .9qu gene product (Rossak et al., 1995). In an .9qu mutant, the novel compound sulfoquinvose-l-O-dihydroxyactone (SQ-DHA) was identified (Rossak et al., 1997). When added to the extracts of R. sphaeroides or spinach chloroplasts, labeled SQ- DHA was not incorporated into SQDG (Rossak et al., 1997). Therefore it is still unclear whether SQ-DHA is a true intermediate of sulfolipid synthesis or whether it is a by- product that is only present in the mutant (Rossak, 1997). Because no water-soluble 24 sulfur-containing compounds accumulated in the .9qu mutant, the biochemical function of the squ gene product remains unclear (Benning, 1998). Orthologs of the squ gene subsequently were cloned from cyanobacteria Synechococcus sp. PCC7942, and synechocystis sp PCC 6803 (Giiler et al., 1996), as well as Arabidopsis (Essigmann et al., 1999), spinach (Shimojima and Benning, 2003) and the green alga C. rein/tardtii (Riekhof et al., 2003). All SQDB orthologs are highly conserved suggesting that the mechanism for UDP-SQ biosynthesis evolved during a single event (Benning, 1998). However no other sequences similar to the other sqd genes fi‘om R. sphaeroides have been found in organisms not belonging to q—group bacteria, indicating that some aspects of the pathway are evolutionarily more divergent than others. By analyzing the sequence flanking the .9qu in Cyanobacteria, a new gene involved in sulfolipid biosynthesis, .9qu was identified. The predicted squ product has a sequence similar to glycosyltransferases (Giiler et al., 2000). The insertional inactivation of squ caused sulfolipid dificiency. This gene product may therefore encode the SQDG synthase in Cyanobacteria. 1.3.1.2. The biosynthesis of UDP-sulfoquinovose Based on the presence of a sulfur containing sugar nucleotide in extracts of Clzlorella (Shibuya et a]. , 1963), UDP-sulfoquinovose (UDP-SQ) was proposed to be the head group donor for SQDG. Recently, more evidence has arisen in support of this hypothesis. For example, chemically synthesized UDP-SQ could stimulate the SQDG biosynthesis by membranes of spinach chloroplasts (Heinz et al., 1989). More direct 25 evidence was obtained from SQDG-deficient mutants. The accumulation of UDP-SQ was observed in mutants of R. sphaeroz‘des inactivated in the .9qu gene (Rossak et al., 1995). UDP-SQ has also been identified in extracts of different unicellular algae and mosses (Benning, 1998). The biosynthesis of UDP-SQ starts from UDP-glucose and a sulfur donor. The involvement of UDP-glucose is based on the stimulation of the incorporation of [3SS]SO4 2‘ into SQDG by UTP and the observation that UDP-[14C]glucose is effectively incorporated into sulfolipid. In addition, the squ mutant of R. sphaeroides did not accumulate a sulfur-labeled water soluble compound suggesting that the SQDB protein catalyzes the reaction before or during the incorporation of sulfur into UDP-SQ (Benning, 1993). Indeed, the SQDB protein can accept UDP-glucose and convert it to a new compound in vitro. The similarity of the SQDB protein to sugar nucleotide- modifying enzymes, UDP-glucose-4-epimerases and nucleotide-hexose-4, 6- dehydratrases suggest that the intermediate, UDP-4-ketoglucose-5-ene, could serve as a sulfur acceptor. The production of UDP-SQ from this intermediate was first suggested by Barber (1963). Furthermore, the incorporation of UDP-[MC] glucose to SQDG was stimulated by the addition of methyl alpha-glucose enide or by addition of an enzyme system known to be forming (although not accumulating) UDP-4-ketoglucose-5-ene.The cloning of the SQD] gene from Arabidopsis made it possible to study the biosynthesis of UDP-SQ in detail. SQDl contains conserved Y-XXX-K and glycine-rich (G-XX-G-XX- G) sequence pattems and appears to be a member of the short-chain dehydrogenase- /reductase (SDR) family (Mulichak et al., 1999). It has been proposed that SQDl catalyzes the change from UDP-glucose to UDP-4—keto-glucose, followed by the formation UDP-4—keto-5,6-glucosene which serves as an acceptor for a sulfur donor 26 (Essigmann et al., 1999). The SQDl protein has been crystalized at 1.6 A (Mulichak et al., 1999). Recombinant SQDl lacking the chloroplast transit peptide forms a dimer, and contains a buried active site with tightly bound NAD+. Co-crystallization with IHDP- Glucose (UDP-Glc) demonstrated directlythe binding of this presumed substrate in the active site (Mulichak et al., 1999). Another intriguing question regarding UDP-SQ biosynthesis was the identity of the sulfur donor. Labeled sulfate can be incorporated into SQDG by isolated chloroplasts, indicating that the sulfur donor is derived from the sulfate reduction pathway in the chloroplast (Haas et al., 1980; J oyard et al., 1986; Kleppinger- Sparace et al., 1985). Intermediates of this pathway, adenosine 5'-phosphosulfate (APS) and 3'-phosphoadenosine-5‘-phosphosulfate (PAPS), were indeed incorporated into SQDG by isolated chloroplasts (Kleppinger-Sparace et a1; 1990). Another intermediate of the sulfate reduction pathway, sulfite, was shown to be incorporated into SQDG by extracts of Chlamydomonas reinhardtii (Hoppe and Schwenn, 1981). However this reaction was linear over time and thus was thought to be nonenzymatic. An in vitro assay for the recombinant SQDI protein has been developed to find the sulfur donor (Sanda et al., 2001). The result showed that the recombinant SQDI protein uses sulfite but not APS to synthesize UDP-SQ, demonstrating that sulfite rather than APS is the sulfur donor (Sanda et al., 2001). Recently, Shimojima and Benning (2003) showed that the Km for sulfite of the native enzyme purified from isolated spinach chloroplasts is at least four times lower than that of the recombinant spinach enzyme. Additionally, size determination by gel filtration indicated that the native form of SQDl is purified as a large complex of approximately 250 kDa, which was more than twice as large as the calculated size for the hornodimer, suggesting a multirneric complex in association with 27 SQDl is required for UDP-SQ biosynthesis. It has been proposed that the function of this complex is to cope with low sulfite concentrations in the chloroplast by increasing the affinity for free sulfite, or substrate channeling (Shimojima and Benning, 2003). 1.3.1.3. The assembly of SQDG The final step of SQDG biosynthesis is catalyzed by a UDP-ot-sulfoquinovosezl, 2- diacylglycerol 3-O—q—D-sulfoquinovosyltransferase designated SQDG synthase (Heinz et al., 1989; Seifert and Heinz, 1992; Rossak et al., 1997; Guler et al., 2000; Benning, 1998). In spinach, this enzyme activity has been characterized in some detail using intact chloroplasts into which radio labeled DAG had been biochemically preloaded as the acceptor for the sulfoquionovose moiety (Heinz et al., 1989; Seifert and Heinz, 1992). Among the different nucleotide sulfoquinovoses tested, UDP-SQ was the best substrate while GDP-SQ resulted in a lower enzyme activity. This enzyme showed the maximum activity at pH 7.5 with a Km for UDP-SQ of lOuM in the presence of 5 mM Mg 2+. The enzyme activity was only observed for osmotically ruptured chloroplasts following the addition of UDP-SQ. This fact together with the co-purification of enzyme activity with the chloroplast inner envelope suggested that SQDG synthase is associated with the inside of the inner envelope (Heinz et al., 1989; Seifert and Heinz, 1992). The R. Sphaeroides .9qu gene belongs to glycosyltransferase gene family GT8 and was proposed to encode a potential SQDG synthase. The accumulation of UDP—SQ in the squ-inactivated mutant is consistent with the anticipated effect of blocking the assembly of SQDG (Rossak et al., 1995). The proposed function of SQDC is to provide 28 substrate specifity for SQDD based on accumulation of SQ-DHA in the .9qu mutant (Rossak et al., 1997). Alternatively, the function of SQDD is to synthesize SQ-DHA followed by the reduction of SQ-DHA and acylation at the 911-] and .9n-2 position of glycerol catalyzed by SQDC and the SQDA product. The involvement of SQDC and SQDD in SQDG assembly suggests that the fully functional SQDG synthase is a protein complex in some organisms. In cyanobaceteria, the SQDG synthase is encoded by the SQDX gene and the gene product belongs to the glycosyltransferase family GT4 (Gtiler et al., 2000). The SQDX protein contains a putative membrane spanning domain. The cyanobaterial SQDX is not similar to the d-group bacterial SQDD protein, suggesting that SQDG assembly may have evolved independently more than one time, while UDP- SQ biosynthesis evolved only once based on the conservation of the SQDB protein in all organisms. However, both GT4 and GT8 enzymes are retaining glycosyltransferases maintaining the anomeric proton configuration. Mechanistically, a two-step processes is involved, beginning with the formation of a glycosyl-enzyme intermediate, followed by the release of the nucleotide diphosphate and the subsequent attack of the glycosyl- enzyrne by the acceptor (Sinnott, 1991; McCarter and Withers, 1994). Therefore SQDX and SQDD probably have similar catalytic mechamsims despite their divergent amino acid sequences. In higher plants, much was known about UDP—SQ biosynthesis. On the contrary, although the activity of the sulfolipid synthase catalyzing the last step of sulfolipid biosynthesis had been fairly well characterized in spinach chloroplast extracts , the protein had not been isolated and the respective gene remained unidentified. 29 1.3.1.4. The biosynthesis of acyl-SQDG Recently, 2'-O—acyl-sulfoquinovosyldiacylglycerol (ASQD), an SQDG derivative, was identified in Creinhardtii (Riekhof et al., 2003). Unlike SQDG in this organisms, the acyl groups of ASQD predominantly are unsaturated fatty acids. Moreover, thel 8— carbon fatty acid with four double bonds directly attached to the head group of ASQD was the dominant acyl group. Inactivation of the C. rez‘nhardtii SQDl ortholog led to the absece of both lipids suggesting that ASQD is synthesized either by acylation of UDP- sulfoquinovose or by acylation of SQDG (Riekhof et al., 2003). It was proposed that SQDG is the substrate for acylation, because UDP-sulfoquinovose synthesis occurs in the plastid and the acyl groups found attached to the sulfoquinovose head group of ASQD appeared to be of extraplastidic origin (Riekhof et al., 2003). 1.3.2. The function of SQDG Since the discovery of SQDG, speculations about its function focused on its role in photosynthesis, based on the association of SQDG and photosynthetic membranes in photosynthetic organisms, combined with the unusual structure of the head group (Barber and Gounaris, 1986; Benning, 1993, 1998). With the SQDG-deficient mutants constructed by insertional inactivation of the sqd genes (Benning, 1992, 1993), as well as a mutant from the unicellular algae Chlamydomonas (Sato et al., 1995), the role of SQDG in photosynthetic organisms could be analyzed in detail. It was also found that SQDG has apparent antiviral and antitumor activities (Gustafson et al., 1989; Shirahashi et al., 1993). Recently, SQDG has been reported as a selective inhibitor of eukaryotic 30 DNA polymerases a and [3 (Ohta et al., 1998), and as an immunosuppressive agent (Matsumoto et al., 2002). 1.3.2.1. The function of SQDG in photosynthetic organisms The availability of SQDG-deficient mutants permitted us to test the correlation between the primary phenotype, SQDG deficiency, and secondary phenotypes such as photo- synthetic activity, growth, and so on (Benning, 1998). The detailed analysis of SQDG -deficient mutants showed that SQDG plays different roles in different organisms. In the oxygenic bacteria R. Sphaeroides, SQDG deficiency has no observed effect on growth of the bacterium under normal growth conditions (Benning et al., 1993). Photosynthetic electronic transfer was also not affected in these mutants. These results suggested that SQDG has no essential role in R. sphaeroidcs under conditions tested (Benning et al., 1993). In a SQDG-deficient mutants of the oxygenic cyanobacteria Synechococcus sp. PCC7942, subtle changes in the photosynthesis—related biochemistry of molecular oxygen were observed as well as a slight increase in the variable room temperature chlorophyll fluorescence yield, but the light response curves for oxygen evolution were not affected (Gfiler et a]. 1996). The growth of this mutant was not affected either. Thus neither the overall electron transport rate nor the optical cross section of oxygen evolution were effected by the deficiency of SQDG (Giiler et a]. 1996). In comparision, SQDG seems to play essential roles in Synechocystis. sp PCC6803, (Aoki et al., 2003). The SQDG~deficient mutant, named SDl, lacked the ability to synthesize SQDG and required SQDG supplementation for growth. After transfer from SQDG-supplemented to SQDG-free medium, SDl showed decreased net photosynthetic 31 and PSH activity on a chlorophyll basis proportionate to the decrease in SQDG (Aoki et al., 2003). The PSH activity of SDI was more sensitive to 3(3,4-dichlorophenyl)-1,1- dimethyurea (DCMU), which is binding to the QB acceptor site of P811 and therefy blocks PSII activity (Aoki et al., 2003). In Arabidopsis, transgenic plants with reduced amounts of SQDG expressing the SQDl cDNA in antisense orientation showed no obvious growth impairment (Essigmann et al., 1998). In the unicellular green alga C. rein/tardtii, SQDG seems to play an intermediate role. The SQDG deficient mutant exhibited a reduced growth rate, impaired photosynthetic activity and was more sensitive to DCMU (Minoda et al., 2002; Riekhof et al., 2003; Sato et al., 2003). The reasons for this discrepancy in the importance of SQDG among different organisms is not clear. One possible explanation is the evolutionary flexibility of photosynthetic membranes in terms of their lipid compositions, which could only require that a given combination of these lipids fomis a functional photosynthetic membrane. Consistent with the analysis of SQDG deficient mutants, biochemical analysis also suggests that SQDG plays different roles in PSII of different organisms. In C. reinhardtii, SQDG is associated with the PSII core complex and is part of light harvesting complex II (LHCII) (Sato et al., 1995; Sigris et al., 1988). In tobacco, SQDG was found in the outer surface of the Dl/D2 heterodimer where it was accessible to antibodies against SQDG (Radunz,and Schmid, 1992). It has also been found that SQDG is located in PSII core dimers in spinach (Murata et al., 1992; Kruse et al., 2000). But in preparations of light harvesting complex II trimers of pea chloroplast, PG in place of SQDG was found, and PG rather than SQDG was required for the crystallization of the complex (NuBberger et al., 1993). 32 Because of the presence of plastids in eukaryotic photosynthetic organisms, SQDG may play additional roles during the development or assembly of the photosynthetic apparatus. In fact, the interaction between SQDG and the transit peptide of a chloroplast protein has been observed. This suggests that the negatively charged lipid SQDG may be essential for protein import into the chloroplast (van’t Hof et al., 1993). It has been speculated that SQDG provided plant resistance to stress-factor action (Taran et al., 2000). It has been found that SQDG accumulated in response to cold temperature in l-year-old of cold temperature tolerant shoot bark of apple varieties and in Siberia apple. In response to high temperature, SQDG accumulated in resistant plants while the proportion of SQDG decreased in sensitive plants. It has been proposed that the role of SQDG in these phenomena could be the stabilization of photosynthetic processes, particularly ATP synthesis and light—harvesting complex 11 functioning, although the possibility that SQDG functions in signaling can not be ruled out either (Taran et al., 20001 1.3.2.2. Function of SQDG in non-photosynthetic organisms SQDG is also present in non-photosynthetic organisms, such as Bacillus acidocaldarius (Langworthy et al., 1976), halophilic bacterium (Sprott et al., 2003), several Caulobact‘er and Brevundimonas ssp and Sinorhizobium meliloti (Cedergreen and Hollingsworth, 1994). This raises the question of the function of SQDG in these organisms. Taking advantage of the cloning of genes involved in SQDG biosynthesis, a reverse genetic approach has been used to explore the function of SQDG in non- photosynthetic organisms. The inactivation of an squ-like gene in Sinorchozobium 33 meliloti led to SQDG deficiency (Weissenmayer et al., 2000). However, the SQDG- deficient mutant was still capable of producing functional nodules and did not show an obvious disadvantage under different laboratory culture conditions. Therefore, no specific roles for SQDG could be asigned in Sinorchizobium meliloti and it was suggested that sulfolipid, may become critical only under growth conditions that still need to be discovered (Weissenmayer et al., 2000). 1.3.3. Other potential applications for SQDG SQDG also possesses biological activities outside its natural environment due to its unique head group and its excellent detergent properties. It has been reported that SQDG purified from cyanobacteria functions as an antiviral agent against HIV (Gustafson et al., 1989) suggesting its potential application in medicine. SQDG also exhibits anti-tumor activities. The proliferation of human gastric cancer cell line SNU-l cells by 72 hours of culture in the presence of 100 ,uM and 1 mM SQDG was inhibited 24 and 100%, respectively, as compared with the number of SNU—l cells cultured in the absence of SQDG (Quasney et al., 2001). Inhibition of cell proliferation by 100 [tM sulfoquino- vosyldiacylglycerol was in part associated with apoptotic cell death. These results suggested that sulfolipid has intriguing potential as a chemopreventive or chemother- apeutic agent (Quasney et al., 2001). In addition, SQDG is a potential inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1. The inhibition was dose-dependent, and complete (more than 90%) inhibition of DNA polymerase or, DNA polymerase 13 and HIV-reverse transcriptase type 1 (HIV-RT) was observed at concentrations of 5, 10, and 30 MM, respectively (Ohta et al., 1998). These results 34 suggested the potential application of SQDG as an inhibitor in DNA polymerase research. Recently Matsumoto et al., (2002) reported the synthetic analogues of sulfolipids B-SQDG(sn-1, 18:0; sn-2, 18:0) could suppress the host's immunocompetence and prolonged the time before rat skin allograft rejection in vivo. There seemed to be no obvious adverse pathohistological effects in these rats. These results suggest that [3- SQDG could act as a new immunosuppressive reagent. 1.4. Phosphate regulation of lipid biosynthesis During their evolution, plants, like other organisms, have developed strategies to cope with various enviromnental stresses. The replacement of phospholipids by nonphosphorous lipids is one way by which plants deal with phosphate deprivation. This phenomena was first discovered in nonphotosynthetic bacteria and later in photo- synthetic organisms (Minnikin et al., 1974; Benning et al., 1993) and Arabidopsis (Essigmann et al., 1998). It has been shown that the acidic lipid SQDG can replace the acidic thylakoid lipid PG. Analyzing the phosphate-deficient p110] mutant of Arabidopsis, it was found that not only SQDG but also DGDG increased after phosphate deprivation. Thus it was proposed that the substitution of phospholipids by nonphosphorous lipids is a more general phenomenon in plants. Consistent with the increase of glycolipids, the genes involved in these lipid's biosynthesis, are also induced by phosphate stress, indicating the presence of a signal transduction cascade to regulate lipid metabolism in response to phosphate-limiting conditions (Essigmann et al., 1998; Kelly and Donnann, 2002). Further analysis showed that DGDG accumulated after phosphate-limitation, was also present in extraplastic membranes and possesed eukaryotic lipid structure (Hartel et 35 al., 2000). This also occurred in the act] mutant defection in the prokaryotic pathway (Hartel et al., 2000). These results suggested an increased contribution of the eukaryotic pathway to DGDG biosynthesis, therefore, a general redirection of galactolipid synthesis from the plastid to the eukaryotic pathway was present in phosphate-deprived plants. Further understanding of the redirection and the regulation of glycolipid biosynthesis could provide new insight into the control of lipid metabolisim. The observation that SQDG has no apparent essential role in some photosynthetic organisms gives rise to the question why the SQDG biosynthetic pathway is maintained in these organisms. One appealing idea is that SQDG is required to help these organisms to cope with specific environmental condition such as nutrient limitation (Benning, 1998). In support of this idea, it has been observed that the growth of SQDG-deficient mutants such as R. sphaeroides (Benning et al., 1993), cyanobacteria Synechococcus sp. PCC7942 (Giller et al., 1996) and C. rein/tardtii (Riekhof et al., 2003) was reduced under phosphate-limitation. Drastic changes in lipid composition also occurred in response to phosphate-limitation in these mutant as compared to wild type. Particularly interesting was the inverse relationship between two acidic/anionic lipids SQDG and PG. In the wild type grown under phosphate-limiting condition, the relative amount of SQDG was increased, while the amount of PG was decreased; in SQDG-deficient mutants, the relative amount of PG was maintained at the same level as under optimal growth conditions, although the changes in the relative amount of lipids other than SQDG were comparable with those of the wild-type. Based on these observations, the sulfolipid- phospholipid substitution hypothesis was proposed (Benning et al., 1993; ; Giller et al., 1996; Benning, 1998; Riekhof et al., 2003). The sum of the two major anionic lipids, 36 SQDG and PG, was proposed to be critical for photosynthetic membrane development and function. Photosynthetic organisms are able, within limitations, to maintain a constant amount of total anionic thylakoid lipids by reciprocally adjusting the sulfolipid and phosphatidylglycerol contents as phosphate availability changes. Considering that one third of organic phosphate is normally bound in the membrane in the wild-type (Poirier et 1., 1991), sulfolipid-deficient null mutants exhaust their phosphate reserves sooner than the corresponding wild types, resulting in reduced growth under these conditions (Benning, 1998). This reverse relationship is not only present under phosphate stress, it also happens under other conditions. For instance, under sulfur-deprived condition, the proportion of PG is increased while SQDG is decreased in C. reinhardtii (Sato et al., 2000). These facts suggest that sulfolipid/phosphatidylglycerol substitution may be ubiquitous to photosynthetic organisms containing these lipids. It should be pointed out that SQDG can only partially substitute for PG which, unlike sulfolipid, is prominently present in the crystal structure of PSI (Jordan et al., 2001). Recently, Aoki et al., (2003) showed that the proportion of PG is increased in the Synechocystis sp PCC6803 SQDG deficient mutant SDl, but it can not fully complement the loss of SQDG suggesting that SQDG also has some unique function in this organism which can’t be replaced by PG. 37 1.5 Overview Many aspects of the biosynthesis and function of thylakoid lipids in higher plants are still unclear. In the current dissertation, efforts to use Arabidopsis as a model system for understanding the biosynthesis and function of two major anionic lipids, SQDG and PG, and for dissecting the role of plastidic lyso-PA acyltransferase (pLPAAT) in plants are described. As discussed above, our understaning of the plant SQDG biosynthetic pathway was previously incomplete due to the unidentified SQDG synthase. Therefore my first goal was to identify the SQDG synthase from Arabidopsis and to propose a complete SQDG biosynthetic pathway. Because plant chloroplast shares a common ancestor with cyanobacteria, it seemed possible that plants possess an ortholog of SQDX, the SQDG synthase of cyanobacteria. Therefore, a combination of reverse genetics and heterozygous expression in E. coli was employed to identify the SQDG synthase in Arabidopsis as described in Chapter 2. It was shown that a reduced level of SQDG has no apparent effect on the growth of Arabidopsis. Thus I postulated that like in bacteria (Benning et al., 1993; Gtiler et al., 1996), SQDG may play an essential role under phosphate-limitation in plants as well. As described in Chapter 2, this hypothesis was tested utilizing an SQDG-deficient mutant. At the beginning of my thesis work, most genes involved in chloroplast PG biosynthesis were not known, and the specific function of PG in photosynthesis was unclear. A forward genetic approach was used to screen for a mutant deficient in PG, which would presumably arise from disrruption of the PG biosynthetic pathway. In 38 Chapter 3, I describe the isolation of such a mutant. This mutant was further employed to dissect chloroplast PG biosynthesis as well as the function of PG in photosynthesis. The observation of the inverse relationship between SQDG and PG led to the hypothesis that the function of SQDG is to replace PG under phosphate-limitation to maintain a constant amount of anionic lipids. To test the SQDG/PG substitution hypothesis and to understand the essential role of a constant proportion of anionic lipids in photosynthesis and chloroplast development, an SQDG-deficient mutant described in Chapter 2 and a partially PG-deficient mutant, described in Chapter 3, were used to construct a double mutant, which was hypothesized to contain a reduced amounts of total anionic lipids . In Chapter 4, I describe the results of my work utilizing this double mutant to address the questions discussed above. Phosphatidic acid is a key intermediate for chloroplast lipid biosynthesis. At the beginning of my thesis work, the gene for plastidic LPAAT had not been identified in Arabidopsis. The in vivo role of LPAAT was also unkown in plants. A reverse genetic approach was used to address this problem. In Chapter 5, the identification of a plastidic LPAAT, and a detailed analysis of the in vivo function of LPAAT are described. Inactivation of a plastidic G-3-P acyltransferase in the ats] (act!) mutant led to the loss of the “prokaryotic” pathway for glycolipid biosynthesis. However, despite the drastic effects on "prokaryotic”glycolipid biosynthesis, PG biosynthesis was only mildly impaired in the ats] (actI) mutant. This observation made the origin of “prokaryotic” PA unclear. The LPAAT-deficient mutant was used to address the origin of “prokaryotic” PA as described in Chapter 5. 39 Reference Andrews J, Mudd JB (1985) Phosphatidylglycerol synthesis in pea chloroplasts. Pathway and localization. Plant Physiol. 79:259-265. Araki S, Eichenberger W, Sakurai T, Sato N. (1991) Distribution of Diacylgly— cerylhydroxyrnethyltrimethylalanine (DGTA) and phosphatidylcholine in brown algae. Plant Cell Physiol. 32:623—628. Awai K, Marechal E, Block MA, Brun D, Masuda T, Shimada H, Takamiya K, Ohta H, Joyard J. (2001) Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and. nonphotosynthetic tissues in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 98:10960-10965. Barber GA. (1963) The formation of uridinediphosphate L-rhamnose by enzymes of the tobacco leaf. Arch. Biochem. Biophys. 103:276—282. Barber J, Gounaris K. (1986) What role does sulpholipid play within the thylakoid membrane? Photosynth. Res. 9:239—249. Benning C, Somerville CR (1992a) Isolation and genetic complementation of a sulfolipid-deficient mutant of Rhodobacter sphaeroides. J. Bacteriol. 174:2352-60. Benning C, Somerville CR. (1992b) Identification of an operon involved in sulfolipid biosynthesis in Rhodobacter sphaeroides. J. Bacteriol. 174:6479—6487. Benning C. (1998) Biosynthesis and function of the sulfolipid sulfoquinovosyl- diacylglycerol. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:53-75. Benning C, Beatty JT, Prince RC, Somerville CR. (1993) The sulfolipid sulfoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances grth under phosphate limitation. Proc. Natl. Acad. Sci. USA 90:1561-1565. Benson AA, Daniel,H, Wiser R. (1959) A sulfolipid in plants. Proc. Natl. Acad. Sci. USA 11:1582-1587. Bertrams M, Heinz E. (1981) Positional specificity and fatty acid selectivity of purified sn-glycerol 3-phosphate acyltransferase from chloroplasts. Plant Physiol. 68: 653— 657. Bhella RS, Mackenzie SL. (1994) Nucleotide sequence of a cDNA from Carthamus tinctorius encoding a g1ycerol-3-phosphate acyltransferase. Plant Physiol. 106: 1713—1714. 40 Bourgis F, Kader J C, Barret P, Renard M, Robinson D, Robinson C, Delseny M, Roscoe TJ ( 1999) A plastidial lysophosphatidic acid acyltransferase from oilseed rape. Plant Physiol. 120:913-922. Browse J, Somervllle C. (1991) Glycerolipid metabolism, biochemistry and regulation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:467-506. Cain BD, Donohue TJ, Shepherd WD, Kaplan S. (1984) Localization of phospholipid biosynthetic enzyme activities in cell-free fractions derived from Rhodopseudomonas sphaeroides. J. Biol. Chem. 259:942-948. Cedergreen RA, Hollingsworth RI. ( 1994) Occurrence of sulfoquinovosyl diacylglycerol in some members of the family Rhizobiaceae. J. Lipid Res. 35:1452—1461 Chang SC, Heacock PN, Clancey CJ, Dowhan W. (1998) The PELl gene (renamed PGSl) encodes the phosphatidylglycerophosphate synthase of Saccharomyces cerevisiae. J. Biol. Chem. 273:9829—9836 Cline K, Keegstra K. (1983) Galactosyltransferases involved in galactolipid biosynthesis are located in the outer membrane of pea chloroplast envelopes. Plant Physiol. 71 2366-3 72. Dembitsky VM, Pechenkina-Schubina EE, Rozentsvet OA. (1991) Glycolipids and fatty acids of some sea weeds and marine grasses from the black sea. Phytochemistry 30:2279—2283. Dembitsky VM, Rozentsvet OA, Pech-enkina EE. (1990) Glycolipids, phospho-lipids and fatty acids of brown algae species. Phytochemistry 29:3417—343 1. Dormann P, Balbo I, Benning C. (1999) Arabidopsis galactolipid biosynthesis and lipid trafficking mediated by DGDl. Science 284:2181-2184. Ddrmann P, Benning C (2002) Galactolipids rule in seed plants. Trends Plant Sci. 7:112- 118. Dome AJ, Block MA, J oyard J and Douce R (1982) The galactolipid:galactolipid galactosyltransferase is located on the outer membrane of the chloroplast envelope. FEBS Lett. 145230-34. Dome AJ, J oyard J, Block MA, Douce R. (1985) Localization of phosphatidylcholine in outer envelope membrane of spinach chloroplasts. J. Cell Biol. 100:1690-1697. Dryden SC, Dowhan W . (1996) Isolation and expression of the Rhodobacter sphaeroides gene (pgsA) encoding phosphatidylglycerophosphate synthase. J. Bacteriol. 178: 1030-1038. 41 Essigmann B, Giiler S, Narang R A, Linke D, Benning C. (1998) Phosphate availability affects the thylakoid lipid composition and the expression of SQD] , a gene required for sulfolipid biosynthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 95:1950-1955. Frentzen M, Griebau R (1994) Biosynthesis of cardiolipin in plant mitochondria. Plant Physiol. 106: 1527-1532. Frentzen M, Heinz E, Mckeon TA, and Stumpf PK. (1983) Specificities and selectivities of glycerol-3 -phosphate acyltransferase and monoacylglycerol-3—phosphate acyltransferase from pea and spinach chloroplasts. Eur. J. Biochem. 129:629—636. Frentzen M, Nishida I, and Murata N. (1987) Properties of the plastidial acyl-(acyl- carrier-protein): glycerol—3-phosphate acyltransferase from the chilling-sensitive plant squash (Cucurbita moschata). Plant Cell Physiol. 28:1195—1201. Froehlich J E, Benning C, Dorrnann P. (2001) The digalactosyldiacylglycerol (DGDG) synthase DGDl is inserted into the outer envelope membrane of chloroplasts in a manner independent of the general import pathway and does not depend on direct interaction with monogalactosyldiacylglycerol synthase for DGDG biosynthesis. J. Biol. Chem. 276:31806-12. Gombos Z, Vérkonyi Z, Hagio M., Iwaki M., Kovacs L., Masamoto K, Itoh S. and Wada H. (2002) Phosphatidylglycerol requirement for the function of electron acceptor plastoquinone QB in the photosystem II reaction center. Biochemistry 41: 3796—3 802. Giiler S, Seeliger A, Harte] H, Renger G, Benning C. (1996) A null mutant of Synechococcus sp. PCC7942 deficient in the sulfolipid sulfoquinovosyldiacylglycerol. J. Biol. Chem. 271:7501-7507. Gtiler S, Essigmann B, Benning C. (2000) A cyanobacteria] gene, squ, required for biosynthesis of the sulfolipid sulfoquinovosyldiacylglycerol. J. Bacteriol.182:543- 545. Gustafson KR, Cardellina JH 2nd, Fuller RW, Weislow OS, Kiser RF, Snader KM, Patterson GM, Boyd MR. (1989) AIDS-antiviral sulfolipids from cyanobacteria (blue-green algae). J. Natl. Cancer. Inst. 81:1254-8. Haas R, Siebertz HP, Wrage K, Heinz E. (1980) Localization of sulfolipid labeling within cells and chloroplasts. Planta 148:238—244 Haines TH. (1973). Sulfolipids and halo-sulfolipids. In Lipids and Biomembranesof Eucaijyotic Microorganisms, ed. J A Er-win, pp. 197—232. New York: Academic press 42 Hagio M, Gombos Z, Varkonyi Z, Masamoto K, Sato N, Tsuzuki M, Wada H. (2000) Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis Plant Physiol. 124:795—804. Harte] H, Essigmann B, Lokstein H, Hoffmann-Benning S, Peters-Kotti g M, Benning C. (1998) The phospholipid-deficient phol mutant of Arabidopsis thaliana is affected in the organization, but not in the light acclimation, of the thylakoid membrane. Biochim. Biophys. Acta 1415: 205-218. Heinz E, Schmidt H, Hoch M, Jung KH, Binder H, Schmidt RR. (1989) Synthesis of different nucleoside 5'-diphospho-sulfoquinovoses and their use for studies on sulfolipid biosynthesis in chloroplasts Eur. J. Biochem. 184:445-53. Heinz E, Roughan G. (1983) Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants. Plant Physiol. 72:273—279 Hoppe W, Schwenn JD. (1981) In vitro biosynthesis of the plant sulpholipid: on the origin of the sulphonate head group. Z. Naturforsch. Teil. C. 362820—26 Ishizaki-Nishizawa O, Azuma M, Ohtani T, Murata N, Toguri T. (1995) Nucleotide sequence of cDNA from Spinacia oleracea encoding plastid glycerol-3—phosphate acyltransferase. Plant Physiol. 108: 1342 Ishizaki O, Nishida I, Agata K, Eguchi G, Murata N. (1988) Cloning and nucleotide sequence of cDNA for the plastid glycerol-3-phosphate acyltransferase from squash. FEBS Lett. 238:424—430. Isono Y, Mohri H, Nagai Y. (1967) Effect of egg sulpholipid on respiration of sea urchin spermatozoa. Nature 214:1336—1338. Jarvis P, Dormann P, Peto CA, Lutes J, Benning C, Chory J .. (2000) Galactolipid deficiency and abnormal chloroplast development in the Arabidopsis MGD synthase 1 mutant. Proc. Natl. Acad. Sci. USA 97:8175-8179. J iang F, Rizavi HS, Greenberg ML. (1997). Cardiolipin is not essential for the growth of Saccharomyces cerevisiae on fermentable or non-fermentable carbon sources. Mol. Microbiol. 26:481-491. Jonson TC, Schneider J C, and Somerville C. (1992) Nucleotide sequence of acyl-acyl carrier protein: glycerol-3-phosphate acyltransferase from cucumber. Plant Physiol. 99:771—772. J orasch P. et al. (2000) Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, g1yc0phospholipids, glycosphingolipids and glycosylsterols. Eur. J. Biochem. 267:3770-3783. 43 Jordan BR, Chow W-S, Baker AJ (1983) The role of phospholipids in the molecular organisation of pea chloroplast membranes: effect of phospholipid depletion on photosynthetic activity. Biochim. Biophys. Acta 725: 77-86. Joyard J, Blee E, Douce R. (1986) Sulfolipid synthesis from 35SO42' and [1-14 C] acetate in isolated spinach chloroplast. Biochim. Biophys. Acta 879:78—87. Joyard J, Block MA, Douce R. (1991) Molecular aspects of plastid envelope biochemistry. Eur. J. Biochem.199:489-509. J oyard J, Block MA, Malherbe A, Marachal E, and Douce R. (1993) Origin and synthesis of galactolipid and sulfolipid head-groups. In Lipid Metabolism in Plants, Moore TS, Jr., ed (Boca Raton, FL: CRC Press), pp. 231-258. Joyard J, Marechal E, Miege C, Block MA, Dome AJ, Douce R. (1998a) Structure, distribution and biosynthesis of glycerolipids from higher plant chloroplasts. In Siegenthaler,P.A. and Murata,N. (eds), Lipids in Photosynthesis: Structure, Function and Genetics. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 21—52. J oyard J, Teyssier E, Miege C, Bemy—Seigneurin D, Marechal E, Block MA, Dome AJ, Rolland N, Ajlani G, Douce R (1998b) The biochemical machinery of plastid envelope membranes. Plant Physiol. 118:715-723. Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, et al. (1996) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. Strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein- coding regions. DNA Res. 3: 109—136. Kawasaki K, Kuge O, Chang SC, Heacock P.N, Rho M, Suzuki K, Nishijima M, Dowhan W; (1999) Isolation of a chinese hamster ovary (CHO) cDNA encoding phosphatidylglycerophosphate (PGP) synthase, expression of which corrects the mitochondrial abnormalities of a PGP synthase-defective mutant of CHO-K1 cells. J. Biol. Chem. 274: 1 828—1 834. Kelly AA, Dormann P. (2002) DGD2, an Arabidopsis Gene Encoding a UDP-Galactose- dependent Di galactosyldiacylglycerol Synthase Is Expressed during Growth under Phosphate-limiting Conditions J. Biol. Chem. 277: 1166 - 1173. Kikuchi S, Shibuya I, and Matsumoto K. (2000) Viability of an Escherichia coli pgsA null mutant lacking detectable phosphatidylglycerol and cardiolipin. J. Bacteriol. 182:371—376. Kinney AJ (1993) Phospholipid head groups. In Moore TS, ed, Lipid Metabolism in Plants. CRC Press, Boca Raton, FL, pp 259-284. 44 Kleppinger-Sparace KF, Mudd JB, Bishop DG. (1985) Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants: the incorporation of 35804 by intact chloroplasts. Arch. Biochem. Biophys. 240:859-865. Kleppinger-Sparace KF, Mudd JB. (1990) Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants. Use of adenosine-50 -phosphosulfate and adenosine-30 -phosphate- 50 -phosphosulfate as precursors. Plant Physiol. 93:256—263. Kopka J, Ludewig M, Mfiller-Rdber B (1997) Complementary DNAs encoding eukaryotic-type cytidine-S'-diphosphate-diacylglycerol synthases of two plant species. Plant Physiol. 113: 997-1002. Kruse O, Hankamer B, Konczak C, Gerle C, Morris E, Radunz A, Schmid GH, Barber J. (2000) Phosphatidylglycerol is involved in the dimerization of photosystem II. J. Biol. Chem. 275:6509 —6514. Kunst L, Browse J, Somerville, C. (1988) Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol phosphate acyltransferase activity. Proc. Natl. Acad. Sci.USA 85:4143-4147. Langworthy TA, Mayberry WR, Smith PF. (1976) A sulfonolipid and novel glucosamidyl glycolipids from the extreme therrnoacidophile Bacillus acidocaldaius. Biochim. Biophys. Acta 431 :550—69. Liu JM., Chen SN, Yan B, Huang XQ, Yang MZ. (1999) Cloning and sequencing of the cDNA coding for glycerol-3-phosphate acyltransferase from Viciafaba. Plant Physiol. 120: 934. Lyons JM. (1973) Chilling injury in plants. Annu. Rev. Plant Physiol. 24:445-466 Marechal E, Block MA, Dome AJ, J oyard J (1997) Lipid synthesis and metabolism in the plastid envelope. Physiol. Plant 100:65-77 Manaf AM, Harwood J L. (2000) Purification and characterisation of acyl-CoA: glycerol 3-phosphate acyltransferase from oil palm (Elaeis guineensis) tissues. Planta 210: 318—328. Matsumoto Y, Sahara H, Fujita T, Hanashima S, Yamazaki T, Takahashi S, Sugawara F, Mizushina Y, Ohta K, Takahashi N, J imbow K, Sakaguchi K, Sato N. (2000) A novel immunosuppressive agent, SQDG, derived from sea urchin. Transplant Proc. 32 :205 1-3. McCarter JD., Withers SG. (1994) Mechanisms of enzymatic glycoside hydrolysis. Curr. Op. Struct. Biol. 4:885-892 45 Miege C. et al. (1999) Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG. Eur. J. Biochem. 265:990-1001. Minnikin DE, Abdolrahimzadeh H, Baddiley J. (1974) Replacement of acidic phosphates by acidic glycolipids in Pseudomonas diminuta. Nature (London) 249:268-269. Minskoff SA, Greenberg ML. (1997) Phosphatidylglycerophosphate synthase from yeast. Biochim. Biophys. Acta 1348:187—191. Mongrand S, Bessoule JJ, Cassagne C. (1997) A re-examination in vivo of the phosphatidylcholine-galactolipid metabolic relationship during plant lipid biosynthesis. Biochem. J. 327:853-8. Mongrand S, Cassagne C, Bessoule J J (2000) Import of lyso-phosphatidylcholine into chloroplasts likely at the origin of eukaryotic plastidial lipids. Plant Physiol. 122:845-852. Moon BY, Higashi S, Gombos Z, Murata N. (1995) Unsaturation of the membrane lipids of chloroplasts stabilizes the photosynthetic machinery against low-temperature photoinhibition in transgenic tobacco plants. Proc. Natl. Acad. Sci USA. 9226219- 6223. Moore TS Jr (1982) Phospholipid biosynthesis. Annu. Rev. Plant Physiol. 33:235-259. Mulichak AM, Theisen MJ, Essigmann B, Benning C, Garavito RM. (1999) Crystal structure of SQDl , an enzyme involved in the biosynthesis of the plant sulfolipid headgroup donor UDP-sulfoquinovose. Proc. Natl. Acad. Sci. USA 96:13097- 13102. Murata N. (1983) Molecular species composition of phosphatidylglycerols from chilling- sensitive and chilling-resistant plants. Plant Cell Physiol. 24:81-86. Muller F, Frentzen M. (2001) Phosphatidylglycerophosphate synthases from Arabidopsis thaliana. FEBS Lett. 509:298-302. Murata N, Higashi S. Fujimura Y.(1990a) Glycerolipids various preparations of photosystem II from spinach chloroplasts. Biochim. Biophys. Acta10192261 —268. Murata N, Ishizaki-Nishizawa O, Higashi S, Hayashi H, Tasaka Y, Nishida I. (1992) Genetically engineered alteration in the chilling sensitivity of plants. Nature 356: 710—713. Murata N, Nishida I (1990b) Lipids in relation to chilling sensitivity of plants. In Chilling Injury of Horticultural Crops, C.Y. Wang, ed (Boca Raton, FL: CRC Press), pp. 181- 199. 46 Murata N, Yamaya J. (1984) Temperature-dependent phase behavior of phosphatidylglycerols from chilling-sensitive and chilling resistant plants. Plant Physiol. 74: 1016-1024. Nussberger S, Dorr K, Wang DN, Kuhlbrandt W. (1993) Lipid-protein interactions in crystals of plant lightharvesting complex. J. Mol. Biol. 234:347—56 Ohlrogge J, Browse J. (1995) Lipid Biosynthesis. Plant Cell 7: 957-970 Ohta A, Waggoner K, Radominska—Pyrek A, Dowhan W. (1981) Cloning of genes involved in membrane lipid synthesis: effects of amplification of phosphatidylglycerophosphate synthase in Escherichia coli. J. Bacteriol. 147:552— 562 Ohta K, Mizushina Y, Hirata N, Takemura M, Sugawara F, Matsukage A, Yoshida S, Sakaguchi K. (1998) Sulfoquinovosyldiacylglycerol, KM043, a new potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1 from a marine red alga, Gigartina tenella. Chem. Pharrn. Bull (Tokyo) 46:684-686. Ostrander DB, Zhang M, Mileykovskaya E, Rho M, Dow-han W. (2001) Lack of mitochondrial anionic phospholipids causes an inhibition of translation of protein components of the electron transport chain. A yeast genetic model system for the study of anionic phospholipid function in mitochondria. J. Biol. Chem. 276225262— 25272. Poirier Y, Thoma S, Somerville C, Schiefelbein J. (1991) Mutant of Arabidopsis deficient in xylem loading of phosphate. Plant Physiol. 97: 1087-1 109 Ponting CP, Kerr ID. (1996) A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: Identification of duplicated repeats and potential active site residues. Protein Sci. 5: 914-922 Pugh CE, Roy AB, Hawkes T, Harwood J L. (1995) A new pathway for the synthesis of the plant sulpholipid, sulphoquinovosyldiacylglycerol. Biochem. J. 3092513—521 Quasney ME, Carter LC, Oxford C, Watkins SM, Gershwin ME, German JB. (2001) Inhibition of proliferation and induction of apoptosis in SNU-l human gastric cancer cells by the plant sulfolipid, sulfoquinovosyldiacylglycerol. J. Nutr. Biochem. 12:3 10-3 15. Radcliffe CW, Broglie RM, Niederrnan RA. (1985) Sites of phospholipid biosynthesis during induction of intracytoplasmic membrane formation in Rhodopseudomonas sphaeroides. Arch. Microbiol. 142:136-40. Raison J K. (1973) The influence of temperature-induced phase changes on the kinetics of respiratory and other membrane associated enzyme systems. Bioenergetics 4:285- 309. 47 Riekhof WR, Ruckle ME, Lydic TA, Sears BB, Benning C. (2003) The Sulfolipids 2'-O -Acy1-Sulfoquinovosyldiacylglycerol and Sulfoquinovosyldiacylglycerol Are Absent fiom a Chlamydomonas reinhardtii Mutant Deleted in SQDl. Plant Physiol. 133:864-874. Rossak M, Tietje C, Heinz E, Benning C. (1995) Accumulation of UDP-sulfoquinovose in a sulfolipid-deficient mutant of Rhodobacter sphaeroides. J. Bio. Chem. 270:25792—25797. Rossak M, Schafer A, Xu NX, Gage DA, Benning C. (1997) Accumulation of sulfoquinovosyl—1-O-dihydroxyace-tone in a sulfolipid-deficient mutant of Rhodobacter sphaeroides inactivated in squ. Arch. Biochem. Biophys. 3402219— 230. Roughan PG, Slack CR. (1982) Cellular organization of glycerolipid metabolism. Annu. Rev. Plant Physiol. 33197-132. Roughan PG. (1985) Phosphatidylglycerol and chilling sensitivity in plants. Plant Physiol. 77:740-746. Sanda S, Leustek T, Theisen MJ, Garavito RM, Benning C. (2001) Recombinant Arabidopsis SQDl converts udp-glucose and sulfite to the sulfolipid head group precursor UDP-sulfoquinovose in vitro. J. Biol. Chem. 276:3941-3946. Sato N, Hagio M, Wada H, Tsuzuki M. (2000) Environmental effects on acidic lipids of thylakoid membranes. Biochem. Soc. Trans. 28:912-914. Sato N, Hagio M, Wada H, Tsuzuki M. (2000) Requirement of phosphatidylglycerol for photosynthetic firnction in thylakoid membranes. Proc. Natl. Acad. Sci. USA 97:10655-10660. Sato N, Sonoike K, Tsuzuki M, Kawaguchi A. (1995) Impaired photosystem H in a mutant of Chlamydomonas reinhardtii defective in sulfoquinovosyldiacylglycerol. Eur. J. Biochem. 234:16—23. Schmid VHR, Cammarata KV, Bruns BU, Schmidt GW (1997) In vitro reconstruction of the photosystem I light-harvesting complex LHCI-730: Heterodimerization is required for antenna pigment organization. Proc. Natl. Acad. Sci. USA 94: 7667- 7672. Seifert U, Heinz E. (1992) Enzymatic characteristics of UDP sulfoquinovose: diacylglycerol sulfoquinovosyltransferase from chloroplast envelopes. Bot. Acta 105:197—205. 48 Shibuya I, Yagi T, Benson AA. (1963) Sulfonic acids in algae. In Microalgae and Photosynthetic Bacteria, ed. Jpn. Soc. Plant Physiol. 627—36. Tokyo: Univ.Tokyo Press Shimojima M, Benning C. (2003) Native uridine 5'-diphosphate-sulfoquinovose synthase, SQDI, from spinach purifies as a 250-kDa complex. Arch. Biochem. Biophys. 413:123-130. Shirahashi H, Murakami N, Watanabe M, Nagatsu A, Sakakibara J, Tokuda H, Nishino H, Iwashima A. (1993) Isolation and identification of antitumor-promoting principles from the fresh-water cyanobacterium Phonnidium tenue. Chem. Pharm. Bull (Tokyo). 41 :1664-1666. Siegenthaler PA, Smutny J, Rawler A (1987) Involvement of distinct populations of phosphatidylglycerol and phosphatidylcholine molecules in photosynthetic electron- flow activities. Biochim. Biophys. Acta. 891:85-93. Sigrist M, Zwilliengerg C,Giroud CH, Eichenberger W, Boschetti A. (1988) Sulfolipid associated with the li ht-harvesting complex associated with photosystem II apoproteins of Chlamydomonas reinhardtii. Plant Sci. 58:15 —23. Sinnott ML. (1991) Catalytic mechanisms of enzymic glycoside transfer. Chem. Rev. 90:1170-1202. Slabas T. (1997) Galactolipid biosynthesis genes and endosymbiosis. Trends Plant Sci. 2:161-162 Slack CR, Roughan PG, Balasingham N. (1977) Labelling studies in vivo on the metabolism of the acyl and glycerol moieties of the glycerolipids in the developing maize leaf. Biochem. J. 162: 289-96. Sprott GD, Larocque S, Cadotte N, Dicaire CJ, McGee M, Brisson JR. (2003) Novel polar lipids of halophilic eubacterium Planococcus H8 and archaeon Haloferax volcanii. Biochim. Biophys. Acta 1633:179-88. Taran N, Okanenko A, Musienko N. (2000) Sulpholipid reflects plant resistance to stress- factor action. Biochem. Soc. Trans. 28:922-924. Tropp BE. (1997) Cardiolipin synthase from Escherichia col. Biochim. Biophys. Acta 1348: 192—200. Tuller G, Hrastnik C, Achleitner G, Schiefthaler U, Klein F, Daum G. (1998) YDL142c encodes cardiolipin synthase (Clslp) and is nonessential for aerobic grth of Saccharomyces cerevisiae. FEBS Lett. 421215-18. 49 van Besouw A, Wintermans J FGM. (1978) Galactolipid formation in chloroplast envelopes. 1. Evidence for two mechanisms in galactosylation. Biochim. Biophys. Acta 529144-53. van’t Hof R, van Klompenburg W, Pi-lon M, Kozubek A, de Korte-Kool G, et al. (1993) The transit sequence mediates the specific interaction of the precursor of ferredoxin with chloroplast envelope lipids. J. Biol. Chem. 268:403 7—4042. Vij ayan P, Browse J. (2002) Photoinhibition in mutants of Arabidopsis deficient in thylakoid unsaturation. Plant Physiol. 129:876-885. V013,R.,Radunz,A, Schmid GH.(1992) Bindin of lipids onto polypeptides of the thylakoid membrane.I.Galacto-lipids and sulfolipid as prosthetic roups of core peptides of the photosystem II complex. Z. Naturforsch 47c2406 —415. Weber S, Wolter F P, Buck F, Frentzen M and Heinz E. (1991) Purification and cDNA sequencing of an oleate-selctive acyl-ACP: sn-glycerol-3-phosphate acyltransferase from pea chloroplasts. Plant Mol. Biol. 17: 1067—1076. Williams JP, Imperial V, Khan MU, Hodson JN (2000).The role of phosphatidylcholine in fatty acid exchange and desaturation in Brassica napus L. leaves. Biochem. J. 349127-349133. Xu C, Fan J, Riekhof W, Froehlich J E, Benning C. (2003) A permease-like protein involved in ER to thylakoid lipid transfer in Arabidopsis. EMBO J. 22:2370-23 79. Yokoi S, Higashi S, Kishitani S, Murata N, Toriyama K. (1998) Introduction of the cDNA for Arabidopsis glycerol-3-phosphate acyltransferase (GPAT) confers unsaturation of fatty acids and chilling tolerance of photosynthesis on rice. Mol. Breed 4: 269—275. Yu B, Xu C, Benning C. (2002) Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth. Proc. Natl. Acad. Sci. USA 99:5732-5737. 50 Chapter 2 Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth The work presented in this chapter has been published: Yu B, Xu C, Benning C. (2002) Proc. Natl. Acad. Sci. USA 99:5732-7. 51 Abstract The sulfolipid sulfoquinovosyldiacylglycerol is one of the three non-phosphorous glycolipids that provide the bulk of the structural lipids in photosynthetic membranes of seed plants. Unlike the galactolipids, sulfolipid is anionic at physiological pH due to its 6- deoxy-6-su1fonate glucosyl (sulfoquinovose) head group. The biosynthesis of this lipid proceeds in two steps, first, the assembly of UDP-sulfoquinovose from UDP-glucose and sulfite and second, the transfer of the sulfoquinovose moiety from UDP-sulfoquinovose to diacylglycerol. The first reaction is catalyzed by the SQDl protein in Arabidopsis. Here we describe the identification of the SQD2 gene of Arabidopsis. We propose that this gene encodes the sulfoquinovosyltransferase catalyzing the second step of sulfolipid biosynthesis. Expression of SQD] and SQD2 in Escherichia coli reconstituted plant sulfolipid biosynthesis in this bacterium. Insertion of a T-DNA into this gene in Arabidopsis led to complete lack of sulfolipid in the respective sqd2 mutant. This mutant showed reduced growth under phosphate-limited growth conditions. The results support the hypothesis that sulfolipid can function as a substitute of anionic phospholipids under phosphate-limited growth conditions. Along with phosphatidylglycerol, sulfolipid contributes to maintaining a negatively charged lipid-water interface, which is presumably required for proper function of photosynthetic membranes. 52 Introduction The photosynthetic membranes of seed plants are rich in non-phosphorous glycolipids. These include the galactolipids mono- and digalactosyldiacylglycerol and the sulfolipid sulfoquinovosyldiacylglycerol. This lipid is unusual, because its polar head group consists of sulfoquinovose, a 6-deoxy-6-sulfonate-glucose. Much has been speculated about the possible function of this lipid in photosynthesis (Barbar and Gounaris, 1986; Benning, 1998), but in recent years the isolation of sulfolipid-deficient bacterial mutants (Benning et al., 1993; GUler et al., 1996) and a mutant of the unicellular algae Chlamydomonas has provided some novel clues (Sato et al., 1995). It has become apparent from these mutants that there is no essential role for sulfolipid in photosynthetic bacteria with anoxygenic or oxygenic photosynthesis, or in eukaryotic cells containing chloroplasts, because all mutants were capable of photoautotrophic growth. The effects on photosynthesis are subtle under normal growth conditions, at best. However, if bacterial sulfolipid-deficient null mutants are starved for phosphate, they cease growth much earlier than the respective wild type (Benning et al., 1993; GUler et al., 1996). Thus, sulfolipid is of conditional importance in these bacteria. Like in bacteria, the ratio of non-phosphorous g1ycolipids-to-phospholipids drastically increases in Arabidopsis upon phosphate deprivation (Essigmann et al., 1998; Harte] et al., 2000). In particular, the relative amount of sulfolipid rises several fold due to an active process based on the increased expression of at least one of the sulfolipid genes, SQD] (Essigmann et al., 1998). Therefore, it seems likely that sulfolipid is of conditional importance also in Arabidopsis and other plants. 53 Figure 2.1. The pathway for sulfolipid biosynthesis in Arabidopsis. Two enzymes, SQDl and SQD2, are specific to this process and catalyze the reactions as indicated. DAG, diacylglycyerol; R, fatty acyl groups; SQDG, sulfoquinovosyldiacylglycerol; UDP-Glc, UDP-glucose; UDP-SQ, UDP—sulfoquinovose. 54 2 o CH,so; HO 0 H0 0H0 H0 \UDP so3 \UDP o o l | R, R1 UDP-GIG UDP-SQ DAG 55 The unique step in sulfolipid biosynthesis is the synthesis of UDP-sulfoquinovse, the sulfolipid head group donor (Fig. 2.1), catalyzed by the SQDl protein in Arabidopsis (Sanda et al., 2001). The crystal structure for this protein is known and a reaction mechanism has been proposed (Essigmann et al., 1999; Mulichak et al., 1999). On the contrary, although the activity of the sulfolipid synthase catalyzing the last step of sulfolipid biosynthesis has been fairly well characterized in spinach chloroplast extracts (Seifert and Heinz, 1992), the protein had not been isolated and the respective gene remained unidentified. Orthologs of the Arabidopsis SQD] gene are highly conserved in different groups of bacteria and even in plants (Benning, 1998). However, two unrelated classes of genes encode the UDP-sulfoquinovose-dependent glycosyltransferases responsible for the final assembly of sulfolipid in the bacteria of the a-group (Rossak et al., 1995) and cyanobacteria (Giller et al., 2000). Because it seemed possible that plants inherited their sulfolipid synthase from the postulated cyanobacterial ancestor of chloroplasts, we searched for an ortholog of the cyanobacterial squ gene in the fully sequenced Arabidopsis genome. As a result of this genomic approach, the correct Arabidopsis ortholog of squ, designated SQD2, was identified, characterized, and exploited to answer open questions about sulfolipid biosynthesis and function in seed plants. 56 Materials and methods Plant Material and Plant Growth Experiments. Surface sterilized seeds of Arabidopsis (A. thaliana, ecotype Wassilewska, WS) were germinated on 0.8% (w/v) agar-solified MS medium (Murashige and Skoog, 1962) supplemented with 1% (w/v) sucrose. The seedlings were kept on agar for 10 days prior to the transfer to pots containing a soil mixture as described (Estelle and Somerville, 1987), except that Sphagnum was replaced with Bactomix (Michigan Peat Company, Houston, TX). Plants were grown in growth chambers (AR-75L, Percival Scientific Inc., Boone, IA) under light of a photosynthetic photon flux density of 80 umol photons m"2 s". A l4-h light/1 O-h dark regime was applied. The day/night temperature was controlled at 22/1 8°C. For growth under phosphate limitation, sterile Arabidopsis medium (Estelle and Somerville, 1987) was used, but at half concentration and containing 0.8% agarose, 1% sucrose and 20 mM MES (pH 6.0), and different concentrations of KHZPO4 as indicated. Plants were grown for 8 days on 0.8% (w/v) agar-solidified MS medium supplemented with 1% (w/v) sucrose and then transferred to the agar plates containing the phosphate-limited medium. For quantitative growth experiments, sucrose was left out of the medium following the transfer. Plant culture dishes for each experiment were placed on a single shelf of a CU-365L chamber (Percival Scientific Inc., Boone, IA). The dishes were covered with two layers of cheese cloth to diffuse the light and maintain a uniform photon flux density of 80 amol photons m'2 s". The dishes were carefully rotated over the shelf to ensure uniformity. At each time point, eight mutant and wild -type plants on matching dish positions were harvested and their aerial parts were weight. At least, 57 three independent biological repeats of the experiment were conducted with similar results. Mutant Isolation. A T-DNA insertion into the SQD2 gene was isolated from the Arabidopsis knock-out collection at the University of Wisconsin (Logemann et al., 1987; Sambrook et al., 1989). The gene-specific primers used for screening of insertions into the SQD2 gene were 5'- AATCTCTCTATACCTCCTCATTTGCTTCC-3' and 5'—CACAAGCTCAATA- TAGGTTGACCCATAAG-3', the T-DNA-specific primer matching the left end of the T- DNA was 5'-CATTTTATAATAACGCTGCGGACATCT AC—3'. PCR fragments obtained using combinations of the T-DNA-specific primer and the two gene-specific primers were subcloned and sequenced at the MSU Genomics Facility to determine the insertion point for the 5.5 kb T-DNA. Cloning of the SQD2 cDNA. The SQD2 open reading frame corresponding to the protein (GeneBank accession CAB69850) predicted from BAC locus F 7J 8_200 (GenBank accession AL137189) was cloned by RT-PCR using the primers 5'-CGGGATCCCCATGACGACTCTTTCT- TCTATA-3' and 5'-AAGGTACCCTACACGTTACCTTCCGGTA-3'. Total leaf RNA was isolated from approximately 20 day old plants (Col-2) according to the method by Logemann et al. (Logemann et al., 1987). Poly A+ mRNA was purified using an oligotex kit from Qiagen (Valencia, CA) according to the instructions. RT-PCR reactions were performed using the ProSTAR HF single tube RT-PCR system from Stratagene (La J olla, 58 CA). The PCR product was cloned into the ligation ready Stratagene vector pPCR-Script Amp SK+ giving rise to pPCR-SQD2. The insert was sequenced and deposited at GenBank (Accession AF454354). Northern blot Analysis. Total RNA isolated as described above was separated by agarose gel electrophoresis and blotted onto Hy‘bond N+ (Amersham Pharmacia Biotech Inc., Piscataway, NJ) membranes using standard procedures (Bechtold and Pelletier, 1998). Hybridization was done under standard conditions (Sambrook et al., 1989). Complementation Analysis. The insert of pPCR-SQD2 containing the full length coding sequence including the transit peptide was amplified by PCR using the primers 5'-AAGGTACCATGACGAC- TCTTTCTTCTATA-3' and 5'-AATCTAGACTACACGTTA CCTTCCGGTA-3'. This fragment was cloned into the binary vector pBINAR-Hyg (Dormann and Benning, 1998) using the KpnI and XbaI sites. The resulting plasmid pBINAR—Hyg/SQD2 was used to transform the squ mutant by vacuum infiltration (Bechtold and Pelletier, 1998). Transgenic plants were selected in the presence of kanamycin (60 ug/mL) and hygromycin B (25 ug/mL) on MS medium lacking sucrose. Expression of SQDI and SQD2 in E. coli. To express SQD2, the BamHI/Kpnl fragment of pPCR-SQD2 containing the transit peptide encoding sequence was cloned in-frame into the expression vector pQE32 from 59 Qiagene (Valencia, CA) giving rise to pSQD2. The expression cassette encoding the SQDl protein lacking the N-tenninal 84 amino acid transit peptide was excised from pSQDl-TP (6) with XhoI/PvuII, and was ligated into pACYC184 (Chang and Cohen, 1978) cut with SalI/EcoRV giving rise to pSQD], which is compatible with pSQD2 in E. coli. Both plasmids were transferred to E. coli host XLl-Blue (Stratagene, La J 0118, CA) and selected for in the presence of 100 ug/mL ampicillin and 34 ug/mL chloramphenicol. Expression was induced according to the Qiagene protocol. Lipid and Fatty Acid Analysis. Lipids were extracted from frozen leaves or E. coli pellets as previously described (D'o'rmann et al., 1999). Lipid extracts were analyzed as described (Essigmann et al., 2000) and lipids were visualized with iodine vapor or with sugar sensitive a-naphthol reagent (Gage et al., 1992) and identified by co-chromatography with lipid extracts of known composition. For quantitative analysis methyl esters were prepared and quantified by GLC (Rossak et al., 1997). For the analysis of 35S-labeled lipids, four leaves of 3- week-old plants were cut midway through the petiole and immediately transferred petiole-down into a polypropylene microfuge tube containing 100 uL of an aqueous solution of carrier free 35S-sulfate (3.7 MBq/mL; NEN Life Science Products, Boston, MA). Lipids were extracted following 1 hour of incubation and separated by two- dimensional TLC as previously described (Bemring et al., 1995). Labeled compounds were visualized by autoradiography. Fast atom bombardment mass spectroscopy (FAB-MS) measurements of sulfolipid (Gage et al., 1992) were done at the MSU-Mass Spectrometry Facility. 60 Chlorophyll Fluorescence Measurements. In vivo Chlorophyll fluorescence was measured at room temperature with an FMS2 fluorometer (Hansatech, King’s Lynn, UK) after 20 min of dark adaptation. The maximal variable fluorescence (Fv) was obtained by subtraction of the initial fluorescence (F0) from the maximum fluorescence (Fm). The ratio of FV/Fm served as a measure of the maximal photochemical efficiency of PSII. The effective quantum yield of photochemical energy conversion in photosystem 11, (1)133“, was determined as described by Harte] et al. (H'artel et al., 1998). Calculation of CIDPSH was according to Genty et al.(1989). 61 Results Identification of an Arabidopsis SQD2 Candidate Gene. Taking advantage of the published Arabidopsis genome , we developed a set of criteria for the identification of an SQD2 candidate gene encoding the sulfolipid synthase of Arabidopsis. First, given the ancestral relationship between chloroplasts and cyanobacteria it seemed likely that SQD2 would be similar to the squ gene of the cyanobacterium Synechococcus strain sp. PCC7942, which was proposed to encode the sulfolipid synthase (GUler et al., 2000). Indeed we, and recently Berg et al. (Berg et' al., 2000), identified Arabidopsis gene F 7] 8_200 (AT5g01220; GenBank accession AL137189.2) on chromosome five as a possible squ ortholog. The originally predicted protein (GenBank accession CAB69850) for this gene showed 37 % identity over 370 amino acid residues with the cyanobacterial Squ protein, sufficient to be considered an ortholog. A wild-type cDNA corresponding to gene F7J8_200 was isolated by RT-PCR and sequenced (GenBank accession AF454354). The open reading frame for this cDNA disagreed with the current protein predictions for the F7J8_‘200 gene (GenBank accessions CAB69850, AAK76635) leading us to revise the exon-intron structure for F7J8_200 as shown in Figure 2.2A. Exon 6 was added and exons 7, 8, and 9 increased slightly in size. The revised protein sequence for the F71 8_200 open reading frame of 510 amino acids is 41% identical over 370 residues to the cyanobacterial Squ protein. As a second criterium for the correct identification of an SQD2 candidate, a protein domain search using the Pfam database (Betemann et al., 2000) revealed a glycosyltransferase group 1 motif (Pfam accession 00534) from amino acids 294 to 458 of the putative SQD2 62 Figure 2.2. Structure of the SQD2 gene carrying the T-DNA insertion (A), induction of SQD2 expression following phosphate deprivation (B), and lack of expression of SQD2 in the squ mutant (C). A, exons are numbered as referred to in the text. A small portion of the BAC F 7J 8 sequence is shown with nucleotide numbers referring to GenBank accession AL137189.2. The arrow indicates the T-DNA insertion point. B, plants were grown on agar solidified medium for approximately 20 days. As loading control one of the rRNA bands stained with ethidium bromide is shown. C, plants were grown on soil for approximately 25 days. An actin cDNA (GenBank accession M20016.1) was used as a probe for control purposes. 63 A -: :T-DNA(-5.5 kb): .‘ ',. ~‘~ 300 bp 1 2 3 4 56 7 8 910 11 12 TAG BAC F7J 8 84444 5' - AGAATTACTTG ACAAGTGTTAACT - 3' 84421 B Phosphate in Medium (mM) 1 06 01 0 C WT sqd2 8002 are rRNA SQD2 Actin 64 protein encoded by the cDNA. According to the glycosyltransferase classification database CAZy (http://afmb.cnrs.mrs.fr/~pedro/ CAZY/db.html) (Herissat et al., 2001), this protein is grouped in with glycosyltransferase family 4, which is mechanistically characterized by “retaining” glycosyltransferases. This classification would be in agreement with the a-anomeric proton configuration of the substrate and product of the sulfolipid synthase reaction. Third, sulfolipid synthase is an enzyme of the inner envelope in chloroplasts (Tiej e and Heinz, 1998). As such, the translation product should contain a chloroplast transit peptide. According to an in silico analysis (Emanuelsson et al., 1999), the putative SQD2 protein contains an 83 amino acid transit peptide. Fourth, we expected that the expression of the candidate SQD2 gene would be induced as phosphate is depleted, because sulfolipid biosynthesis and the expression of SQD] is induced under these conditions (Essigmann et al., 1998). As shown in Figure 2.2B, the expression of the candidate SQD2 gene followed this prediction. With all four prediction criteria satisfied, a series of experiments was conducted to determine, whether Arabidopsis gene F7J8_200 indeed encodes a sulfolipid synthase. Isolation of an squ T-DNA Insertion Mutant. To obtain corroborating evidence that the gene F7J8_200 encodes a protein essential for sulfolipid biosynthesis in Arabidopsis, the isolation of a T—DNA insertion mutant from the University of Wisconsin-Madison collection (Sussman et al., 2000) was pursued. One homozygous mutant line, which lacked sulfolipid was isolated. Sequencing PCR-products obtained using gene-specific and T—DNA specific primers, it was determined that the T-DNA was inserted after base pair 84434 of the F7J 8 BAC sequence 65 (Gen Bank accession AL137189.2). This places the insertion point into the second intron ofthe predicted F7J8_200 gene as shown in Figure 2.2A. A comparative Northern analysis between the wild type and the homozygous sqd2 mutant (Fig. 2.2C) suggested complete lack of expression. Homozygosity was determined at the molecular level using combinations of gene-specific and T-DNA-specific primers to generate PCR products diagnostic for mutant and wild—type chromosomes. Following a cross between the homozygous mutant and the wild type, genetic segregation was determined in the F2 generation. Approximately one quarter of the plants were kanamycin sensitive (93 of 379 total) and approximately one third of the kanamycin resistant plants (31 of 100) were sulfolipid deficient (see below). The observed segregation ratios agreed with a single T- DNA insertion into the F7J8_200 locus, with kanamycin resistance scoring as a dominant and sulfolipid-deficiency as a recessive marker. To ensure that the observed T-DNA insertion was indeed causing the lipid phenotype, the cDNA derived from F7J8_200 was ligated into a binary expression vector, and transferred into the homozygous mutant (sulfolipid-deficient, kanamycin resistant) by Agrobacterium-mediated transformation. A hygromycin B (transgene T-DNA) and kanamycin resistant (Ko—T-DNA) transgenic line was recovered. This line produced sulfolipid (data not shown) and showed wild-type growth characteristics (see below), thereby demonstrating complementation. Therefore, disruption ofthe F7J8_200 locus was responsible for the sulfolipid lipid deficiency in the squ mutant and growth impairment as discussed below. This locus was designated SULFOQUINOVOSYLDIA CYLGLYCEROL 2 (SQD2). 66 The sqd2 Mutant is Completely Devoid of Sulfolipid. Lipids extracted from the sqd2 mutant and the wild type were separated by two- dimensional TLC. No sulfolipid was detected in the mutant extracts by iodine staining (Fig. 2.3) or by highly sensitive isotope labeling (Fig. 2.3, inserts). Based on this result it appears that sulfolipid biosynthesis is completely inactivated in this mutant due to a sqd2 null-allele. To investigate compensatory effects in the sqd2 mutant, the leaf lipid composition was compared for extracts from mutant and wild-type (WS) plants grown in the presence and absence of phosphate (Table 2.1). Within the experimental error and besides the complete lack of sulfolipid, the leaf composition of the mutant showed no drastic differences compared to the wild type. Given that the relative amount of sulfolipid under phosphate-sufficient conditions does not exceed 2 mol%, this result was not surprising. However, under phosphate-deficient growth conditions, under which relative sulfolipid amounts increased and phosphatidylglycerol amounts decreased in the wild type, phosphatidylglycerol did not decrease in the sulfolipid-deficient Arabidopsis mutant. The same had been observed for sulfolipid-deficient bacterial mutants (Benning et al., 1993; GUler et al., 1996) and was interpreted as a requirement to maintain the overall amount of anionic lipids in the thylakoid membrane. Taking sulfolipid and phosphatidylglycerol out of consideration, the g1ycolipid-to-phospholipid ratio increased in similar ways in the sqd2 mutant and the wild type in response to phosphate starvation (Tab. 2.1). 67 Figure 2.3. The sqd2 mutant is SQDG deficient. Two dimensional separation of lipids extracted from wild-type (A) and sqd2 mutant (B) leaves. The lipids were visualized by iodine. Inserts represent autoradiographs in the area of the sulfolipid spots. Leaves were incubated with 35S-sulfate to label sulfolipid. Lipids were separated as in the main panel. Small circles in the lower right comers indicate the origins, the arrows point towards the position of the sulfolipid. DGDG, digalactosyldiacyl glycerol; MGDG, rnonogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacyl glycerol. 68 First Dimension .- DGDG ‘ SQDG ,PG-t “ ' 1' Pc PE -5 ., I114 I /~’/‘ 1 r. , ‘ '3‘» ' ‘9: . . e4- “! . A. \"i ‘.'~ -. 1. H ‘1 . .. .1. . '1. 9:" ai"~: . ' .‘ "/ Second Dimension 69 Table 2.1 Quantitative analysis of leaf lipid extracts from wild type (WS) and the sqd2 mutant of A. thaliana grown on medium with or without phosphate for 20 days a 1 mM Phosphate in medium no Pi in medium Lipid WT (WS) sqd2 WT (WS) sqd2 MGDG 34.8 d: 0.9 32.2 i 3.3 38.4 :1: 0.7 37.7 :t 0.2 PG 10.5 i 0.5 12.8 i 1.1 3.2 d: 0.3 11.2 i 0.3 DGDG 16.5 i 1.0 16.6 i 0.7 39.9 i 2.0 37.5 i 1.0 SQDG 1.3 3: 0.2 n.d. 5.7 i 0.7 n.d. P1 2.0 i 0.2 2.8 i 0.2 0.8 d: 0.4 1.7 i 1.1 PE 15.1 i 0.1 15.6 d: 1.1 4.1 i 0.3 4.9 i 0.2 PC 19.8 i 1.2 20.2 :1: 0.6 7.6 :t 0.9 6.9 i 0.4 a Values (mol %) represent means of three measurements. Lipid symbols as in Fig. 2.1 and Fig. 2.3; n.d. , not detected. 70 Expression of Arabidopsis SQDI and SQD2 in E. coli. To demonstrate directly that SQD2 encodes the sulfolipid synthase and to test whether the two proteins SQDl and SQD2 of Arabidopsis represent the biosynthetic machinery specific to sulfolipid biosynthesis in plants, these two genes were coexpressed in E. coli. This bacterium does not normally contain sulfolipid. A truncated version of the SQD] protein lacking the chloroplast transit peptide had been previously expressed in E .coli, and the purified recombinant protein was shown to catalyze the formation of UDP- sulfoquinovose from UDP-glucose and sulfite in vitro (Sanda et al., 2001). To express both proteins, we followed a two plasmid strategy as was used for the reconstitution of plant galactolipid biosynthesis in E. coli (Chang and Cohen, 1978). Both constructs are shown in Figure 2.4A. No new glycolipid bands were visible by a-naphthol staining in lipid extracts from E. coli cells containing only one of each of the two plasmids. However, when induced E. coli cells were analyzed that contained both constructs, a new band appeared, which cochromatographed with authentic plant sulfolipid from an Arabidopsis leaf lipid extract (Fig. 2.4B). This new lipid was isolated from induced E. coli cultures using TLC purification and was analyzed by negative fast atom "bombardment mass spectrometry. Two diagnostic molecular ion peaks (M-H) at 791 m/z and at 819 m/z were detected which agreed with the predicted M-H mass for sulfoquinovosyldiacylglycerol containing 16:0/16:1 and 16:0/18:1 fatty acids respectively. Gaschromatographic analysis of fatty acid methylesters derived from the newly produced lipid revealed 16:0, 16:1, and 18:1 fatty acids as the predominant fatty acids. Taken together, all data were in agreement with the formation of authentic sulfolipid in E. coli following the expression of the 71 Figure 2.4. Expression of Arabidopsis SQDI and SQD2 in E. coli. Plasmid constructs used for the expression of the SQD] and SQD2 genes in E. coli (A), and separation of lipids from different E. coli strains (B). A, the employed vector plasmid is indicated in brackets for each construct. The open boxes indicate the His tag, the black boxes plant sequences. P, promoter; T, terminator. B, the four left lanes represent extracts from E. coli harboring no plasmid or plasmids as indicated. An Arabidopsis leaf lipid extract was included for lipid identification purposes. The lipids were visualized by or-naphthol, which primarily stains glycolipids. Other lipids are visualized due to light charting. Abbreviations as in the legend of Fig. 2.3 72 A B pSQD1(pACYc—184) pSQDZ (pQE32) 300 be ‘1 6900989 - ’\ 6°“ 900 oorL oOK 8°C 6 Q Q9 Q9 P can. .- - - - - — PG - — SQDG — PE Arabidopsis SQD] and SQD2 genes. These data also suggested that SQD2 indeed encodes the UDP-sulfoquinovosyl:diacylglycerol sulfoquinovose transferase from Arabidopsis. The effect of sulfolipid deficiency on the sqd2 mutant. The sqd2 sulfolipid null-mutant of Arabidopsis described here, provides an excellent tool for the in vivo analysis of sulfolipid function in seed plants. Thus far, the molecular and genetic analysis of the mutant described above has not provided any evidence for multiple T-DNA insertions into the genome or other secondary mutations, which can occur in T-DNA tagged mutant lines. Therefore, comparison of mutant and wild. type should be a valid approach towards the elucidation of sulfolipid function. Comparing the growth of the mutant and the wild type (WS) on agar plates in a controlled growth chamber environment on the basis of fresh weight did not reveal any difference, when the plants were grown in the presence of phosphate-replete medium (Fig. 2.5A). However, the growth rate of the sqd2 mutant was reduced when plants were phosphate depleted during growth on agar medium lacking phosphate (Fig. 2.5B). To eliminate the possibility that this subtle difference in growth was due to a secondary mutation, we repeated one time point midway through the experiment comparing the homozygous mutant (sqd2) with the homozygous mutant line expressing the wild-type SQD2 cDNA (sqd2/SQD2 cDNA), lacking and containing sulfolipid, respectively. These two lines are nearly isogenic, but the second line contains an additional mutation due to the insertion of an additional T-DNA. Again, no difference between the wild type and the mutant was observed under normal growth conditions (Fig. 2.5A, open symbols), although, the onset 74 Figure 2. 5. Growth curves for wild type (filled diamonds, solid lines) and sqd2 mutant (filled circles, broken lines) on agar-solidified medium with 1 mM (A) or no (B) phosphate added. Eight plants were weight for each time point. To rule out effects of secondary mutations, one time point (12 days) was repeated with an sqd2 mutant line transformed with the SQD2 cDNA (open diamonds) containing sulfolipid and the original sulfolipid-deficient sqd2 mutant (open circles). At least 15 plants are represented in each point in this experiment. Standard errors are indicate where they exceed the size of the symbols. 75 Fresh weight (mg) 120 100‘ 6'8'8’8'13'75'1‘8 Time after transfer (d) 76 Table 2.2. Pigment content (mg g'1 Fresh Weight) 3 and chlorophyll fluorescence b of wild type and sqd2 mutant. WT sqd2 Chlorophyll a 0.91 i 0.06 0.93 d: 0.03 Chlorophyll b 0.29 i 0.02 0.28 d: 0.02 Chlorophyll a+b 1.21 :1: 0.07 1.21 i 0.05 Chlorophyll a/b 3.14 :I: 0.12 3.32 i 0.14 Carotenoids 0.40 :t 0.02 0.42 i 0.01 Fv/Fm 0.82 i 0.01 0.80 i 0.01 CDPSH 0.77 :l: 0.01 0.70 i 0.01 3 Samples were taken from fully expanded leaves of 20-to-30 day old plants grown on agar solidified medium containing 1 mM phosphate. Pigment values represent means (E SE) of 3 independent determinations. b Actinic light for fluorescence measurements was 80 amol photons m'2 3'], the same as used for raising the plants. Values represent means (3: SE) of 10 independent determinations. 77 of the exponential growth phase in this completely independent experiment was delayed, as visible in the slight off-set between the two data point sets (Fig. 2.5A open and closed symbols). More importantly, the mutant lacked behind the complemented mutant line under phosphate-limited growth conditions (Fig. 2.5B, open symbols). Thus, secondary mutations as cause for the observed growth difference between the mutant and wild type under phosphate limiting conditions can be ruled out. Using noninvasive chlorophyll fluorescence measurements it was determined that the maximum quantum yields for photosystem II photochemistry (F v/Fm) were very similar for the mutant and the wild type (Table 2.2). The effective quantum yield of photochemical energy conversion in photosystem II ((DPS”) was slightly, but significantly reduced in the mutant grown under normal conditions (Table 2.2). Pigment content was not altered in the mutant (Table 2.2). Based on this basic analysis of photosynthesis, an essential fimction for sulfolipid in photosynthesis of seed plants can be ruled out. However, the current results do not exclude more subtle or conditional roles for sulfolipid in photosynthesis as were observed for Chlamydomonas and cyanobacteria (GUler et al., 1996; Sato et al., 1995). It should be pointed out that chlorophyll fluorescence parameters were indistinguishable for the mutant and the wild type under phosphate limitation (data not shown). Apparently, phosphate deficiency by itself has more pronounced effects on chlorophyll fluorescence (Hartel et al., 1998) than sulfolipid-deficiency. 78 Discussion More than 40 years after the discovery of the “Plant Sulfolipid” sulfoquinovosyl- diacylglycerol by AA. Benson (1959), the quest for the enzymes involved in the biosynthesis of sulfolipid in seed plants has come to an end. Only two Arabidopsis proteins, SQDl and SQD2 are required for sulfolipid biosynthesis (Fig. 2.1), when simultaneously produced in E. coli. The first, SQDl, has been shown to catalyze the biosynthesis of UDP-sulfoquinovose from UDP-glucose and sulfite (Sanda et al., 2001) in vitro. The successful reconstitution of sulfolipid biosynthesis in E. coli suggests that the proper in vivo sulfolipid donor is present in this bacterium, but is not specific to organisms naturally capable of sulfolipid biosynthesis. Thus, this results supports the hypothesis that sulfite as product of the general sulfur assimilation pathway acts also as the in vivo sulfur donor. The second required plant enzyme, SQD2, is highly similar to glycosyltransferases (Berg et al., 2001) and it is proposed that this protein represents the sulfolipid synthase. Thus far, this enzyme has only been described in envelope preparations from spinach chloroplasts (Seiftert and Heinze, 1992). The availability of an expression system for the production of SQD2 in E. coli provides the means to study the enzymatic reaction catalyzed by this protein in detail. The high similarity between the two Arabidopsis proteins SQDl and SQD2, and the cyanobacterial sulfolipid proteins Squ and Squ (Giller et al., 1996; Gliler et al., 2000) and their ability to functionally reconstitute sulfolipid biosynthesis in E. coli suggests that these are true orthologs. The isolation of the sulfolipid-deficient sqd2 null-mutant of Arabidopsis, allowed us to address another fundamental question, the role of sulfolipid in photosynthetic membranes of seed plants. Although the sqd2 mutant has not yet been studied in greater 79 detail, our initial analysis of photosynthesis and growth suggests that sulfolipid does not have a crucial role in plant photosynthesis. The sqd2 mutant did not show any obvious signs of altered morphology or growth and photosynthetic parameters were only mildly affected under normal laboratory growth conditions. A similar conclusion can be drawn for bacterial mutants (Benning et al., 1993, GUler et al., 1996) and a sulfolipid deficient mutant in Chlamydomonas (Sato et al.,1995). Although the later mutant appears to be more severely impaired in photosynthesis than the Arabidopsis sqd2 mutant or the bacterial mutants, effects due to secondary mutations cannot be ruled out. Unlike the Chlamydomonas mutant, the Arabidopsis sqd2 mutant and its nearly isogenic complementation line should provide the ideal material to probe further for specific roles of sulfolipid in Chloroplastic photosynthesis. If sulfolipid is not essential to photosynthesis, why did plants maintain this pathway during the course of evolution? One answer to this question is that sulfolipid is of importance under conditions under which phosphate becomes limiting for membrane biosynthesis. As in bacterial null-mutants (Benning et al., 1993; Gt'Jler et al., 1996), sulfolipid deficiency in the Arabidopsis sqd2 mutant led to growth impairment under phosphate-limiting growth conditions (F ig.2.5). Although the effect was subtle, it was clearly measurable under highly controlled laboratory conditions and one would assume that sulfolipid-deficiency may be of even greater disadvantage under naturally occurring conditions. Remarkably, the wild type is able to maintain, within limitations, the relative amount of total anionic thylakoid lipids by reciprocally adjusting the sulfolipid and phosphatidylglycerol contents as phosphate availability changes (Essigmann et al., 1998). The lipid profiles for the bacterial and Arabidopsis sulfolipid-deficient null-mutants share 80 a common feature, phosphatidylglycerol amounts are not decreasing under phosphate- limiting conditions. Therefore, sulfolipid-deficient null-mutants exhaust their phosphate reserves sooner than the corresponding wild types resulting in reduced growth under these conditions. The fact that this effect occurs in bacteria and plants suggests that sulfolipid/phosphatidylglycerol substitution may be ubiquitous to photosynthetic organisms containing these lipids. It should be pointed out, though, that sulfolipid cannot fully substitute phosphatidylglycerol, which unlike sulfolipid is prominently present in the crystal structure of photosystem I (Jordan et al., 2001) and may have specific functions beyond sulfolipid. However, photosynthetic membranes generally seem to need a certain amount of anionic lipids for proper function and these can be provided either by phosphatidylglycerol or by sulfolipid. One component of the underlying regulatory process involves changes in the transcription of the SQD] (Essigmann et al., 1998) and the SQD2 genes (Fig. 2.1) in response to phosphate availability. It will be interesting to see, whether these lipid genes are controlled by a more general phosphate sensing regulatory pathway, or by a regulatory pathway sensing biophysical properties of membranes, such as the ratio of anionic to neutral lipids. 81 Acknowledgments Financial support was provided in parts by the US Department of Energy, and the MSU Center for Novel Plant Products. We would also like to acknowledge the Arabidopsis Stock Center at Ohio State University for providing the seeds for this study, and the Arabidopsis Knock-Out facility at the University of Wisconsin-Madison, for enabling us to isolate the sqd2 mutant. 82 References Barber J, Gounaris K. (1986) What role does sulpholipid play within the thylakoid membrane? Photosynth. Res. 92239—249. Bateman A, Bimey E, Durbin R, Eddy SR, Howe KL, Sonnhammer EL. (2000). The Pfam protein families database. Nucleic Acids Res. 28:263-266. Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. In J Salinas, J M Martinez- Zapater, eds, Arabidopsis Protocols, Methods in Molecular Biology, Vol. 82. Humana Press, Totowa, NJ, pp 259-266 Benning C, Huang ZH, Gage, DA. (1995) Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation Arch. Biochem. Biophys. 317:103-111. Benning C. (1998) Biosynthesis and function of the sulfolipid sulfoquinovosyl- diacylglycerol. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:53-75. Benning C, Beatty JT, Prince RC, Somerville CR. (1993) The sulfolipid sulfoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances grth under phosphate limitation. Proc. Natl. Acad. Sci. USA 90:1561-1565. Berg 8, Edman M, Li L, Wikstrom M, Wieslander A (2001) Sequence properties of the 1,2-diacylglycerol 3-glucosy1transferase from Acholeplasma laidlawii membranes. Recognition of a large group of lipid glycosyltransferases in eubacteria and archaea. J. Biol. Chem. 276:22056-22063. Benson AA, Daniel,H, Wiser R. (1959) A sulfolipid in plants. Proc. Natl. Acad. Sci. USA 11:1582-1587. Chang AC, Cohen SN. (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134:1141-1156. Dormann P, Balbo I, Benning C. (1999) Arabidopsis galactolipid biosynthesis and lipid trafficking mediated by DGDl. Science 284:2181-2184. Dormann P, Benning C (2002) Galactolipids rule in seed plants. Trends Plant Sci.7:112- 8. Emanuelsson O, Nielsen H, von Heijne G. (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci. 8:978-984. 83 Essigmann B, Hespenheide BM, Kuhn LA, Benning C. (1999) Prediction of the active- site structure and NAD(+) binding in SQDl, a protein essential for sulfolipid biosynthesis in Arabidopsis. Arch. Biochem. Biophys. 369:30-41. Essigmamr B, Giller S, Narang R A, Linke D, Benning C. (1998) Phosphate availability affects the thylakoid lipid composition and the expression of SQD] , a gene required for sulfolipid biosynthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 95, 1950-1955. Estelle MA, Somerville C. (1987) Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. Mol. Gen. Genet. 206:200-206 Gage DA, Huang ZH, Benning C. (1992) Comparison of sulfoquinovosyl diacylglycerol from spinach and the purple bacterium Rhodobacter spaeroides by fast atom bombardment tandem mass spectrometry. Lipids 27:632-636. Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87—92 Biochem. Biophys. Acta 990287-92. Giiler S, Essigmann B, Benning C. (2000) A cyanobacterial gene, squ, required for biosynthesis of the sulfolipid sulfoquinovosyldiacylglycerol. J. Bacteriol. 182:543- 545. Gtiler S, Seeliger A, Hartel H, Renger G, Benning C. (1996) A null mutant of Synechococcus sp. PCC7942 deficient in the sulfolipid sulfoquinovosyldiacylglycerol. J. Biol. Chem. 271 :7501-7507. Hattel H, Dormann P, Benning C. (2000) DGDl-independent biosynthesis of extraplastidic galactolipids after phosphate deprivation in Arabidopsis. Proc. Natl. Acad. Sci. USA 97:10649-10654. Hartel H, Reinhardt I, Grimm B. (1998) Relationship between energy-dependent fluorescence quenching and xanthophyll-cycle-pigments in transgenic chlorophyll- deficient tobacco grown under different light intensities J. Photochem. Photobiol. B Biol. 43:136-145. Hartel H, Essigmann B, Lokstein H, Hoffinann-Benning S, Peters-Kottig M, Benning C. (1998) The phospholipid-deficient phol mutant of Arabidopsis thaliana is affected in the organization, but not in the light acclimation, of the thylakoid membrane. Biochim. Biophys. Acta 1415: 205-218. Henrissat B, Coutinho PM, Davies GJ. (2001) A census of carbohydrate-active enzymes in the genome of Arabidopsis thaliana. Plant Mol. Biol. 47:55-72. Jordan P, Fromme P, Witt HT, Klukas O, Saenger W, Krauss N. (2001) Three- dimensional structure of cyanobacterial photosystem I at 2.5 A resolution. Nature. 84 41 11909-917 Logemann J, Schell J, Willmitzer L. (1987) Improved method for the isolation of RNA from plant tissues. Anal. Biochem. 163:16—20. Mulichak AM, Theisen MJ, Essigmann B, Benning C, Garavito RM. (1999) Crystal structure of SQDI, an enzyme involved in the biosynthesis of the plant sulfolipid headgroup donor UDP-sulfoquinovose Proc. Natl. Acad. Sci. USA 96:13097-13102. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15: 473-497 Rossak M, Tietje C, Heinz E, Benning C. (1995) Accumulation of UDP-sulfoquinovose in a sulfolipid-deficient mutant of Rhodobacter sphaeroides. J. Bio. Chem. 270:25792—25797. Rossak M, Schafer A, Xu NX, Gage DA, Benning C. (1997) Accumulation of sulfoquinovosyl—1-O-dihydroxyace-tone in a sulfolipid-deficient mutant of Rhodo- bacter sphaeroides inactivated in squ. Arch. Biochem. Biophys. 340:219—230. Sambrook, J ., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, NY). Sanda S, Leustek T, Theisen MJ, Garavito RM, Benning C. (2001) Recombinant Arabidopsis SQDl converts udp-glucose and sulfite to the sulfolipid head group precursor UDP-sulfoquinovose in vitro. J. Biol. Chem. 276:3941-3946. Sato N, Sonoike K, Tsuzuki M, Kawaguchi A. (1995) Impaired photosystem II in a mutant of Chlamydomonas reinhardtii defective in sulfoquinovosyldiacylglycerol. Eur. J. Biochem. 234:16-23. Seifert U, Heinz E. (1992) Enzymatic characteristics of UDP sulfoquinovose: diacylglycerol sulfoquinovosyltransferase from chloroplast envelopes. Bot. Acta 105:197—205. Sussman MR, Amasino RM, Young J C, Krysan PJ, Austin-Phillips S. (2000) The Arabidopsis Knockout Facility at the University of Wisconsin-Madison. Plant Physiol. 124:1465-1467. Tietje C, Heinz E. (1998) Uridine-diphospho-sulfoquinovose: diacylglycerol sulfoquinovosyltransferase activity is concentrated in the inner membrane of chloroplast envelopes. Planta 206:72-78. Weissenmayer B, Geiger O, Benning, C. (2000) Disruption of a gene essential for sulfoquinovosyldiacylglycerol biosynthesis in Sinorhizobium meliloti has no detectable effect on root nodule symbiosis. Mol. Plant. Microbe. Interact. 13:666- 672. 85 Chapter 3 The pgpI Mutant Locus of Arabidopsis Encodes a Phosphatidylglycerolphosphate Synthase with Impaired Activity The work presented in this chapter has been published: Xu C, Harte] H, Wada H, Hagio M, Yu B, Eakin C, Benning C. (2002) Plant Physiol. 1292594-604. 86 Abstract ’ Phosphatidylglycerol is a ubiquitous phospholipid that is also present in the photosynthetic membranes of plants. Multiple independent lines of evidence suggest that this lipid plays a critical role for the proper function of photosynthetic membranes and cold acclimation. In eukaryotes, different subcellular compartments are competent for the biosynthesis of phosphatidylglycerol. Details on the plant-specific pathways in different organelles are scarce. Here we describe a phosphatidylglycerol biosynthesis—deficient mutant of Arabidopsis, pgp] . The overall content of phosphatidylglycerol is reduced by 30%. This mutant carries a point mutation in the CDP-alcohol phosphotransferase motif of the phosphatidylglycerolphosphate synthase (E.C.2.7.8.5) isofonn encoded by a gene on chromosome 2. The mutant shows an 80% reduction in plastidic phosphatidylglycerol- phosphate synthase activity consistent with the plastidic location of this particular isoforrn. Mutant plants are pale green and their photosynthesis is impaired. This mutant provides a promising new tool to elucidate the biosynthesis and function of plastidic phosphatidylglycerol in seed plants. 87 Introduction Phosphatidylglycerol (PG) is one of the most common phosphoglycero-lipids found in nature. It is the only major phospholipid present in the thylakoid membranes of plant chloroplasts (Marechal et al., 1997) and the only phospholipid in cycanobacteria, which strikingly resemble plant chloroplasts in their lipid composition (Murata and Nishida, 1987). Indeed, a large body of correlative and direct evidence suggests that PG is critical for the structural and functional integrity of the thylakoid membrane. Thus, the presence of specific molecular species of PG in photosynthetic membranes correlates well with low-temperature-induced photoinhibition and chilling sensitivity of plants and cyanobacteria (Murata et al., 1992; Somerville, 1995). Light harvesting pigment protein complexes of photosystem II are specifically enriched in PG (Murata et al., 1990; Tremolieres et al., 1994). Moreover, PG is crucial for the in vitro trimerization of the major peripheral light-harvesting pigment-protein complexes (NuBberger et al., 1993; Hobe et al., 1994; Ktihlbrandt, 1994) and the dimerization of the reaction/center core pigment-protein complexes of photosystem II (Kruse et al., 2000). It is also an integral component of the phototsystem I reaction center (Jordan et al., 2001) and required for the in vitro reconstitution of the light harvesting pigment protein complexes of photosystem I (Schmid et al., 1997). Thylakoid membranes treated with phospholipase A2 are PG depleted and are inhibited in their photosynthetic electron transport activities (Jordan et al., 1983; Siegenthaler et al., 1987). Furthermore, the anionic lipid PG interacts with the transit peptide of chloroplast precursor proteins during protein import into chloroplasts (van't Hofet al., 1993). 88 Recently, PG-deficient auxotrophic mutants of the cyanobacterium Synechocystis sp. PCC6803 have been isolated which are severely impaired in photosynthesis (Hagio et al., 2000; Sato et al., 2000). Two Arabidopsis mutants, pho] and ats](act]), are known that show, among other phenotypes, a decreased relative amount of PG (Hartel et al., 1998a; Kunst et al., 1988; Poirier et al., 1991). However, because both mutants are affected in multiple aspects of lipid metabolism, their usefulness for the investigation of specific PG functions in photosynthetic membranes of seed plants is limited. Two enzymatic reactions are specific to PG biosynthesis in plants, the formation of phosphatidylglycerolphosphate (PGP) from CDP-diacylglycerol and glycerol-3- phosphate catalyzed by the PGP synthase (EC. 2.7.8.5), and the subsequent dephosphorylation by PGP phosphatase (EC. 3.1.3.27) (Moore, 1982; Kinney, 1993; Andrews and Mudd, 1986). Labeling studies indicate that enzymatic activity involved in PG biosynthesis is associated with the inner mitochondrial membrane, the endoplasmic reticulum, and the chloroplast (Moore, 1982; Kinney, 1993) suggesting that at least three isoforms of PGP synthase and PGP phosphatase exist in plant cells. None of them has been purified to homogeneity and studied in vitro. Here we describe a chemically induced, PG-deficient mutant of Arabidopsis, pgp], with a mutation in one isoforrn of PGP synthase. 89 Materials and methods Plant Material Surface sterilized seeds of Arabidopsis (A. thaliana, ecotype Col-2, or Ler-O), the multiple-times-backcrossed pgp] , ats](act]) (Kunst et al., 1988), and pho] (Poitier et al., 1991) mutants were germinated on 0.8% (w/v) agar-solidified MS medium (Murashige and Skoog, 1962) supplemented with 1% (w/v) sucrose. The seedlings were kept on agar for 10 days prior to the transfer to pots containing a standard soil mixture (equal parts of Bacto Soil, Michigan Peat Company, Houston, TX; medium vermiculite; and perlite) drenched with half-strength Arabidopsis nutrient solution (Estelle and Somerville, 1987). Plants were grown in growth chambers (AR-75L, Percival Scientific Inc., Boone, IA) under light of a photosynthetic photon flux density (400-700 nm) of 75 umol m"2 s". A l4-h light/ 10-h dark regime was applied. The day/night temperature was controlled at 23/180C. Genetic and Molecular Procedures The pgp] mutant was isolated by TLC of lipid extracts of individual M2 plants as previously described for the isolation of the dgd] mutant (Dormann et al., 1995). The pgp] mutant locus was mapped in a small F2 population (42 homozygous pgp] plants) derived from a cross of pgp] (Col-2) to a standard Landsberg erecta (Ler-O) line employing single sequence polymorphic markers evenly spaced on the Arabidopsis genome map (http://www.arabidopsis.org) under conditions described by Bell and Ecker (1994). At least two markers per chromosome were tested. Standard sequence searching 90 tools (Altschul et al., 1990) were used at the National Center for Biological Information web site (http://www.ncbi.nlm.nih. gov) to identify putative PGP synthase genes in the Arabidopsis genomic sequence (The Arabidopsis Genome Initiative, 2001). References to the respective DNA and amino acid sequences are provided as GenBank accession numbers throughout the text. New sequences were obtained at the MSU Genomics Facility by automated sequencing. For Southern blot analysis, Arabidopsis DNA was extracted from 4-week-old plants (Rogers and Bendich, 1994). The digested DNA was separated on a 1% agarose gel and transferred onto a Hybond N+ membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Blots were hybridized at 65°C in standard hybridization solution (250 mM sodium phosphate buffer, 7% (w/v) SDS, 1 mM EDTA, 1% (w/v) BSA, lOmg/mL autoclaved herring sperm DNA). Blots were repeatedly washed for 15 min in 2 X SSC and 0.1% SDS. Cloning of the Mutant and Wild-type cDNAs and Expression in E. coli The PGP] cDNA was amplified in three steps from an Arabidopsis Agtll cDNA library derived from leaves. An internal fragment of the Arabidopsis PGP] cDNA was amplified by PCR using the primers 5'-CGCTGCAGGTCTGGCTTCGTTAAT-3' and 5'-CGCTGCAGCTACTTCATTAGTACTTT-3'. The 5'-terminal region of the cDNA was obtained by the 5'-rapid amplification of cDNA ends (5'-RACE) method (5'-Full RACE Core Set; Takara, Shiga) and the 3'—terminal region by amplification using an internal primer and oligo dT. The amplified cDNAs were subcloned into pCR2.1 (Original TA Cloning Kit; Invitrogen, Carlsbad, CA, USA) and their nucleotide sequences were 91 determined. The sequence fragments were combined using appropriate restriction sites to obtain the full-length cDNA as deposited in GenBank (AB048535). The E. coli mutant YA5512 defective in PGP synthase (Asai et al., 1989; Usui et al., 1994) was provided by K. Matsumoto (Saitama University, Japan). A region of the Arabidopsis PGP] cDNA encoding the presumed mature form of PGP synthase (first 93 amino acids removed) was amplified by PCR using the primers 5'-AAGGTACCCTT CACCGCCTCCGT-3' and 5'-AACTGCAGCTACTTCATTAGTACTTTCCA-3'. The corresponding pgp] mutant sequence was cloned by RT-PCR using the same set of primers. For this purpose, total leaf RNA was isolated from approximately 20-day-old plants according to the method by Logemann et al. (1987). Poly A+ mRNA was purified using an oligotex kit from Qiagen (Valencia, CA) according to the instructions. RT-PCR reactions were performed using the Omniscript RT-PCR system from Qiagen. The PCR product was cloned into the ligation ready Stratagene vector pPCR—Script Amp SK+ (La J olla, CA). The presence of the C to T mutation in pgp] cDNA was confirmed by DNA sequencing. The amplified DNA fragments from the wild type and the mutant were digested with KpnI and PstI, and ligated into the KpnI and PstI sites of the expression vector pQE32 from Qiagen. The resulting plasmids expressing the wild-type ( pQE32- PGPI) and mutated protein (pQE32-pgp]) were used to transform E. coli YA5512. The constructs were confirmed by sequencing. YA5512 was also transformed with the vector pQE32, for control purposes. The transformants were grown at 37 ° C in LB medium supplemented with 100 ug mL'1 ampicillin. A 3-mL overnight culture was centrifuged and the cells were washed and resuspended in 10 mL of the same medium with antibiotic. The culture was incubated at 37°C for 3 h and then isopropyl-l-thio-B-galactoside was 92 added to a final concentration of 0.4 mM and the culture was further incubated for 3 h at 37°C. Cells were collected by centrifugation (10 min, 5000 g). The resulting cell pellet was resuspended in 50 mM Tris, pH7.2, 20 % (v/v) glycerol and stored at —80 °C. For enzyme assays, the cells were disrupted by one freeze/thaw cycle and ultrasonification (three times for 20 s). The sonicated suspension was centrifuged at 15,000g and the supernatant (cell-free extract) was used for the assay of PGP synthase activity as described below. Complementation Analysis The full length coding sequence of wild type PGP synthase was amplified by RT- PCR using the primer 5’-AAGGTACCATGCTCAGATCCGGTCTGGCT-3’ and 5’- AACTGCAGCTACTTCATTAGTACTTTCCA-3’ as described above for the pgp] mutant sequence. This fragment was ligated into the Stratagene pPCR-Script Amp SK+ vector and the nucleotide sequence was determined. The insert was excised with KpnI taking advantage of the site in the vector and in one of the primers, and was subsequently cloned into the respective site of the binary vector pBINAR-Hyg (Dormann and Benning, 1998). Arabidopsis wild-type (Col-2) plants were transformed in planta (Bechtold and Pelletier, 1998). Transformants were selected on agar-solidified MS medium containing 1% sucrose and 70 ug/mL hygromycin B. The lipid phenotype was determined by thin layer chromatographic analysis of lipid extracts (see below). The presence of the transgene construct was confirmed by PCR using the primers 5'-AAGGTACCATGCT CAGATCCGGTCTGGCT-3' and 5'-ACCATAAGCTTATCAGCAACTGGATTCAA-3'. 93 Lipid Analysis Rosette leaves were immediately frozen in liquid nitrogen upon harvesting, and lipids were extracted as previously described (Dormann et al., 1995). Bacterial cells were extracted as described by Benning and Somerville (1992). Lipid extracts were analyzed on activated ammonium sulfate impregnated silica gel TLC plates using a solvent system of acetone/toluene/water (91 :3027, v/v/v). Lipids were visualized with iodine vapor and identified by co-chromatography with lipid extracts of known composition. For quantitative analysis, individual lipids were isolated from TLC plates and used to prepare fatty acid methyl esters. The methyl esters were quantified by GLC using myristic acid as the internal standard (Rossak et al., 1997). Chloroplast Isolation and PGP Synthase Assay Chloroplasts were isolated according to Joy and Mills (1987) from 4-week-old Arabidopsis plants grown on agar-solidified MS medium supplied with 1% sucrose. Typically, the equivalent of 5 g of rosette leaves was used in a single preparation. The chloroplast PGP synthase activity was measured by the incorporation of radiolabled glycerol 3-phosphate into the chlorofonn-soluble product PGP according to Mudd and coworkers (Mudd et al., 1987). The reaction mixture contained 25 mM HEPES (pH 7.3), 2 mM MgC12, 0.1 mM CDP-diacylglycerol prepared as described (Mudd et al., 1987), 0.5 mM [MC]-DL-glycerol 3-phosphate (1 ttCi/ISO nmol), 0.050/0 Triton x—100 (v/v) and chloroplast suspension equivalent to 150 pg chlorophyll in a final volume of 150 uL. The reaction mixture for the determination of galactolipid synthase activities in isolated chloroplasts contained the same buffer except that 0.5 aCi UDP-[14C] galactose (325 94 mCi/mmol; Amersham) was added instead of CDP-diacylglycerol and labeled glycerol-3- phosphate. Furthermore, this assay was performed in a total volume of 120 uL containing chloroplasts equivalent to 150 ug chlorophyll. The incubations were performed at 27°C for 60 min, and terminated by the addition of 0.5 mL of 0.1 M HCl in methanol. The lipids were extracted with 2 mL of chloroform and 3 mL of 1 M MgClz. The chloroform phase was evaporated under a stream of nitrogen and then redissolved in 0.2 mL chloroform. A 25-uL aliquot was taken for the determination of radioactivity by scintillation counting. The remainder was used for lipid analysis by TLC as described above. Chlorophyll Fluorescence Measurements and Pigment Analysis In vivo room temperature chlorophyll fluorescence was monitored with plants, which were dark adapted for 1 h as described (Hartel et al., 1998c). The fluorescence parameters used in this study are as previously defined (Genty et al., 1989; van Kooten and Snel, 1990). Pigments were determined as described by Lichtenthaler (1987). 95 Results Isolation of a new phosphatidylglycerol—deficient mutant of Arabidopsis To isolate mutants of Arabidopsis deficient in the biosynthesis of complex lipids, we designed a brute force screening procedure based on thin-layer-chromatographic analysis of lipid extracts from leaves of a chemically mutagenized Arabidopsis M2 population. Screening 3,000 Mz-plants from 20 independent batches for lipid alterations by thin layer chromatography (TLC) of lipid extracts, we were not only able to isolate a galactolipid- deficient dgd] mutant as previously described (derann et al., 1995), but also two mutant lines with a slight reduction in PG content. One of these mutant lines was dark green, small, and resembled the pho] mutant in appearance, while one mutant was pale green in color. Due to phosphate deprivation of the pho] mutant, in which the primary defect is in the translocation of phosphate from the root to the shoot system (Poirier et al., 1991), the relative content of phospholipids, including PG, is reduced while the content of non-phosphorous lipids is increased (Essigmamr et al., 1998; Hartel et al., 1998a). Unlike the pale green mutant, the dark green mutant was reduced in leaf phosphate content and Yves Poirier confirmed by genetic complementation analysis that it carries a mutant allele of the pho] locus (Y. Poirier, personal communication). The pale green mutant, designated pgp] and backcrossed three times, is shown in Fig. 3.1. Its growth was slightly reduced and, particularly under photoautotrophic growth conditions, developing young leaves in the center of the mutant rosette were yellowish in color (Fig. 3.1 C). Thin layer chromatography of lipid extracts from expanded leaves followed by quantitative analysis of lipid composition of the pgp] mutant showed that the PG content 96 Figure 3.1. Growth and morphology of Arabidopsis wild type and pgp] mutant. A, six week old plants grown for 10 days on agar-solidified MS medium with 1% sucrose followed by photoautotrophic growth on soil. B, three week old plants grown on agar- solidified MS medium with 1% sucrose. C, three week old plants grown on soil. Note the yellow tissues in the center of the mutant rosette. 97 98 Table 3.1. Leaf Lipid Composition (mol%) of Arabidopsis Wild Type and Mutants a WT pgp] ats] (actI) pho] MGD 48.5 i 1.3 45.9 i 3.3 41.0 i 0.9 48.4 i 1.1 G PG 8.4 :1: 0.6 5.6 d: 0.8 5.5 d: 0.6 4.9 i 0.3 DGD 16.3 3: 0.8 14.7 i 1.9 13.3 :1: 1.7 28.6 :1: 0.3 G SQD 1.2 i 0.4 1.0 i 0.3 1.1 i 0.4 5.8 d: 0.3 G P1 1.8 :l: 0.5 1.5 i 0.4 0.9 i 0.4 0.7 i 0.3 PE 9.1 i 0.3 13.1 i 1.2 13.8 :1: 0.7 4.3 i 1.2 PC 14.7 ml: 1.4 18.3 i 2.4 24.4 :1: 0.7 7.3 i 0.7 a Values represent means of three measurements. Plants were 25-to-30 day old. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycero1; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol. 99 was reduced by approximately 30% (Table 3.1). For comparison, we also included the analysis of the pho] and the ats](actI) mutants. In all three cases the reduction in PG content was similar. However, contrary to pgp] and ats](act]), the pho] mutant showed an increase in the relative amounts of the sulfolipid sulfoquinovosyldiacylglycerol and the galactolipid digalactosyldiacylglycerol, a phenotype characteristic for pho] (Hartel et al,1998a) The primary defect in the ats](actl) mutant is a reduction in the activity of the plastidic g1ycerol-3-phosphate:acyl-ACP acyltransferase (Kunst et al., 1988). As a consequence, the plastid pathway of lipid assembly (Browse et al., 1986; Browse and Somerville, 1991; Heinz and Roughan, 1983) in this plant is shut down and molecular species of monogalactosyldiacylglycerol containing 16:3 fatty acids (16 carbons, three double bonds) are strongly reduced. This phenotype was not visible in the fatty acid composition of monogalactosyldiacylglycerol of pgp] , which had an overall fatty acid composition similar to wild type (data not shown). Furthermore, morphology and growth of ats](act1) are similar to wild type, unlike observed for pgp] (Fig. 3.1). This phenoty- pic analysis suggested that pgp] was not a new allele of pho] or ats](act]), but a new locus affecting the biosynthesis of PG as was confirmed by genetic mapping (see below). The pgp] locus maps in the proximity of a putative PGP synthase gene on chromosome 2 To narrow down the search for a candidate gene affected in pgp] , genetic mapping of the mutation leading to PG-deficiency in pgp] was employed. For this purpose we crossed pgp], which is in the Col-2 genetic background, to a PGP] wild-type line in the Ler-O 100 genetic background. In the F1 generation, all plants were phenotypically indistinguishable from wild type with regard to PG content based on semiquantitative TLC analysis with an example shown in Fig. 3.2A ( top, left two lanes). However, in the F2 generation of 233 plants screened, 54 (23%) plants were recovered with a reduced amount of PG, as shown for the homozygous pgpl mutant in Fig. 3.2A, while lipid extracts from all other plants were indistinguishable from wild-type extracts. Without exception, only these 54 plants were also reduced in growth. The F3 progeny of these plants did no longer segregate suggesting that they represent the homozygous mutant fraction. The nearly 3-to-1 segregation observed for the F2 plants was in agreement with a single nuclear recessive mutation that is responsible for the PG-deficiency in the pgp] mutant. A small mapping population of 42 homozygous pgp] mutants derived from this cross was used to map simple sequence polymorphic markers (SSLPs) (Bell and Ecker, 1994) relative to the pgp] locus. Only markers on chromosome 2 at approximately 70 cM showed linkage to pgp] . Recombination frequencies and calculated map distances for this region are shown in Fig. 3.3A. Two minimally overlapping bacterial artificial chromosomes (BACs) sequenced by “The Arabidopsis Genome Initiative” (2001), one containing the most tightly linked SSLP marker nga168 as derived from “The Arabidopsis Information Resource” web page (www.arabidopsis.org), are shown in Fig. 3.3B. The nucleotide sequence of T16B24 (GenBank AC004697) contains an open reading frame (BAC locus T16B24.7, nucleotides 78,883-80,690; gene At2g3920) with a translated amino acid sequence (Genbank AAC28995) annotated as CDP-diacylglycerol glycerol-3-phosphate 3-phosphatidyltransferase (PGP synthase). The predicted intron-exon structure for this gene is shown in Fig. 3.3C and was later confirmed following the isolation of the cDNA 101 r‘igure 3.2. Lipid phenotype of pgp] and molecular defect. Sections of the lipid :hromatogram (top panel) and the a DNA gel of PCR products (bottom panel) of wild ype, the pgp] mutant and two independent transgenic pgp] lines transformed with the 9GP] wild-type cDNA (pgp]/CPGPI) are shown. Lipids were analyzed by thin layer :hromatography and stained by exposure to iodine. Monogalactosyldiacylglycerol MGDG),serving as loading control, and phosphatidylglycerol (PG) are shown. B, 'estriction length polymorphism in the pgp] mutant locus. Sequence comparison of the vild-type (PGP!) and mutant (pgp]) locus showing the mutated BamHI site (top). The rucleotide number refers to the GenBank accession number of the BAC clone T16B24 AC004697). Genomic Southern blot of the wild type and the pgp] mutant probed with he PGP] gene (bottom). Restriction digests are indicated. Numbers indicate the size (kb) )f the fragments marked by arrows. 102 fl “ a“ m —MGDG -PG - 850 bp _ ,,,,, _ - 526 bp BamHl PGP] 79,747 GLuiirbuibLL i 1 pgpl 79,747 ULbl bu u lllllbuflllLHUl BamH l/Xbal BamHl ’\ ’\ {é QQQ $4 QQ’Q “.9145 .- N 1.14—— 4.6 103 (see below). Searching the amino acid sequence (Altschul et al., 1990) of PGP synthase from E. coli (GenBank P06978) against all amino acid sequences of Arabidopsis confirmed this open reading frame on chromosome 2 as putative PGP synthase and revealed two additional putative PGP synthase genes of Arabidopsis, one on chromosome 3 (BAC locus T15C9.30, gene At3g55030) and one on chromosome 4 (BAC locus T4821 .19, gene At4g04870).Targeting signal analysis (Emanuelsson et al., 2000) suggested that of all three putative PGP synthases, only the one encoded on chromosome 2 contained a putative chloroplast transit peptide. The pgpl mutant is recognizable by a distinct RFLP in BAC locus T16BZ4.7 To determine the exact molecular defect in the pgp] mutant, a detailed analysis of the presumed PGP synthase locus on chromosome 2 was conducted in the mutant and the wild type. By chance we discovered during the restriction analysis of cloned fragments derived from the mutant that the T16B24.7 equivalent locus in the pgp] mutant is missing a BamHI restriction site resulting in a restriction fragment length polymorphism (RFLP). Fig. 3.2B shows a comparison of wild type and pgp] genomic DNA restricted with BamHI/Xbal or just BamHI and probed with a 987 bp genomic DNA fragment from the beginning of the open reading frame to the BamHI site present in the wild type (GenBank AC004697, nucleotides 78,777 to 79,764). In both digests the size of the hybridizing fragment is increased in the pgp] sample as predicted from the genomic sequence for the loss of a specific BamHI site (GenBank AC004697, nucleotide 79,764). Sequence analysis of the entire coding region of the respective gene isolated by PCR 104 from wild-type and mutant genomic DNA confirmed a mutation altering the BamHI site (Fig. 3.28). Expression of a cDNA derived from wild-type locus T168247 restores PG biosynthesis in the pgpl mutant background To obtain conclusive evidence that the deficiency in PG biosynthesis in the pgp] mutant is indeed caused by the observed mutation in the T168247 locus, the respective wild-type cDNA was isolated by RT—PCR and cloned into a T-DNA binary vector. This construct was used to transform pgp] mutant plants by vacuum infiltration. Several hygromycin B resistant transformants were isolated and their lipid composition was examined by semiquantitative TLC. Lipid extracts from two independent transformants are shown in Fig. 3.2A (top). Their lipid composition as well as their macroscopic appearance was indistinguishable from wild type. The presence of the transgene construct was confirmed by PCR as shown in Fig. 3.2A (bottom). The genomic sequence gives rise to a diagnostic fragment of 850 bp, while the transgene appears as 525 bp fragment. The intermediate size fragment was isolated and analyzed, but its sequence was unrelated to PGPl or any of its paralogs (data not shown). Thus, introduction of the wild-type cDNA corresponding to T168247 into the mutant background rescued lipid and morphological phenotypes, thereby confirming that the observed mutant phenotypes are caused by a single mutation in the T168257 locus. 105 BAC locus T168247 encodes a fully functional PGP synthase in the wild-type but not in the pgpl mutant The mutation in pgp] that abolished the BamHI site mentioned above is located in the third exon of the gene (Fig. 3.3C). Accordingly, the predicted protein carries a Pro170 to Ser mutation. The alignment of the predicted protein with the two other Arabidopsis paralogs and known bacterial PGP synthases (Fig. 3.4A) revealed that this proline residue is part of a conserved active site motif for CDP-alcohol phosphotransferases (Williams and McMaster, 1998). At the beginning of this study, no experimental data were available for any of the three putative PGP synthase genes of Arabidopsis. To confirm that the wild-type locus T168247 indeed encodes a fully functional PGP synthase, the respective full length wild type cDNA (GenBank A8048535) was isolated as described in the methods section. In addition, the corresponding cDNA from the pgp] mutant was cloned by RT-PCR. The 1210 bp long full-length wild—type cDNA encodes a 296 amino acid protein with a molecular mass of 32.2 kD. To test PGP synthase activity for the respective wild-type and mutant proteins, we expressed the cDNA for the predicted mature proteins (with the 93- N-terminal amino acids missing) in the E. coli mutant YA5512. This mutant is deficient in PGP synthase activity (Asai et al., 1989) and carries a Thr°° to Pro replacement in the coding region of the pgsA gene (Usui et al., 1994). As a consequence this strain has very low residual PGP synthase activity leading to PG deficiency. In addition, the content of cardiolipin is very low because PG is a precursor for this lipid. Total lipids extracted from lines of YA5512 transformed with vector control (pQE32), the wild-type cDNA (pQE32-PGPI) or mutant cDNA (PQE32-pgp1) expression constructs were quantified. Both, wild type and mutant constructs partially 106 Figure 3.3. The PGP] locus on chromosome 2 of Arabidopsis. A, genetic map showing SSLP markers (positions on map), experimentally determined map distances, and recombinant chromosomes/total chromosomes analyzed for each marker. 8, BACs from the genome sequencing project and markers. C, structure of the PGP] gene (top) and cDNA (bottom). The grey box represents the exon carrying the mutation in pgp] . A, poly A tail; ATG , start codon; TAG, stop codon; TP, transit peptide. 107 Athbi02b (76.4) A nga361(630) ngal68(738) l l l ' 6.7 +/- 3.7 I 15.6 +/- 4.4 I 3/48 0/70 11/84 /' "a- _ 2 CM / I “\ / ‘a. PGPS(PGP1) _ 30 Kb B ATG nga168 T16BZ4 __; v v I ' Q \ l I , I o o \ s ' ' a ,' ’ o I" o' o“ y \ l I o '0 0 O ‘ ‘ . ' l I ' O ' O ‘ ‘ I O ' ' ’ 'Ol" " ~ \ ' ' I ' o 'o ' O ‘ ‘ l I, "o .l.‘ '0 ‘x ‘1 n. I . 108 Figure 3.4. Active site mutation of PGP synthase in the pgp] mutant. A, alignment of different Arabidopsis PGP] paralo gs (PGP2 and PGP3) and bacterial PGP synthase orthologs. GenBank accession numbers (Arabidopsis gene numbers) for the respective protein sequences are: Arab-PGP], AAC28995 (At2g3920); Arab-PGP2, CAB82698 (At3g55030); Arab-PGP3, CAB80852 (At4g04870); E. coli, P06978; Rhodobacter spit; AAC44003; Bacillus sub., 139950; Synechocystis, 576208. The arrow indicates the proline (Prom) to Set change in the pgp] mutant. 8, reduced specific activity of the PGPl-S170 mutant protein. Total activity in extracts of the E. coli YA5512 PGP synthase- deficient mutant expressing wild type PGP] cDNA (squares) or mutath pgp] cDNA (circles) was determined. The two graphs represent single representative experiments. Equal amounts of protein were used. In addition, the mean specific activity (+/— SE) for three independent PCR constructs is provided. The PGP synthase activities in YA5512 and YA5512 containing pQE32 were not detectable. 109 A 1 Arab -pgp1 SAFGAFLDSVADKLMVAATLI LLCTK Arab-PGPl SAFGAFLDPVADKLMVAATLILLCTK Arab-PGP2 SEFGAFLDPVADKLMVAATLILLCTK Arab-PGP3 SVVGSYLDPLADKVLIGCVAVAMVQK E.Coli -RFGAFLDPVADKVLVAIAMVLVTEH Rhodobacter sph. SKFGAMLDPIADKAMVVIALVIITGY Bacillus sub. TNFGKFLDPLADKLLVSAALIILVQF Synechocystis -——GKFLDPLVDKLLVLAPLLILLTW CD 180 - PGP1: 3.0 +/- 0.4 pmol/min/mg Prot. Conversion of G3P (pmol) pgpl: 1.1 +/- 0.2 pmol/min/mg Prot. l I l I l o 5 10 15 20 25 30 Time(min) 110 restored the lipid composition of the E. coli mutant, but the pgp] mutant construct to a lesser extent suggesting reduced activity (data not shown). Based on this result it was concluded that PGP] encodes a functional PGP synthase. Because mutant and wild proteins are expressed to the same extent in E. coli as confirmed by gel electrophoresis of total proteins (data not shown), the result suggested that the activity of the pgp] mutant is impaired, but not abolished. This was directly confirmed by enzymatic assays under saturating substrate conditions in the linear range of the assay of crude protein extracts from the two E. coli strains as shown in Fig. 3.48. Three independent clones were tested for each construct and averaged. The specific activity of the mutant protein was reduced to approximately one third of wild type activity. The PGP synthase activity was not detectable in the crude protein extracts from YY5512 or YY5512 hosting the empty vector pQE32 (data not shown). Plastidic PGP synthase activity is strongly reduced in the pgp] mutant To determine the effect of the P170 to S mutation in the pgpl mutant, PGP- synthase activity was determined in isolated chloroplasts. When extracts from isolated wild-type chloroplasts were incubated with CDP-diacylglycerol and labeled glycerol-3- phosphate in the dark, two major lipids were labeled, PGP and PG, which was presumably derived from PGP due to the action of PGP phosphatase (Fig. 3.5). In chloroplast extracts from the pgp] mutant, the incorporation of label into PGP was nearly abolished and the labeling of PG was markedly reduced (Fig. 3.5). In comparison, incorporation of labeled UDP—galactose into galactolipids was not affected in the pgp] mutant (Fig. 3.5). The labeling pattern was more complex when the PGP synthase assay 111 Figure 3.5. Chloroplastic PGP synthase and galactolipid biosynthetic activities in wild type and pgp] mutant. Isolated and ruptured chloroplast were either incubated with labeled glycerol-3-phosphate (Gro-3—P) and CDP-diacylglycerol (PGP synthase assay, left two lanes), or with labeled UDP-galactose (galactolipid biosynthesis, right two lanes). Shown is an autoradiograph of a thin layer chromatogram of labeled lipid extracts. Identified lipids were: DGDG, Di galactosyldiacylglycerol; MGDG, monogalactosyl- diacylglycerol; PG, phosphatidylglycerol; PGP, phosphatidylglycerolphosphate. 112 Gro-3-P UDP-Gal WTpgp1 WT pgpt . . .,,.,...-_..,,,_1 MK I- MGDG i! T c - DGDG M (———-PGP O c 113 Table 3.2 Pigment content (mg g'1 Fresh Weight)a and chlorophyll fluorescenceb of Wild-type and mutant fully expanded leaves WT pgpl Chlorophyll a 1.48 :E 0.08 1.17 d: 0.11 Chlorophyll b 0.49 d: 0.03 0.36 i 0.03 Chlorophyll a+b 1.97 i 0.11 1.55 i 0.14 Chlorophyll a/b 3.01 i 0.02 3.29 i 0.07 Carotenoids 0.25 :l: 0.02 0.19 :l: 0.02 Chlorophyll/Carotenoid 7.77 i 0.26 7.99 :E 0.27 FV/Fm (n=25) 0.79 :t 0.02 0.74 i 0.01 [:1 p3” (n=10) 0.55 i 0.01 0.49 i 0.01 FV’/Fm’ (n=10) 0.69 :t 0.01 0.60 :1: 0.01 3 Samples were taken from fully expanded leaves of 25-to-30 day old plants harvested after 8 h light exposure. Plants were grown under a l6-h-light/8-h-dark regime at a PPFD of 75 umol m'2 s'1 in soil. Pigment values represent means (i SE) of six independent determinations. b Data were acquired at a PPFD of 75 umol m'zs'z. 114 was performed in the light (data not shown). Apparently, fatty acid biosynthesis and de novo lipid biosynthesis are stimulated in the light and lead to the incorporation of label from glycerol-3-phosphate into the diacylglycerol moieties of other lipids. We therefore quantified the PGP synthase activity in dark incubated chloroplasts by determining the amount of label in the total lipid extracts derived from the assay mixture. In the pgp] mutant, the Chloroplastic PGP synthase activity was reduced to approximately 19% of wild-type activity from 24.4 d: 4.4 amol glycerol-3-phosphate min’1 mg'l chlorophyll to 4,7 :1: 1.7 umol glycerol-3- phosphate min’1 mg'l chlorophyll. Photosynthesis is impaired in the pgp] mutant Although the loss of total PG in leaves of the pgp] mutant does not exceed 30%, the growth of the mutant is reduced and the mutant plants appear pale green consistent with an impairment in photosynthesis (Fig. 3.1). This initial observation was confirmed by quantification of the pigment content of fully expanded leaves (Table 3.2). Chlorophyll a and b as well as carotenoid amounts were reduced in the mutant and the ratio of chlorophyll a/b was increased. This result points to an alteration in the structure of the photosynthetic apparatus in the mutant and explains the pale green color of pgp] leaves. To investigate the functionality of the photosynthetic apparatus in the pgp] mutant, we used non-invasive chlorophyll fluorescence measurements. This technology is Well established and was successfully used to investigate photosynthetic competency and light utilization of other Arabidopsis lipid mutants (Hartel et al., 1997; Hartel et al., 19983; Harte] et al., 1998b). Measurements under steady state conditions at a Photosynthetic photon fluence density comparable to that used for the growth of the 115 plants revealed a marked decrease in the quantum yield of linear electron transport through photosystem II ((Dpsu) and in the efficiency of open photosystem II units (FV’/Fm’) (Table 3.2). A small (approximately 6%), but significant decrease in the maximum photochemical efficiency of photosystem II (Fv/Fm) was observed for the mutant following a 1 h dark adaptation period. For comparison, the initial Fv/Fm ratios in the ats](actI) and phol mutant were 0.79i0.01 and 0.78i0.02, respectively. Taken together, these alterations in the pigment composition and photosynthetic light energy utilization in the pgp] mutant underline the importance of PG for the proper structure and function of photosynthetic membranes in seed plants. 116 Discussion The majority of PG in green plant tissues is localized in the chloroplasts and the biosynthesis of PG in green plants has been studied in greatest detail using intact or broken chloroplasts from pea leaves (Andrews and Mudd, 1986). Based on pulse chase labeling experiments, it was proposed that PG biosynthesis proceeds in chloroplasts as worked out for bacteria and yeast. First, CDP-diacylglycerol is formed from CTP and phosphatidic acid through the action of CDP-diacylglycerol synthase (EC. 3.1.3.4). Plant cDNAs encoding this enzyme have been recently identified and expressed in E. coli (Kopka et al., 1997). Although this reaction is required for PG biosynthesis, it is not specific for this process, because CDP-diacylglycerol also serves as precursor in the biosynthesis of other phospholipids (Kinney, 1993).The second step of PG biosynthesis involves the transfer of glycerol-3-phosphate onto CDP-diayclglyerol giving rise to PGP and CMP. This reaction is catalyzed by PGP synthase (E. C. 2.7.8.5). Third, PGP is hydrolized to PG and inorganic phosphate by PGP phosphatase (EC. 3.1.3.27). While the latter two reactions had been observed in pea chloroplasts (Andrews and Mudd, 1986), no further biochemical analysis of the respective proteins has been performed. A mutant deficient in one of the two enzymes involved in plastidic PG biosynthesis would serve at least two purposes. First, it would permit the study of PG function in plastid membranes, and second, it would provide experimental evidence that the pathway described above indeed represents the major route of PG biosynthesis in plastids. Based on our analysis, pgp] is just such a mutant, because it severely affects plastidic PGP synthase activity. The experimental evidence for this conclusion can be summarized as follows: first, pgp] maps within one cM of BAC T168247 annotated as chloroplast targeted PGP synthase; 117 second, complementation analysis employing a PGP synthase deficient E. coli mutant confirmed the gene product of BAC locus T168247 is indeed a PGP synthase; third, introduction of wild type cDNA corresponding to T168247 into the mutant background rescued lipid and morphological phenotypes; forth, a mutation in the third exon of T168247 in pgp] gives rise to an RFLP at the DNA level, and at the protein level to a Pro 1 7° to Ser substitution in a highly conserved motif characteristic for CDP-alcohol phosphotransferase; and fifth, PGP synthase activity in isolated chloroplasts of pgp] is markedly reduced consistent with the other findings. Taken together, these observations provided an unambiguous logical link between the molecular defect and the biochemical phenotype in pgp], as well as a firm gene-product relationship between T168247 and the encoded PGP synthase. While the Pro170 to Ser mutation in pgp] affects a highly conserved proline in the active site (Williams and McMaster, 1998), the activity of the recombinant mutant protein was only reduced by approximately two thirds. Thus, the mutation in pgp] is “leaky”. Despite the fact that the relative amount of PG was only reduced by 30% in the Pg}?! mutant, this biochemical defect led to a visible phenotype. More detailed analysis revealed that pigment composition and photosynthetic light utilization are affected in this mutant, thereby explaining the slow growth and the pale green appearance of pgp] . Chlorophyll b is exclusively located in the light—harvesting pigment-protein complexes of phOIOSYStem II and photosystem 1. Therefore, a decrease of total chlorophyll content associated with an increase in the chlorophyll a/b ratios in the pgpl mutant (Table 3.2) Indicates both, a decrease in the total number of photosystems and a stronger decline in hgilt—harvesting antenna complexes relative to the chlorophyll a-containing reaction 118 center/core complexes. Chlorophyll fluorescence measurements on intact leaves revealed that the chlorophyll loss was associated with a decline in the quantum efficiency of photosystem II photochemistry. In particular, (Dpsn and Fv’lFm’ were markedly reduced in the mutant. Taken together, the chlorophyll fluorescence and pigment data for the pgp] mutant most likely reflect the importance of PG in photosynthesis of seed plants. This is consistent with previous observations on cyanobacterial mutants which are deficient in PG biosynthesis (Hagio et al., 2000; Sato et al., 2000). These cyanobacterial mutants have an absolute requirement for PG supplementation in their medium and deficiencies in photosynthetic competence correlate in severity with the amount of PG in cell membranes, which can be manipulated in this system through external supply. What is surprising about the pgp] mutant in comparison to ats](act]), is the fact that the latter mutant does not show a striking effect on growth and a less severe effect on photosynthesis in spite of a similar reduction in overall PG content (Kunst et al., 1988). While we currently do not know the basis for this difference, we expect that the detailed analysis of the pgp] mutant and a future reexamination of ats] (actI) mutant may provide Clues towards a deeper understanding of PG biosynthesis and function in plant Chloroplasts. While this work was under review, Mtiller and Frentzen (2001) published the expression of the PGP] cDNA in yeast and demonstrated a mitochondrial localization of the enzyme in this heterologous system. Although the data presented here, clearly show that PGPI is the major Chloroplastic isoform of phosphatidylglycerolphosphate synthase, We currently cannot rule out that PGP] is also associated with mitochondria in “ U abidopsis cells. -___—__L4 References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ .(1990) Basic local alignment search tool. J. Mol. Biol. 215:403-410 Andrews J, Mudd JB. (1986) Phosphatidylglycerol synthesis in pea chloroplasts. Plant Physiol. 79:259—265 Asai Y, Katayose Y, Hikita C, Ohta A, Shibuya I. (1989) Suppression of the lethal effect of acidic-phospholipid deficiency by defective formation of the major outer membrane lipoprotein in Escherichia coli. J. Bacteriol. 171:6867-6869 Bechtold N, Pelletier G. (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. In J Salinas, J M Martinez- Zapater, eds, Arabidopsis Protocols, Methods in Molecular Biology Vol 82. Humana Press, Totowa, NJ, pp 259-266 Bell CJ, Ecker JR. (1994) Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics 192137-144 Benning C, Somerville CR. (1992) Isolation and genetic complementation of a sulfolipid- deficient mutant of Rhodobacter sphaeroides. J. Bacteriol. 174:2352-2360 Browse J, Somerville C. (1991) Glycerolipid biosynthesis: Biochemistry and regulation. Annu Rev Plant Physiol Plant Mol. Biol. 42:467-506 Browse J, Warwick N, Somerville CR, Slack CR. (1986) Fluxes through the prokaryotic and eukaryotic pathways of lipid synthesis in the '16:3' plant Arabidopsis thaliana. Biochem. J. 235225-31 Dormann P, Hoffinann-Benning S, Balbo I, Benning C. (1995) Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacylglycerol. Plant Cell 721801-1810 Dérmann P, Benning C. (1998) The role of UDP-glucose epimerase in carbohydrate metabolism of Arabidopsis. Plant J. 132641-652 Emanuelsson O, Nielsen H, Brunak S, von Heijne G .(2000) Predicting subcellular localization of proteins based on their N- terminal amino acid sequence. J. Mol. Biol. 300:1005-1016 ESSigmann 8, Giiler S, Narang RA, Linke D, Benning C. (1998) Phosphate availability affects the thylakoid lipid composition and the expression of SQD], a gene required for sulfolipid biosynthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 95:1950-1955 120 Estelle MA, Somerville C. (1987) Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. Mol. Gen. Genet. 206:200-206 Genty B, Briantais J M, Baker NR. (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochem. Biophys. Acta 990:87-92 Hagio M, Gombos Z, Varkonyi Z, Masamoto K, Sato N, Tsuzuki M, Wada H. (2000) Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis. Plant Physiol. 124:795-804 Hartel H, Essigmann 8, Lokstein H, Hoffrnann-Benning S, Peters-Kottig M, Benning C. (19988) The phospholipid-deficient pho] mutant of Arabidopsis thaliana is affected in the organization, but not in the light acclimation, of the thylakoid membrane. Biochim. Biophys. Acta 1415:205-218 Hal-tel H, Lokstein H, Ddrmann P, Grimm 8, Benning C. (1997) Changes in the composition of the photosynthetic apparatus in the galactolipid-deficient a’ga'] mutant of Arabidopsis thaliana. Plant Physiol.1 15 :1 175-1 184 Ham-.1 H, Lokstein H, Dormann P, Trethewey RN, Benning C. (1998b) Photosynthetic light utilization and xanthophyll-cycle activity in the galactolipid deficient dgd] mutant of Arabidopsis thaliana. Plant Physiol. Biochem. 362407-417 Héirtel H, Reinhardt I, Grimm 8. (19980) Relationship between energy-dependent fluorescence quenching and xanthophyll-cycle-pigments in transgenic chlorophyll- deficient tobacco grown under different light intensities. J. Photochem. Photobiol. 8. Biol. 43:136-145 Heinz E, Roughan G. (1983) Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants. Plant Physio. 72:273-279 Hobe S, Prytulla S, Kuhlbrandt W, Paulsen H. (1994) Trimerization and crystallization of reconstituted 1i ght-harvesting chlorophyll a/b complex. EMBO J. 13:3423-3429 JOrdan 8R, Chow W-S, Baker AJ. (1983) The role of phospholipids in the molecular organisation of pea chloroplast membranes. Effect of phospholipid depletion on photosynthetic activity. Biochim. Biophys. Acta 725277-86 J01‘dan P, Fromme P, Witt HT, Klukas O, Saenger W, Krauss N. (2001) Three- dimensional structure of cyanobacterial photosystem I at 2.5 A resolution. Nature 411:909-917 Joy KW, Mills WR. (1987) Purification of chloroplasts using silica sols. Methods Enzymol. 148:179-199 121 Kinney AJ. (1993) Phospholipid head groups. In TS Moore, ed, Lipid Metabolism in Plants, CRC Press, Boca Raton, FL, pp 259-284 Kopka J, Ludewig M, Mtiller-Rdber 8. (1997) Complementary DNAs encoding eukaryotic-type cytidine-5'-diphosphate- diacylglycerol synthases of two plant species. Plant Physiol. 1 13 2997-1002 Kruse O, Hankammer 8, Konczak C, Gerle C, Morris E, Radunz A, Schmid GH, Barber J. (2000) Phosphatidylglycerol is involved in the dimerization of photosystem II. J. Biol. Chem. 275: 6509-6514 Kuhlbrandt W. (1994) Structure and function of the plant light-harvesting complex, LHC-II. Curr. Opin. Struct. Biol. 4:519-528 Kunst L, Browse J, Somerville C. (1988) Altered regulation of lipid biosynthesis in a mutant Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity. Proc. Natl. Acad. Sci. USA 85:4143-4147 Lichtenthaler HK. (1987) Chlorophylls and carotenoids: Pigments of photosynthetic membranes. Methods Enzymol. 148:350-382 Lo gemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal. Biochem. 163:16-20 Marechal E, Block MA, Dorne A-J, Joyard J. (1997) Lipid synthesis and metabolism in the plastid envelope. Physiol. Plant 100165-77 Moore TS. (1982) Phospholipid biosynthesis. Annu. Rev. Plant Physiol. 33:235-259 Mudd JB, Andrews JE, Sparace SA. (1987) Phosphatidylglycerol synthesis in chloroplast membranes. Methods Enzymol. 148:338-345 Miiller. F, Frentzen M. (2001) Phosphatidylglycerolphosphate synthases from Arabidopsis thaliana. FEBS Let. 509:298-302 M urashige T, Skoog F. (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant 152473-497 M ul‘ata N, Fujimura Y, Higashi S. (1990) Glycerolipids in various preparations of photosystem II from spinach chloroplasts. Biochim. Biophys. Acta. 1019:261-268 1\/II—11‘ata N, Ishizaki-Nishizawa O, Higashi S, Hayashi H, Tasaka Y, Nishida I. (1992) Genetically engineered alteration in the chilling sensitivity of plants. Nature 356:710-713 IVIuI‘ata N, Nishida I. (1987) Lipids of blue-green algae (cyanobacteria). In PK Stumpf, ed, The Biochemistry of Plants, Vol 9. Academic Press, New York, NY pp 315-347 122 N uBberger S, D'o'rr K, Wang DN, Kuhlbrandt W. (1993) Lipid-protein interactions in crystals of plant light-harvesting complex. J. Mol. Biol. 234:347-356 Poirier Y, Thoma S, Somerville C, Schiefelbein J. (1991) A mutant of Arabidopsis deficient in xylem loading of phosphate. Plant Physiol. 97:1087-1093 Rogers SO, Bendich AJ. (1994) Extraction of total cellular DNA from plants, algae and fungi. In SB Gelvin, RA Schilperoort, eds, Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 1-8 Rossak M, Schafer A, Xu N, Gage DA, Benning C. (1997) Accumulation of sulfoquinovosyl-1-O-dihydroxyacetone in a sulfolipid-deficient mutant of Rhodobacter sphaeroides inactivated in squ. Arch. Biochem. Biophys. 3402219- 230 Sato N, Hagio M, Wada H, Tsuzuki M. (2000) Requirement of phosphatidylglycerol for photosynthetic function in thylakoid membranes. Proc. Natl. Acad. Sci. USA 97: 10655-10660 Schmid VHR, Cammarata KV, Bruns 8U, Schmidt GW. (1997) In vitro reconstruction of the photosystem I light-harvesting complex LHCI—730: Heterodimerization is required for antenna pigment organization. Proc. Natl. Acad. Sci. USA 94:7667-7672 S i e genthaler P-A, Smutny J, Rawler A. (1987) Involvement of distinct populations of phosphatidyl glycerol and phosphatidylcholine molecules in photosynthetic elctron- flow activities. Biochim. Biophys. Acta. 891:85-93 Somerville C. (1995) Direct tests of the role of membrane lipid composition in low- temperature-induced photoinhibition and chilling sensitivity in plants and cyanobacteria. Proc. Natl. Acad. Sci. USA 92:6215-6218 The Arabidopsis Genome Initiative. (2001) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796-815 Tremolieres A, Dainese P, Bassi R. (1994) Heterogenous lipid distribution among chlorophyll-binding proteins of photosytem II in maize mesophyll chloroplasts. Eur. J. Biochem. 221 :721-730 Usui M, Sembongi H, Matsuzaki H, Matsumoto K, Shibuya I. (1994) Primary structures of the wild-type and mutant alleles encoding the phosphatidylglycerophosphate synthase of Escherichia coli. J. Bacteriol. 176:3389-3392 Van't Hof R, van Klompenburg W, Pilon M, Kozubek A, de Korte-Kool G, Demel RA, Weisbeek PJ, de Kruij ff 8. (1993) The transit sequence mediates the specific interaction of the precursor of ferredoxin with chloroplast envelope membrane lipids. J. Biol. Chem. 268:4037-4042 123 Van Kooten O, Snel JFH. (1990) The use of fluorescence nomenclature in plant stress physiology. Photosynth. Res. 25: 147-150 Williams JG, McMaster CR. (1998) Scanning alanine mutagenesis of the CDP-alcohol phosphotransferase motif of Saccharomyces cerevisiae cholinephosphotransferase. J. Biol. Chem. 273:13482-13487 124 Chapter 4 Anionic lipids are required for chloroplast structure and function in Arabidopsis The work presented in this chapter is in press: B in Yu and Benning. The Plant Journal 125 Abstract Photosynthetic membranes 0 f plants primarily contain non-phosphorous glycolipids. The exception is phosphatidylglycyerol (PG), which is an acidic/anionic phospholipid. A second major anionic lipid in chloroplasts is the sulfolipid sulfoquinovosyldiacyl glycerol (SQDG). It is hypothesized that under severe phosphate limitation SQDG substitutes for PG, ensuring a constant proportion of anionic lipids even under adverse conditions. A newly constructed SQDG and PG-deficient double mutant supports this hypothesis. This mutant, sqd2 pgp]-1, carries a T-DNA insertion in the structural gene for SQDG synthase (SQD2) and a point mutation in the structural gene for phosphatidylglycerolphosphate synthase (PGPl). In the sqd2pgp1-I double mutant the fraction of total anionic lipids is reduced by approximately one third resulting in pale yellow cotyledons and leaves with reduced chlorophyll content. Photoautotrophic growth of the double mutant is severely compromised and its photosynthetic capacity is impaired. In particular, photosynthetic electron transfer at the level of photosystem II is affected. Besides these physiological Changes, the mutant shows altered leaf structure, a reduced number of mesophyll cells and ultrastructural change of the chloroplasts. All observations on the sqd2 pgp]-I mutant lead to the conclusion that the total content of anionic thylakoid lipids is limiting f or chloroplast structure and function, and is critical for overall photoautotrophic growth and plant development. 126 Introduction The two major anionic/acidic lipids of photosynthetic membranes are phosphatidyl— glycerol (PG) and the non-phosphorous glycolipid sulfoquinovosyldiacylglycerol (SQDG). The phospholipid PG is common in the biological membranes of animals, plants and microorganisms and was found to be essential in photosynthetic organisms (Hagio et al., 2000; Sato et al., 2000; Hagio et al., 2002; Babiychuk et al., 2003). In vitro experiments showed that PG is enriched in 1i ght-harvesting pi gment-protein complexes of photosystem II (PSII) (Murata et al., 1990; Tremolieres et al., 1994) and that it is essential for the dimerization of the P811 reaction center core pi gment-protein complex (Kruse et al. , 2000). Furthermore, the recently published crystal structure of photosystem I contained three molecules of PG in tight coordination with the core and the antenna complex (Jordan et al., 2001). A PG-deficient mutant of the cyanobacterium Synechocystis sp. PCC6803 is no longer able to grow photoheterotrophically and PSII activity was severely compromised (Hagio et al., 2000; Sato et al., 2000). In addition, a pale green mutant of ArabidOpsis, pgp]-1, carrying a leaky mutation in the gene for phosphatidylglycerolphosphate synthase (PGPl) was recently described (Xu et al., 2002). In this mutant, a reduction in the content of PG of only 30% resulted in the impairment of photosynthesis. Moreover, complete inactivation of the PGP] gene by T-DNA insertion led to the abolishment of chloroplasts and loss of photoa‘utotrophy (Hagio et al., 2002; Babiychuk et al., 2003). These results suggested that PG was essential for chloroplast development and function in seeds plants. 127 The second anionic lipid found in thylakoid membranes, the sulfolipid SQDG, is not essential under normal growth conditions. Mutants of photosynthetic bacteria and Arabidopsis completely lacking this lipid showed only subtle impairments in photosynthesis and growth unless they are phosphate starved (Benning et al., 1993; Giiler et al., 1996; Yu et al. , 2002). As a common phenomenon in photosynthetic organisms, the relative amount of total anionic thylakoid lipids is maintained by reciprocally adjusting SQDG and PG contents as phosphate availability changes. Typically the relative content of PG decreases and that of SQDG increases following phosphate limitation. In SQDG—deficient mutants, the proportion of PG does not decrease under phosphate-limitation and these mutants become phosphate starved sooner than the respective wild types. Based on these observations, it was suggested that one of the main function for SQDG is to substitute for PG under phosphate limitation to maintain the proper balance of anionic charge in the thylakoid membrane (Benning et al., 1993; Gtiler et al., 1996; Yu et al., 2002). To further test this SQDG/PG substitution hypothesis by endogenously manipulating the total amount of anionic/acidic thylakoid lipids, we constructed an sqd2pgp1-1 double mutant of Arabidopsis lacking SQDG completely and PG partially. The sqd2 mutant carries a T-DNA insertion into the sulfolipid synthase gene SQD2 (Yu et al., 2002). The weak allele, pgp]-I, for the PGP synthase gene PGP] was preferred in this study, because plants carrying stronger pgp] homozygous alleles are all“eady completely non-photosynthetic (Hagio et al., 2002; Babiychuk et al. , 2003) and additive effects of SQDG-deficiency cannot be revealed. Here we describe the CCDrlstruction and characterization of the sqd2 pgp]-I double mutant. 128 Materials and methods Plant material, growth experiments and DCMU treatment Arabidopsis wild-type and mutants were grown under a photosynthetic photon flux density (PPFD) of 70-80 :mol m'zs’l at 22/18°C(day/night) with a 14-h li ght/ 10-h dark period. Surface-sterilized seeds were germinated on 0.8% (w/v) agar-solidified Murashige and Skoog (MS) medium (Murashige et al., 1962) supplemented with 1% sucrose. Seedlings were grown for 8 days on agar before transfer to soil or plates containing Arabidopsis medium (half strength without sucrose) (Estelle et al., 1987). For phosphate limitation experiments, KH2PO4 was omitted from the medium and substituted by MES buffer (Hartel et al., 2000). Quantitative growth experiments were done as described (Yu et al., 2002). Briefly, plants were grown under a uniform PPFD of 80 :mol m‘2 8‘. At each time point 7-to-10 plants per line were harvested and their aerial parts were weighed. For DCMU treatment, half strength Arabidopsis medium without sucrose was used with addition of different concentrations of DCMU as indicated. Plants were grown on MS medium for 8 days and then transferred to the DCMU containing agar plates. After 7 days of growth the fresh weight of 4-to-5 plants was averaged and relative growth was plotted as the ratio of the fresh weight per plant of DCMU untreated-to- DCMU treated samples. Construction of the sqd2 pgp]-I double mutant To generate the sqd2 pgp]-1 double mutant, sqd2 (Yu et al., 2002) was used as the po 1 len donor in a cross to pgp]-1 (Xu et al., 2002). The ecotypes were Wassilewskija for 129 the sqd2 mutant and Columbia-2 for pgp]-1. Nearly isogenic lines were generated by inbreeding through 8 generations. Following each round of selfing, seeds of 20 individual plants were collected, 100-200 seeds per plant were plated and the segregation of young seedlings was phenotypically scored. This was accomplished by visual examination of the plants, as homozygous pgp]-1 seedlings, double homozygous mutants and wild type were clearly distinguishable from each other and other genotypes based on their tint and size (Fig. 4.1). Descendants from a double heterozygous line were analyzed again in the next generation until the four required lines were recovered from a single F7 double heterozygous plant. Analysis of lipids from leaves and chloroplasts Leaves were harvested and frozen immediately in liquid nitrogen, and lipids were extracted as previously described (Dormann et al., 1995). Lipid extracts were analyzed on activated, ammonium sulfate-impregnated silica gel TLC plates (Hartel et al., 2000) using running solvent of acetone/toluene/water (93:30:8 v/v/v). Lipids were visualized with iodine vapor and identified by cochromatography with lipid extracts of known composition. For quantitative analysis, methylesters were prepared and quantified by GLC (Rossak et al., 1997). For chloroplast lipid analysis, plastids were isolated as described (Hartel et al., 2000). The chloroplasts were immediately resuspended in 200 :l Chloroform/methanol/formic acid (10:10:1, v/v/v), followed by the addition of 100 :l 0.2 M H3PO4 and 1 M KCl. In all quantitative experiments Student’s t—test was used to assess the significance of differences between samples. 130 Transmission electron microscopy and light microscopy Three leaves from each line were prepared for light and transmission electron microscop-y. The leaves were fixed with 2.5% (v/v) glutaraldehyde and 2% paraformaldehyde (v/v) in 0.1 M sodium phosphate buffer (PH 7.4) by infiltration under vacuum and continually incubated at ambient pressure and 4 °C for 24 h. The samples were post fixed with1% osmium tetroxide in the same buffer for 2 h at room temperature. This was followed by a serial dehydration with 50%, 70%, 80%, 88%, 95%, and 100% (v/v) of acetone in water. The specimens were infiltrated with a series of 33%, 50%, 75%, and 100% (v/v) of poly/bed resin (Polyscience Inc., Warrington, PA) in acetone. After embedding in poly/bed resin, three blocks each of wild-type and mutants were sectioned and the sections stained with 1% toluidine blue and examined with a Zeiss Pascal confocal laser scanning microscope (Carl Zeiss, Germany). The thin sections were stained with 2% uranyl acetate and lead citrate before viewing in a J EOL 100CX transmission electron microscope (J EOL Inc., Peabody, MA). Chlorophyll Fluorescence and pigments measurements Pigments were determined as described (Lichtenthaler, 1987). In vivo chlorophyll fluorescence at room temperature was determined with an F MS2 fluorometer (Hansatech Instruments, Pentney King’s Lynn, UK) as previously described (Hartel et al., 1998). Fluorescence parameters used were as defined by Genty et al., and van Koten and Snel (Genty et al., 1989; van Kooten et al., 1990). 131 Results Growth of the sqd2pgp1-I double mutant is reduced The sqd2 pgp]-I double mutant was constructed by crossing the two respective single mutant lines (Yu et al., 2002; Xu et al., 2002). These lines were of different ecotypes (sqd2, Wassilewskija and pgp]-1, Columbia-2), a fact which might have posed a serious problem for the interpretation of comparative data as discussed below. To create nearly isogenic lines for the four genotypes compared in this study, sqd2/sqd2 PGPI/PGP] (sqd2 mutant), SQD2/SQD2pgp1-I/pgpI-I (pgp]-I mutant), SQD2/SQD2 PGPI/PGP] (wild type), and sqd2/sqd2pgpI-1/pgp1-I (homozygous double mutant), a recombinant inbred line was generated by repeated selfing of single double heterozygous descendants from the original cross through 8 generations. According to theory (Reiter et al., 1992), the four different homozygous lines isolated in the F3 generation should be nearly (99.2%) isogenic, eliminating effects from genetic variations at loci different from those under direct consideration. In agreement with recessive mutations, the F1 progeny were phenotypically wild-type. Thin-layer chromatography (TLC) of lipid extracts from 3 1 5 segregating F2 plants showed that 64 (approximately 3/16) had a reduced proportion of PG (homozygous for pgp]-1), 60 were SQDG-deficient (homozygous for sqd2), and 2 O (approximately1/16) lacked SQDG and showed a reduced proportion of PG. These latter were the expected homozygous sqd2pgp1-I double mutants. The cotyledons and true leaves of sqd2 pgp]-1 double mutants were paler green than those of the pgp]-1 mutants as shown in Figure 4.1. The true leaves of the double mutant were smaller 132 Figure 4.1. Appearance and growth of wild-type and mutant lines. (a), six week-old plants grown in soil under standard conditions; (b), plants grown for 8 d on agar- solidified MS medium with 1% (w/v) sucrose followed by 12 d of growth on a half concentration Arabidopsis medium without sucrose. (c), close up of leaves from plants shown in (a); (d), seedlings 2-3 days after germination grown on agar-solidified MS medium with 1% (w/v) sucrose. 133 sqd2 pgp1- 1 WT» 1-1 sqd2 pgp sqd2pgp1-1 134 Figure 4.2. Growth curves for wild-type (open diamond), sqd2 (filled diamond), pgp]- ] (open square), and sqd2pgp1-I (filled square) lines on agar-solidified half-strength Arabidopsis medium with 1mM(a) or no (b) phosphate added. At each time point, the aerial part fresh weights of at least 7 plants were determined and averaged (iSE). 135 IAvD... .. /V// / / ) lbw // H /. 1'5 20 1'0 8 Time after transfer (d) 70- 60- 50- q o 4 14 . . 0 3 A o. 2 m6 1 u .. a. 007654321 1 V 2963 fish. 0 136 and its growth rate was reduced. As shown in Figure 4.28, the fresh weight of the aerial part of the double mutant was reduced to approximately one third compared to the other lines at three weeks of grth on phosphate-replete medium. The double mutant was able to survive on soil and on agar-solidified medium lacking sucrose, but the growth rate was greatly reduced compared to plants grown in the presence of sucrose (data not shown). These results suggested that the sqd2 pgp]-1 double mutant is capable of photoauto- trophic growth, but its growth is severely limited by a compromised photosynthetic apparatus. Under phosphate-limited conditions, the growth of the double mutant was reduced compared to the wild-type and the pgp] mutant, but to nearly the same extent as observed for the sqd2 mutant (Fig. 4.2b). The proportion of total anionic lipids in the double mutant is decreased Total leaf lipid analysis of the sqd2pgp1-I double mutant as shown in Figure 4.33 revealed that the relative proportion of PG was approximately 11 mol%, higher than in the pgp]—1 mutant. Because PG is also found in extraplastidic membranes, this result might reflect a decrease in the absolute amount of chloroplast membranes. Indeed, the relative proportion of the exclusively extraplastidic lipid phosphatidylethanolamine (PE), Was also increased in the double mutant compared to the pgp]—1 mutant, in agreement with an overall increase in the ratio of extraplastidic-to-plastidic membrane lipids. Comparing the lipid composition of isolated chloroplasts from the four different lines as shown in Figure 4.3b, a similar decrease in the relative amount of PG was observed for the pgp]-1 mutant and the sqd2pgp1-1 double mutant corroborating the 137 Figure 4.3. Quantitative analysis of polar lipids. Lipids were extracted from leaves (a), or isolated chloroplast (b). Plants in (a) and (b) were grown on medium with lmM phosphate. Leaf lipid extracts of plants grown on medium without phosphate are shown in (c). Values represent means (:l:SE) of three independent measurements. DGDG, digalactosyldiacylglyeerol; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol. 138 [E sqd2 -WT 5°jra> m pgp1-1 [:1 sqd2 pgp1-1 . w . m . a. . m. 3.63 @0099. m .mm mam“ b C ( /\ 4 1 d I I d. 36.0.5 35: ion do :88qu 0. PC PE MGDG PG DGDG SQDG Pl 139 hypothesis that the ratio of extraplastidic—to-plastid lipids is increased in the double mutant. Summing the amount of the anionic lipids SQDG and PG in chlor0plast extracts (Figure 4.3b, inset), it was apparent that the total amount of anionic lipids in the two single mutants remained similar to that of wild type, but was reduced in the sqd2 pgp]-1 double mutant by approximately one third (99% confidence level based on Student’s t- test). In the pgp]-I mutant the relative increase in SQDG and in the sqd2 single mutant the relative increase in PG appeared to reciprocally compensate the respective deficiency in the alternate anionic lipid. These compensatory mechanisms were also visible when total leaf lipid extracts were examined following phosphate-limited growth (Figure 4.30). The relative amount of PG in the two SQDG-deficient lines was similarly maintained under these conditions, explaining the similar growth reduction of the sqd2 and sqd2 pgp]-1 double mutant following phosphate deprivation (Figure 4.2b). In addition to these changesin relative anionic lipid content, the proportion of the galactolipid DGDG increased following phosphate limitation as was previously observed for Arabidopsis (Hartel et al., 2000). Photosynthetic activity is compromised in the double mutant The pale green leaves of the sqd2pgp1-1 double mutant suggested a reduction in chlorophyll content and a possible impairment in photosynthetic activity. Indeed, the amounts of chlorophylls a and b, and carotenoids were decreased in the double mutant to approximately one third of that observed for wild type and sqd2 mutant, and to one half of that of the pgp] -] mutant (Table 4.1). The chlorophyll a/b ratio was higher than for wild type and sqd2, but was similar to that of the pgp]-1 mutant. Taken together, these 140 -1 Table 4.1 Pigment content (mg g fresh weight)* of wild-type and mutant lines WT sqd2 pgp] -1 sqd2 pgp] -] chlorophyll a 0.86 i 0.04 0.99 :l: 0.07 0.61 :1: 0.07 0.32 :l: 0.03 chlorophyll b 0.29 i 0.01 0.36 i 0.02 0.18 i: 0.02 0.09 i 0.01 :Tgmphy“ 1.14 a 0.05 1.34 a 0.1 0.80 a 0.08 0.42 a 0.04 chlorophyll a/b 2.98 3: 0.03 2.76 i 0.03 3.3 i 0.1 3.54 d: 0.16 Carotenoids 0.25 i 0.01 0.27 i— 0.02 0.18 i 0.02 0.10 i 0.08 *Samples were taken from fully expanded leaves of 20-30 day—old plants grown on agar- solidified medium containing 1 mM phosphate. Pigment values represent means (:E SE) of at least four independent determinations. 141 results suggested that the ratio of light-harvesting antenna-to-reaction core complex was similarly altered in the pgp]-1 single and the sqd2pgp1-1 double mutant, but that the total amount of pigment protein complexes and therefore the extent of total thylakoid membrane in the double mutant was strongly reduced. Photosynthesis in plants of the four lines included in this study was investigated in greater detail using pulse amplitude- modulated chlorophyll fluorescence analysis, a sensitive and non-invasive method (Krause et al., 1991). This method provides an indication of the overall photosynthetic competence of leaves and indirectly permits conclusions with regard to the electron- transport reactions at the thylakoid membranes. As shown in Figure 4.4a and b, there was no obvious difference in the maximum intrinsic efficiency of PSII in the light adapted state (F V’/F M’) over a wide range of photosynthetic photon flux densities (PPFD), but the quantum yield of linear electron transfer through PSH (CDps 11) was reduced in the double mutant (95% confidence level based on t-test), especially in the low PPFD range as compared to wild type, sqd2, and pgp]-I . The deduced fluorescence parameter l-qp, which reflects the fraction of “open” PSII reaction center provides an indication of the reduction state of the primary electron acceptor of PSH, QA. This parameter was increased in the sqd2pgp1-1 double mutant (99% confidence level), while no obvious differences were visible for the wild type and the two single mutant lines sqd2 and pgp]- ] . Therefore, it seemed likely that PSII is more reduced in the squ pgp]-1 double mutant in accordance with a decreased quantum yield of PSII (CDpsu). To verify this hypothesis using an independent approach, we tested the effect of 3 (3, 4-dichlorophenyl)-1, 1- dimethylurea (DCMU) on the growth of plants. This herbicide binds at the secondary electron acceptor (QB) site of PSII, and blocks the electron transfer from QA to QB 142 Figure 4.4. Light dependence of steady-state chlorophyll fluorescence in fully expanded leaves of the wild-type (open diamond), sqd2 (filled diamond), pgp]-1 (open square), and sqd2 pgp]-1 (filled square). (a), CDPSH, effective quantum yield of linear electron transport through PSII calculated as (Fm’-FS’)/Fm’; (b) FV’/Fm’, maximum photochemical efficiency of PSII calculated as (Fm’-F0’)/Fm’; (0), QA photoreduction state (l-qp) estimated as 1 -(FS’—FO’)/(Fm’-FO’). Values represent means (:ESE) of 6 independent determinations. 143 (18d 08- (bPSH QA- 0.2 -1 0.0 5 0.8- PV IF' g: c: a> a: l L I Q A reduction (1 -q P) O i F’ N 1 A 00 500 1000 Y 1500 I 2000 PPFD (Timon m'2 s") 144 2500 Figure 4.5. Sensitivity to DCMU of wild-type and mutant lines. Plants were grown for 12 days on MS medium followed by 7 days on plates with half strength Arabidopsis medium containing the herbicide; (a) no DCMU; (b) 121 nm DCMU. (c), relative growth of plants on agar with different DCMU concentrations. Relative growth was calculated as fresh weight ofthe aerial part of plants at a specific DCMU concentration divided by fresh weight ofthe aerial part of plants grown in the absence of DCMU. Wild type (open diamond), sqd2 (filled diamond), pgp]-1 (open square), and sqd2pgp1-I (filled square). 145 Relative Growth ' 200 ' 400 ' 600 800 '10'0011'200' 14 DCMU in medium (nM) 146 00 (Oettmeier et al., 1983). As shown in Figure 4.5, the sqd2pgp1-I double mutant was considerably more sensitive to DCMU than the two single mutant lines and the wild type in agreement with an increased reduction pressure at the QA site of PSH in the double mutant. After one week in the presence of 13.5 nM DCMU, the relative growth of the double mutant was reduced to approximately 50% (99% confidence level), while the other three lines were still growing normally. A concentration of 405 nM DCMU led to a complete cessation of growth of the sqd2pgp1-I double mutant. At even higher concentrations of DCMU, the single mutants also showed increased sensitivity to the herbicide compared to the wild type, suggesting that complete SQDG deficiency or a decrease of the PG content by 30% were sufficient to cause subtle changes in PS 11 activity, however, with no apparent effect on growth under normal laboratory conditions (Figure 4.2a). Leaf structure and chloroplast ultrastructure are altered in the double mutant The biochemical analysis of the sqd2pgp1-1 double mutant described above provided indirect evidence for an increased ratio of extraplastidic-to-plastidic lipids. To corroborate this hypothesis in more direct ways, the leaf structure of the different lines was compared by light microscopy of thick sections (Figure 4.6). The sqd2pgp1-1 dou- ble mutant showed larger intercellular spaces and. a reduction in mesophyll cell numbers. The epidermal cells and mesophyll cells were enlarged. Mesophyll cells with chloroplasts Were only present adjacent to vascular structures. Chloroplast numbers per cell cross section were reduced in the sqd2 pgp]-I double mutant to approximately 5 0% of wild tyIJe. Averaging at least 30 sections per sample, numbers of chloroplasts per cell cross 147 Figure 4. 6. Light microscopy of the leaf sections of wild-type (a), sqd2 (b),pgpI-I (c) and sqd2 pgp]-I ((1). Bar = 100 :m. Sections ofthe first true leaves oftwo week—old plants are shown. The sections were stained with 1% toluidine blue 148 8.91835 s ‘2 31' r! . *a'x‘ A ‘. ,A v 1“.er a 1,4. 1 r T»! f 3’» 3:“ '3’": .11? ‘1’" 1 5“ 91-9.» 53- '3’“? 7.1} v 3;} ‘5'.- f‘?‘ .3; 7“; a: \\ L :2 'H ’w "r u,” 'Rzn’r- . . ,. \ x. v 1‘ (\a -4. .V'}. .H ’1; .14 .41“ t \‘a \ - . - . rub—3”,}- , Cf 149 Figure 4.7. Ultrastructure of chloroplasts from mesophyll cells. Wild type (a), and sqd2pgp1-1 (b) from emerging (youngest true) leaves. Wild type (c), sqd2 (d), pgp]-I (e), sqd2pgp1-1 (i) from first true leaves. The plants were grown for two weeks in soil. Bar=1um. 150 151 section i SD were for wild type, 8.0 i 0.3; sqd2, 7.9 a: 0.2;pgp1-I, 8.0 :I: 0.3, and sqd2 pgp]-I, 3.9 i 0.3. The ultrastructures of chloroplasts in emerging (not yet expanded) leaves of the wild type and the double mutant are shown in Figure 4.7a and b. The wild- type chloroplasts contained well developed thylakoid membranes contrary to the double mutant chloroplasts , which nearly lacked grana stacks. Figure 4.7 panels c, d and e depict chloroplasts of the expanded first true leaves from wild-type, sqd2, and pgp]—1 . Chloroplasts in these samples were generally indistinguishable. However, in chloroplasts of the double mutant, the thickness of the thylakoid grana stacks (Figure 4.70 appeared to be reduced(WT, 0.22 i 0.03 :m; sqd2, 0.20 :l: 0.03 :m;pgp]-], 0.21 i 0.03 :m; sqd2 pgpl-1, 0.11 i 0.02 :m; n=25). 152 Discussion The analysis of genetic mutants with biochemical defects provides a powerful tool to understand the function of the affected pathway and the molecule in question. As more biochemical mutants of Arabidopsis become available, opportunities arise to combine these mutants in single lines, thereby revealing the interaction of two and more pathways and compounds. Combining mutations in different pathways in a single line might reveal redundancies and limit the plant’s adaptive responses. An example is the dgd] act] double mutant, which is unable to grow photoautotrophically, while the parental mutants are much less affected in their growth (Ddrrnann et al., 1999). The phenotype was interpreted as a reduction of flux through the endoplasmic reticulum and plastid-based pathways of thylakoid lipid biosynthesis, which are partially redundant in Arabidopsis (Klaus et al., 2002). Plants are well known for their broad biochemical repertoire enabling them to adapt to and survive under adverse environmental conditions. A mutation in a single biochemical pathway of mostly conditional importance might present no phenotype at all, or might have only mild effects on the development or physiology of the plant under standard laboratory conditions. Sulfolipid-deficient mutants are a particularly good example. When the first sulfolipid-deficient mutants of bacteria became available, their growth was generally not affected by the mutation under optimal growth conditions (Benning et al., 1993; Giiler et al., 1996). In the case of the cyanobacterium Synechococcus sp. PCC7942, subtle changes in PSII photochemistry were observed. However, these changes were not growth-limiting (Gtiler et al., 2000). Arabidopsis completely lacking sulfolipid also did not show a growth impairment under standard 153 laboratory growth conditions (Yu et al., 2002), which may not necessarily duplicate conditions encountered by wild plants in nature. However, when additional metabolic pressure was applied to sulfolipid-deficient mutants, they began to lag behind wild type in growth. Phosphate-limitation brings about a metabolic impasse, to which bacteria and plants respond by decreasing the ratio of phosphorous—to-non-phosphorous lipids (Benning et al., 1995 ; Hartel et al., 2000). Sulfolipid-deficiency limits the ability of photosynthetic organisms to remodel their thylakoid membrane, partially replacing PG with SQDG. This inability to adjust was proposed as the primary cause of reduced growth of sulfolipid-deficient mutants under phosphate limitation (Benning et al., 1993; Guler et al., 1996; Yu et al., 2002). A different independent way of demonstrating the conditional importance of sulfolipid is to study SQDG-deficiency in a double mutant impaired also in the biosynthesis of PG. Two issues had to be addressed for the construction of such a double mutant and the corresponding control lines. First, severe PG-deficiency leads to a complete lack of photosynthesis making it impossible to study the more subtle effects of SQDG in such a line (Hagio et al., 2002). This issue was overcome by choosing the weak allele, pgp]-I, which causes only a 30% reduction in PG content. However, the choice of this allele posed a second problem, namely that the sulfolipid-deficient mutant of Arabidospis, sqd2, carrying a null allele caused by T-DNA insertion was of a different ecotype. Because the conclusions of this study were expected to be based on subtle differences between the double mutant, the two single mutants, and the wild type, it was prudent to generate nearly isogenic lines by inbreeding through 8 generations. This 154 strategy resulted in four strains with the required homozygous sqd2 or pgp]-1 alleles in a 99.2% identical genetic background in all four lines. The sqd2pgp1-I double mutant analysis demonstrated that SQDG-deficiency can be growth-limiting if the PG-based compensatory mechanism is eliminated. The two biochemical defects in this mutant caused a reduction in the overall anionic thylakoid lipid content. This was not the case for the single mutants, which compensate by adjusting the amount of the alternate anionic lipid (Fig. 4.3b). It is proposed that the overall reduction in anionic lipids limits the development of fully functional photosynthetic membranes, and therefore photoautotrophic growth. Different independent phenotypes of the double mutant were consistent with a limitation in thylakoid membrane lipid biosynthesis leading to an increase in the ratio of extraplastidic-to-plastidic lipids in the sqd2pgp1-1 double mutant: 1. The relative proportions of PE, which exclusively occurs in extraplastidic membranes, and of PG present in plastidic and extraplastidic membranes were increased (Fig. 4.3); 2. Pigments exclusively associated with the photosynthetic membranes were reduced on a fresh weight basis (Tab.4. 1), and most directly, mesophyll cell numbers, chloroplasts per cell and the amount of thylakoid membranes within chloroplasts were reduced (Fig. 4.6, 4.7). Beyond the inability of the sqd2pgp1-1 double mutant to assemble thylakoid membranes at a rate required for nomral growth, lack of SQDG and reduction in PG appeared to have also more direct effects on the photosynthetic apparatus. The double mutant clearly showed altered chlorophyll fluorescence consistent with a reduced electron transport rate through PSII in the double mutant, as compared to the single mutants or the wild type (Fig. 4.4). This effect was further aggravated by additions of the 155 herbicide DCMU, which blocks the electron transfer from PSII to the QB plastoquinone acceptor (Oettmeier et al. , 1983). Contrary to the control lines, growth of the double mutant was 3 orders of magnitude more sensitive to DCMU (Fig. 4.5). Studying a sulfolipid-deficient mutant of Chlamydomonas reinhardtii, Sato et al. and Minoda et al., (Minoda et al., 2002; Sato et al., 2003) have observed that this mutant is sensitive to DCMU and shows other signs of photosynthetic impairment. In the case of Arabidopsis as studied here, there is only a slight increase in sensitivity towards DCMU in the SQDG- deficient and PG-deficient mutants suggesting that the sensitivity of the double mutant is not specifically due to SQDG-deficiency, but a consequence of the reduction of anionic lipids in general. The alga C. reinhardtii contains a relatively high proportion (~12 mol%)of SQDG(Sato et al., 1995), compared to Arabidopsis (~3 mol%), and SQDG- deficiency may sufficiently reduce the amount of anionic thylakoid lipids to cause an impairment in photosynthesis by itself. This effect became more aggravated at elevated temperature (Sato et al., 2003) suggesting structural roles for SQDG in association with pigment protein complexes of the photosynthetic membrane. In general, the findings made with the C. reinhardtii mutant and the Arabidopsis sqd2pgp1-1 double mutant agree, suggesting a role of SQDG as an anionic lipid in the assembly and proper function of the photosynthetic membrane. In the case of Arabidopsis, which contains only small amounts of SQDG, it takes the additional reduction in PG content to clearly bring about the effects of sulfolipid deficiency under normal growth conditions. However, under phosphate-limited growth conditions, when the relative amount of SQDG is increased in the wild type, the inability to synthesize SQDG as a substitute for PG quickly becomes limiting for growth in an SQDG-deficient mutant. At first glance it seemed puzzling that 156 under phosphate-limited conditions, the sqd2pgp1-I double mutant showed no additional reduction in growth in comparison to sqd2 (Fig. 4.2). Upon closer examination of the lipid composition in the double mutant and sqd2 (Fig. 4.3), it is apparent that the relative PG content in these two lines under phosphate limitation was similar, and therefore no further reduction of anionic lipids in the double mutant occurred. Presumably, the PG- biosynthetic activity provided by the enzyme encoded by the pgp]-1 allele was sufficient under these conditions to keep up with the demand of PG biosynthesis. Whatever the specific biochemical or molecular roles that anionic lipids might fill in the photosynthetic membrane, i.e. providing a proton conducting pathway at the surface of the thylakoid membrane (Haines, 1983), or as boundary lipids and integral components of photosynthetic complexes (Barber et al., 1986), the double mutant analysis described here led to the conclusion that the total amount of the anionic lipids PG and SQDG is most critical. These findings also support the general concept of substituting one anionic lipid for another under phosphate—limited conditions. While SQDG and PG both play an important role in this adaptive mechanism to maintain the proper balance of anionic lipids in the thylakoid membrane, PG might have additional and more specific functions, as the greater impairment of the PG-deficient single mutants suggests (Hagio et al., 2002; Babiychuk et al., 2003). 157 Acknowledgments We thank Dr. Alicia Pastor for her help with light and transmission electron microscopy and Dr. Changcheng Xu, for the sqd2pgp1-1 F 1 seeds. This work was supportedin parts, by grants from the US Department of Energy Bioscience Program and the MSU Center for Novel Plant Products. 158 References Babiychuk E, Mtiller F, Eubel H, Braun HP, F rentzen M, Kushnir S. (2003) Arabidopsis phosphatidylg1ycerophosphate synthase 1 is essential for chloroplast differentiation, but is dispensable for mitochondrial function. Plant J. 33:899-909. Barber J, Gounaris K. (1986) What role does sulfolipid play within the thylakoid membrane? Photosynthes. Res. 9:239-249. Benning C, Beatty JT, Prince RC, Somerville CR. (1993) The sulfolipid sulfoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances growth under phosphate limitation. Proc. Natl. Acad. Sci. USA. 90:1561-1565. Benning C, Huang ZH, Gage DA. (1995) Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation. Arch. Biochem. Biophys. 317:103-111. Dormann P, Balbo I, Benning C. (1999) Arabidopsis galactolipid biosynthesis and lipid trafficking mediated by DGD]. Science 284:2181-2184. Dormann P, Hoffrnann-Benning S, Balbo I, Benning C. ( 1995) Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacylglycerol. Plant Cell 7: 1801-1810. Estelle MA, Somerville C. (1987) Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. Mol. Gen. Genet. 206:200-206. Genty B, Briantais JM, Baker NR. (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochem. Biophys. Acta 990287-92. Guler S, Essigmann B, Benning C. (2000) A cyanobacterial gene, squ, required for biosynthesis of the sulfolipid sulfoquinovosyldiacylglycerol. J. Bacteriol. 182:543- 545. Giiler S, Seeliger A, Harte] H, Renger G, Benning C. (1996) A null mutant of Synechococcus sp. PCC7942 deficient in the sulfolipid sulfoquinovosyl diacylglycerol. J. Biol. Chem. 271:7501-7507. Hagio M, Gombos Z, Varkonyi Z, Masamoto K, Sato N, Tsuzuki M, Wada H. (2000) Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis. Plant Physiol. 124:795-804. Hagio M, Sakurai I, Sato S, Kato T, Tabata S, Wada H. (2002) Phosphatidylglycerol is essential for the development of thylakoid membranes in Arabidopsis thaliana. Plant Cell Physiol. 43: 1456-1464. 159 Haines TH. (1983) Anionic lipid headgroups as a proton-conducting pathway along the surface of membranes: a hypothesis. Proc. Natl. Acad. Sci. USA 80:160-164. H'artel H, Dormann P, Benning C. (2000) DGDl-independent biosynthesis of extraplastidic galactolipids following phosphate deprivation in Arabidopsis. Proc. Natl. Acad. Sci. USA 97:10649-10654. Hartel H, Reinhardt I, Grimm 8. (1998) Relationship between energy-dependent fluorescence quenching and xanthophyll-cycle-pigments in transgenic chlorophyll- deficient tobacco grown under different light intensities. J. Photochem. Photobiol. Biol. 43:136-145. Jordan P, Fromme P, Witt H, Klukas O, Saenger W, Krauss N. (2001) Three-dimensional structure of cyanobacterial photosytem I at 2.5 A resolution. Nature 411:909-917. Klaus D, Hartel H, Fitzpatrick L, F roehlich J, Hubert J, Benning C, Dormann P. (2002) Digalactosyldiacylglycerol synthesis in chloroplasts of the Arabidopsis thaliana dgd] mutant. Plant Physiol. 128:885-895. Krause GH, Weis E. (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:313-349. Kruse O, Hankamer B, Konczak C, Gerle C, Morris E, Radunz A, Schmid GH, Barber J. (2000) Phosphatidylglycerol is involved in the dimerization of photosystem II. J. Biol. Chem. 275:6509-6514. Lichtenthaler HK. (1987) Chlorophylls and carotenoids: Pigments of photosynthetic membranes. Methods Enzymol. 148:350-382. Minoda A, Sato N, Nozaki H, Okada K, Takahashi H, Sonoike K, Tsuzuki M. (2002) Role of sulfoquinovosyl diacylglycerol for the maintenance of photosystem II in Chlamydomonas reinhardtii. Eur. J. Biochem. 269:2353-2358. Murashige T, Skoog F. (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant 15:473-497. Murata N, Fujimura Y, Hi gashi S. (1990) Glycerolipids in various prepaprations of photosytem II from spianch chloroplasts. Bichim. Biophys. Acta 1019:261-268. Oettmeier W, 3011 HJ. (1983) Competition between plastoquinone and DCMU at the acceptor side of photosystem II. Bichim. Biophys. Acta 724:287-290. Reiter RS, Young RM, Scolnik PA. (1992) Genetic linkage of the Arabidopsis genome: methods for mapping with recombinant inbreds and Random Amplified Polymorphic DNAS (RAPDS). In Methods in Arabidopsis Research (Koncz, C., Chua, N.—H., Schell, J ., eds). River Edge, NJ: World Scientific Publishing, pp 170-190. 160 Rossak M, Schafer A, Xu N, Gage DA, Benning C. (1997) Accumulation of sulfoquinovosyl-1-O-dihydroxyacetone in a sulfolipid- deficient mutant of Rhodobacter sphaeroides inactivated in squ. Arch. Biochem. Biophys. 340:219- 230. Sato N, Aoki M, Maru Y, Sonoike K, Minoda A, Tsuzuki M. (2003) Involvement of sulfoquinovosyl diacylglycerol in the structural integrity and heat-tolerance of photosystem II. Planta 217:245-251. Sato N, Hagio M, Wada H, Tsuzuki M. (2000) Requirement of phosphatidylglycerol for photosynthetic function in thylakoid membranes. Proc. Natl. Acad. Sci. USA 97:10655-10660. Sato N, Sonoike K, Tsuzuki M, Kawaguchi A. (1995) Impaired photosystem H in a mutant of Chlamydomonas reinhardtii defective in sulfoquinovosyl diacylglycerol. Eur. J. Biochem. 234216-23. Tremolieres A, Dainese P, Bassi R. (1994) Heterogeneous lipid distribution among chlorophyll-binding proteins of photosystem II in maize mesophyll chloroplasts. Eur. J. Biochem. 221:721-730. van Kooten, O., Snel, J .F .H. (1990) The use of fluorescence nomenclature in plant stress physiology. Photosynthes. Res. 25:147-150. Xu, C, Hartel H, Wada H, Hagio M, Yu 8, Eakin C, Benning C. (2002) The pgp] locus of Arabidopsis encodes a phosphatidylglycerol synthase with impaired activity. Plant Physiol. 129:594-604. Yu 8, Xu C, Benning C. (2002) Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth. Proc. Natl. Acad. Sci. USA 99:5732-5737. 161 Chapter 5 Loss of plastidic lysophosphatidic acid acyltransferase causes embryo- lethality in Arabidopsis The work presented here has been submitted to The plant Journal: Bin Yu, Setsuko Wakao, Jilian Fan, Christoph Benning (2003) 162 Abstract Phosphatidic acid is a key intermediate for chloroplast membrane lipid biosynthesis. De novo phosphatidic acid biosynthesis in plants occurs in two steps: First the acylation of the sn-1 position of glycerol-3-phosphate giving rise to lysophosphatidic acid; Second, the acylation of the sn-2 position of lysophosphatidic acid to form phosphatidic acid. The second step is catalyzed by a lysophosphatidic acid acyltransferase (LPAAT). Here we describe the identification of the AT 52 gene of Arabidopsis encoding the plastidic isoform of this enzyme. Introduction of the AT 52 cDNA into E. coli J C 201, which is temperature-sensitive and carries a mutation in its LPAAT gene plsC, restored this mutant to nearly wild-type at high temperature. A green-flourescent protein fusion with ATS2 localized to the chloroplast. Disruption of the AT S2 gene of Arabidopsis by T- DNA insertion caused embryo lethality. The development of the embryos was arrested at the globular stage concomitant with a transient increase in AT 52 gene expression. Apparently, plastidic LPAAT is essential for embryo development in Arabidopsis during the transition from the globular to the heart stage when chloroplasts begin to form. 163 Introduction The thylakoid membrane of photosynthetic organisms contains four major glycerolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG), phosphatidylglycerol (PG), and the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). Two pathways contribute to the biosynthesis of membrane lipids in many plants including Arabidopsis (Roughan and Slack, 1982). According to this “two pathway hypothesis”, fatty acids de novo synthesized in the chloroplast and bound to the acyl carrier protein (acyl-ACP) are either directly incorporated into glycerolipids in the chloroplast envelopes or are exported to the endoplasmic reticulum (ER). At the ER, they serve in the form of acyl-CoAs as precursors for extraplastidic glycerolipids. A fraction of the diacylglycerol moieties derived from phosphatidylcholine is returned to the chloroplast and enters thylakoid lipid biosynthesis. As a consequence, diacylglycerol (DAG) moieties of plastidic and extra- plastidic origin are found in the glycerolipids of the thylakoid membranes of Arabidopsis. A critical intermediate of both pathways in plants is phosphatidic acid (PA), which is also essential for the biosynthesis of glycerolipids and triacylglycerol in bacteria, yeast and animals. A deficiency for PA biosynthesis is lethal in E. coli consistent with an essential role for PA in this bacterium (Coleman, 1990). Unlike animals and yeast, in plants the glycerol-3-phosphate (G3P) pathway is thought to be the only pathway for the de novo PA biosynthesis (Athenstaedt and Daum, 1999). Two acylations are involved: First the transfer of an acyl group from either acyl-ACP or acyl-CoA to the sn-1 position of G3P catalyzed by a G3P acyltransferase leading to the formation of 1-acyl-sn-G3P (lysophosphatidic acid, LPA). This intermediate is further acylated to PA by an l-acyl- sn-G3P acyltransferase (LPAAT). In plants, LPAAT activity is associated with multiple 164 membrane systems, including chloroplasts, ER, and the outer membrane of mitochondria, suggesting the presence of several different isoforms. Genes encoding the G3P acyltransferase have been isolated from different plant species. Generally, the sn-1 position of PA typically contains 16 or 18 carbons indicating that this enzyme does not discriminate between these two fatty acid substrate classes. However, in some plant species, G3P acyltransferase seems to be more specific. For instance, in Arabidopsis, the ATSl(ACT1) protein, the plastidic isoform of G-3-P acyltransferase, has considerable substrate preference for 18-carbon fatty acids (Yokoi et al., 1998). Of the enzymes acting on LPA, the plastidic LPAAT prefers 16-carbon saturated fatty acids and the ER form 18-carbon unsaturated fatty acids. This substrate specificity provides the means to distinguish thylakoid lipid species derived from the plastid or ER-pathways based on the fatty acids at the sn-2 position. Furthermore, the distinct substrate specificities of the different LPAAT isofomrs are a critical factor in determining the overall lipid acyl composition in plants. To date, several cDNAs encoding plant LPAATs have been isolated from coconut (Davies et al., 1995; Knutzon et al., 1995), the immature embryo of meadow foam (Brown et al., 1995; Hanke et al., 1995), the maize endosperm (Brown et al., 1994) and from Brassica napus (Bourgis et al., 1999). Among these enzymes, BAT2 of B. napus has been shown to be a plastid- localized isoform. However, the in vivo roles for this enzyme are still unknown due to the lack of lines with altered activity. In Arabidopsis, inactivation of the plastidic G3P acyltransferase in the ats] (actI) mutant led to the loss of the plastid pathway for glycolipid biosynthesis (Kunst et al., 1988). A surprising observation was that despite the drastic effects on plastidic glycolipid 165 biosynthesis, PG biosynthesis was only mildly impaired in the ats] (actI) mutant. However, it is not clear whether the currently described alleles for ats] (act]) are leaky, a caveat that makes it difficult to draw definitive conclusions regarding the function of the enzyme and leaves the interpretation of the mutant phenotype ambiguous. This study was conducted to gain a better understanding of the origin of PA in plants and to determine the in vivo function of plastidic LPAAT. As will be described in detail, the respective null-mutant leads to embryo-lethality, an unequivocal demonstration of the essential function of plastidic LPAAT in Arabidopsis. 166 Materials and methods Plant materials and growth Surface-sterilized seeds were genninated on 0.8% (w/v) agar-solidified Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 1% sucrose. Seedlings (IO-days-old) of Arabidopsis wild-type and mutants were transferred to soil drenched with half-strength Arabidopsis nutrient solution (Estelle and Somerville, 1987), and grown under a photosynthetic photon flux density (PPFD) of 70-80 mol m '2 s '1 at 22/18 °C (day/night) with a 14-h light/1 O-h dark period. Bioinformatics For routine sequence comparison, 8LAST2 was used (Altschul et al., 1997). Multiple sequence alignment was done by CLUSTALW software (Thompson et al., 1994) at The Biology Work Bench (http://workbench.sdsc.edu/). Prediction of chloroplast transit peptides was accomplished using ChloroP (Emanuelsson et al., 1999). Transmembrane spanning helices were predicted using TMHMM (Sonnhammer et al., 1998). T-DNA insertion analysis, ATSZ cDNA cloning and expression in transgenic plants The T-DNA insertional mutants, atsZ—I (SALK_073445) and ats2-2 (SALK_108812) were obtained from the Arabidopsis Stock Center (www.arabidopsis.org). The T-DNA left border primer P1 5'—GTTCACGTAGTGGGCCATCG-3', and gene-specific primers P2 5’-CAG G T ACCTTAG A G A TCCATTGATTCTGC-3’ and P3 5’-GAGGATC- CAGTGAAAAATTTATGGGCGA-3’ were used to screen the plants for T-DNA 167 insertions (cf. Fig. 4). The T-DNA-bordering DNA fragments were ligated into the plasmid pPCR-script AMP SK(+) (Stratagene, La J olla, CA), and sequenced to determine the localization of the T-DNA insertion. The AT 52 open reading frame corresponding to Arabidopsis gene At4g30580 (GenBank accession no. NP_194787 ) was predicted from the DNA sequence of BAC F17123_80 (GenBank accession no. AF 1601 82) and amplify- ed by reverse transcription-PCR using the primers 5'-GAGGATCCATGGA-TGTCG- CTTCTGCTCG-3' and 5'-CAGGTACCTTAGAGATCCATTGATTCTGC-3'. For this purpose, total leaf RNA was isolated from 20-day-old plants (Col-2) according to the method by Logemann et al. (Logemann et al., 1987). Reverse transcription-PCR was performed by using the ProSTAR HF system from Stratagene (La J olla, CA). The PCR product was inserted into the ligation-ready Stratagene vector pPCR-Script Amp SK(+) giving rise to pATS2. For plant transformation, the insert of pATSZ containing the full- 1ength coding sequence of A T S2 including the transit peptide was amplified by PCR us- ing the primers 5 '-GGACTAGTGATGTCGCTTCTGCTCGGA-3' and 5'-GGACTAGT- GAGATCCATTGATTCTGCA-3'. This fragment was inserted into the binary vector pCAMBIA1304 (www.cambia.org) using the SpeI restriction site to give plasmid pCAT- S2. Based on the nature of pCAMBIA1304, this construct led to the expression of an ATS2-GFP (green fluorescent protein) fusion protein and the resulting plants were used in the complementation and localization analysis. Stable transformation of Arabidopsis was achieved using the vacuum infiltration method (Bechtold and Pelletier, 1998). Transgenic plants were selected in the presence of hygromycinB (25ug/ml) on MS medium lacking sucrose. 168 Semi-quantitative RT-PCR Total RNA was extracted from plant tissues other than developing siliques with an RNA extraction kit (RNeasy, QIAGEN, Valencia, CA). Silique RNA was extracted from frozen tissue (50-100 mg) wich was ground in 1.5mL tubes. To the powder 350 1.1L of pre-heated (80 °C) extraction buffer (0.02 M sodium borate, 30 mM EDTA, 30 mM EGTA, 1% sodium dodecylsulfate, 2% deoxycholate, 2% polyvinylpolypyrrolidone, 2% polyvinylpyrrolidone 40K, 100 mM dithiothreitol, 100 mM a-mercaptoethanol) were added. This mixture was incubated at 80 °C for 1min, then chilled on ice. After addition of 0.15 mg of Proteinase K, the extract was incubated at 37 °C for 1hr. Following the clearing by centrifugation, the supernatant was extracted twice with phenol/choloroform (1:1), and once with chloroform/isoamyl (24:1). Total RNA was precipitated with isopropanol, resolved in water, then precipitated with 2 M LiCl overnight, and washed with 70% ethanol. Following DNase treatment (DNase I, Roche, Indianapolis, IN), cDNA was synthesized from 300 ng of RNA using a reverse transcriptase kit (Omnis- cript, QIAGEN, Valencia, CA) and IU of Taq DNA polymerase (Roche, Indianapolis, IN). The 3’ region of the AT S2 gene was amplified for gene expression analysis using the primers 5'-ACGCTAATGGGAACAGGCA-3' and 5'-AAGATCTCAACATTTAATTC- TTC-3’. The coding region of a translation elongation factor EFla (GenBank Accesion no. NM_125432) was used as a control (primers: 5'-ATGCCCCAGGACATCGTGATT- TCAT-3' and 5'-TTGGCGGCACCCTTAGCTGGATCA-3') (Boisson et al., 2001). Cycle numbers of 28 and 30 were applied for detection of EF 1 01 and AT S2 respectively. The amplified fragments were quantified using Quantity One (BioRad, Hercules, CA). Each AT S2 signal was normalized to the EF] 0 signal for calculation of relative amounts. 169 Heterologous complementation The AT S2 open reading frame lacking the predicted transit peptide was generated by RT- PCR using the primers 5’-GAGGATCCAGTGAAAAATTTATGGGCGA-3’ and 5'- CAGGTACCTTAGAGATCCATTGATTCTGC-3'. The open reading frame was fused by blunt end ligation to the lacZ gene under the control of an IPTG-inducible promoter in the predigested plasmid pPCR-script AMP SK(+) (Stratagene, La J olla, CA) giving rise to plasmid pATS2-s. The plasmid pATS2-s was used to complement the temperature- sensitive phenotype of E. coli JC201 ((Coleman, 1990); genotype: plsC thr-I ara-14 A (gal-attit)-99 hisG4 rpsL136 xyl—5 mil-1 lacY] tsx- 78 eda-50 rfbD] thi-I).This strain is unable to grow at 42°C due to the deficiency in lysophosphatidic acid acyltransferase encoded in the wild type by plsC, but can grow at 30°C. The control strain J C200 was isogenic to JC201 with exception of the plsC wild-type allele in J C200. Microscopy For the GFP fusion protein localization study, leaf samples of transgenic lines were direc- tly examined using a Meridian Instruments Insight confocal laser scanning microscope (Okemos, MI). Excitation light was provided by an argon laser at 488 nm. GFP fluores- cence was obsered with a band-pass filter of 520-560 nm and chlorophyll fluorescence with a 670 nm cut-off filter. Siliques of different developmental stages from hetero- zygous AT SZ/ats2 plants were dissected with hypodermic needles. Ovules from indivi- dual siliques were mounted on microscope slides in a clearing solution (chloral hydrate, water, glycerol, 8:2:1 v/v) and cleared for 1 hour at 4°C before microscopy. The ovule was observed with a Leica DMLB microscope (Leica Microsystems, Wetzlar, Germany). 170 RESULTS Identification of an Arabidopsis ATSZ candidate gene Taking advantage of the published Arabidopsis genome, we identified an Arabidopsis gene At4g30580 (GenBank accession no. NP_194787 ) on chromosome 4 as a putative ortholog of BA T 2 from B. napus. A wild-type cDNA corresponding to gene At4g305 80 was isolated by reverse transcription-PCR and sequenced. This cDNA encoded 356 amino acids in agreement with the protein predicted from the genomic sequence. The protein was designated ATSZ following the gene designation at TAIR (www.Arabidopsis.org) for the plastidic G3P acyltransferase in Arabidopsis, ATS1(ACT1). Using the BLAST2 sequence alignment software (Altschul et al., 1997), it showed 80% identity, and 85% similarity over 344 amino acid residues with BAT2 (Fig.5.1a). Like for BAT2, three putative membrane spanning domains were predicted for ATS2 by the TMHMM software (Somrhammer et al., 1998). To explore the evolutionary origin of LPAATs, we performed a phylogenetic analysis with ATSZ and other putative and bonafide LPAATs. The experimentally verified LPAATs included enzymes from Escherichia coli (Coleman, 1992), human (AGPATl, AGPAT2) (Eberhardt et al., 1997; West et al., 1997), Limnanthes douglasii (LATl, LAT2) (Brown et al., 1995) and L. alba (meadowfoam) (Lassner et al., 1995), coconut (Knutzon et al., 1995), B. napus (BATl and BAT2) (Bourgis et al., 1999), Yeast (Nagiec et al., 1993), and maize (Brown et al., 1994). Predicted LPAATs were those from A. thaliana (LPAT1-4), Oryza sativa (LPAT1-2), B. napus (BAT3), Schizosaccharomyces pombe, the bacteria Rhodobacter sphaeroides and Sinorhizobium 171 Figure 5.1. Evolutionary relationship between LPAATs. Sequence alignment of BAT2 and ATS2 (a). Identical amino acids are indicated by black boxes, similar ones are shaded grey. (b) Phylogenetic relationship of LPAATs. The following proteins were ncluded in the analysis (GenBank accession 110.): Arabidopsis. thaliana AT S2 NP_194787); A. thalianaLPA T1 (NP_567052); A. thaliana LPAT2 (NP_175537); A. ‘halianaLPA T3 (NP_565098); Athaliana LPAT4 (NP_188515); Brassica napus BATl :CAA90019); Brassica napus BAT2(AF1 l l 161); Brassica napus BAT3 (CA809138); Oryza sativa LPATl (CAE03516); Oryza sativa LPAT2 (AAL58271); Limnanthes alba :Q42868); Limnanthes douglasii LAT2 (CAA86877); Limnanthes douglasii LATl :CAA88620); Cocos nucifera (Q42670); Synechococcus sp. WH 8102 (NP_898339); Vostoc sp. PCC 7120 (NP_484285); Synechocystis sp. PCC 6803 (NP_441924); Saccharomyces cerevisiae (NP_010231); Schizosaccharomyces pombe (NP_595192); Escherichia coli (NP_417490); Homo sapiens AGPATl (AAH03007); Homo sapiens AGPAT2 (AAH07269); Sinorhizobium meliloti (NP_386764); Rhodobacter sphaeroides :ZP_00008109). 172 BAT2 ATSZ BAT2 ATSZ BAT2 ATSZ BAT2 ATSZ BAT2 ATSZ BAT2 ATs2 BAT2 ATSZ BAT2 ATSZ (b) 1 1 51 51 87 101 137 151 187 201 237 251 287 301 337 351 MDVASARGH PYYSKPICSSQSSLIRIPISKG SSNL TSLHA MDVASARSI SYYGKPICSSQSSLIRISRDKV G ISNGTTSPTT A339 TR ------- Tye“ CY I Fppggvu NS ------- RT? SL3, PS R36: SN L anxgrL KSPRSSQLARD' SDLSGAATPE TYPEPEIKLSSRLRGIC LVAGISAIVLIVLMIIGHP ADLSGAATPD SFPEPEIKLSSRLRGIF wVAGISATPLIVLMIIGHP FVLLFDRYRRKFHHFIAKLWASISIYPFYKTD QGLENLPSSDTPC FVLLFDPYRRKFHHFIAKLWASISIYPFYKIN EGLENLPSSDTP‘ NHQSFLDIYTLLSLGQ YKFISKTGIFVIP IGWAMSMMGVVPLKRMDPR NHQSFLDIYTLLSLGK FKFISKTGIFVIPIIGWAMSMMGVVPLKRMDPR SQVDCLKRCMELVKKGASVFFFPEGTRSKDGRLGPFKKGAFTIAAKTGVP SQVDCLKRCMELLKKGASVFFFPEGTRSKDGRLGSFKKGAFT AAKTGVA VVPITLMGTGKIMPTGSEGILNHGDVRVIIHKPIYGSKADVLCEEARNKI VVPITLMGTGKIMPTGSEGILNHG VRVIIHKPIHGSKADVLCNEARSKI N D_- O. sativa (LPAT1) C. nucifera r—" | r' L. alba * l. L. douglasii (LAT2) H. sapiens (AGPAT2) H. sapiens (AGPAT1) E. coli S. cerevisiae N. PCC 7120 S. PCC 6803 S. WH 8102 I'__A' thaliana (ATSZ) I L— B. napus (BAT2) ' o. sativa (LPAT2) B. napus (BAT3) B. napus (BAT1) A. thaliana (LPAT1) Z. mays L. douglasii (LAT1) I A. thaliana (LPAT2) A. thaliana (LPAT4) A. thaliana (LPAT3) S. pombe R. shaeroides "T I S. meliloti 173 meliloti and different cyanobacteria as indicated. Of these LPAATs, LPAT2 from 0. sativa is a putative plastidic isoform based on the predicted presence of a chloroplast transit peptide. Figure 5.1b shows a phylogenetic tree derived from protein sequence alignments. The ATS2 protein clustered with two plastidic LPAATS on a branch alongside the cyanobacterial cluster. ATS2 appears to represent a highly conserved plastidic LPAAT. It shared 68% and 91% identity, respectively, with 0. sativa and B. napus over 207amino acids representing the domain aligning with the bacterial sequence encoded by plsC. Expression of the ATSZ cDNA complements an E. coli LPAAT mutant To demonstrate directly that AT S2 encodes an LPAAT, we tested if the expression of the AT 52 cDNA rescued the temperature-sensitive phenotype of E. coli strain J C201. This strain is unable to grow at 42°C but grows well at 30°C due to the deficiency of LPAAT activity (Coleman, 1990). Thus, the test is based on the restoration of its growth at the non-permissive temperature. This strategy was successfully used to isolate LPAATs from many other organisms (Brown et al., 1994; Davies et al., 1995; Eberhardt et al., 1997; West et al., 1997; Bourgis et al., 1999). A truncated version of the AT S2 cDNA lacking the sequence encoding the putative chloroplast transit peptide was fused to the lacZ gene which was under the control of an inducible promoter. The result shown in Fig. 5.2 indicates that the vector expressing AT 52, but not the empty vector rescued the temperature sensitivity of E. coli J C201. This result suggested that AT S2 of Arabidopsis indeed encodes an LPAAT that can substitute for the inactive bacterial LPAAT in the mutant. 174 / JC201+ATS2 '/ jC201+VeCf0r 8 z‘. E + s? 1:: JC200+vector C200+vector / \, V. Figure 5.2. Heterologous complementation of E. coli JC201 by expression of ATS2 of Arabidopsis. Strain JC200 is the E. coli wild-type plsC control strain isogenic to J C201. 175 Localization of the ATSZ protein The ATS2 protein sequence has a predicted 56 amino acid N-terminal chloroplast transit peptide (ChloroP) (Emanuelsson et al., 1999). To experimentally verify the subcellular localization of ATSZ, the AT S2 cDNA was fused to the N- terminus of the green fluorescent protein (GFP) cDNA. The resulting construct was expressed in transgenic wild-type plants under the control of the 35S cauliflower mosaic virus (CMV) promoter. The GFP fluorescence in transgenic plants was observed using confocal microscopy. As shown in Figure 5.3, the GFP fluorescence was exclusively associated with chloroplasts. In control plants expressing only the GFP cDNA, green fluorescence was not associated with chloroplasts but appeared to be diffuse in the cell. Based on these results, it was concluded that ATS2 is localized in the plastid. These results were also consistent with findings of proteomics studies detecting the presence of this protein in chloroplast preparations (F erro et al., 2002; F erro et al., 2003). Isolation of mutants with T-DNA insertion into ATSZ In an attempt to investigate the function of ATS2 in the chloroplast, two T-DNA insertion lines, SALK_073445 and SALK_108812, (alleles ats2-1 and ats2-2 respectively) were identified in theSalk T-DNA insertion population (Alonso et al., 2003). The T-DNA insertion sites were determined by sequencing PCR products obtained with a combination of gene-and T-DNA-specific primers as shown in Figure 5.4. The ats2-1 allele carried a T-DNA insertion in the fourth exon of the predicted At4g30580 gene (Fig. 5.48) at base pair 22022 of the sequence of BAC F17123 (GenBank accession no. AF 1601 82) and the ats2-2 allele in the fifth exon at base pair 22390 (Fig. 5.4b). Despite extensive 176 Figure 5.3. Subcellular localization of the ATSZ protein. (a) ATSZ-GFPF fusion protein, (b) chlorophyll fluorescence, (c) overlay of (a) and (b). 177 Figure 5.4. Isolation and characterization of ATS2 T-DNA insertion lines. (a) Insertion into the AT S2 gene in the heterozygous AT S2-I/at82-1 allele and (b) into the AT S2—2/ats2-2 allele. Black boxes represent exons, open boxes introns. The exact insertion site (based on the sequence of BAC F17I23 GenBank Accession no. AF 160182) is indicated by a vertical arrow. Horizontal arrows labeled P1, P2, P3 represent the binding site locations for PCR primers. (c) PCR results for genomic DNA isolated from the wild type (A T SZ/A T S2) and the two allelic heterozygous mutant lines (A T S2-1/ats2-l and AT S2-2/ats2—2). The primer combination P2-P3 is diagnostic for the endogenous gene, the combination P1 -P3 for the T-DNA flanking genomic DNA as indicated in (a) and (b). (d) Dissected green siliques of heterozygous mutant lines (alleles as indicated) showing the segregation of clear white seeds. 178 BAC F17|23 22015 S'-C llCAGG AGCTGCAACCCCTGAC-S' 220038 (13) P1 __> I LB 277 bp 179 Table5.1. Segregation and complementation analysis of the two allelic heterozygous AT SZ/ats2 lines. Multiple siliques from single plants were analyzed. The heterozygous state of A T S2/at52 was confirmed by genotyping using PCR (cf. Fig. 5.4c). Green seeds White seeds x2(P< 0.1) ATS2-1/ats2-1 413 135 0.04 (3:1) A T S2-2/at52-2 353 120 0.03 (3: 1) ATS2-1/at52-I (ATS2 cDNA) 498 35 0.09 (15:1) A TS2-2/ats2-2 (A T SZ cDNA) 540 38 0.10 (15:1) 180 screening, no homozygous plants could be identified. Combinations of gene-specific and T-DNA—specific primers to generate PCR products diagnostic for mutant and wild—type chromosomes and a typical result for wild-type and AT S2/ats2-1 and AT SZ/atsZ-2 heterozygous lines are shown in Figure 5.4c. Both mutant lines showed insert-specific as well as wild-type genomic DNA fragments. Furthermore, when immature siliques were opened which grew on heterozygous plants as confirmed by genotyping, approximately 25% of the seeds were clear white (Fig. 5.4d), indicative for the arrest of embryo development at an early stage. This observation raised the possibility that a homozygous atsZ null—mutant was embryo-lethal. To quantify this phenomenon, one or two siliques from 10 heterozygous plants were opened and the ratio of viable to aborted seeds in all the siliques examined was obtained as shown in Table 5.1. These data were consistent with a 3:1 segregation ratio as expected for a recessive mutation in an essential nuclear gene. When viable plants derived from AT SZ/atsZ-I heterozygous lines or AT S2/ats2-2 were analyzed, approximately two thirds (39 out of 61 for AT S2/ats2-I and 42 out of 62 for AT S2/ats2-2) produced aborted seeds consistent with a 2—to-1 ratio of heterozygous to wild-type genotypes among the surviving plants. Complementation of the embryo-lethal phenotype was tested by introduction of the wild-type AT S2 cDNA under the control of I the 3SS—CMV promoter into AT S2/ats2 heterozygous plants. In the T1 generation, transgenic plants were selected on hygromycinB containing medium and tested for GFP expression. Three classes were expected among the transgenic plants: homozygous mutants (atsZ/ats2), heterozygous plants (A TS2/(1152) and homozygous wild-type plants (A T SZ/A T 52). Because a single copy of the transgene would behave like a second (dominant) genetic marker in this experiment, complementation would be in effect if 181 homozygous mutant lines segregated 1/4 white seeds, heterozygous lines 1/ 16 white seeds, and homozygous wild-type lines no white seeds. All three classes were observed among 10 transgenic plants tested, indicating complementation. Because transgenic heterozygous plants were most informative, one plant for each allele was analyzed in detail. Genotyping by PCR (cf. Fig. 5.4c) confirmed these plants to be AT S2/ats2 heterozygous and analysis of seeds in immature siliques indicated the presence of 1/16 white seeds (Tab.5.l) as expected for complementation. Because of the embryo lethality, no further phenotypic analysis was feasible on homozygous lines. When heterozygous lines were analyzed, no changes in growth or leaf fatty acid composition were observed (data not shown). A transient increase in ATSZ expression in developing siliques in the wild type corresponds to the arrest of embryo development in the mutant Analyzing the expression of the AT SZ gene, the mRNA was detected in all tissues tested (Fi g. 5.5), as would be expected for a gene involved in an essential process. Interestingly, AT 52 mRNA abundance was reduced in silique walls compared to leaves and the abundance was higher in RNA samples from intact siliques. This result suggested an increased expression of A T S2 in developing seeds. Because it is exceedingly difficult to obtain sufficient amounts of mRNA from very young embryos, a time course of developing intact siliques was analyzed. Comparison to the silique wall sample (5 days after flowering) should provide an indication of mRNA abundance in the developing seed. Interestingly, AT SZ expression transiently increased in siliques four days after flowering (Fig. 5.5). 182 Figure 5.5. Expression ofATSZ in various tissues and during silique development. Semi-quantitative RT—PCR was used and products for ATS2 are shown in comparison to the control gene EFIa (GenBank accession no. NM_125432). Relative levels ofATSZ normalized to EFla are shown in the bottom panel. Lanes from left to right: L, leaf; S, stern; SW, silique wall (age 5 days after flowering); R, root; B, bud; F, flower; 2-12, siliques of different development as indicated (days after flowering). 183 .L. 33W R43 F 2 4,6 81012 135% Jim: wig-17:. . LI_I_____I_-___-.¥ __ 184 Figure 5.6. Arrest of embryo development. Four siliques of different developmental age growing on heterozygous AT S2/ats2-I -1 plants were dissected. (a, e), two representative embryos from the youngest silique, in which all embryos were at the globular stage and indistinguishable; (b, i), older silique with most embryos at the heart stage (b) and with about 25 % of the embryos arrested at the globular stage (t); (c) developing early torpedo stage embryo and (g) arrested embryo from the same silique at the globular stage; (d), oldest silique with developing embryo at the late torpedo stage and (h) globular arrested embryo from the same silique. 185 186 Embryo development was compared by Nomarski microscopy in different developing seeds of siliques growing on heterozygous plants (Fig. 5.6). Before the onset of visible chlorophyll accumulation at the transition from globular to the early heart stage (Mansfield et al., 1991), the embryos were indistinguishable. However in older siliques, seeds with arrested development became visible to the naked eye and closer examination revealed that the clear white seeds contained embryos arrested at the globular stage. Presumably, these seeds corresponded to dark brown aborted seeds apparent in mature siliques. It should be noted that the transient increase in AT 52 expression observed for the wild type four days after flowering (Fig. 5.5) corresponded to the transition in embryo development from the globular to the heart stage, when the developmental arrest occurs in a quarter of the seeds developing in siliques on heterozygous plants. 187 Discussion With the availability of the Arabidopsis genome sequence (The Arabidopsis Genome Initiative, 2001) it has become feasible to predict genes encoding enzymes involved in lipid metabolisms in Arabidopsis based on sequence similarity to enzymes with known functions from bacteria and other plants. Ohlrogge and coworkers have used this approach to assemble a catalog of lipid metabolism genes in Arabidopsis (Beisson et al., 2003). As we now know based on the present study, they correctly annotated Arabidopsis gene At4g30580 to encode a plastidic LPAAT. Functional proof was provided by heterologous complementation of the E. coli LPAAT-deficient plsC mutant (Fig. 5.2). Furthermore, subcellular localization of the ATSZ-GFP fusion (Fig. 5.3) agreed with a plastidic localization of the protein as predicted. Independent experimental evidence for a plastid localization of ATSZ was also obtained by Ferro and colleagues (Ferro et al., 2002; F erro et al., 2003). When confirmed and putative LPAAT protein sequences from different organism were aligned and grouped based on the amino acid sequence similarity, ATS2 of Arabidopsis clustered with BAT2 from B. napus and LPAT2 from 0. sativa, both plastidic LPAATs (Fig. 5.3). Furthermore, these plastidic LPAATs formed a subcluster with cyanobacterial LPAATs providing supportive evidence for the cyanbacterial endosymbiont origin of chloroplasts. Four other putative Arabidopsis LPAATs were grouped in a distinct cluster representing microsomal LPAATs (Fig. 5.1) suggesting that their evolutionary origin is different from that of plastidic isoforms. Although in case of the well studied class of LPAATS the analysis of genomic information led to precise predictions, for which the experimental verification as shown seemed trivial, one should keep in mind that most of the information in current genome 188 databases is based only on conjecture. Corroborating genomic predictions is only the beginning to a broader understanding of the biological (physiological) function of gene products, in this case ATSZ. One of the most versatile resources to shed light on the function of a particular gene in Arabidopsis is the broad availability of sequenced T-DNA insertion lines (Alonso et al., 2003). In case of A T SZ, two independent alleles were available, both of which gave rise to a striking phenotype, embryo-lethality. The presence of two independent alleles and restoration of embryo development following the transgenic expression of the AT S2 cDNA (genetic complementation) linked the embryo defect to the T-DNA inactivation of the gene encoding plastidic LPAAT in the two allelic mutants. The result was surprising, because a mutant in the plastidic G3P acyltransferase gene, AT S] (A C T 1 ), affecting the step prior to ATSZ in the pathway, had no effect on embryo development, despite the fact that the plastid pathway of thylakoid lipid biosynthesis was strongly impaired in this mutant (Kunst et al., 1988). However, it should be noted that it is not clear at this time to which extent the ats](actl) mutants is “leaky”, if at all, because plastidic PG, which is assumed to be synthesized by the plastid pathway of membrane lipid biosynthesis, was only moderately reduced in the mutant. The complete loss of PG in the ats2 mutants could not be the cause for the observed embryo arrest, because homozygous null-alleles of the PGP] gene encoding plastidic phosphatidylglycerolphosphate synthase gave rise to seedlings lacking plastidic PG (Hagio et al., 2002; Babiychuk et al., 2003). However, these mutant seedlings were completely white and non-photosynthetic. Therefore, either the absence of plastidic PA as a critical intermediate or signaling molecule, or the accumulation of lyso-PA harmful to membrane integrity might be the cause of the observed embryo lethality in the ats2 189 mutants. The fact that embryo development was arrested just at the transition from the globular to the heart stage, when thylakoid membranes start to develop and a corresponding peak in AT S2 gene expression was observed (Fig.5.5), suggests that the lack of PA as a central intermediate of lipid metabolism might be the main cause of embryo arrest in the ats2 mutants. However, other possibilities cannot be ruled out at this time. While embryo-lethality is an easily observable phenotype, the respective null- mutants provide no detailed information on the role of plastidic ATS2, because homozygous lines could not be further analyzed and heterozygous lines lacked any phenotype. Mutations with leaky alleles in AT S2 giving rise to viable plants with milder phenotypes will have to be isolated and studied to better understand the role of PA in plant metabolism. Acknowledgements We thank Hongbo Gao for his help with fluorescence microscopy. We would like to thank Dr. David S. Stephens for the E. coli strains J C201 and J C200. This work was supported in parts, by grants from the US Department of Energy Bioscience Program and the MSU Center for Novel Plant Product 190 References Alonso J M, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Baraj as P, Cheuk R, Gadrinab C, Heller C, J eske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H. Geralt M, Hazari N, Horn E, Kames M, Mulholland C, Ndubaku R, Schmidt 1, Guzman P, Aguilar- Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653-657. Altschul, SF, Madden, TL, Schaffer AA., Zhang J, Zhang Z, Miller W, Lipman DJ. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic. Acid. Res. 25:3389—3402. Athenstaedt K, Daum G. (1999) Phosphatidic acid, a key intermediate in lipid metabolism. Eur. J. Biochem. 26621-16. Babiychuk E, Muller F, Eubel H, Braun HP, Frentzen M, Kushnir S. (2003) Arabidopsis phosphatidylglycerophosphate synthase 1 is essential for chloroplast differentiation, but is dispensable for mitochondrial function. Plant J. 33:899-909. Bechtold N, Pelletier G (1998) In plant Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. In Arabidopsis Protocols (Martinez-Zapater, J ., Salinas, J ., eds). Totowa, NJ: Humana Press, pp 259-266. Beisson F, Koo AJ, Ruuska S, Schwender J, Pollard M, Thelen JJ, Paddock T, Salas JJ, Savage L, Milcamps A, Mhaske VB, Cho Y, Ohlrogge JB. (2003) ArabidOpsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database. Plant Physiol. 132:681-697. Boisson M, Gomord V, Audran C, Berger N, Dubreucq B, Granier F, Lerouge P, Faye L, Caboche M, Lepiniec L. (2001) Arabidopsis glucosidase I mutants reveal a critical role of N-glycan trimming in seed development. EMBO J. 20:1010—1019. Bourgis F, Kader J C, Barret P, Renard M, Robinson D, Robinson C, Delseny M, Roscoe TJ. (1999) A plastidial lysophosphatidic acid acyltransferase from oilseed rape. Plant Physiol. 120:913-921. Brown AP, Brough CL, Kroon J TM, Slabas AR. (1995) Identification of a cDNA that encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes Douglasii. Plant Mol. Biol. 29:267-278. Brown AP, Coleman J, Tommey AM, Watson MD, Slabas AR. (1994) Isolation and characterization of a maize cDNA that complements a 1-acyl sn-glycerol-3- 191 phosphate acyltransferase mutant of Escherichia Coli and encodes a protein which has similarities to other acyltransferases. Plant Mol. Biol. 26:211223. Coleman J. (1990) Characterization of Escherichia coli cells deficient in l-acyl-sn- glycerol-3-phosphate acyltransferase activity. J. Biol. Chem. 265:17215-17221. Coleman J. (1992) Characterization of the Escherichia coli gene for l-acyl-sn-glycerol-3- phosphate acyltransferase (PLSC). Mol. Gen. Genet. 232:295-303. Davies HM, Hawkins DJ, Nelsen J S. (1995) Lysophosphatidic acid acyltransferase from immature coconut endosperm having medium-chain length substrate-specificity. Phytochemistry 39:989-996. Eberhardt C, Gray PW, Tjoelker LW. (1997) Human lysophosphatidic acid acyltransferase - cDNA cloning, expression, and localization to chromosome 9q34.3. J. Biol. Chem. 272:20299-20305. Emanuelsson O, Nielsen H, von Heijne G. (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci. 8:978-984. Estelle MA., Somerville C. (1987) Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. Mol. Gen. Genet. 206:200-206. F erro M, Salvi D, Brugiere S, Miras S, Kowalski S, Louwagie M, Garin J, J oyard J, Rolland N. (2003) Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana. Mol. Cell. Prot. 22325-345. Ferro M, Salvi D, Riviere-Rolland H, Vermat T, Seigneurin-Bemy D, Grunwald D, Garin J, J oyard J, Rolland,N. (2002) Integral membrane proteins of the chloroplast envelope: Identification and subcellular localization of new transporters. Proc. Natl. Acad. Sci. USA 99: 1 1487-1 1492 Hagio M. Sakurai I, Sato S, Kato T, Tabata S, Wada H. (2002) Phosphatidylglycerol is essential for the development of thylakoid membranes in Arabidopsis thaliana. Plant Cell Physiol. 43:1456-1464. Hanke C, Wolter FP, Coleman J, Peterek G, Frentzen M. (1995) A plant acyltransferase involved in triacylglycerol biosynthesis complements an Escherichia coli sn-l- acylglycerol-3-phosphate acyltransferase mutant. Eur. J. Biochem. 232:806-810. Knutzon DS, Nelsen J S, Lardizabal KD, Bleibaum J L, Hawkins DJ, Davies HM, Metz JG. (1995) Purification and molecular cloning of a medium chain—preferring lysophosphatidic acid acyltransferase from immature coconut endosperm. Plant Physiol. 108:999—1006 192 Kunst L, Browse J, Somerville C. (1988) Altered regulation of lipid biosynthesis in a mutant Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity. Proc. Natl. Acad. Sci. USA 85:4143-4147. Lassner MW, Levering CK, Davies HM, Knutzon DS. (1995) Lysophosphatidic acid acyltransferase from meadowfoam mediates insertion of erucic-acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil. Plant Physiol. 10921389-1394. Logemann J, Schell J, Willmitzer L. (1987) Improved method for the isolation of RNA from plant tissues. Anal. Biochem. 163:16-20. Mansfield SG, Briarty LG, Erni S. (1991) Early embryogenesis in Arabidopsis thaliana. I. The mature embryo sac. Can. J. Bot. 447-460. Murashige T, Skoog F. (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15:473-497. Nagiec MM, Wells GB, Lester RL, Dickson RC. (1993) A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that resembles an Escherichia coli fatty acyltransferase. J. Biol. Chem. 268222156- 22163. Roughan PG. Slack CR. (1982) Cellular organization of glycerolipid metabolism. Ann. Rev. Plant Physiol. 33297-132. Sonnhammer EL, von Heijne G, Krogh A. (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. Proc. Int. Conf. Intell. Syst. Mol. Biol. 6: 175-1 82. The Arabidopsis Genome Initiative (2001) Analysis of the genome sequence of the flowering plant ArabidOpsis thaliana. Nature 408:796-815. Thompson JD, Higgins DG, Gibson TJ. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position- specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680. West J, Tompkins CK, Balantac N, Nudelman E, Meengs B, White T, Bursten S, Coleman J, Kumar A, Singer J W, Leung DW. (1997) Cloning and expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells. DNA Cell Biol. 162691701. Yokoi S, Hi gashi S, Kishitani S, Murata N, Toriyama K. (1998) Introduction of the cDNA for Arabidopsis glycerol-3-phosphate acyltransferase (GPAT) confers unsaturation of fatty acids and chilling tolerance of photosynthesis on rice. Mol. Breeding 4:269-275. 193 Chapter 6 Conclusions and Future Directions 194 While our knowledge of chloroplast lipid biosynthesis has been advanced in the past several decades by biochemical and genetic analysis (Browse and Somerville, 1991; Dormann and Benning 2002; Ohlrogge and Browse, 1995, Joyard et al., 1998 a, b), many aspects still need further investigation. The current dissertation, with the use of Arabidopsis (A. thaliana) as a model system, is mainly focused on identification of enzymes involved in SQDG and PG biosynthesis, using reverse or forward genetic approaches, and on dissecting the functions of these two anionic lipids by analyzing the corresponding single or double mutants. During the course of this research, effort was also put on the isolation of the plastidic LPAAT responsible for PA biosynthesis, and the elucidation of the function of this LPAAT in lipid biosynthesis. A gene, SQD2 involved in SQDG biosynthesis, was identified (Chapter 2). Several lines of evidence indicated that SQD2 encodes the SQDG synthase catalyzing the transfer of SQ from UDP-SQ to DAG. First, the deduced SQD2 protein sequence showed 41% similarity to that of SQDX, a cyanobacterial SQDG synthase. Second, inactivation of SQD2 led to the deficiency of SQDG in Arabidopsis. Third, stable transformation of sqd2 with SQD2 cDNA restored the biosynthesis of SQDG. Finally, the biosynthetic pathway of SQDG was reconstituted in E. coli by co-expressing SQDl and SQD2. The isolation of the sulfolipid-deficient sqd2 null mutant of Arabidopsis allowed me to address another fundamental question about the role of sulfolipid in photosynthetic membranes of seed plants. My initial analysis of photosynthesis and growth suggests that sulfolipid does not have a crucial role in plant photosynthesis. The sqd2 mutant did not show any obvious signs of altered morphology or growth and photosynthetic parameters were affected only mildly under normal laboratory growth conditions. Like some bacteria, SQDG deficiency 195 in the Arabidopsis sqd2 mutant led to growth impairment under phosphate-limiting growth conditions, indicating the conditional importance of SQDG in seed plants. The wild type is able, within limitations, to maintain the relative amount of total anionic thylakoid lipids by increasing its sulfolipid content, when phosphatidyl glycerol content is decreased by reduced phosphate availability. In comparison, phosphatidylglycerol amounts are not decreased under phosphate—limiting conditions in the Arabidopsis sulfolipid-deficient mutant, due to a requirement for the constant amount of anionic lipid. Considering that one-third of the organic phosphate is normally bound in the membranes of wild-type plants, the conclusion can be drawn that a sulfolipid-deficient null mutant exhausts its phosphate reserves sooner than the corresponding wild type, resulting in reduced growth under these conditions. Based on these observations, 1 concluded that one function of SQDG is to help plants to cope with phosphate—limited growth conditions. Analyzing the correlation between the relative amount of SQDG of different plant species and phosphate availability in different geographic areas should provide verification of this conclusion. SQDG obviously plays different roles in different cyanobacterial species (Aoki et al., 2003). This phenomenon gives rise to the question of whether SQDG also has different function in different higher plants. Clarifying this question will not only increase our understanding of the evolution and the function of SQDG, but also shed new light on how different plants maintain a functional photosynthetic membrane by adjusting their lipid composition. An approach to solve this question is to analyze SQDG-deficient mutants either artificially constructed or naturally occurring in other species. 196 The SQD2 protein belongs to the glycosyltransferase gene family GT4, which represents retaining glycosyltransferases. The catalytic mechanism for these glycosyltransferases is still unclear. Further experiments toward understanding the SQD2 catalytic mechanism will provide new insights into SQDG biosynthesis, and will providing understanding of the GT4 family enzyme in general. An attractive approach is to determine the protein’s three-dimensional structure by X-ray diffraction using a crystallized SQD2 protein. Some enzymes in this gene family have been studied focusing on the substrate-binding and catalytic sites (Edman et al., 2003; Abdian et al., 2000). Utilizing information from these studies, I would predict the amino acids of the SQD2 protein involved in substrate—binding and catalytic reaction, followed by experimental verification of the function by site-directed mutagenesis and enzyme activity assays. Using a forward approach, we isolated a PG deficient mutant, pgp] (Xu et al., 2002), and identified the phosphatidylglycerolphosphate synthase 1 (PGPI). Three facts confirmed that PGP] encodes phosphatidylglycerolphosphate synthase. The first was that the pgp] mutant has a reduced PGP synthase activity in isolated chloroplasts. Second, stable transformation of PGP cDNA into the pgp] mutant rescued the pgp] mutant phenotype. Third, the expression of PGP] complemented the E. coli mutant YA5512 deficient in PGP synthase activity (Asai et al., 1989). The pgp] mutant plant was pale green, and its photosynthesis was impaired. In addition, the complete inactivation of PGP] by T-DNA knockout led to the arrest of chloroplast development, and the mutant plants could not survive on soil (Hagio et al., 2002; Babiychuk et al., 2003). These facts suggested that phosphatidyl glycerol is essential for the development of the photosynthetic thylakoid membranes in A. thaliana. 197 Similar to pgp]-1, the overall content of PG in the act] mutant is also reduced by 30%. However, unlike pgp]-1, the act] mutant showed no obvious growth phenotype. One possible explanation for this difference is that pgp]-1 is a leaky mutant. The activity of the PGPl enzyme is reduced by 70%, therefore it is possible that PG content is reduced more than 30% at the beginning of chloroplast development, which may lead to the phenotype of pgp] -] . This hypothesis is supported by the fact that developing leaves of pgp]-1 have more severe phenotype than mature leaves. To further verify this possibility, the overall content of PG in pgp]-1 and act] can be compared at different develOpmental stages. Alternatively, the phenotype of pgp] -1 could be caused by the accumulation of PA, which is known to be a signaling molecule, or lyso-PA, which is toxic, due to the deficiency of PGPl. The act] pgp] double mutant showed the phenotype of act], suggesting this possibility (Xu and Benning, unpublished data). Further comparision of the amount of PA or lyso-PA of pgp] -] and act] may provide some clues regarding to this hypothesis. Photosynthesis is impaired in the pgp]-1 mutant. An obvious reason for this is the decrease of PG, which has been shown to play an important role in photosynthesis. However, the reduction of mesophyll cell numbers in pgp]-1 suggested that the decrease in photosynthetic activity might be a secondary effect. Further study of the photosynthesis with isolated chloroplast may provide some clue as to which is the correct interpretation. The reduction of mesophyll cell numbers in pgp]-I also suggested a possible function of PG in development, although this function may be indirect, i.e. caused by chloroplast impairment. One interesting question to be addressed in further studies is how PG affects cell number. One approach would be to look for suppressors or 198 enhancers of pgp] -1 in terms of cell number by screening EMS-mutagenised pgp]-1 seeds. It was hypothesized that under severe phosphate limitation SQDG substitutes for PG, ensuring a constant amount of anionic lipids even under adverse conditions (Benning, 1998). Taking advantage of the SQDG deficient—mutant, sqd2, and partially PG- deficient mutant, pgp]-1, an SQDG and PG-deficient double mutant was constructed to test this hypothesis (Chapter 4; Yu et al., 2003). In the sqd2pgp1-I double mutant, the fraction of total anionic lipids was reduced by approximately one third resulting in pale yellow cotyledons and leaves with reduced chlorophyll content. Photoautotrophic growth of the double mutant was severely compromised and its photosynthetic capacity was impaired. In particular, photosynthetic electron transfer at the level of photosystem II was affected. Besides these physiological changes, an altered leaf structure, a reduced number of mesophyll cells and ultrastructural changes in the chloroplasts were observed. All observations on the sqd2pgp1-1 mutant led to the conclusion that the total content of anionic thylakoid lipids is limiting for chloroplast structure and function, and is critical for overall photoautotrophic growth and plant development. The reciprocal regulation of SQDG and PG gives rise to questions of how the plant senses the decrease of anionic lipid, and what the signal transduction network is, which is involved in this reciprocal regulation. Therefore, future experiments should be focused on the identification of genes involved in the regulation of PG and SQDG biosynthesis. For instance, a forward screening of EMS mutagenized sqd2 can be conducted to look for mutants, which have reduced PG content under phosphate stress. Alternatively, one can look for mutants in which SQDG does not respond to phosphate regulation by screening 199 EMS mutagenized wild-type. Isolation of genes in the corresponding mutants by map- based cloning should provide new insight into the regulation of lipid biosynthesis. Phosphatidic acid (PA) is a key intermediate for chloroplast lipid biosynthesis. The de novo PA biosynthesis ocurres by a two-step reaction: The first step is the sn-1 acylation of G-3-P by glycerol-3-phosphate acyltransferase leading to the formation of lyso- phosphatidic acid (LPA), the second is the sn-2 acylation of LPA to form PA catalyzed by l-acylglycerolphosphate acyltransferase (LPAAT) (Athenstaedt and Daum, 1999). 1 confirmed the AT S2 gene of Arabidopsis and determined that AT S2 encodes a LPAAT by heterozygous complementation of E. coli mutant J C 201, which is a temperature sensitive mutant and deficient in the LPAAT, encoded by plsC (Coleman, 1990, 1992). A green fluorescent protein fusion with ATS2 localized to the chloroplast. Thus, A T S2 encodes a plastidic isoform of LPAAT of Arabidopsis. Insertion of a transfer DNA into this gene in Arabidopsis led to embryo lethality. The development of the embryo was arrested at the globular stage. These results indicated the essential role of de novo plastidic PA biosynthesis in embryo development. One possible reason for the lethality is that PA is required for chloroplast development. Due to the embryo lethality, the functions of ATS2 in chloroplast development and chloroplast lipid biosynthesis could not be further analyzed using the null mutants. Partial down regulation of A T S2 in wild-type plants by RNAi or antisense approach may give rise to viable plants, which can be studied in great detail to clarify the function of PA in chloroplast biogenesis. 200 References Abdian PL, Lellouch AC, Gautier C, Ielpi L, Geremia RA. (2000) Identification of essential amino acids in the bacterial alpha -mannosyltransferase aceA. J. Biol. Chem. 275:40568-40575 Asai Y, Katayose Y, Hikita C, Ohta A, Shibuya I. (1989) Suppression of the lethal effect of acidic-phospholipid deficiency by defective formation of the major outer membrane lipoprotein in Escherichia coli. J. Bacteriol. 171:6867-6869 Babiychuk E, Miiller F, Eubel H, Braun HP, Frentzen, M, Kushnir S. (2003) Arabidopsis phosphatidylglycerophosphate synthase 1 is essential for chloroplast differentiation, but is dispensable for mitochondrial function. Plant J. 33:899-909. Benning C. (1998) Biosynthesis and function of the sulfolipid sulfoquinovosyl- diacylglycerol. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:53-75 Browse J, Somervllle C. (1991) Glycerolipid metabolism, biochemistry and regulation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:467-506. Coleman J. (1990) Characterization of Escherichia coli cells deficient in l-acyl-sn— g1ycerol-3-phosphate acyltransferase activity. J. Biol. Chem. 265:17215-17221. Coleman J. (1992) Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3- phosphate acyltransferase (PLSC). Mol. Gen. Genet. 232:295-303. Dormann P, Benning C (2002) Galactolipids rule in seed plants. Trends Plant Sci. 7:112- 8. Edman M, Berg S, Storm P, Wikstrom M, Vikstrom S, Ohman A, Wieslander A. (2003). Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae. J Biol. Chem. 278:8420-8428 Hagio M, Sakurai I, Sato S, Kato T, Tabata S, Wada H. (2002) Phosphatidylglycerol is essential for the development of thylakoid membranes in Arabidopsis thaliana. Plant Cell Physiol. 43:1456-1464. Joyard J, Marechal E, Miege C, Block MA, Dome AJ, Douce R. (1998a) Structure, distribution and biosynthesis of glycerolipids from higher plant chloroplasts. In Siegenthaler,P.A. and Murata,N. (eds), Lipids in Photosynthesis: Structure, Function and Genetics. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 21——52. 201 J oyard J, Teyssier E, Miege C, Bemy-Seigneurin D, Marechal E, Block MA, Dorne AJ, Rolland N, Ajlani G, Douce R (1998b) The biochemical machinery of plastid envelope membranes. Plant Physiol. 118:715-723. Ohlrogge J, Browse J. ( 1995) Lipid Biosynthesis. Plant Cell 72957-970. Xu, C, Harte] H, Wada H, Hagio M, Yu B, Eakin C, Benning C. (2002) The pgp] locus of Arabidopsis encodes a phosphatidylglycerol synthase with impaired activity. Plant Physiol. 129:594-604. Yu B, Xu C, Benning C. (2002) Arabidopsis disrupted in SQDZ encoding sulfolipid synthase is impaired in phosphate-limited growth. Proc. Natl. Acad. Sci. USA 99:5732-5737. 202 :1} ”a. , r i u. 5.3.... e in. run. ...