> .. . . '.7 *- “mam-:- .\"r'.'\-rf. . . ,my 5”??? . d ' u" ‘. :fvixr‘. n 172:}??? 71%.?!“ .4 “4i . _' v o ’ . ‘ » '5‘ ' ' -r H“ ' JR}. " I _ . :t [‘5‘ War” I " .’.‘%‘V“ f '13" . ‘- ',_. x , . p .2" d . A: ny-‘tF-g -‘f- ‘1 ' 9"{137'id‘y ’ NE. ‘«.‘.~.‘..;._<>:.. , I ' '. . J '32}13"'L .' 3'." .‘. . r ' - Ema; .aUW v' ' \f'. _ " U a ‘3.;,(:‘_-'.32'. . {5}":“5. A: . 53>:- . . ,. $755: H’.\-,- - ‘\ ‘-'.‘ "JV-4 I 4 ‘ ‘A 1“ IV 0" .v :{t'sz‘uh'd' ‘IJF‘.‘"|-ll A". "f n. A- "25's <2“ I;..x:._..':§ : ’6’"; .. .V ‘g‘ I "3.5: 5" Jr- ‘ L”; "- -‘f§'~’"~'-‘5K""7.3“1 31.4w”- {554 . -_. awuvg.:\.,.‘. \ ‘4 “3-34.?" ,7“. ' - " ‘ ‘- ‘.. \ . 0.1‘ . ' - it}: “ ., "7‘." ‘.\ “-9-“ r '~ 't'. "c' ' Ki. . 91;: .' ,w; ’.’o‘ll“ ‘: '41, in; . ,— f. ’1'. 1" ‘17!" 3C." _. r. ‘ . AH ~ 1‘: .7; ti" I. - "N . ' '|E§?uu‘r‘a .1", fry}. '3‘.“ ‘ 9‘ . ~ .5 ‘-'k- V .a'..5.-.‘., ‘4 ' v '~ 4. H ‘ "41, ",.l.....'l_ 1‘, .4 .,. . *‘. '- ' ‘ -‘-..’4 .' ‘. I‘. \..'(\.". V. -.. '91., . I, "i - '4". ’5', v | .’.‘I.“...‘ ‘;... ”‘3': "I. . , .c‘ . < v .. .‘. . ‘. “NAP/v.74. c » .x 5 , .- . . .--: "rd-y i'" 4- ‘ . V ....,. A . 9.1." ”gut . ' I .r 7. v "v _ rpm-Gm . a .- 03. E133“: . h] .-’ _ .. .- m .. , . _ ._ . __.- -. 4--...J; , '4“.—‘,'.° . .. .. ”-5... ~ .3)“, 51.7 ' ‘0 1&7??qu llfllll‘flllillfilllllllllg gm 3 1293 0055 M LIBRAR 1’ Michigan State University IBRAR l l H llll This is to certify that the dissertation entitled Mutants of Arabidopsis thaliana (L.) HeyT. With Altered Leaf Membrane Lipid Composition presented by Lj erka Kuns t I has been accepted towards fulfillment ' of the requirements for Ph. D Botany ’ degree in Major professor Date 6-22—1988 urti.’....1m......> 1‘ r1 .n .1, . . 042771 MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. MUTANTS OF ABABLQQESIS IflALlAflA (L.) HEYNH. WITH ALTERED LEAF MEMBRANE LIPID COMPOSITION By Ljerka Kunst A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1988 5059 "v (.‘ .. (- f: a, J V’ ABSTRACT MUTANTS OF W IHALIANA (L.) HEYNH. WITH ALTERED LEAF MEMBRANE LIPID COMPOSITION By Ljerka Kunst In plant cells each membrane has a characteristic lipid composition. However, the specific roles of lipids and fatty acids in proper functioning of membranes are not well understood. In an attempt to examine the functional significance of chloroplast fatty acid composition, a collection of mutants of the crucifer AW 1113113113 (L.) Heynh. was isolated from an ethyl methane sulfonate mutagenized population. The mutants were selected by direct analysis of leaf fatty acid composition using gas chromatography. This dissertation describes biochemical and physiological characterization of two of these mutants. The first mutant analyzed was deficient in the activity of the chloroplast glycerol-B-phosphate acyltransferase, due to a single nuclear mutation at a locus designated 3511. This lesion in the prokaryotic pathway of glycerolipid biosynthesis results in a redirection of fatty acids towards the eukaryotic pathway in the endoplasmic reticulum. The increased synthesis of lipids by the eukaryotic pathway provides, with the exception of phosphatidylglycerol, almost normal amounts of lipids required for chloroplast biogenesis. Since the acyltransferases associated with the two lipid biosynthetic pathways exhibit different substrate Ljerka Kunst specificities, the fatty acid composition of chloroplast membrane lipids is altered. As a consequence, pronounced changes in chloroplast ultrastructure were observed. The number of stacked membrane regions per chloroplast is increased, but there is a corresponding reduction in the average number of thylakoid membranes in the appressed regions. The analysis of Chl fluorescence emission spectra of the m1 mutant revealed a slight decline in the excitation energy transfer from the light harvesting Chl a/b protein complex to P511 and PSI. However, the changes in chloroplast ultrastructure and Chl fluorescence emission do not affect the overall photosynthetic performance of the mutant. The second mutant studied lacks polyunsaturated lG-carbon fatty acids, and shows a corresponding increase in the levels of the 16:0 acyl group. These changes suggest that a single nuclear mutation at the Ladfi locus causes a specific deficiency in the activity of a chloroplast n-9 desaturase. The mutation affects the fatty acid composition of both chloroplast and extrachloroplast lipids, but it does not appear to have any major functional effects on photosynthesis. However, fadB-related changes in leaf lipid composition seem to confer an enhanced thermal stability upon chloroplast membranes of the mutant. To George and my family for their love, patience and support ii ACKNOWLEDGEMENTS There are many people I would like to thank for helping me in the past several years. First of all Chris Somerville, for his guidance and enthusiasm, teaching me to think critically and to be patient, for giving me a lot of freedom and providing the most wonderful project for me to work on. I also thank the rest of my guidance committee: Jan Zeevaart, Barb Sears and Shelagh Ferguson-Miller for their advice, support and reviewing this dissertation. special thanks to Peter McCourt, who convinced me that lipids are interesting, and all the members of Chris Somerville’s lab (past and present) for making me feel at home not only in the lab, but in this country as well. Thanks for that also go to Cathy Chia and Sylvia Darr, my good friends, and John Fitchen. They were there whenever I needed assistance, encouragement, or just somebody to talk to. I will always be grateful to my first teacher, dr. Mercedes Hrischer, for introducing me to the fascinating world of science. Finally, thank you Ivo and George for caring. TABLE OF CONTENTS £39: List of tables ........................... x List of figures .......................... xii List of abbreviations ....................... xv CHAPTER 1. Literature review .................... 1 Lipid structure ........................ 1 Glycerolipid synthesis in leaf cells ............. 7 16:3 plants ....................... 8 18:3 plants ....................... 13 Fatty acid desaturation ................. l3 Lipid composition of chloroplast membranes .......... 16 Literature cited ....................... 18 CHAPTER 2. Mutant isolation and genetic characterization ...... 22 Introduction ......................... 22 Agtl mutants ......................... 24 Egg mutants .................... . ...... 27 Double mutants ........................ 37 Summary ............................ 39 Literature cited . ,. ..................... 41 CHAPTER 3. Altered regulation of lipid biosynthesis in a mutant of Arabjdggsis deficient in chloroplast glycerol phosphate acyltransferase activity ................ 43 Abstract ........................... 43 Introduction ......................... 44 iv 2393 Material and methods ..................... 47 Plant material ...................... 47 Chemicals ........................ 48 Lipid analysis ...................... 48 Chloroplast isolation .................. 49 Chloroplast labeling ................... 51 Enzyme assays ...................... 51 Results ............................ 52 Genetic analysis ..................... 52 Biochemical characterization ............... 52 Labeling of leaves .................... 56 Lipid composition .................... 58 Discussion .......................... 62 Synthesis of glycerolipids ................ 62 Altered ratio of 16C/18C fatty acids ........... 63 Synthetic capacity of the eukaryotic pathway ....... 64 Synthesis of PG . . . . . . . . . . . . . . . . ..... 64 Evolutionary implications ................ 65 Literature cited ....................... 66 CHAPTER 4. Alterations in chloroplast ultrastructure caused by changes in membrane lipid composition in a mutant of Arabigggsis deficient in plastid G3P acyltransferase activity ........................ 69 Abstract ........................... 69 Introduction ......................... 70 Materials and methods. . .l .................. 71 Plant material and growth conditions ........... 71 V has Measurements of growth rate ............... 72 Extraction and analysis of Chl, proteins and lipids . . . 72 Pigment-protein electrophoresis ............. 73 L-[355]-Methionine labeling of thylakoid proteins and protein extraction .................... 73 Two-dimensional gel electrophoresis ........... 74 Isolation of chloroplast membranes ............ 75 Measurements of relative fluidity ............ 75 Photosynthetic electron transport measurements ...... 76 Room temperature Chl fluorescence ............ 77 Low temperature (77K) fluorescence ............ 77 Gas exchange ....................... 78 Electron microscopy ................... 78 Chloroplast copy number ................. 79 Results ............................ 79 Effects of temperature on relative growth rate ...... 79 Effects of high temperature on stability of chloroplast membranes ........ _ ................ 81 Membrane fluidity .................... 85 Effects of membrane lipid composition on Chl and protein content ......................... 85 Photosynthetic characteristics .............. 91 Chl fluorescence measurements .............. 91 Chloroplast ultrastructure and number .......... 98 Discussion .......................... 98 Lipid composition and enhanced thermal tolerance ..... 98 Chloroplast membrane function .............. 102 vi £398 ‘Chloroplast ultrastructure ................ 104 Why do 16:3 plants exist? ................ 106 Literature cited ....................... 106 CHAPTER 5. A mutant of Anahidggsis that accumulates palmitic acid in leaf lipids ..................... 110 Abstract . . . .' ....................... 110 Introduction ......................... 111 Materials and methods ..................... 113 Plant material ...................... 113 Reagents ......................... 113 Lipid analysis ............. ' ......... 114 Labeling of plants .................... 114 Monoacyl-G3P acyltransferase assay ............ 115 Results ............................ 115 Genetic analysis ..................... 115 Biochemical characterization ............... 115 Fatty acid composition of individual lipids ....... 120 Labeling of leaves .................... 121 Discussion .......................... 123 Literature cited ....................... 126 Chapter 6. Enhanced thermal tolerance in a mutant of Arabidggsis deficient in palmitic acid unsaturation ......... 128 Abstract ........................... 128 Introduction ......................... 129 Material and methods ..................... 131 Plant material and growth conditions ........... 131 Measurements of growth rate ............... 131 vii Ease Extraction and analysis of Chl, proteins and lipids . . . 132 Isolation of chloroplast membranes ............ 132 Pigment-protein electrophoresis ............. 132 L-[3551-Methionine labeling of thylakoid proteins, protein extraction and two-dimensional gel electrophoresis ..................... 133 Fluorescence polarization measurements .......... 133 Photosynthetic electron transport measurements ...... 133 Chl fluorescence measurements .............. 134 Effects of temperature on Chl fluorescence ........ 134 Gas exchange ....................... 134 Electron microscopy ................... 135 Chloroplast number determination ............. 135 Results ............................ 136 Effects of temperature on growth ............. 136 Effects of high temperature on fluorescence ....... 136 Effects of temperature on photosynthetic electron transport ........................ 139 Membrane fluidity .................... 142 Effect of lipid membrane composition on Chl and protein content ......................... 142 Photosynthetic characteristics .............. 145 Chl fluorescence measurements .............. 148 Chloroplast ultrastructure and number .......... 152 2-dimensional SDS-polyacrylamide gel electrophoresis. . . 154 Discussion .......................... 154 Literature cited ....................... 158 viii £392 CHAPTER 7. Concluding remarks ................... 161 Summary ............................ 161 Future directions ....................... 164 Additional mutants .................... 164 Fatty acid desaturation ................. 165 Chloroplast morphogenesis ................ 166 Chilling sensitivity ................... 167 Thermal tolerance .................... 168 Literature cited ....................... 169 ix l-I. l-II. 2-I. 2-II. 2-III. Z-IV. 2-V. 2-VI. 2-VII. Z-VIII. 2-IX. Z-X. 3-1. 3-11. 4-1. 4-11. 4-III. LIST OF TABLES Page The major fatty acids in leaves .............. 2 Polar lipid composition of thylakoid membranes ...... l7 Fatty acid composition of total leaf lipids of Arabigggsis mutants at 25C ...................... 25 Fatty acid composition of total lsaf lipids of mutant and wild type Arabidopsis grown at 22C ............ 26 F2 linkage analysis of actl ................ 28 Fatty acid composition of total leaf lipids of mutant and wild type Arapiggnsis grown at 22 C ............ 32 Fatty acid composition of total lsaf lipids of mutant and wild type Arabidopsis grown at 22C ............ 33 F2 linkage analysis of fadB ................ 34 F2 linkage analysis of fadC ................ 35 Localization of fiadfl on chromosome 3 ........... 36 Localization of £319 on chromosome 4 ........... 38 Fatty acid composition of total Teaf lipids (in mol 0%) of wild type and mutant Arabidopsis plants grown at 22°C.. . 40 Enzyme activities in the stromal fraction and leaf extracts of Arabidgpsis chloroplasts ........... 55 Fatty acid composition of leafolipids from wild type and mutant Arabidopsis grown at 22 C ............. 60 Relative amounts of lipid, chl and protein in mutant and wild type Arabidopsis leaves and chloroplast membranes . . 88 Photosynthetic activities in mutant and wild type Arabidopsis ........................ 93 Room temperature fluorescence induction and low temperature (77K) fluorescence of isolated thylakoids. . . 97 Labia 4-IV. 5-1. 5-11. 6-1. 6-11. 6-111. . Page Morphometric analysis of chloroplasts from mutant lines and wild type Arabidopsis ................. 100 Fatty acid composition of leafolipids from wild type and mutant Arabidopsis grown at 22 C ............. 118 Fatty acid distribution in MGD from wild type and mutant Arabiggpsls established by degradation with Bhizgggs arrhizgs lipase ...................... 119 Relative amounts of lipid, chl and protein in mutant and wild type Arabidopsis leaves and chloroplast membranes . . 144 Photosynthetic activities in mutant and wild type Arghiggggis ........................ 147 Room temperature fluorescence induction and low temperature (77K) fluorescence of isolated thylakoids. . . xi 1-1. 1-2. 1-3. 1-4. 1-5. 2-1. 2-2. 3-1. 3-2. 3-3. 4-1. 4-2. 4-3. 4-4. 4-5. LIST OF FIGURES Acyl glycerols of leaf membranes .............. Glycolipids of leaf membranes . . . ............ Major phospholipids of leaf membranes ........... Lipid biosynthesis in 16:3 plants ............. Lipid biosynthesis in 18:3 plants ............. Gas chromatography tracing of fatty acid methyl esters from a wild type leaf ...................... Position of 3511, raga and fadC loci on chromosomes 1, 3 and 4, respectively, with estimated recombination percentages ........................ An abbreviated scheme for lipid biosynthesis in the leaves of a 16:3 species ..................... The distribution of radioactivity among the polarllipids of mutant 0825 and wild type Arabidopsis following ( C)-G3P labeling of isolated chloroplasts ............. The distribution of radioactivity in leaf lipids of mutant 9325 and wild type Anahigggsis after labeling with C-acetate ........................ Effect of temperature on the relative growth rate of wild type and mutant Arabigggsjs, ................ Temperature induced fluorescence enhancement yield of wild type and mutant leaves ................... Effect of temperature on whole chain photosynthetic electron transport of thylakoid membranes from wild type and mutant Arabidopsis ................... Photosynthetic electron transport activity in chloroplast membranes from wild type and mutant Arabidopsis preincubated at 40 C for various times indicated ...... Effect of temperature on DPH fluorescence polarization by thylakoid membranes from wild type and mutant Arabidopsis . xii SOON-FE 14 23 30 45 53 57 8O 82 83 84 86 4-6. 4-7. 4-9. 4-10. 4-11. 5-2. 6-1. 6-2. 6-4. 6-5. Bags Autoradiographs of 35S-Methionine labeled proteins of chloroplast membranes from wild type and mutant Arabidopsis separated by 2-dimensional SDS-polyacrylamide gel electrophoresis ...................... 89 Chl-protein complexes of chloroplast membranes from wild type and mutant Arabidggsis ................. 90 Absorption spectra of Chl-protein complexes of chloroplast membranes of the wild type and mutant Arabidopsis ..... 92 Light response curves for a) whole chain, b) PSI and c) PSII electron transport by wild type and mutant Arabldggsis ........................ 94 Room temperature induction transients of isolated chloroplast membranes of the wild type and mutant Arghiggnsis in the absence of DCMU ............. 96 Transmission electron micrographs of chloroplasts from wild type and mutant rosette leaves of Arabidgpsis ....... 99 The distribution of radioactivity among the polar lipids following [ C]-16:0-CoA labeling of isolated chloroplast envelopes of the mutant 0867 and wild type Arabidopsis. . . 117 The distribution of radioactivity in leaf lipids of (A) wild type and (B) 0867 mutant of Arabigggsis after labeling with [ C]-acetate ..................... 122 Effect of temperature on the relative growth rate of wild type and mutant Arabidgngis ................ 137 Temperature induced fluorescence enhancement yield of wild type and mutant leaves ................... 138 Effect of temperature on photosynthetic electron transport in chloroplast membranes from wild type and mutant Arabidggsis ........................ 140 Photosynthetic electron transport activity in chloroplast membranes from wild type and mutant Arabidopsis preincubated at 40 C for various times indicated ...... 141 Effect of temperature on DPH fluorescence polarization of chloroplast membranes from wild type and mutant Argpiggngig ........................ 143 Chl-protein complexes of chloroplast membranes from wild type and mutant Arabidopsis ................ 146 xiii 6-9. 2393 Light response curves for (a) whole chain, (b) PSI and (c) PSII electron transport by isolated chloroplast membranes from wild type and mutant Arabidopsis ........... 149 Chl fluorescence spectra of chloroplast membranes from wild type and mutant Arabidopsis in the absence of MgCl2 . . . . 151 Transmission electron micrographs of chloroplasts from rosette leaves of (A) wild type and (8) mutant Arabidgpsis. 153 6-10. Autoradiographs of [35$]-Methionine labeled proteins of chloroplast membranes from wild type and mutant Arabidopsis separated by two-dimensional SDS-polyacrylamide gel electrophoresis ...................... 155 xiv ACP BSA Chl CoA CPI CPla CPa DCMU DGD DPH EDTA 63? LHCP LHCP1 and LHCP LHCP3 LPA 2 LIST OF ABBREVIATIONS acyl carrier protein symbol for a gene controlling the activity of glycerol-3-phosphate acyltransferase bovine serum albumine chlorophyll coenzyme A PSI reaction center Chl-protein complex oligomer of CPI PSII reaction center Chl-protein complex 3-(3,4-dichlorophenyl)-1,1-dimethylurea digalactosyldiacylglycerol 1,6-diphenyl-1,3,5-hexatriene ethylenediaminetetraacetic acid symbol for a gene controlling the activity of an n-9 fatty acid desaturase initial fluorescence variable fluorescence maximum fluorescence gas liquid chromatography glycerol-3-phosphate light harvesting Chl a/b protein complex oligomeric forms of LHCP monomeric form of LHCP lysophosphatidic acid XV MES MOPS MGD MV PA PC PE PEP P6 P1 PS SOS SL HT 2[N-morpholino]-ethane sulfonic acid 3[N-morpholino]-propane sulfonic acid monogalactosyldiacylglycerol methyl viologen fatty acid containing n carbons and x double bonds phosphatidic acid phosphatidylcholine phosphatidylethanolamine phosphoenolpyruvate phosphatidylglycerol phosphatidylinositol photosystem sodium dodecyl sulfate sulfolipid wild type xvi CHAPTER 1 LITERATURE REVIEW Lipid structure The largest group of acyl lipids present in the photosynthetic tissue is based on glycerol, and includes acyl glycerols, glycoglycerolipids and phosphoglycerolipids. In all cases, sn-l and sn-2 positions of the glycerol backbone are esterified with fatty acids, while the sn-3 position contains sugar or phosphate moieties, known as the head group. The head group is the polar part of the lipid molecule and faces out into the aqueous environment when in a bilayer. The major fatty acids in leaves are even numbered, unbranched, monocarboxylic acids, which can be classified into two distinct groups: the saturated and unsaturated fatty acids. Saturated fatty acids contain no double bonds in their hydrocarbon chain. The most common representatives of this group are palmitic (hexadecanoic) acid and stearic (octadecanoic) acid, comprising approximately 12% and 3 %, respectively, of the total fatty acid content of the leaf (Harwood, 1980; Table l-I). 0n the other hand, unsaturated fatty acids contain one or more double bonds, and are by far the most abundant leaf fatty acids. For example, linolenic (c1; 9,12,15 octadecanoic) acid, with Table 1-1. The major fatty acids in leaves. Symbol Systematic name Common name Saturated Unsaturated 16:0 18:0 16:1 (7c) A 16:1 (3t) 16:2 (7c 10c) 16:3 (7c 10c 13c) 18:1 (9c) 18:2 (9c 12c) 18:3 (9c 12c 15c) Hexadecanoic acid Octadecanoic acid Hexadecenoic acid trans-hexadecenoic acid Hexadecadienoic acid Hexadecatrienoic acid Octadecenoic acid Octadecadienoic acid Octadecatrienoic acid Palmitic acid Stearic acid Palmitoleic acid Oleic acid Linoleic acid Linolenic acid 3 th three double bonds between the 9th and 10th, 12 and 13th and 15th and 16th carbons of the chain, counting from the carboxyl end, may comprise up to 80% of total fatty acids of the leaf in some plant species. A shorthand nomenclature is commonly used for fatty acids. It consists of two numbers separated by a colon. The first number corresponds to the carbon chain length, while the number after the colon denotes the number of double bonds (Table l-I). Unless otherwise specified it is assumed that the double bonds are $15. On the basis of the head group attached to position sn-3 of glycerol, we can distinguish three major categories of leaf lipids: acyl glycerols, with unesterified sn-3 hydroxyl groups, glycolipids, whose third position is occupied with a sugar residue, and phospholipids, containing a phosphatidic acid derivative for a head group. Acyl glycerols, 1,2-diacylglycerol (DAG) and monoacylglycerol (Figure 1-1), are only minor constituents of the photosynthetic tissue and do not accumulate to any significant amount. However, they are important metabolic intermediates. One of the most striking features of leaf lipid composition is an extremely high proportion of glycolipids. The three principal glycolipids, monogalactosyldiacylglycerol (MGD), digalactosyldiacylglycerol (DGD) and sulfoquinovosyldiacylglycerol (SL) (Figure 1-2), are predominantly found in chloroplasts where they account for more than 70% of total lipids of the leaf cells (Harwood, 1980; Barber and Gounaris, 1986). The commonly occurring phospholipids in plant tissues include: phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) Their structures are shown in Figure 1-3. PC is the most important phospholipid in the cwon "(f-OH ”2°"?wa 0 1 -monoacylglycerol cwon Hé-o Hzc-o O 1,2-diacylglycerol (DAG) Figure 1-1. Acyl glycerols of leaf membranes. 0" O—CH OH | 2 ' Hc-o - OH I I Hzc-o Monogalactosyldiacylglycerol (MGD) CH20H OH (, OH ‘0”: (”‘0 (JO-CH H I 2 "1-0 H o rho-o Dlgalactosyldlacylglycerol (DGD) 012803" H OH o-CH2 H I Hc-o o rec-o Sulfoqulnovosyldlacylglycerol (SL) Figure 1-2. Glycolipids of leaf membranes. 6 CH3 0- CH—Nl't-CH CH -o-I'>-o 3 l 5— 2 I'- CH3 o—CH2 H - Phosphatidylcholine (PC) Hzc-o S’- c'izHi-o-IJ-o-cH2 "(f—OH O "(f-0 Phosphatldylglycerol CH20H "20. (PG) '+ .. .. _.._ O "(Ii-0 Phosphatidylethanol- Hzc-O anflne(PE) (3 CH1 CH1 OH _ 8’ O-f—O-Cflz 0" 0 "(Lo Phosphaaig’yllnositol PEG-OW +NH3 0' _ l OOC-(E-CHfO-fi-O-CHz H O HIE-O Phosphatidylserlne I (PS) Hzc-o Figure 1-3. Major phospholipids of leaf membranes. 7 majority of plant membranes, except the thylakoid membranes, where PG predominates. PC and PI that are found in the chloroplast are located only in the outer chloroplast envelope. 0n the other hand, PE and PS have not been detected in the chloroplast, and are primarily found in the mitochondrion (Harwood, 1980). Glycerolipid synthesis in leaf cells Isolated intact chloroplasts readily incorporate added acetate into long chain fatty acids in the light (Slack, 1977; Roughan et al. 1979; Roughan and Slack, 1982). This observation, in conjunction with the localization of acyl carrier protein (ACP) (Ohlrogge et al., 1979) and acetyl-CoA synthetase (Kuhn et al., 1981) exclusively within chloroplasts, led to the conclusion that these organelles are the only sites of fatty acid synthesis in the photosynthetic tissue. The mechanism of fatty acid synthesis involves three steps: (1) the carboxylation of acetyl-CoA to form malonyl-CoA, (2) the repeated condensation of malonyl-CoAs with a growing acyl chain attached to ACP to make 16:0-ACP, and (3) the elongation of 16:0-ACP to 18:0-ACP. The condensation reactions are catalyzed by fatty acid synthase (FAS), that consists of six loosely associated enzymes, partially purified and characterized by Shimakata and Stumpf (1982). In contrast, fatty acid synthase activity in animal tissues is localized in the cytosol, and it is associated with a single enzyme, a homodimer with 6-7 active site domains (Stumpf, 1981). Most of the 18:0-ACP produced in the plant chloroplast is desaturated to 18:1-ACP by a highly active ferredoxin 8 dependent stromal desaturase (McKeon and Stumpf, 1982). Therefore, the main products of fatty acid synthesis are 16:0- and 18:1-ACPs. These thioesters can be used directly in the chloroplast by the prokaryotic pathway of lipid synthesis, or may be hydrolyzed to free fatty acids and exported to the cytoplasm and eventually to the endoplasmic reticulum (ER), where the enzymes of the eukaryotic pathway are located. The partitioning of fatty acids between these cell compartments, and their respective pathways of lipid synthesis, depends on the plant species and results in differences in lipid composition of their chloroplast membranes. Detailed analyses of leaf lipid composition of a variety of plants has led to their classification into two major groups: those containing hexadecatrienoic acid ("16:3 plants"), and those that do not contain 16:3 acyl groups (“18:3 plants“; Roughan and Slack, 1984). 161191101: 16:3 plants include families like: Solanaceae, Brassicaceae, Chenopodiaceae and Apiaceae, and typically contain up to 20% 16:3 fatty acid in their leaf lipids. This acyl group is found only on MGD and 060 molecules synthesized in the chloroplast through the prokaryotic pathway. The prokaryotic pathway is initiated by the sequential acylation of glycerol-3-phosphate (GBP) using acyl-ACPs (Figure 1-4). The final product of these reactions, catalyzed by two acyl transferases, is phosphatidic acid (PA). The sn-l specific acyl transferase is a soluble stromal protein which preferentially utilizes 18:1-ACP as a substrate (compared to 16:1-ACP) (Frentzen et al., 1983) and generates lysophosphatidic acid (LPA). This lipid enters the inner .35: 3: E £35595 3...: .I 23: 3.0: OS. : C”. i, L LIL 40 000 \OOI / as: «HOP “"0— O-MGD + UDP MGD + UDP-Gal ---->DGD + UDP. The enzyme responsible for DGD formation would be UDP-GaleGD galactosyltransferase. However, this enzyme has not been identified in isolated chloroplasts, envelope membranes or microsomes. The latter pathway was proposed by Van Besouw and Wintermans (1978), who discovered another enzymatic activity in the spinach chloroplast envelope, producing DGD in the absence of UDP-Gal. This galactolipid:galactolipid galactosyltransferase transfers the galactose moiety between two MGD molecules to produce DGD and DAG: MGD + MGD ---->-DGD + DAG. This enzyme was localized on the outer surface of the outer envelope membrane, and it is, at the moment, a more likely candidate for DGD synthesis. Despite considerable attention, the biosynthetic pathway of SL also remains uncertain (Barber and Gounaris, 1986). Hhen spinach leaves were labeled with 14C02, 14C was found in 16:0 at position sn-Z, and in both 16:0 and 18-carbon fatty acids at position sn-l (Siebertz et al., 1979). These features, shared with prokaryotic DAG, suggest a f 14C-acetate biosynthetic relationship 1g vivo. The incorporation 0 into SL by isolated chloroplasts (Roughan et al., 1979, Roughan et al., 1980) also indicates that chloroplasts are probably autonomous in SL synthesis. 13 mm Short term labeling experiments of intact leaves with 14C02 and 14C-acetate (Roughan, 1970; Slack and Roughan, 1975; Williams et al., 1976) have shown that plants of certain plant families such as: Cucurbitaceae, Fabaceae, Asteraceae and Poaceae distribute their newly synthesized fatty acids predominantly into PC, and only a minor proportion into PG (Figure 1-5). Since PC is exclusively made by the eukaryotic pathway, this labeling pattern indicated that the ER is the major site of glycerolipid synthesis in these plants. A detailed analysis of leaf fatty acid composition demonstrated that they do not contain a 16:3 acyl group, which is a specific marker of MGD and DGD made through the prokaryotic pathway. It is replaced mostly by linolenic acid (18:3), hence these species were named 18:3 plants (Heinz and Roughan, 1983). Direct assays of chloroplast enzymes confirmed a relatively low activity of prokaryotic acyl transferases (Heinz and Roughan, 1983) and PA phosphatase, and led to the conclusion that the only product of the prokaryotic pathway in 18:3 plants is PG (Andrews and Mudd, 1985). WEE!!! Each lipid class constituent of higher plant membranes has a characteristic acyl group composition in terms of chain length, as well as degree, position and stereochemistry of unsaturation. Typically, plant fatty acids contain 0-3 c1; double bonds (Frentzen, 1986). The first double bond is always introduced at position 9, counting from the carboxyl end of the acyl group, the second at position 12, while the third occurs at position 15. The most common exception to this general l4 .ma=~_a mum_ =_ ”.mogucsmoen uaaas_.m-~ weaned 66: «8. one, «6. «a. «a: «6. IL ILIIL TILL .3 /aoo§o=\\ no: a". p N“. « II[ 20 fi 665/ as: 05w 03¢ «3p «3p 9— 0.. Pain 0: O.— z 0 a3 93 6 0.... \ c J a: to. due 06. ..r... . .50 H K036. 1153. :8 93 :2 LL 0.. one. :3 t ‘5 n I 3&3 p (00.6qu rm. Us. \ (t h 03.100 1' 2 .C 330:0 15 rule is the acyl group 3- trans 16:1 which is always esterified to the sn-2 position of PG (Dubacq and Tremolieres, 1983). Besides these observations, the desaturation reactions in plants are not well understood. It is not known, for example, how many desaturase enzymes are involved in the desaturation process, and their compartmentation has not been precisely established. The only desaturase partially purified and characterized in some detail so far, is the 18:0-ACP desaturase, a soluble stromal enzyme (McKeon and Stumpf, 1982). All the other attempts to isolate chloroplast desaturases have not been successful, since chloroplasts lose the ability to synthesize polyunsaturated fatty acids when broken (Roughan, 1979), or when they are exposed to hypotonic conditions (Andrews and Heinz, 1987). Microsomal desaturases have not been purified either, but it is possible to measure the 18:1 and 18:2 desaturase activity in microsomal preparations (Browse and Slack, 1981). Despite the described difficulties associated with the characterization of plant desaturases, substantial information concerning fatty acid desaturation is available from 1n,yiyg labeling experiments. Analysis of 14C labeled acyl groups suggested that PC is the major site of 18:1 desaturation for lipids made by the eukaryotic pathway (Slack et al., 1977). This conclusion was confirmed by showing that l4C-oleoyl-CoA is first esterified to lyso-PC before desaturation occurs (Murphy et al., 1983). On the other hand, the major substrate for 18:2 desaturation for eukaryotic lipids seems to be MGD (Hawke and Stumpf, 1979; Roughan and Slack, 1982). This lipid also serves as a major site for 18:1 and 18:2 desaturation of prokaryotic lipids. However, the desaturation reactions of lipids made through the l6 14C-18zl-PG prokaryotic pathway are not confined to MGD. For example, synthesized by the chloroplast is sequentially converted to labeled 18:2- and 18:3-PG (Roughan, 1985). A similar conclusion was reached by analyzing the fadfl mutant of Arabigggsis (Browse et al., 1986). This mutant is deficient in the activity of a chloroplast 18:2 desaturase, due to a single nuclear mutation. The observation that all the chloroplast polar lipids are affected by the fadn mutation, clearly indicates that they are all substrates for 18:2 desaturation, or that substantial transfer of acyl groups occurs between these lipids. In addition, the £310 mutant provided important, although indirect information concerning 18:2 desaturase function. This enzyme introduces the final double bond in acyl chains with no apparent specificity for the chain length (16- or 18-carbon), or their point of attachment to the glycerol backbone (sn-I or sn-2). Lipid composition of chloroplast membranes A detailed survey of lipid composition of various plant tissues revealed major differences among membranes of different cell types, and even organelles within a single cell type. Basically, every single membrane in the cell has a unique lipid composition with respect to both the head group and the acyl chains (Harwood, 1980). However, the functional significance of lipid diversity has not been established. In comparison with other eukaryotic membranes, the acyl lipid composition of the photosynthetic membrane is remarkably constant in a wide variety of species (Table 1-11; Harwood, 1980; Douce and Joyard, Table 1-11. Polar lipid composition of thylakoid membranes. Values are weight %. Plant MGD DGD SL PG PC Source Spinach 52 26 7 10 5 1 Wheat 45 35 8 10 2 1 Barley 54 24 8 9 5 2 Tomato 50 25 3 22 0 2 White clover 48 28 4 21 0 1 Broad bean 52 28 5 9 6 1 Arabidgnsis 48 28 6 l3 5 3 1 Harwood JL 1980 2 Douce R, 0 Joyard 1980 3 This dissertation 18 1980). The principal chloroplast polar lipids are MGD and DGD, accounting for more than 70 % of total lipid fraction of this organelle. The additional 30 %.is made up mostly of PG, PC and SL. Another interesting feature of thylakoid membranes is an unusually high proportion of polyunsaturated fatty acids. Depending on the plant species, trienoic acids (18:3 and 16:3) represent up to 80 % of fatty acyl groups of this membrane. In an attempt to examine the structural and functional roles of individual lipid classes, and distinct fatty acid composition of chloroplast membranes, we have isolated a number of mutants of. Arabidgpsls thallana (L.) Heynh. with altered leaf lipid metabolism. Two of the mutants have already been investigated in detail. One is deficient in trans-16:1 synthesis (Browse et al., 1985, McCourt et al., 1985), while the other lacks a specific desaturase responsible for introducing n-3 double bond in both 18-carbon and 16-carbon fatty acids (Browse et al., 1986, McCourt et al., 1987). This work describes the genetic and biochemical characterization of the two additional mutants, as well as the consequences of specific changes in fatty acid composition on photosynthetic properties of these plants. Literature cited Andrews 0, K Keegstra 1983 Acyl-CoA synthetase is located in the outer membrane and acyl-CoA thioesterase in the inner membrane of pea chloroplast envelopes. Plant Physiol 72: 735-740 Andrews 0, B Mudd 1985 Phosphatidylglycerol synthesis in pea chloroplasts. Plant Physiol 79: 259-265 19 Andrews 0, 08 Ohlrogge, K Keegstra 1985 Final step of phosphatidic acid synthesis in pea chloroplasts occurs in the inner envelope membrane. Plant Physiol 78: 459-466 Andrews 0, E Heinz 1987 Desaturation of newly synthesiszed monogalactosyldiacylglycerol in spinach chloroplasts. 0 Plant Physiol 133: 75-90 Barber 0, K Gounaris 1986 What role does sulfolipid play in the thylakoid membrane? Photosynthesis Res 9: 239-250 Block MA, A0 Oorne, J Joyard, R Douce 1983 The phosphatidic acid phosphatase of the chloroplast envelope is located on the inner envelope membrane. FEBS Lett 164: 111-115 Browse 0, CR Slack 1981 Catalase stimulates linoleate desaturase activity in microsomes from developing linseed cotyledons. FEBS Lett 131: 111-114 Browse 0, P McCourt, CR Somerville 1985 A mutant of Arabjgggsis lacking a chloroplast specific lipid. Science 227: 763-765 Browse 0, P McCourt, CR Somerville 1986 A mutant of deficient in C18,3 and C16,3 leaf lipids. Plant Physiol 81: 859-864 Coves 0, MA Block, 0 Joyard, R Douce 1986 Solubilization and partial purification of UDP-galactose diacylglycerol galactosyl transferase activity from spinach chloroplast envelope. FEBS Lett 208: 401-407 Douce R, 0 Joyard 1980 Plant galactolipids. In PK Stumpf, EE Conn, eds, The Biochemistry of Plants, Vol 4, Academic Press, New York, pp 321-362 Dubacq J-P, A Tremolieres 1983 Occurrence and function of phosphatidylglycerol containing 3-trans-hexadecenoic acid in photosynthetic lamellae. Physiol Veg 2: 293-312 Frentzen M, E Heinz, TA McKeon, PK Stumpf 1983 Specificities and selectivities of glycerol-3-phosphate acyltransferase and monoacylglycerol-3-phosphate acyltransferase from pea and spinach chloroplasts. Eur 0 Biochem 129: 629-636 Frentzen M, W Hares, A Schiburr 1984.Properties of the microsomal glycerol-3-phosphate and monoacylglycerol-3-phosphate acyl transferase from leaves. In PA Siegenthaler, W Eichenberger, eds, Structure, Function and Metabolism of Plant Lipids, Elsevier, Amsterdam pp 105-110 Frentzen M 1986 Biosynthesis and desaturation of the different diacylglycerol moieties in higher plants. 0 Plant Physiol 124: 193-209 Harwood JL 1980 Plant acyl lipids: Structure, distribution and analysis. In PK Stumpf, EE Conn, eds, The Biochemistry of Plants, Vol 4, Academic Press, New York, pp 2-48 20 Hawke 0C, PK Stumpf 1980 The incorporation of oleic and linoleic acids and their desaturation products into galactolipids of maize leaves. Arch Biochem Biophys 203: 296-306 Heemskerk JWM, G. Bogemann, 0FGM Wintermans 1985 Spinach chloroplasts: localization of enzymes involved in galactolipid metabolism. Biochim Biophys Acta 835: 212-220 Heinz E, PG Roughan 1983 Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants. Plant Physiol 72: 273-279 Kleppinger-Sparace K, 0M Mudd, OG Bishop 1985 Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants. Arch Biochem Biophys 240: 859-865 Kuhn DN, M Knauf, PK Stumpf 1981 Subcellular localization of acetyl-CoA synthetase in leaf protoplasts of Sginacja glgragga. Arch Biochem Biophys 209: 441-450 McCourt P, 0 Browse, 0 Watson, C0 Arntzen, CR Somerville 1985 Analysis of photosynthetic antenna function in a mutant of Arabigggsis thaliaga (L.) lacking trans-hexadecenoic acid. Plant Physiol 78: 853-858 McCourt P, L Kunst, 0 Browse, CR Somerville 1987 The effects of the reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabigggsis. Plant Physiol 84: 353-360 McKeon TA, PK Stumpf 1982 Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. 0 Biol Chem 257: 12141-12147 gggrgsgs 1982 Phospholipid biosynthesis. Ann Rev Plant Physiol 33: Murphy 00, IE Woodrow, E Latzko, KD Murkherjee 1983 Solubilization of oleoyl-CoA thioesterase, oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. FEBS Lett 162: 442-446 Neufeld EF, CW Hall 1964 Formation of glactolipids by chloroplasts. Biochem Biophys Res Commun 14: 503-508 Ohlrogge 08, ON Kuhn, PK Stumpf 1979 Subcellular localization of acyl carrier protein in leaf protoplasts of Sninagia glgnagga. Proc Natl Acad Sci USA 76: 1194-1198 Ohnishi 0, M Yamada 1982 Glycerolipid synthesis in Aygna leaves during greening of etiolated seedlings 111. Plant Cell Physiol 23: 767-773 Ongun A, 08 Mudd 1968 Biosynthesis of galactolipids in plants. 0 Biol Chem 243: 1558-1566 21 Roughan PG 1970 Turnover of the glycerolipids in pumpkin leaves. The importance of phosphatidylcholine. Biochem J 117: 1-8 Roughan PG, R Holland, CR Slack 1979 On the control of long chain fatty acid synthesis in isolated intact spinach (ngnagia plenagga) chloroplasts. Biochem J 184: 193-202 Roughan PG, R Holland, CR Slack 1980 The role of chlorgplasts and microsomal fractions in polar lipid synthesis from [1- C]-acetate by cell free preparations from spinach (Spinagia glgragga) leaves. Biochem J 188:17-24 Roughan PG, CR Slack 1982 Cellular organization of glycerolipid metabolism. Ann Rev Plant Physiol 33: 97-132 Roughan PG, CR Slack 1984 Glycerolipid synthesis in leaves. Trends Biochem Sci 9: 383-386 Roughan PG 1985 Cytidine triphosphate-dependent acyl-CoA-independent synthesis of phosphatidylglycerol by chloroplasts isolated from spinach and pea. Biochim Biophys Acta 835: 527-532 Shimakata T, PK Stumpf 1982 Fatty acid synthetase from Sginagig plenacga leaves. Plant Physiol 69:1257-1262 Siebertz M, E Heinz 1977 Galactosylation of different monogalactosyldiacylglycerols by cell free preparations from pea leaves. Hoppe Seyler’s Physiol Chem 358: 27-34 Siebertz HP, E Heinz, M Linscheid, 0 Joyard, R Douce 1979 Characterization of lipids from chloroplast envelopes. Eur J Biochem 101: 429-438 Slack CR, PG Roughan 1975 The kinetics of incorporation in vivo of [ C]-acetate and CO into the fatty acids of glycerolipids in developing leaves. Bithem 0 152: 217-228 Slack CR, PG Roughan, N Balasingham 1977 Labeling of glycerolipids in the3cotyledons of developing oil seeds by [ C]-acetate and [2- H]-glycerol. Biochem J 162: 289-296 Stumpf PK 1981 Plants, fatty acids, compartments. Trends Biochem Sci 6: 173-176 Van Besouw A, 0FGM Wintermans 1978 Galactolipid formation in chloroplast envelopes. FEBS Lett 102: 33-37 Williams 0P, GR Watson, S Leung 1976 Galactolipid synthesis in leaves.II. Formation and desaturation of long chain fatty acids in PC, PG and the galactolipids. Plant Physiol 57: 179-184 CHAPTER 2 MUTANT ISOLATION AND GENETIC CHARACTERIZATION Introduction The analysis of microbial mutants has shown that there are many changes in lipid composition which are incompatible with the survival of the organism (Clark and Cronan, 1981; Rock and Cronan, 1985). In particular, major changes in the amounts of different head groups are severely deleterious, hence all of these mutants have a detectable phenotype. On the other hand, some E; ggli mutants are completely defective in the synthesis of cyclopropane fatty acids, but show no impairment of growth under a variety of environmental conditions (Taylor and Cronan, 1976; Gorgan and Cronan, 1986). This observation suggested that fatty acid composition of membrane lipids might not be essential for the organism. Therefore, a rapid direct screening method was designed that involves the preparation of fatty acid methyl esters (FAME) from single leaves followed by gas chromatography (GC) (Browse et al., 1985b). A typical GC tracing of fatty acids obtained from a wild-type leaf is shown in Figure 2-1. This method was employed to analyze leaf fatty acid composition of individual mutagenized Arabidgpsis plants. From approximately 2000 plants examined in the 22 23 18:3 1 8:0 Detector response 16x3 1 8:2 ‘1'. O F O L l I 1 l n l__ O 4 8 12 Elutlon time (min) Figure 2-1. Gas chromatography tracing of fatty acid methyl esters from a wild type leaf. 24 initial screen, and 2000 analyzed in several subsequent searches, we isolated 9 lines with stably inherited changes in fatty acid composition of leaf glycerolipids (Table 2-I). These mutants provide an opportunity to directly address questions concerning the enzymology, regulation and functional significance of desaturation, as well as the control of cellular lipid metabolism. Furthermore, since 75 % of all fatty acyl groups in Arabjgggsis leaves are constituents of chloroplast membranes, mutant analysis can contrubute to our understanding of the relationship between specific fatty acid composition of thylakoids and chloroplast structure and function. A911 mutants Four mutant lines (083, 0825, 0828 and LK8) were isolated from two independently mutagenized populations because of the deficiency in 16:3 acyl group. The absence of 16:3 is compensated for by increases in 18:1, 18:2 and 18:3 fatty acids (Table 2-11). Otherwise the mutants were indistinguishable from the wild type in appearence. Complementation analysis has shown that the four lines have a lesion at the same locus designated actl (acyl transferase 1). Therefore, the genetic basis of the phenotype was determined only for the line 0825 by crossing the mutant with the wild type as maternal parent. The leaf fatty acid composition of F1 progeny, measured by GC, was identical to the wild type, suggesting a recessive mutation (Table 2-11). The frequency of the homozygous mutant phenotype in the F2 25 Table 2-1. Fatty acid composition of total leaf lipids of Azahiggnsjs mutants at 25 C. Each value is a mean of 10 plants. mmmmmn Mutantficnc mmmmmmmmmmrce Linc mm ' 0860 1315 18 tr. . - tr. 12 1 3 I9 47 1 0827 fgdA l7 tr. - tr. I3 I 3 19 47 2 0867 .fgdfl 24 - 2 - - 1 2 14 56 3 LK3 fad; 15 11 2 - - 1 21 14 36 3 081 fadfl 15 2 2 12 3 l 3 26 36 2 LK9 £310 13 2 3 12 4 2 3 32 29 3 0812 fadfi 15 tr. 4 tr. IO 1 27 4 36 4 0825 3931 " 13 tr. 2 tr. tr. 1 8 23 53 3 LK8 3911 12 tr. 2 tr. tr. 1 9 23 53 3 WT 15 tr. 2 tr. 12 1 3 18 48 3 1 Browse et al. 1985a 2 McCourt P0, Ph. D. Thesis 3 This dissertation 4 Browse 0A, personal communication 26 Table 2-11. Fatty acid composition of total leaf lipids of mutant and wild type Arabidopsis grown at 22 C. Values are mol % 1 SD (n-10) Fatty Wild type F1(WT x 0825) 0825 acid. 16:0 14.1 1 0.5 14.0 1 0.5 12.6 1 0.3 16:1 915 1.6 1 0.1 1.6 1 0.5 1.4 1 0.4 16:1 Iran; 1.6 1 0.3 2.3 1 0.4 2.0 1 0.4 16:2 0.5 1 0.1 0.9 1 0.4 0.5 1 0.3 16:3 11.4 1 0.3 10.5 1 0.6 1.5 1 0.3 18:0 1.7 1 0.1 1.6 1 0.1 0.8 1 0.2 18:1 3.0 1 0.3 3.2 1 0.4 8 4 1 0.8 18:2 13.4 1 0.5 14.2 1 0.8 17.8 1 0.8 18:3 52.5 1 0.4 51.7 1 1.7 55.1 1 1.5 27 generation was also analyzed. Of 271 F2 plants, 64 had no detectable levels of 16:3. This is a good fit to the 3:1 hypothesis (X2-0.36, P>0.5) indicating that the alteration in fatty acid composition is due to a single nuclear mutation. ' The 3911 mutation was mapped to a chromosome by F2 mapping from a cross of the W-lOO strain to 0825. W-IOO contains 2 visible markers for each of the five Axgpidgggig chromosomes. All the markers and the 1911 mutation segregated 3:1, as expected for a simple mendelian trait. 5111 assorted independently of all markers, except for an. A significant departure from 9:3:3:1 ratio is indicated by a high X2 value (Table 2-III). This aberrant independent assortment was used to assign 3511 mutation to chromosome 1, 34.8 map units (cM) away from the an marker (Figure 2-2). Egg mutants The majority of Arghidgpgig mutants identified by direct GC assay of leaf fatty acid composition, showed specific changes in the levels of unsaturation of their acyl groups. The responsible mutations map to five different loci designated fgdA i.e., fatty acid desaturation gene A, fgdfi, fadC, fgdu and 131: (Table 2-1, Figure 1-4). In all the mutants described so far (1115: Browse et al., 1985a, McCourt et al, 1985; fadn: Browse et al. 1986, McCourt et al., 1987) the enzymatic lesions were due to single nuclear mutations. I have recently determined that the same is true for the fadB and .1119 mutants. The lines 0867 carrying a fgdfl mutation, and LK3 with a 28 Table 2-III. F2 linkage analysis of actl. "4001311 321 91 e1: 1112-911 1212 + + + + + + 0825 (+ 93.112 4. (A) Single locus goodness of fit tests E an 90 + 206 an]. 81 + 211 g1 53 + 242 g: 67 + 225 M2 70 + 227 all 67 + 230 I 74 222 73 219 74 221 73 219 74 223 74 223 x20) 4.61 1.17 7.78 0.66 0.32 0.94 +13 .ffii .FEE -+§; +0 rt- g—e mm; + + 42 247 74 217 67 224 100 190 60 235 X 72 217 73 218 73 218 73 217 74 221 expected segregation for a single recessive nuclear trait (3:1) x20) 16.88 0.03 0.61 13.91 3.42 29 (8) Joint segregation for pairs of loci Chromosome 1 ' 13 Obs Exp Obs Exp 2 21 Obs Exp e: Obs Exp 3 hyz Obs Exp 911 Obs Exp 4 hp Obs Exp gen: Obs Exp 5 113 Obs Exp m; Obs Exp 2 * Indicates a significant difference 2 +/+ 151 167 170 167 193 165 179 163 182 166 183 166 198 161 171 163 175 163 146 162 +/- 83 56 55 55 41 55 54 54 53 55 52 55 33 54 61 54 57 54 85 54 actl/+ 53 56 51 55 48 55 44 54 45 55 46 55 47 54 44 54 48 54 43 54 18 actl/- 7 19 19 18 11 18 13 18 15 18 14 18 9 13 18 9 18 14 18 x2 12.74* 0.0003 0.004 3.03 expected segregation for two recessive nuclear traits assorting independently (9:3:3:1) 3O 1 :3 ‘4 . CHLen . lithflbzi 14.8“ 54 f d 31 [10.80139 34. 37.51 " ' 23.6 24.7” 323° .1 L34_3...¢t1 1 39.9 . '39'6W9'4 42.00cer-21 L “34110.? “fadC 0 58.7oap-2J helm-5 Figure 2-2. Position of 1111, fgdB and fadC loci on chromosomes 1, 3 and 4, respectively, with estimated recombination percentages. Circle with a range indicates the position of the centromere. 31 lesion at the 13d; locus were crossed to the wild type as maternal parent, and the fatty acid composition of F1 and F2 progeny was analyzed. All the F1 plants from the WT x 0867 cross had higher levels of 16:0 than the wild type (Table 2-IV), while Fl progeny from the WT x LK3 cross had higher levels of 16:1 and 18:1 acyl groups than wild-type plants (Table Z-V). These results suggest codominant mutations and indicate that levels of c16:1, as well as 16:2 and 18:2 levels are probably regulated by the amount of active enzyme present. Leaf fatty acid composition of 308 F2 plants from the WT x 0867 cross, and 221 plants from the WT x LK3 cross was also determined. The 3:1 segregation pattern (78 1395 : 230 WT, x2-9.013, P>O.9; 51 {Egg : 170 v1, xz-o.44, P>0.4) obtained for both mutants is consistent with the presence of single nuclear mutations at the fagfl (0867) and fad; (LK3) loci. 0867 and LK3 mutant lines were also crossed to line MKI which carries 5 visible chromosome markers. Linkage analysis was carried out on 417 F2 plants from the MKI x 0867 cross, and 228 F2 progeny from the MKI x LK3 cross (Tables 2-v1 and 2-v11). High x2 value obtained for 911, fgdfl pair of loci clearly indicated that these two mutations are closely linked on chromosome 3 (23.6 map units away, Figure 2-2). The same reasoning was used to assign the 11d; allele to chromosome 4, 10.3 map units away from the $112 marker (Figure 2-2). In order to determine the position of 11518 on the map more accurately, I analyzed the F2 progeny of a cross between fQQB/fagg and the strain MSU 22, homozygous recessive for 3 marker genes: 1112/1112, gl;1/gl;1,and 1115/1115 (Table 2-VIII). Joint segregation for pairs of loci (Table 2-VIII) and recombination frequencies (Figure 2-2) were obtained using a computer program (Linkagel) written by K.A. Suiter, 32 Table 2-IV. Fatty acid composition of total leaf lipids of mutant and wild type Arabiggg§1§ grown at 22 C. Values are mol %,1 SD (n-IO). Fatty Wild type F1(WT x 0867) 0867 acid 16:0 13.0 1 0.8 16.4 1 0.8 24.1 + 0.9 16:1 91; 1.5 1 0.2 1.1 1 0.2 1.5 1 0.3 16:1 1115; 3.6 1 0.4 2.8 1 0.3 2.8 1 0.4 16:2 1.7 1 0.5 l 0 1 O 3 0.3 1 0.1 16:3 15.7 1 1.0 12 5 1 0 7 0.3 1 0.2 18:0 0.7 1 0.2 l 2 1 0 2 1.2 1 0.4 18:1 2.4 1 0.4 3.1 1 0.4 2.5 1 0.5 18:2 12.3 1 0.6 13.6 1 0.3 17.1 1 1.0 18:3 49.1 1 1.4 48.2 1 1.4 50.21 1 1.5 33 Table Z-V. Fatty acid compositionoof total leaf lipids of mutant and wild type Arabidopsis grown at 22 C. Values are mol % 1 SO (n-IO) Fatty Wild type F1(WT x LK3) LK3 acid 16:0 13.0 1 0.8 12.3 1 0.5 13.9.+ 0 3 16:1 11; 1.5 1 0.2 3.7 1 0.3 11 2 1 0 7 16:1 1Lan§ 3.6 1 0.4 2.5 1 0.3 3.8 1 0.5 16:2 1.7 1 0.5 0.3 1 0.1 0 5 1 0.2 16:3 15.7 1 1.0 14.0 1 1 3 0.2 1 0.1 18:0 0.7 1 0.2 0 7 1 0 2 0 8 1 0 2 18:1 2.4 1 0.4 5.8 1 0.8 16.1 1 1 0 18:2 12.3 1 0.6 16 7 1 0.5 16.5 1 0 2 18:3 49.1 1 1.4 43.9 1 1.7 37.0 1 1 5 34 Table 2-VI. F2 linkage analysis of fadB. MKlIan 2191131221115 +) x ++ +1398) (A) Single locus goodness of fit tests +an+nx+911+cccz+ms +£adB Obsl 286 91 289 85 270 106 277 96 266 105 301 73 Exp 283 94 281 94 282 94 280 93 278 93 280 94 X2 0.15 1.03 2.04 0.11 2.16 5.99 1 expected segregation for a single recessive nuclear trait (3:1) (8) Joint segregation for pairs of loci Chromosome +/+ +/- fadB/+ fadB/- X2 1 19 Obs2 221 62 80 11 4.23 Exp 211 70 70 23 2 91 Obs 232 55 68 17 0.03 Exp 209 70 70 23 3 911 Obs 199 69 102 4 23.34* Exp 211 70 70 23 4 99:2 Obs 223 54 77 18 0.01 Exp 211 70 70 23 5 m; Obs 209 57 90 15 2.46 Exp 208 70 70 23 2 Expected segregation for two recessive nuclear traits assorting independently (9:3:3zl) * Indicates a significant difference 35 Table 2-VII. F2 linkage analysis of fadC. MKl Ian 01 911 sex: ms + ) x LK3 (+ + + (A) Single locus goodnes of fit tests + an + 01 + 911 + cert + ms + actl Obsl 179 42 169 46 173 48 159 56 151 56 177 44 Exp 166 55 161 54 166 55 161 54 155 52 166 55 2 4.24 1.49 1.27 0.13 0.47 3.05 1 X expected segregation for a single recessive nuclear trait (3:1) (8) Joint segregation for pairs of loci Chromosome +/+ +/- fadC + fadC/- X2 1 an Obs2 142 36 34 8 0.03 Exp 124 41 41 14 2 91 Obs 136 33 36 10 0.11 Exp 121 40 4O 13 3 911 Obs 134 39 43 5 3.47 Exp 124 41 41 14 4 99:2 Obs 90 56 68 l 30.89* Exp 121 40 4O 13 5 m9 Obs 121 30 46 10 0.11 Exp 116 39 39 13 2 Expected segregation for two nuclear recessive traits assorting independently (9:3:3:1) * Indicates a significant difference 36 Table 2-VIII. Localization of 1999 on chromosome 3. MSU 22 (9112 9111 1115, +) x 0867 (+ + (A) Single locus goodness of fit tests + 01:2 + 91:1 + 11:5 05sl 93 44 97 45 109 32 Exp 105 35 105 35 105 35 x2 2.71 3.39 0.40 + £393) + £398 125 17 106 36 12.85 1 Expected segregation for a single recessive nuclear trait (3:1) (8) Joint segregation for pairs of loci +/+ +/- fadB/+ hy-Z Obs2 64 39 38 Exp 80 27 27 gl-l Obs 83 42 17 Exp 80 27 27 11;§ Obs 96 27 13 Exp 79 26 26 +/+ +/- g]-1/+ hy-Z Obs 76 21 22 Exp - 80 27 27 fad8/- x2 1 15.72* 9 1 4.50 9 4 0.003 9 91-1,- 23 11.13* 9 2 Expected segregation for two nuclear recessive traits assorting independently (9:3:3zl) * Indicates a significant difference 37 0.F. Wendel and 0.5. Case (1983). All the values were corrected for double cross-overs with the Kosambi mapping function: D - 251n (lOO+2r)/(100-2r) where D - distance in centiMorgans (cM), and r - estimated recombination percentage (Koornneef et al., 1983). The precise location of the £999 locus was estimated by S. Hugly on the basis of the F2 analysis resulting from a cross between 1999/1999 and the line MSU 15 carrying recessive marker genes 99:;2/99912, 99/99 and 9912/9912 (Table Z-IX). The linkage data (Table 2-IX) and recombination percentages (Figure 2-2) were obtained as described for 1993 gene. Oouble mutants Double mutants were constructed for two reasons: determination of epistatic relationships between various mutations and generation of more severe phenotypes with respect to the overall changes in fatty acid composition. An epistatic relationship between the two mutations can provide detailed information about the sequence of steps affected by mutations in a biosynthetic pathway. Since both the line 0825 (9911) and the line 081 (1999) have altered 16:3 levels, it was of interest to check their epistatic relationship. To do this we have first crossed the two mutants, and analyzed 49 F2 plants from the 081 x 0825 cross. The mutations segregated independently (27 EAQQ -/- A_C_‘L1 : 5 EDD 9c_tl/- 9911 : 15 fadO ACTl/fadO - : 1 fadD actyfadO actl, x’-(3)-5.59 38 Table 2-IX. Localization of 199; on chromosome 4. MSU 15 (9992 hp 9912 +) x LK3 (+ + + 1999) (A) Single locus goodness of fit tests +9522 +110 +113 +1911; 05sl 123 38 125 35 122 39 127 34 Exp 121 40 121 40 121 40 121 40 x2 0.17 0.50 0.05 1.29 1 Expected segregation for a single recessive nuclear trait (3:1) (8) Joint segregation for pairs of loci +/+ +/- fadC/+ fadC/- x2 cer-2 05s2 89 38 34 0 13.32* """ Exp 91 30 30 10 99 Obs 94 33 31 3 4.55* Exp 91 3o 30 10 ap-z Obs 88 39 34 0 13.77* Exp 91 3O 30 10 +/+ +/- bp/+ 99/- X2 cer-2 Obs 102 18 20 18 17.92* Exp 89 3O 30 9 +/+ +/- an:Z/+ 10.24 - cer-2 Obs 112 11 10 28 65.29* ““‘ Exp 91 30 3o 10 1 Expected segregation for two recessive nuclear traits assorting independently (9:3:3zl) * Indicates a significant difference 39 P>O.9) confirming that 9911 and 1990 loci are unlinked. An individual designated LIPI homozygous for £999 and 9911 was identified from the F2 population on the basis of fatty acid compositional analysis, and the result was verified by backcrossing the double mutant to both parents. As in the 0825 parent, LIPI had no detectable 16:3 acyl group at all growth temperatures examined. The 16:3 fatty acids occur only on the sn-2 position of MGD made by the prokaryotic pathway. Therefore it can be concluded that the 9911 mutation affects one of the steps of the prokaryotic pathway, and that it precedes the step marked by the 1999 mutation. On the other hand, LIPI also has reduced amounts of 18:3, but only at 27°C, a trait described for mutant line 081 (Table 2-X). If the 9911 mutation is epistatic to 1990, one would not expect a deficiency in 18:3 levels, since the 9911 mutation has no major effect on 18:3 synthesis. This result suggests the involvement of another (eukaryotic) pathway in 18:3 synthesis, that is affected only by the mutation at 1990 locus. Summary The mapping of genes on a linkage map is becoming increasingly important with the development of molecular genetic procedures that allow the cloning of genes by chromosome walking if they are located in close proximity to those DNA sequences that are already available. 9999199991; 19911999 is especially suitable for this approach because of the small size of its genome (Meyerowitz and Pruitt, 1985). Therefore, the linkage data integrated in Figure 2-2, which show 40 Table 2-X. Fatty acid composition of total leaf lip3ds (in mol %) of wild type and mutant Arabidopsis plants grown at 22 C. *Plants grown at 27 C. Each value is a mean of 10 plants. ND, not detected. Fatty Wild type 0825 081* LIPl LIP1* acid 16:0 13.6 13.3 13.4 12.3 13.6 . 16:1 919 0.9 0.7 3.1 1.5 ND 16:1 1999; 2.3 2.7 2.7 2.8 2.0 16:2 0.9 0.3 10.6 1.2 NO 16:3 15.7 1.5 2.0 ND ND 18:0 1.1 0.8 1.4 1.6 1.1 18:1 2.2 6.2 9.3 9.4 6.0 18:2 13.3 19.7 38.3 31.0 58.2 18:3 50.0 54.8 19.2 40.2 19.1 41 estimated recombination percentages between different pairs of loci, might prove useful in future efforts to isolate the genes marked by 9911,,1993 and £999 mutations. Literature cited Browse J, P McCourt, CR Somerville 1985a A mutant of lacking a chloroplast specific lipid. Science 227: 763-765 Browse J, P McCourt, CR Somerville 1985b Overall fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152: 141-146 Browse J, P McCourt, CR Somerville 1986 A mutant of 99991999919 «deficient in C18,3 and C16,3 leaf lipids. Plant Physiol 81: 859-864 Clark DP, JE Cronan 1981 Bacterial mutants for the study of lipid metabolism. Meth Enzymol 72: 693-707 Grogan DH, JE Cronan 1986 Characterization of £9999919h19 9911 mutants completely defective in synthesis of cyclopropane fatty acids. J Bacteriol 166: 872-877 Koornneef M, J van Eden, CJ Hanhart, P Stam, FJ Braaksma, NJ Feenstra 1983 Linkage map of 59991999919p19911999. J Hered 74: 265-272 McCourt PJ 1986 Ph. D. Thesis, Michigan State University McCourt P, J Browse, J Hatson, CJ Arntzen, CR Somerville 1985 Analysis of photosynthetic antenna function in a mutant of a (L.) lacking 1:999-hexadecenoic acid. Plant Physiol 78: 853-858 McCourt P, L Kunst, J Browse, CR Somerville 1987 The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of 59991999919. Plant Physiol 84: 353-360 Rock CO, JE Cronan 1985 Lipid metabolism in procaryotes. In DE Vance, JE Vance, eds, Biochemistry of Lipids and Membranes, Benjamin/Cummings Publishing Company Inc., pp 73-115 Suiter KA, JF Hendel, JS Case 1983 LINKAGE-1: a PASCAL computer program for the detection and analysis of genetic linkage. J Hered 74: 203-204 42 Taylor F, JE Cronan 1976 Selection and properties of 99999919919 99li mutants defective in the synthesis of cyclopropane fatty acids. J Bacteriol 125: 518-523 CHAPTER 3 ALTERED REGULATION OF LIPID BIOSYNTHESIS IN A MUTANT OF 93991992519 DEFICIENT IN GLYCEROL PHOSPHATE ACYLTRANSFERASE ACTIVITY Abstract The leaf lipids of many plant species, including 99991999919 19911999 (L.) Heynh., are synthesized by two complementary pathways which are located in the chloroplast and the endoplasmic reticulum. By screening directly for alterations in lipid acyl group composition we have identified several mutants of 99991999919 which lack the plastid pathway because of a deficiency in activity of glycerol-3-phosphate acyltransferase, the first enzyme in the plastid pathway of glycerolipid synthesis. The lesion does not cause the accumulation of precursors within chloroplasts, but results in a redirection of fatty acids towards cytoplasmic sites of lipid synthesis. The increased synthesis of lipids by the cytoplasmic pathway compensates for the loss of the plastid pathway and provides, with the exception of phosphatidylglycerol, normal amounts of the various lipids required by the chloroplasts. However, the fatty acid composition of the membrane lipids of the mutant is altered because the acyltransferases associated with the two pathways normally exhibit different substrate 43 44 Specificities. The remarkable flexibility of the system indicates the existence of regulatory mechanisms which allocate lipids for membrane biogenesis. Introduction In the present model of glycerolipid metabolism in higher plants, (Figure 3-1) two pathways contribute to the synthesis of chloroplast glycerolipids in leaf cells (Slack and Roughan, I975; Roughan, I975; Roughan et al., 1980; Roughan and Slack, 1982; Heinz and Roughan, 1983; Roughan and Slack, 1984; Gardiner et al., 1984). The chloroplast is the sole site of 99 9999 fatty acid synthesis ( Ohlrogge et al., 1979) and the main products of this process are 16:0- and 18:1-ACPs (Soll and Roughan, 1982). These fatty acids either enter the prokaryotic pathway through acylation of glycerol-B-phosphate within the chloroplast (Frentzen et al., 1983), or are exported as CoA thioesters (Roughan and Slack, 1982; Andrews and Keegstra, 1983) to enter the eukaryotic pathway at extrachloroplast sites, particularly in the endoplasmic reticulum (Roughan et al., 1980; Roughan and Slack, 1982). Most of the enzymes of the prokaryotic pathway are located in the inner membrane of the chloroplast envelope and can lead to the synthesis of PG, MGD, DGD and SL which are the major glycerolipids of the thylakoid membranes (Heinz, 1977; Joyard and Douce, 1977; Andrews ans Mudd, 1985). The eukaryotic pathway is responsible for the synthesis of the glycerolipids found in extrachloroplast membranes including PC, PE and PI (Roughan and Slack, 1984). In addition, however, diacylglycerol 45 (IYTTJPIJUSAI -—~o—-¢—<-—Plvol 9‘ F“: 9‘5 ‘o-FUA-a»-on—o 4 L9— 18:1-CoA —Q-T LymaJWA LymadWA 0 0 L!— G... __'_T 1 6:0-CoA 1 8:1 -CoA f Acotato—t16:0-ACP —D 18: O-ACP —D18:1-ACP ®T® I C) Lymodfll‘§———1k—{P FKid-Nt-INA'—-—--D owns 1 1 SL MGD DGD 1F—_J CHLOROPLAST Figure 3-1. An abbreviated scheme for lipid biosynthesis in the leaves of a 16:3 species. The enzymes identified by numbers are [1] fatty acid synthetase; [2] elongase; [3] stearoyl-ACP desaturase; [4] G3P acyltransferase; [5] monoacyl-G3P acyltransferase; [6] G3P acyltransferase; [7] monoacyl-G3P acyltransferase. defect in the 9911 mutant is indicated by a break in the pathway at reaction 4. The enzymatic 46 moieties from PC are returned from the endoplasmic reticulum to the chloroplast where they are used for further production of M60, 060 and SL (Douce and Joyard, 1979; Roughan et al., 1979; Murphy and Stumpf, 1980; Ohnishi and Yamada, 1980; Heinz and Roughan, 1983). In the majority of higher plants P6 is the only product of the prokaryotic pathway and the remaining chloroplast lipids are synthesized entirely by the eukaryotic pathway (Roughan and Slack, 1982; Roughan and Slack, 1984). These species are known as 18:3 plants. However, there are angiosperms such as 99991999919 19911999 in which both pathways contribute to the synthesis of M60, 060 and SL (Browse et al., 1986b). They characteristically contain substantial amounts of hexadecatrienoic acid (16:3) which is found only at the sn-2 position of MGD and 060 molecules produced by the prokaryotic pathway. These species have been termed 16:3 plants to distinguish them from 18:3 plants whose galactolipids contain predominantly a-linolenate (Roughan and Slack, 1982; Browse et al., 1986b). It is not known what regulates the allocation of fatty acids between the two pathways in 16:3 plants. Cooperation between the two pathways of lipid biosynthesis is apparent from the results of labeling experiments with whole leaves which indicate that as much as half of the acyl groups exported to the cytoplasm are reimported into chloroplasts for M60, 060 and SL biosynthesis (Roughan et al., 1979; Murphy' and Stumpf, 1980; Ohnishi and Yamada, 1980). The G3P acyltransferases associated with the chloroplast and endoplasmic reticulum have different substrate specificities, resulting in the 99-2 position of the glycerolipid being occupied exclusively by 18- or 16-carbon fatty acids in eukaryotic or prokaryotic lipids, respectively 47 (Roughan and Slack, 1984). Thus, the origin of chloroplast glycolipids can be determined by the characteristic fatty acid positional distribution (Roughan and Slack, 1982; Heinz and Roughan, 1983). This has been exploited to compile a detailed account of the relative contribution of the two pathways in 99991999919 (Browse et al., 1986b). He have previously described the isolation of a number of mutants of 99991999919 with altered fatty acid composition. The mutants were identified by direct analysis of leaf fatty acid composition of individual mutagenized plants by GLC. Several of these mutants have previously been characterized as being deficient in specific desaturases (Browse et al., 1985a; Browse et al., 1986a). Here we describe the biochemical characterization of a new class of mutants which lacks 16:3 acyl group due to a deficiency of chloroplast G3P acyltransferase. Because these mutants lack the activity of the first enzyme of the prokaryotic pathway, the mutation effectively converts a 16:3-plant into an 18:3 type. Thus, the mutant offers a unique opportunity to examine both the effects of this change on the regulation of glycerolipid metabolism and the physiological significance of the 16:3-18:3 dimorphism. Materials and methods mm The lines of 99991999919 19911999 (L.) Heynh. described here were descended from the Columbia wild type. The mutant lines 083, J825, J828 and LK8 were isolated following mutagenesis with ethyl methane 48 sulfonate (Haughn and Somerville, 1986). Before being used for experiments, the line J825 was backcrossed to the wild type at least three times and an individual with the mutant phenotype was reselected from a segregating population. Plants were grown under continuous fluorescent illumination (100-150 uE In"2 s'l) at 22°C on a perlite : vermiculite : sphagnum (1:1:1) mixture irrigated with mineral nutrients (Haughn and Somerville, 1986). 9.9910153]; Sodium (“q-acetate (54 mCi/nlnol) and NaHl4C03 (55.5 mCi/nlnol) were obtained from Research Products International Corporation, Mt. Prospect, IL, (14C)l6:0-CoA (53 mCi/Illnol) and (14C)18:1-CoA (53 mCi/mmol) were purchased from DuPont, Hilmington, DE, and (”Q-glycerol-3-phosphate (30 mCi/mol) from ICN Radiochemicals, Irvine, CA. (”cnszo-Acp (55 mC‘l/mol) and (14C)18:1-ACP (55 mCi/nlnol) were a generous gift from Dr. J.B. Ohlrogge, Department of Botany and Plant Pathology, Michigan State University. Sodium methoxide was prepared as described previously (23). Methanolic HCl reagent was prepared by diluting 3 M solution (Supelco) to 1 M with methanol (Browse et al., 1985b). LipJAJnalsz; Plants were frozen in liquid N2, then extracted with chloroform : methanol : fbrmic acid (10:10:1 by vol.) (Browse et al., 1986b). Following centrifugation, the supernatant was decanted and the tissue reextracted with chloroform : methanol : water (5:5:1 by vol.). The extracts were combined and washed with 0.2 M H3P04/2 M KCl (Hajra, 49 I974). Lipids were recovered in the chloroform phase, dried under N2 and taken up in a small volume of chloroform. Lipids were separated by thin layer chromatography on silica gel coated plates (Baker). For one dimensional chromatography a solvent system of chloroform : acetone : methanol : acetic acid : water (100:40:20:20:10 by vol.) was used. Hhen the lipids were chromatographed in two dimensions a solvent system of chloroform : methanol : ammonia (65:25:2 by vol.) was used in the first development, and chloroform : methanol : acidic acid : water (85:15:10:3 by vol.) in the second development. Individual lipids were identified by comparing their Rf values with those of reference standards and transmethylated with either sodium methoxide or hot methanolic HCl after the addition of a known amount of C17:o methyl ester as an internal standard. The resulting methyl esters were quantified by' gas chromatography as described (Browse et al., 1985b). The kinetics of lipid biosynthesis was followed by labeling intact Arabidopsis plants with (14C)-acetate as described previously (Browse et al., 1986b). Wm Chloroplasts used directly in labeling experiments with (14C)-acetate and (14C)-glycerol-3-phosphate were obtained by grinding 20 g of leaf tissue in 200 ml of homogenization medium containing 0.45 M sorbitol, 20 mM Tricine-KOH (pH 8.4), 10 mM EDTA, 10 mM NaHCO3 and 0.1 x BSA. The extract was passed through Miracloth, centrifuged at 270 x g for 90 sec and resuspended in 0.3 M sorbitol, 20 mM Tricine-KOH (pH 7.6), 5 mM MgCl2 and 2.5 mM EDTA. Chloroplast suspension was then 50 transferred to Percoll gradients prepared by centrifuging 50% Percoll in resuspension buffer at 43,000 x g for 30 min in a Sorvall SS-34 rotor (Cline et al., 1981). The overlayered gradients were centrifuged at 13,000 x g for 6 min in a Sorvall HB-4 rotor. Intact chloroplasts, which form a band near the bottom of the gradient, were recovered, diluted with resuspension buffer, pelleted at 3000 x g for 90 sec in a Sorvall HB-4 rotor and resuspended in the resuspension medium. Chloroplasts used for fractionation and enzyme assays were isolated from protoplasts of 99991999919 leaves by a modified procedure of Somerville et al. (1981). Intact leaves were vacuum infiltrated with a medium containing 0.5 M sorbitol, 10 mM MES-KDH (pH 6), 1 mM CaClZ, 1.6 X (w/v) Macerase and 1.6 % (w/v) Cellulase, and incubated for one hour at room temperature in the same medium. Protoplasts were passed through several layers of cheesecloth to remove debris, harvested by centrifugation at 4°C for 5 minutes at 100 x g, and resuspended in cold 0.5 M sorbitol, 10 mM MES-KDH (pH 6), 1 mM CaClz. The suspension was transferred into test tubes containing 50% Percoll (v/v), 0.5 H sorbitol, 10 mM HES-KOH (pH 6), 1 mM CaClz, and intact protoplasts were banded by centrifugation at 100 x g for 10 minutes. Protoplasts were resuspended in 0.3 M sorbitol, 20 mM Tricine-KOH (pH 8.4), 10 mM EDTA, and gently lysed by passing through a 15 um mesh net. Chloroplasts were centrifuged at 270 x g for 90 seconds and resuspended in cold 0.3 M sorbitol, 20 mM Tricine-KOH (pH 7.6), 5mM MgClz, 2.5 mM EDTA. The percentage of intact chloroplasts was determined by measuring oxygen evolution in the presence of ferricyanide before and after osmotic shock (Lilley et al., 1975). 5] Wm Intact chloroplasts (400 ug chlorophyll/ml) were incubated with shaking at 25°C in a medium containing 0.33 M sorbitol, 25 on Hepes-NaOH (pH 7.9), 10 mM NaHC03, 2 mM EDTA, 1 mM MgCl2, 1 mM MnClz, and 0.4 mM sn-glycerol-3-phosphate with 0.15 «M (”Q-acetate (54 mCi/nlnol), or 0.4 nfl ("Q-glycerol-3-phosphate (30 mCi/nlnol) with 0.15 mM Na-acetate, for 20 min under illumination (150 uE m'z s'l) and then in darkness for 20 min. Reactions were stopped by adding chloroform/methanol (1:1 v/v) and lipids were recovered from the chloroform layer after partitioning against 0.2 M H3P04/2 M KCl (Hajra, 1974). Enzxme_assaxs Crude leaf extracts were prepared by lysing intact protoplasts in 20 mM Tricine-KOH (pH 8.4) and 10 m" EDTA. Chloroplast stromal extracts were prepared by resuspending intact chloroplasts in 10 mM Tricine-KDH (pH 7.6), 1 mM MgCl2. This causes rupture and detachment of envelope membranes and liberation of the stroma (Joyard and Douce, 1977). The chloroplast components were then layered on density step gradients composed of 0.93 H and 0.6 M sucrose in 10 mM Tricine-NaOH, pH 7.6/4 mH HgCl2 and were centrifuged for l h in a swinging-bucket rotor at 72,000 x g. The envelopes were collected from the interface of the 0.93 M and 0.6 M sucrose layers. The fraction containing the chloroplast stromal components was recentrifuged at 130,000 x g for 2 h to remove any remaining membranes and was used immediately for enzyme assays. G3P acyltransferase and monoacyl-G3P acyltransferase activities were assayed at 22°C essentially as described (Bertrams and Heinz, 52 1981, Frentzen et al., 1983), with [14C]acyl CoA as the substrate for chloroplast extracts and [14C]acyl-ACP for whole-cell extracts. The 80 ul G3P acyltransferase assay mixtures contained 250 mM Mops-NaOH (pH 7.4), 50 ug of bovine serum albumin, 5 uM acyl-ACP of acyl-CoA, 2 mM L-G3P, and 25-50 ug of chloroplast stromal protein or 250-300 ug of protein from whole-cell extracts. The same mixture was used for monoacyl-G3P acyltransferase assays except that l-oleoyl-GBP was used instead of G3P and 50-80 ug of chloroplast envelope protein was used instead of the stromal extract. Ribulosebisphosphate carboxylase (Pierce et al., 1982) and phosphoenolpyruvate carboxylase (Stit et al., 1978) were assayed essentially as described. Protein concentrations were measured by the methods of Bradford (1976), or Markwell et al., (1981) with BSA as standard. Results 91031154931151; A genetic analysis of the 9911 mutants is described in Chapter 2. WW 0n the basis of exploratory labeling studies with intact plants and with isolated chloroplasts using [14CJ-glycerol-3-phosphate (Figure 3-2A) we inferred that the mutant had a lesion at an early step of the prokaryotic pathway. However, the distribution of radioactivity among polar lipids from the mutant and wild type was identical in [14C]-acetate labeled chloroplasts (Figure 3-28). Therefore, in order 53 WT .825 WT .825 'H «MGD - w ' "' - m ~ ‘4‘ PA ~ < PG .6! .. < LPA Figure 3-2. The distribution of radioactivity among the polar lipids of mysant J825 and wild type Arabidopsis following A) ( C)-G3P and B) ( C)-acetate labeling of isolated chloroplasts. The same amount of radioactivity was applied to each lane. 54 to determine whether the prokaryotic pathway is affected by the mutation, we assayed the chloroplast enzymes involved in PA synthesis by measuring the incorporation by stromal extracts of (14C)18:1-CoA and (14C)16:0-CoA into lipids. Although acyl-ACP is the normal substrate for these reactions, the CoA esters are also readily accepted by the chloroplast acyltransferases (Bertrams and Heinz, 1981). Because of the presence in crude extracts of both chloroplast and microsomal acyltransferases we first purified chloroplasts and then assayed stromal extracts for activity. The results of this experiment (Table 3-1) indicated that the mutant exhibits only 3.8% of the wild type activity of glycerol-3-phosphate acyltransferase, the first enzyme of the prokaryotic pathway (Figure 3-1). Since the chloroplasts were slightly contaminated with protoplasts (1.9% PEP carboxylase activity was detected in J825 stromal extract), some of the residual activity in the mutant is due to contamination of the chloroplast fraction by cytoplasmic enzymes. The mutant had wild-type levels of the chloroplast enzyme ribulosebisphosphate carboxylase (Table 3-1) and appears to have normal levels of monoacylglycerol-3-phosphate acyltransferase activity. The independently isolated mutant line LK8 also had a specific deficiency in G3P acyltransferase activity (Table 3-1). Therefore, it seems very likely that the 9911 locus specifically controls the activity of the plastid isozyme of glycerol-3-phosphate acyltransferase. Chloroplast GBP acyltransferase can use acyl-ACP as well as acyl-CoA for the acylation reaction, but if both are present, ACP thioesters are exclusively used as substrate (Frentzen et al., 1983). Since ACP thioesters are confined to the chloroplasts, we wanted to 55 Table 3-1. Enzyme activities in the stromal fraction and leaf extracts of Arabidopsis chloroplasts. Values are the mean of 3-5 assays. Specific activity % activity (nmol/mg protein/min) STROMA (14C)18:I-C0A labeling §3£.esxliran§£erese HT 0.011 100 0325 0.00042 3.3 ueneasxl;§3£.asxl;& Iransfenase HT 0.17 100 0325 0.20 113 Bu31§£Q HT 400 100 0325 490 122.5 E££.carhex111§e HT 0.224 100 0325 0.239 105.7 LEAF EXTRACT (14C)18:I-ACP labeling §3£.asxltransferase HT 0.116 100 0325 0.0057 4.9 HT + 0325 0.054 55.2 LK8 0.0039 3.4 Mengacxl;§3£.9sxl; transferase HT 0.0034 100 0325 0.0035 ' 102 LK8 0.0034 100 3151590 HT 570 100 0325 550 97 E£2,93r90xxla§e HT 10.2 100 0825 12.6 124 56 determine whether they can be used as acyl donors by the other leaf acyltransferases. Therefore, we assayed crude leaf extracts of the wild type and 0825 mutant by measuring the incorporation of (14C)18:1-ACP into lipids. Parallel assays were performed in leaf extracts with (14C)18:1-C0A as substrate. This experiment has shown that ACP thioesters in the wild type plants are predominantly incorporated in LPA, PA and PG, while the mutant exhibits less than 5% of GBP acyltransferase activity (Table 3-I). 0n the other hand, when CoA esters are used as donors, the total G3P acyltransferase activity in the mutant reaches 82% of the activity of wild-type plants. This result indicates that acyl-ACP thioesters are probably not the substrates for extraplastid acyltransferases. 1.3123110941133112; In order to investigate the consequences of the enzyme defect on lipid biosynthesis in the mutant (14C)-acetate was applied to leaves of mutant and wild-type plants and the redistribution of radioactivity in polar lipids was followed during the subsequent 142 hours. From the results of this experiment it can be seen that the mutation causes dramatic differences in the pattern of (14C)-acetate incorporation in J825 when compared to the wild type (Figure 3-3). As we have discussed previously (Browse et al., 1986b) the labeling kinetics for wild-type plants demonstrate the parallel operation of the two pathways of lipid synthesis. Flux 'through the prokaryotic. pathway ‘leads to the substantial labeling of M60 at early times while the subsequent transfer of 14C from PC to MGD and DGD occurs via the eukaryotic pathway. In contrast the mutant contained the label primarily in PC at Radioactivity incorporated (96) 7!) 6!) 5!) 3() 2C) 1() 2C) 1() 1C“) Time (h) 1() - 11) 100' Figure 3-3. The distribution of radioactivity in leaf lipids of (A) wild type and (B) J825 mutant 99991999919 after labeling with C-acetate. Symbols:o, PC;|, MGD;[J, DGD;A. SL;A *, PI. . PG;C). PE; 58 short times, whereas MGD contained less than 3% of the total counts. During the course of the experiment there is a steady and substantial decline of radioactivity in PC, which is accompanied by increased label in M60, DGD and SL so that by the end of the experiment the distribution of 14C among the various polar lipids is similar to the wild type. These kinetics demonstrate a precursor-product relationship between PC and the chloroplast glycolipids and indicate that MGD in J825 is made entirely by the eukaryotic pathway. In these respects the labeling kinetics are extremely similar to those observed in analogous experiments with 18:3 plants in which all the chloroplast glycerolipids except P0 are derived from the eukaryotic pathway (Slack and Roughan, 1975; Roughan and Slack, 1982; Roughan and Slack, 1984). 18:3 plants synthesize PA and PG by the prokaryotic pathway but synthesis of other chloroplast lipids is precluded because chloroplast PA is not converted to diacylglycerol (Heinz and Roughan, 1983; Roughan and Slack, 1985). The deficiency in the chloroplast acyl-ACP:glycerol-3-P acyltransferase found in J825 would be expected to block PG synthesis by the prokaryotic pathway. However PG does become labeled in the f 14 mutant although the extent 0 C incorporation into this lipid is only about half of that found in the wild type (Figure 3-3). W In wild-type 99991999919 the prokaryotic pathway is responsible for producing approximately 70% of the total leaf M60, 12% of the 060, 63% of the SL and 85% of the PG (Browse et al., 1986b). Nevertheless, the deficiency of acyl-ACP:glycerol-3-P acyltransferase in the mutant does not lead to a dramatic reduction in the amount of any of these 59 lipids. The total lipid content of leaves from the mutant was the same as the wild type on the basis of both fresh weight and chlorophyll (data not shown) and the most pronounced effect on any individual lipid was the 30% reduction in the proportion of PG in the mutant (Table 3-11). There is also a small decrease in the amount of MGD and a corresponding increase in the amount of PC which is the precursor of MGD in the eukaryotic pathway. The similar proportions of each lipid in the mutant and wild type (Table 3-11) together with the data from the labeling experiment (Fig. 3-3), indicate that the lack of synthesis of MGD, DGD and SL by the prokaryotic pathway is compensated for by increased production of these lipids via the eukaryotic pathway. The differential effect of the 9911 mutation on the amounts of the various chloroplast-specific lipids reflects the various degrees to which these lipids are normally produced by the prokaryotic pathway (Browse at al., 1986b). The increased amounts of PC and PE are consistent with (but not proportional to) the increased flux through the eukaryotic pathway. In order to determine if PG in the mutant had the characteristic structure of a product of the prokaryotic pathway, the purified lipid was digested with 99199999 lipase and the fatty acid composition of the lyso-PG and free fatty acid was determined (Browse et al., 1986b). This analysis indicated that in the mutant 75% of the fatty acid at the 99-2 position of PG was C15. In the wild type the 99-2 position was 83% C16. In contrast, other polar lipids in the mutant contained more than 90% C18 fatty acids at 99-2 of the glycerol, indicating that they were produced by the eukaryotic pathway. c.m o.m m.o~ o.c~ ~.o~ m.- ~.m ".0 o.~ w.~ o.m~ ~.m_ o.¢n c.om u 6O ~.o~ m.n~ m.¢~ ~.o~ «.vn c.ov m.- o.m~ m.vm ~.m¢ m.om m.om m.om ~.~o mum" m.v~ o.m~ o.~v ~.on ~.v~ m.c~ o.~ o.m ~.m n.¢ m.n ~.m ~.m o.~ Nun" ~.o~ ~.~ o.m~ ~.n ~.m~ ~.m o.n c.~ m.m ~.~ N." ~.~ ¢.~ n mum“ ~.m ~.m m.~ v.~ m.H o.~ w.~ ¢.~ m.~ ~.~ m.o «.0 ~.o u oum~ i u u u o a u u a i m.o n." a.“ c.mm muo— i n u i a n i u a u u i i a." «flag 1 u u i 1 u m.m~ o.- u i u i i i ufiuog m.sv m.~v ~.m~ o.~m H.0N m.- o.mm o.~m m.o~ m.mv o.~ o.- ~.~ ~.H can" mwma h: mwma h: mwma h: mwoa h: mwma h: mwaa h: mwma h: ~m um um an an awe no: .vouuouou no: me: naoea —Au~ mg» «as» ouou—vcv magmas .u pas one voucomosn mos—~> .uomu as green upmmoe.aas< acuual use unsung—.3 lac» «v.n.— poop we =o_u—moasou u—ua Anus; ._~-n o_nop 61 The chloroplast-specific acyl group 16:3 which is characteristic of prokaryotic MGD is virtually absent from the lipids of the mutant and is replaced by 18-carbon fatty acids (primarily 18:3). The I3-fold reduction in the amount of 16-carbon fatty acids in MGD is not accompanied by an increase in 16-carbon fatty acids in any other lipid. Thus the 16:0 excluded from the prokaryotic pathway appears to be elongated and desaturated to 18:1 before being exported from the chloroplast to enter the eukaryotic pathway. The implication is that the export of 16:0 is regulated not by the availability of 16:0-ACP but by some other mechanism. In a detailed analysis of wild-type 99991999919 we have previously shown (Browse et al., 1986b) that for every 1,000 fatty acids made in the chloroplast 615 enter the eukaryotic pathway (117 C16 + 498 C18). A similar analysis on the mutant shows that the increase in f1ux through the eukaryotic pathway (to 950 per 1000) is made up almost entirely of C18 fatty acid chains (126 C16 + 824 C18). The ratio of C16:C18 fatty acids in PC, PE and P1 are the same as in the corresponding lipids of the wild type (Table 3-11). In contrast the C16:C18 ratio in HGD, DGD and SL of the mutant is in each case less than the ratio calculated for these lipids synthesized by the eukaryotic pathway in wild-type 99991999919 (Table 4, Browse et al., 1986b). Thus, the additional C18 fatty acids entering the eukaryotic pathway in the mutant are found specifically in the additional quantities of chloroplast lipids (MGD, DGD and SL) which are produced by the eukaryotic pathway in response to the loss of the prokaryotic pathway. 62 The mutation causes an increase in the amount of 18:1 fatty acids and a decrease in the amount of 18:3 in all of the extrachloroplast (PC, PE, PI) lipids of the mutant. There was little or no effect on the amount of 18:2 in these lipids (Table 3-II). The data indicate a 10-30% reduction in the extent of 18:1 desaturation in these lipids in the mutant relative to the wild type. It seems likely that this is caused by the inability of the endoplasmic reticulum c18:1 desaturase to completely metabolize the increased flux of lipid through the eukaryotic pathway in the mutant. Discussion In the mutant J825 a single recessive nuclear mutation at the 9911 locus causes a specific deficiency in the activity of the acyl-ACP:glycerol-3-P acyltransferase. Three other mutants, J83, JBZB and LK8, are allelic to 0825. These other mutants show all of the changes in lipid and fatty acid composition which have been described here for J825 indicating that all the changes are direct consequences of the deficiency in acyltransferase activity. Our analysis of the mutant provides a general outline of the features of the controls which regulate lipid metabolism to maintain suitable glycerolipid and fatty acid compositions in cellular membranes. WWW; Both the labeling data (Fig. 3-3) and the lipid analysis (Table 3-11) indicate that loss of the acyltransferase activity does not 63 result in the accumulation of precursors (18:1- and 16:0-ACPs) upstream of the block but in redirection of lipid metabolism so that the eukaryotic pathway predominates in the mutant. Suprisingly, this redirection has little effect on the proportion of each lipid synthesized or indeed on'the t0tal glycerolipid content of the tissue. Thus not only is the flux through the eukaryotic pathway increased in the mutant, but the proportion of individual lipids synthesized by this pathway changes to produce a complement of leaf lipids which is similar to that of wild-type plants. It would appear from these observations that demand for a particular lipid is a major factor regulating synthesis. There is, at present, no evidence pertaining to the mechanism by which this redistribution of acyl groups might be accomplished. W919; In wild-type 99991999919, C16 fatty acids represent half of the acyl chains found in lipids made by the prokaryotic pathway. However, loss of the prokaryotic pathway in the mutant does not result merely in redirection of the C16 chains into the eukaryotic pathway. Instead the 16:0-ACP is elongated and desaturated to 18:1-ACP before export from the chloroplast. Thus, the overall ratio of C16 to C18 chains produced by fatty acid synthesis is reduced from 0.3 in the wild type to 0.18 in the mutant (TabTe 2-II). It is noteworthy in this respect that the primarily extrachloroplastic lipids (PC, PE and PI) have relatively normal levels of 16:0. The implication is that the amount of export of 16:0 is not regulated simply by the availability of 16:0. This suggests that elongase activity is regulated by availability of 64 substrate (16:0) and that this is determined by competition between alternative pathways of 16:0 utilization. W The two-fold increase in the flux through the eukaryotic pathway obviously challenges the synthetic capabilities of the enzymes involved. Two sets of evidence suggest that in the mutant the eukaryotic pathway is operating near the limit of its ability to produce chloroplast lipids. First, the amount of PC is increased from 21.8 to 26.2% of the leaf lipids while the amount of MGD is slightly decreased. This suggests that transfer of acyl groups to the chloroplast, or further metabolism of PC may be limiting in the mutant. Secondly the extent of desaturation of 18:1 in PC and the other extrachloroplast lipids PE and PI is decreased by 10-20% compared with the wild type. This may well be caused by the inability of the endoplasmic reticulum 18:1 desaturase to match the increased throughput of the eukaryotic pathway. W Evidence from studies by Andrews and Mudd (1985) indicates that isolated chloroplasts are able to synthesize PG at rates which are sufficient to meet the requirements for chloroplast membrane biogenesis and that the pathway of synthesis of PG involves the same pools of LPA and PA used for the synthesis of prokaryotic MGD, DGD and SL. It is puzzling, therefore, that the mutant contains at least 75% as much PG as the wild type, even though it exhibits less than 4% of the wild-type level of G3P acyltransferase activity. One possibility is that the 65 amount of PA made in the mutant with the residual 4% of chloroplast acyltransferase activity is adequate to meet most of the requirement for PG synthesis and is utilized preferentially for PG synthesis. A reduction in the size of the LPA and PA pools might explain why these compounds did not accumulate radioactivity during [14C]G3P labeling of isolated chloroplasts (Figure 3-2). Since only very small amounts of prokaryotic MGD are found in the mutant (Table 3-11) this explanation would require that PG synthesis from PA is efficiently maintained at the expense of DAG synthesis. The other possibility seems to be that an alternative source of PA is used for PG synthesis. However, the predominance of 16-carbon fatty acids on the 99-2 position of PG in the mutant is most consistent with this lipid being derived from the prokaryotic pathway in the chloroplast rather than from any other source. Weary—130.115.111.011; A preliminary physiological characterization of the mutant suggests that the loss of the prokaryotic pathway is not deleterious. The general appearance of the mutant plant is similar to the wild type, and it is not impaired in growth or development under standard growth conditions. Thus, the question that inevitably arises concerns the dispensibility of the prokaryotic pathway for glycerolipid synthesis in the mutant, as well as in naturally occurring 18:3 plants and in the fruits of 16:3 species (Hhitaker, 1986). In the course of evolution, the majority of higher plants have abandoned the prokaryotic pathway so that it persists to varying degrees only in less advanced genera (Jamieson and Reid, 1971). Since a single mutation can eliminate the 66 prokaryotic pathway, it seems reasonable to suggest that there must be both some physiological advantages and disadvantages associated with the loss of prokaryotic lipids from chloroplast membranes. He anticipate that further analysis of the mutants described here will provide unique insights into why both 16:3-plants and 18:3-plants coexist. Literature cited Andrews J, K Keegstra 1983 Acyl-CoA synthetase is located in the outer membrane and acyl-CoA thioesterase in the inner membrane of pea chloroplast envelopes. Plant Physiol 72: 735-740 Andrews J, B Mudd 1985 Phosphatidylglycerol synthesis in pea chloroplasts. Plant Physiol 79: 259-265 Bertrams H, E Heinz 1981 Positional specificity and fatty acid selectivity of purified 99-glycerol 3-phosphate acyltransferases from chloroplasts. Plant Physiol 68: 653-657 Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248-254 Browse J, P McCourt, CR Somerville 1985a A mutant of r lacking a chloroplast specific lipid. Science 227: 763-765 Browse J, P McCourt, CR Somerville 1985b Overall fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152:141-146 Browse J, P McCourt, CR Somerville 1986a A mutant of 99991999919 deficient in C18,3 and C16,3 leaf lipids. Plant Physiol 81: 859-864 Browse J, N Harwick, CR Somerville, CR Slack 1986b Fluxes through the prokaryotic and eukaryotic pathways of lipid synthesis in the 16:3 plant 99991999919 19911999. Biochem J 235: 25-31 Cline K, J Andrews, 8 Hersey, EH Newcomb, K Keegstra 1981 Separation and characterization of inner and outer envelope membrane of pea chloroplasts. Proc Natl Acad Sci USA 78: 3595-3599 67 Douce R, J Joyard 1979 Structure and function of the plastid envelope. Adv Bot Res 7: 1-116 Frentzen M, E Heinz, TA McKeon, PK Stumpf 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 Gardiner SE, E Heinz, PG Rougqan 1984 Rates and products of long-chain fatty acid synthesis from [1- C]acetate in chloroplasts isolated from leaves of 16:3 and 18:3 plants. Plant Physiol 74: 890-896 Hajra AK 1974 On extraction of acyl and alkyl dihydroxyacetone phosphate from incubation mixtures. Lipids 9: 502-505 Haughn GH, CR. Somerville 1986 Sulfonurea-resistant mutants of 99991999919 19911999. Hol Gen Genet 204: 430-434 Heinz E 1977 Enzymatic reactions in galactolipid biosynthesis. In M Tevini, HK Lichtenthaler, eds, Lipids and Lipid Polymers in Higher Plants. Springer Verlag, Heidelberg, pp 102-120 Heinz E, PG Roughan 1983 Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants. Plant Physiol 72: 273-279 Jamieson GR, EH Reid 1971 The occurrence of hexadeca-7,10,13-trienoic acid in the leaf lipids of angiosperms. Phytochemistry 10: 1837-1843 Joyard J, R Douce 1977 Site of synthesis of phosphatidic acid and d;§cylglycerol in spinach chloroplasts. Biochim Biophys_Acta 486: 2 -285 Lilley RMcC, MP Fitzgerald, KG Rienits, DA Halker 1975 Criteria of intactness and the photosynthetic activity of spinach chloroplast preparations. New Phytol 75: 1-10. Markwell MAK, SM Haas, NE Tolbert, LL Bieber 1981 Protein determination in membrane and lipoprotein samples. Meth Enzymol 72: 296-303 Murphy DJ, PK Stumpf 1980 19 9199 pathway of oleate and linoleate desaturation in developing cotyledons of 9999919 9911999 19 seedlings. Plant Physiol 66: 666-671 Ohlrogge JB, DN Kuhn, PK Stumpf 1979 Subcellular localization of acyl carrier protein in leaf protoplasts of 59199919 91999999. Proc Natl Acad Sci USA 76: 1194-1198 Ohnishi J, M Yamada 1980 Glycerolipid synthesis in 99999 leaves during greening of etiolated seedlings II.a-linolenic acid synthesis. Plant Cell Physiol 21: 1607-1618 68 Pierce JH, SD McCurry, RM Mulligan, NE Tolbert 1982 Activation and assay of ribulose-l,5-bisphosphate carboxylase/oxygenase. Meth Enzymol 89: 47-55 Roughan PG 1975 Phosphatidyl choline: donor of 18-carbon unsaturated fatty acids for glycerolipid biosynthesis. Lipids 10: 609-614 Roughan PG, JB Mudd, TT McManus 1979 Linoleate and cx-linolenate synthesis by isolated spinach (59199919 91999999) chloroplasts. Biochem J 184: 571-574 Roughan PG, R Holland, C Slack 1980 The role of chloroplasts and microsomal fractions in polar-lipid synthesis from [1- C]acetate by cell-free preparations from spinach (59199999 91999999) leaves. Biochem J 188: 17-24 Roughan PG, CR Slack 1982 Cellular organization of glycerolipid metabolism. Annu Rev Plant Physiol 33: 97-132 Roughan PG, CR Slack 1984 Glycerolipid synthesis in leaves. Trends Biochem Sci 9: 383-386 Slack CR, PG Roughaq41975 The kinetics of incorporation 19 9199 of [ C]acetate and [ C]carbon dioxide into the fatty acids of glycerolipids in developing leaves. Biochem J 152: 217-228 Soll J, PG Roughan 1982 Acyl-acyl carrier protein pool sizes during steady-state fatty acid synthesis by isolated spinach chloroplasts. FEBS Lett 146: 189-192 Somerville, CR, SC Somerville, HL Ogren 1981 Isolation of photosynthetically active protoplasts and chloroplatss from 99991999919 19911999. Plant Sci Lett 21: 89-96 Stitt M, PV Bulpin, T ap Rees I978 Pathway of starch breakdown in photosynthetic tissues of 21999 9911999. Biochim Biophys Acta 544: 200-214 Hhitaker BD (1986) Fatty-acid composition of polar lipids in fruit and leaf chloroplasts of '16:3'- and "18:3'-plant species. Planta 169: 313-319 CHAPTER 4 ALTERATIONS IN CHLOROPLAST ULTRASTRUCTURE CAUSED BY CHANGES IN MEMBRANE LIPID COMPOSITION IN A MUTANT OF 93991992919 DEFICIENT IN PLASTID GLYCEROL-S-PHOSPHATE ACYLTRANSFERASE ACTIVITY Abstract A mutant of 99991999919 19911999 has altered chloroplast membrane composition due to a deficiency in chloroplast glycerol-3-phosphate acyltransferase activity. The most pronounced effect of the mutation is an increase in the number of appressed regions per chloroplast and a corresponding decrease in the average number of thylakoid membranes in each appressed region. These changes were not associated with a significant alteration in the amount of Chl a/b binding proteins, suggesting that the model for membrane appression based on the properties of light harvesting Chl a/b protein complex (LHCP) is incorrect, or incomplete. The changes in leaf lipid composition do not affect growth or development of the mutant under standard conditions (22°C, 100-150 uE In.2 s'l). Similarly, photosynthetic electron transport, net C02 fixation and room temperature fluorescence of the mutant are comparable to wild-type plants. However, at temperatures above 28°C the mutant grows more rapidly. Measurements of 69 7O temperature-induced fluorescence yield enhancement and the delayed inactivation of whole chain electron transport in isolated chloroplast membranes at high temperatures suggest an increased thermal stability of the photosynthetic apparatus of the mutant. A comparison of 77K fluorescence emission spectra of thylakoid membranes from the mutant and wild type indicated a slight decline in excitation energy transfer from LHCP to P511 and PSI in the mutant. These changes may be due to altered structural organization of 9911 chloroplasts. Introduction Glycerolipid synthesis in leaves of higher plants is thought to involve two biosynthetic routes designated ’prokaryotic’ and ’eukaryotic’ pathways (Roughan and Slack, 1982). Fatty acids synthesized 99 9999 in the chloroplasts may either enter the prokaryotic pathway in the chloroplast envelope, or be exported to the endoplasmic reticulum where they are incorporated into lipids through the eukaryotic pathway. In ’16:3 plants’ such as 99991999919 19911999 both pathways are involved in the production of chloroplast lipids. However, the majority of higher plants uses the prokaryotic pathway only for the synthesis of phosphatidylglycerol (PG), while the remaining chloroplast lipids are made by the eukaryotic pathway. They include more advanced angiosperm genera and are known as ’18:3 plants’. Since ’18:3 plants’ have abandoned the prokaryotic pathway for the synthesis of chloroplast glycolipids, the question that arises concerns the role of this pathway in ’16:3 plants’. It seems reasonable to 71 suggest that an operational prokaryotic pathway confers some selective advantage, because ’16:3’ and ’18:3 plants’ coexist. We have recently described the isolation and biochemical characterization of a class of mutants of 99991999919 that lack the activity of the first enzyme of the prokaryotic pathway, glycerol-3-phosphate acyltransferase (Kunst et al. l988). The mutation responsible (9911) effectively converts a ’16:3’ into an ’18:3 plant’ and offers a unique opportunity to examine the functional significance of the prokaryotic pathway. As a direct consequence of the deficiency in the prokaryotic pathway, the mutant lines J825 and LK8 show specific alterations in composition of leaf membrane lipids (Kunst et al. 1988). These involve a 15-20% reduction in PG, a 9% decrease in MGD, a 12% decrease in DGD, and a 30% decrease in SL content, while the amounts of PC and PE are increased 12% and 10%, respectively. The mutation also results in greatly reduced levels of 16:3 acyl group, characteristic of prokaryotic MGD, and a corresponding increase in 18-carbon fatty acids. Since the prokaryotic pathway provides lipids specifically for chloroplast biogenesis, we have investigated the effects of the changes in membrane composition in the 9911 mutant on chloroplast structure and function. Materials and methods l w h n All lines of 99991999919 described here are descended from the Columbia wild type. The isolation and biochemical characterization of 72 the mutant lines J825 and LK8 has been described (Kunst et al., 1988). Both lines carry a single recessive nuclear mutation at a locus designated 9911. Before being used for experiments reported here, the lines J825 and LK8 were backcrossed to the wild type five times and two times, respectively, and individuals with the mutant phenotype were reselected from segregating populations. Unless otherwise indicated, plants were grown at 22°C under continuous fluorescent illumination (100-150 uE m'2 s'l) on a perlite : vermiculite : sphagnum mixture irrigated with mineral nutrients (Haughn and Somerville, 1986). WW1: Plants were germinated at 22°C and grown as described above for seven days. After that the temperature was adjusted to that mentioned in the text. Samples of four plants were randomly harvested at three day intervals for the next 12 days, and their fresh weight was measured. The relative growth rate (w'l) was determined as the slope of the natural logarithm of the average fresh weight (of 4 samples) plotted against days since the temperature adjustment. WNW Leaves were harvested at the rosette stage (3 weeks old plants) and their fresh weight was determined prior to homogenization in cold 20 mM Tricine-KOH (pH 8.4), 5 mM MgCl2 and 2.5 mM EDTA. Insoluble matter was removed by centrifugation at 100 x g for 10 min and aliquots of the extract were used for Chl, protein and lipid determinations. Chlorophyll was assayed by the method of MacKinney (1941), protein measurements were performed using a modified Lowry assay (1978), and 73 lipids were quantified by gas chromatography using a known amount of 14:0-methyl ester as an internal standard (Browse et al.,1985a). Fatty acid composition of total leaf lipids was determined after preparation of fatty acid methyl esters as described (Browse et al., I985b. E1gment:nretein_electrenheresis Chloroplast membranes were isolated as described above. Pigment-protein electrophoresis was performed according to the method of Andersson et al. (1982), except that the sodium dodecyl sulfate (SDS, Sequanal Grade, Pierce, Rockford, IL) to Chl weight ratio was adjusted to 3.75 : 1. L-[3sSl-Methionine labelingof thylakoid proteins and protein sztrastien L-[3SS]-Methionine (DuPont, NEN, Boston, MA, 1033 Ci mmol'l) was diluted to 0.5 mCi ml"1 with 0.025% Triton X-100 and applied onto both leaf surfaces of 15 days old plants. Twenty-four h after application of the label, aerial portions of 5 plants were harvested and homogenized in 30 ml of medium containing 450 mM sorbitol, 20 mM Tricine-KOH (pH 8.4), 10 mM NaCl, 10 mM EDTA and 0.1% (w/v) BSA. The extract was filtered through Miracloth (Calbiochem, La Jolla, CA), centrifuged for 10 min at 3000 x g, and resuspended in 1 ml of the homogenization buffer. The chloroplast suspensions were then transferred to Eppendorf tubes, repelleted, and suspended in 200 ul of a buffer containing 700 mM sucrose, 500 mM Tris-KOH (pH 9.4), 50 mM EDTA, 100 mM KCl, 2% 2-mercaptoethanol (v/v) and 2 mM phenylmethyl-sulfony fluoride (PMSF) (Hurkman and Tanaka, 1986). An equal volume of phenol was added to the 74 suspension, and proteins were extracted for 10 min. The phases were separated by centrifugation and the phenol phase was recovered. Proteins were precipitated from the phenol phase by the addition of 1 ml of 100 mM ammonium acetate in methanol and incubation at -20°C overnight. The precipitate was washed twice with 20% acetone and dried for l h at room temperature. MW Isoelectric focusing tube gels (3 mm x 120 mm) contained 9.5 M urea, 3% Triton X-100 (w/v), 2.24% ampholines (4-6), 0.96% ampholines (5-8), 0.8% ampholines (3-10), 4% acrylamide (w/v), 0.2% bis-acrylamide (wyv), 0.01%.(w/v) ammonium persulfate and 0.07% (v/v) TEMED. Protein samples were solubilized in a medium containing 9.5 M urea, 1.25% 508 (w/v), 2% 2-mercaptoethanol (v/v), 2% ampholines (3-10), 6% Triton X-100 (w/v) and a trace amount of bromophenol blue. Aliquots (80 ug protein) were loaded at the cathodic end of the gels and overlaid with 6% Triton X-100 (w/v) and 2% ampholines (3-10). The upper (cathode) buffer was 50 mM NaOH, and the lower (anode) buffer was 25 mM H3PO4. Isoelectric focusing was conducted at 200 V for 30 min, 300 V for 30 min, and finally 500 V for 24 h at room temperature. The second dimension slab gels (160 x 180 x 1.5 mm) consisted of an 8% acrylamide (w/v) resolving gel and a 5% acrylamide (w/v) stacking gel prepared according to Laemmli (1970). Focusing tube gels were equilibrated for 45 min in 2 x 10 ml of stacking gel buffer containing 0.1% SDS (w/v) and 2% 2-mercaptoethanol (v/v), and sealed on the slab gel apparatus with 0.8% (w/v) agarose in stacking buffer. Electrophoresis was performed at 15 mA per gel. Proteins were then 75 electrophoretically transferred to nitrocellulose filters (Hybond-C, Amersham, Arlington Heights, IL) (Towbin et al., 1979), and detected by autoradiography. Was; Chloroplast membranes were prepared by grinding leaves in cold 450 mM sorbitol, 20 mM Tricine-KOH (pH 8.4), 10 mM NaCl, 10 mM EDTA and 0.1% (w/v) BSA. The extract was filtered through Miracloth (Calbiochem, La Jolla, CA) and centrifuged at 3000 x g for 5 min. The pellet was washed with 10 mM Hepes (pH 7.9), 10 mM NaCl 5 mM EDTA and resuspended in a buffer containing 300 mM sorbitol, 20 mM Hepes (pH 7.9), 10 mM NaCl, 2 mM MgCl2, 2.5 mM EDTA and 0.1%.(w/v BSA). In the membrane preparations for SDS-PAGE the buffers lacked BSA, while MgClZ was omitted from the resuspension buffer in some Chl fluorescence measurements and fluorescence polarization measurements, as noted in the text. W The relative fluidity of thylakoid membranes was determined by steady state fluorescence polarization after the addition of the hydrophobic fluorophore DPH (Aldrich) (Barber et al., 1984). DPH (3mM stock in tetrahydrofuran) was mixed with membranes (50 ug Chl ml'l) to a final concentration of 5 uM and incubated in the dark at room temperature for 40 min. The membranes were then pelleted at 3000 x g for 5 min and diluted in 100 mM sorbitol, 20 mM Hepes (pH 7.9), 10 mM NaCl to a final concentration of 10 ug ml'l Chl and 1 uM DPH. The measurements were carried out on an SLM 4048 spectrofluorometer (SLM 76 Instruments, Urbana, IL) in a T-format. Excitation was provided by light at 360 nm with a half-bandwidth of 16 nm. Fluorescence was monitored at 460 nm with a half-bandwidth of 8 nm. The degree of polarization (P) was calculated by an on line Hewlett-Packard 9825 computer. WWW Hhole chain and PS1 dependent electron transport activities were assayed at 25°C in the presence of 0.1 mM NaN3, using water and 0.5 mM N,N,N’,N’-tetramethyl-p-phenylenediamine (reduced with 2.5 mM ascorbate) as donors, respectively, by monitoring the 02 consumption by 0.1 mM methyl viologen in a Rank oxygen electrode. Chloroplast membranes (20-30 ug Chl) were added to the reaction mixture which contained 300 mM sorbitol, 20 mM Hepes (pH 7.9), 10 mM NaCl, 2 mM MgCl2, 2.5 mM EDTA, 0.1 % w/v BSA, 0.1 uM gramicidin-D and 1 mM NH4Cl. The PSI assay also contained 1 uM 3-(3,4-dichlorophenyl)-1,1-dimethylurea to inhibit PSII activity and 10 ug/ul superoxide dismutase. Saturating white light illumination (1200 2 5'1) was provided by a high intensity microscope lamp. PSII uE m' mediated 2,6-dichlorophenolindophenol reduction was measured at 580 nm using a Hitachi 100-60 spectrophotometer as described (Steinback et al., 1979). PSII light response was determined in the oxygen electrode as 02 evolution with 1.5 mM K3Fe(CN)6 as the electron acceptor. L,D-glyceraldehyde was also added (10 mM) to inhibit C02 dependent oxygen evolution. 77 WW Room temperature fluorescence induction transients of isolated chloroplast membranes were measured in the presence of 10 uM DCMU (Paterson and Arntzen, I982). Membranes were diluted in resuspension buffer to a final concentration of 5 ug/ml Chl and dark adapted for 5 min before use. The actinic light was provided by a microscope illuminator through a broadband blue optical filter (Corning 4-96), with onset of illumination controlled by an electronic shutter (Vincent Associates, Rochester, NY). Fluorescence was measured through a Corning 2-64 red filter by a photodiode placed 90° to the incident light as described (Paterson at al., 1982). Transients were recorded on a Nicolet Explorer II digital oscilloscope. WWW Aliquots of chloroplast membranes in resuspension buffer lacking MgCl2 were diluted to a concentration of 10 ug/ml Chl in 60 % glycerol (v/v) and sodium fluorescein was added as an internal standard to a final concentration of 2 mM (Krause et al., 1983). Samples were then frozen in liquid N2 in capillary tubes (0.5 mm i.d.). Fluorescence emission spectra were acquired using an SLM 4048 spectrofluorometer. Excitation was provided by light at 440 nm with a half-bandwidth of 4 nm. Fluorescence emission was scanned in 1 nm increments from 470 to 800 nm with a half-bandwidth of 1 nm. Storage and mathematical manipulations of spectra were performed by an on-line Hewlett-Packard 9825 computer. 78 W Short term gas exchange determination on single intact plants has been described (Somerville and Ogren, 1982). Plants were placed in a glass chamber with the roots submerged in a vial containing water. The chamber was immersed in a temperature controlled waterbath at 25°C and connected by two ports to a source of gas of a desired composition, and an infrared gas analyzer (Analytical Development Company Series-225). The CD2 concentration of the entering and exiting gas stream was continuously monitored with the gas analyzer in the differential mode. Measurements of dark respiration and photosynthesis at 100 and 300 uE m'2 s'1 were performed for each plant. After the completion of each experiment, fresh weight and Chl content (MacKinney, 1941) of the aerial portion of the plants were determined. W Rosette leaves of three week old plants were fixed in 2% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h, followed by an 1 h incubation in 1% (v/v) osmium tetroxide in the same buffer. The specimens were dehydrated in a graded ethanol series and embedded in Spurr’s epoxy resin (1969). Both fixation steps and dehydration were done at 4°C. Thin sections were stained with uranyl acetate and lead citrate and examined in a JEOL IOOCX electron microscope. Quantitative data of chloroplast membrane profiles on electron micrographs were obtained using a map measurer from 20 chloroplast sections from two separate embeddings of wild type and mutant plant material grown at different times. 79 99W Chloroplast copy number per cell was determined in isolated protoplasts prepared as described (Kunst et al., 1988). Aliquots (10-20 ul) of protoplast suspension were pipetted on microscope slides and the protoplasts were flattened by the coverslip application, so that the chloroplasts formed a monolayer within cells and could be easily counted (McCourt et al., 1987). Results W19 In order to determine the effects of the altered leaf membrane lipid composition on growth of the 9911 mutant line J825, we measured the rate of increase in fresh weight of mutant and wild type plants growing at different temperatures (IO-34°C). The optimal growth temperature for both the mutant and the wild type was approximately 27°C, and their relative growth rates were very similar at temperatures ranging from 10-30°C (Figure 4-1). At temperatures greater than 30°C the mutant J825 grew slightly more rapidly than the wild type. However, after about 6 days at high temperatures the growth slows down, and eventually both the mutant and wild-type plants turn chlorotic. In any case, this experiment indicates that there are no significant deleterious effects of the 9911 mutation, or other mutations in the background genotype, on the growth of line J825. These observations also suggest that the altered lipid composition of the 9911 mutants improves the thermal tolerance of this race of 99991999919. 80 r I I I I I I I I I 0.5 - - o 0325 0 WT .. 0.4 - .. ,. l .3. 0 H 2 0.3 - - '5 5 a 0.2 - - .2 H .‘E 0 a: 0.1 - 0 I I I I I I I I l I 10 13 16 19 22 25 28 31 34 37 Temperature (’0) Figure 4-1. Effect of temperature on the relative growth rate of wild type and mutant 99991999919. 81 . , .I .1.- , A . .. .. . . . .. . “~1. 1.- To examine the thermal tolerance of the 9911 mutant further, the heat-induced changes in Chl fluorescence in mutant and wild-type leaves was measured (Figure 4-2). The rise in Chl fluorescence is thought to indicate an inhibition of excitation energy transfer from the LHCP antenna to PSII reaction centers, due to separation of LHCP from the P511 core (Armond et al., 1978). It is a sensitive indicator of the photosynthetic membrane stability, which is thought to depend on the membrane lipid composition (Berry and Bjorkman, 1980). The experiment was conducted by heating detached leaves from 25-57°C at a rate of 1°C min'1 , and measuring the fluorescence continuously (Schreiber and Berry, 1977). The fluorescence yield enhancement was observed at 43°C in wild-type leaves (Figure 4-3), while the fluorescence did not rise in the mutant until 45°C. This difference in the threshold temperature suggests greater thermal stability of the chloroplast membranes of the mutant. In an effort to extend the fluorescence measurements, we compared the rates of photosynthetic electron transport by mutant and wild type chloroplast membranes incubated for 10 min at various temperatures ranging from 25-45°C (Figure 4-3). The activities of both mutant and wild-type membranes rapidly declined following incubation at temperatures above 30°C. Although the membranes of the mutant were more resistant than the wild type to thermal inactivation, the effect was subtle. The higher resistance of the mutant was also apparent when membranes were incubated for various times at 40°C (Figure 4-4). Thus, these results suggest that the changes in composition of chloroplast membrane lipids in the mutant result in a slightly enhanced stability 82 O J825 IDVVT' _1ro~ Relative fluorescence 25 35 45 55 Temperature (°C) Figure 4-2. Temperature induced fluorescence engancement yield of wild type and mutant leaves. Plants were grown at 22 C. The arrows indicate estimates of threshold temperatures at which fluorescence is enhanced. Each point represents the mean 1 SD (n23). 83 t l I l I l O a. 0.1825 2 100- N .5 :0: 80- " ‘3? 0.2 60- ‘ 73'5 0“ fag 40" a: g E. 20- - Q 0 3 4: 0" 7 O. I I I I I 25 3O 35 4O 45 Temperature (°C) Figure 4-3. Effect of temperature on whole chain photosynthetic electron transport of thylakoid membranes from wild type and mutant 99991999919. Activity is expressed relaBive to that obtained with membranes preincubated in darkness at 4 C for 10 min. 19e miximal rates for the mutant and wild type were 169.4 umol 0; mg Chl h and 156.7 e gmol 02 mgChl' h' , respectively. Each point presents the mean i 50 n-4). 84 g l l l l l I a. _ 0J825 _ 2 10° owr 2 fl 3 80" .— .3? 2% 60— — o .9 ~23 40L 4 QV .: ‘E .1 > 20- CI .9 2 °- - o. l l J J l l 0 2 4 6 8 10 Time(min) Figure 4- 4. Decline in photosynthetic electron transport activity in chloroplast membrages from wild type and mutant r i preincubated at 40 C. Activity is expressed gelative to that obtained with membranes preincubated in darkness at 4 C for 10 min. Th? mgximal rates for the mutant_ind_ wild type were 169. 4 umol 02 mg Chl and 156.7 umol 02 mg Chl , respectively. Each point represents the mean 9 SD (n-4). 85 of the membranes at high temperatures. However, because of the changes in ultrastructure, noted later, it is not possible to ascribe this effect specifically to the lipid composition. Memhmeflmm It has been suggested that the high proportion of polyunsaturated fatty acids in thylakoid membranes plays a major role in maintaining an extremely fluid matrix necessary for lateral movement of photosynthetic components (Quinn and Williams, 1983). The absence of 16:3 and concomitant changes in fatty acid composition due to the 9911 mutation might be expected to change thylakoid membrane fluidity. Therefore, fluorescence polarization (P) measurements were made on isolated membranes containing the hydrophobic fluorophore DPH, to determine the relative fluidity of thylakoids from mutant and wild type leaves. The relatively low P values obtained for wild-type 99991999919 thylakoids were in agreement with those of other plant species (Barber et al., 1984), reflecting a highly fluid lipid bilayer (Figure 4-5). As with similar measurements made on the membranes from other mutants with altered lipid composition (McCourt et al., 1987, Chapter 6), the polarization values of the thylakoid membranes from mutant line J825 were slightly higher at the majority of temperatures tested, but the difference was not statistically significant. f o ‘1'-II‘ 0.0 0900.101101 ._d0ro - on Under all growth conditions examined i.e., growth at various temperatures in the range of 10-34°C and 100-200 uE m'z 5'1, leaves of the mutant J825 were always slightly lighter green than those of the ' 86 p h I I 1?— Fluorescence polarization (P) o o 14 n: l l l l l l l l O 10 2O 30 4O 50 Temperature (°C) 0 Figure 4-5. Effect of temperature on DPH fluorescence polarization by thylakoid membranes from wild type and mutant 99991999919. Each point represents the mean i SD (n-lO). 87 wild type, due to a 10% reduction in chlorophyll per unit fresh weight (Table 4-I). The analysis of 60 F2 plants from a cross of HT x J825 suggested that this phenotype is related to the 9911 mutation. All of the 16 plants with altered fatty acid composition exhibited the chlorotic phenotype, whereas all of the 44 plants with normal fatty acid composition had normal Chl content. Thus, the 16:3 deficiency in J825 cosegregates with reduced Chl levels. The same conclusion was reached from a similar experiment with the independent allelic mutant LK8 (results not presented). All the chlorophyll present in higher plants is thought to be bound to proteins of the thylakoid membrane (Markwell et al.,1979). Therefore, a decrease in chlorophyll content suggests a reduction in the amount of one or more Chl-protein complexes. Chloroplast membrane polypeptides of the mutant and wild type were separated by 2-dimensional SDS-polyacrylamide gel electrophoresis and examined in detail (Figure 4-6). The polypeptide pattern was very similar for both genotypes, but several differences were observed. No major polypeptides were absent from 9911 chloroplasts, although_some were present in lower amounts than in the wild type. On the other hand, some other proteins were more abundant in the membranes of the mutant. There is also a small increase in Chl a/b ratio in the J825 mutant, due to a preferential loss of Chl a. This result suggests that the stoichiometry of LHCP versus PSII and PSI might not be maintained. In order to determine whether this is the case, we compared the Chl-protein complexes from the mutant and the wild-type chloroplast membranes separated on SDS-polyacrylamide gels (Figure 4-7). Absorption spectra obtained for each of the Chl-protein complexes resolved in this manner 88 Table 4-1. Relative amounts of lipid, chl and protein in mutant and wild type 99991999919 leaves and chloroplast membranes. Values are means 1 SD (n-3). Hild type J825 LEMS Chl/fwt (mg/g) 1.53 1 0.08 1.40 1 0.05 Chl a/b 2.93 1 0.06 3.12 1 0.08 Lipid/Chl (g/g) 2.48 1 0.07 2.52 1 0.01 Protein/Chl (g/g) 38.46 1 0.15 38.38 1 1.20 Protein/Lipid (g/g) 15.56 1 0.90 15.24 1 0.60 WW Lipid/Chl (g/g) 2.12 1 0.08 2.45 1 0.05 Protein/Chl (g/g) 12.27 1 0.50 12.65 1 0.18 Protein/Lipid (g/g) 5.79 1 0.24 5.16 1 0.07 89 Figure 4-6. Autoradiographs of 35S-Methionine labeled proteins of chloroplast membranes from wild type and mutant 99abid999i9 separated by 2-dimensional SDS-polyacrylamide gel electrophoresis. The numbers at the top represent apparent pH, and those at the left apparent molecular mass. fl , proteins which are more abundant in the mutant than in the wild type; 0 , proteins which are more abundant in the wild type than in the mutant. 90 WT .825 .“ - F 8 0 is“ 300 .1 '9 E 2 o 3.200- - c .2 ‘5 E 100- : 0.1325 2 OTNT o o o 1 l 1 I I o" o 250 500 750 1000 1250 lrradlanceutE/mzls) (c) N 8 I .4 J 150- d on O l O J825 0 WT 02 evolution (p. moles/mg chl/h) 8 I l I I I 250 500 750 1000 1 250 1500 Irradlance (uE/m2/S) O O Figure 4-9. Light response curves for a) whole chain, b) PSI and c) PSII electron transport by wild type and mutant 99991999919. Each point represents the mean 1 SD (n-3). 95 excitation energy is trapped by the reaction centers. Following onset of illumination, Chl fluorescence intensity rises to a maximal level FIn which is reached when all the primary acceptors of PSII are reduced. Variable fluorescence (Fv-F - F0) is a measure of time required for m the turnover of all the PSII reaction centers and it reflects the number of Chl molecules associated with these reaction centers. The shape of the room temperature fluorescence transients obtained from J825 and wild type thylakoid membranes were indistinguishable (Figure 4-10). There was also no difference in variable fluorescence values (expressed as Fv/Fo) (Table 4-III). These results considered in conjunction with almost identical PSII electron transport rates of mutant and wild-type plants suggest that their PSII antennas are structurally and functionally indistinguishable. Low temperature (77K) Chl fluorescence emission spectra were used to compare the excitation energy distribution between Chl-containing components of thylakoid membranes isolated from mutant JB25 and wild type. In chloroplast membranes of higher plants, the component with an emission maximum at 685 nm is ascribed to LHCP of PSII, the 695 nm emission is attributed to PSII reaction center core complex, and the fluorescence emitted at 734 nm originates from PSI. The F685/F734 ratio obtained with chloroplast membranes from the mutant was higher than the wild type (Table 4-111) in the presence or absence of MgClz. By normalizing to the magnitude of emission from the internal standard, fluorescein, it was apparent that the Change in F685/F734 ratio is caused by lower fluorescence emission at 734 nm, accompanied by increased Chl fluorescence at 685 nm. This result was confirmed using the independently isolated mutant line LK8 (Table 4-III). The results 96 Relative Fluorescence 011 V o 1 2 3 4 Time of illumination (sec) Figure 4-10. Room temperature fluorescence induction transients of isolated chloroplast membranes of the wild type (-—-) and mutant (---) 99991999919 in the absence of DCMU. 97 Table 4-III. Room temperature fluorescence induction and low temperature (77K) fluorescence of isolated thylakoids. Hild type J825 LK8 ROOM TEMPERATURE FLUORESCENCEi F0 1130 1 33 1127 1 11 - Fm 3221 1 132 3245 1 55 - I’V/Fo 1.35 1 0.07 1.33 1 0.02 - 77K FLUORESCENCEQ F535/F734 +Mg2+ 0.73 1 0.001 0.35 1 0.002 0.34 1 0.004 2+ F535/F734 -M9 0.55 1 0.005 0.75 1 0.005 0.73 1 0.004 a Room temperature fluorescence was measured in the presence of 10 uM DCMU, n-lO; Values are expressed in arbitrary units. b 77K fluorescence was measured in the presence and absence of 5 mM MgClz, n-6. 98 of this experiment imply that the peripheral antenna of PS1 in the mutant is structurally different, or that the efficiency of energy transfer between PSII and PSI is reduced in mutant chloroplasts. W931: In order to examine the effects of changes in leaf lipid composition in the 9911 mutants on chloroplast structure, a morphometric analysis was compiled from electron micrographs of chloroplasts from the wild type and the allelic mutant lines J825 and LK8. Even without a detailed analysis, a difference was apparent in the arrangement of chloroplast membranes between the two genotypes (Figure 4-11). The most striking was the alteration in the membrane appression in 9911 mutants. Quantitative analysis showed that the average number of thylakoid membranes per granum is decreased from 6.2 in the wild type to 3.8 in the mutant (Table 4-IV). This change is accompanied by a large increase in the number of grana per chloroplast, so that the total amount of appressed membranes in the mutant is close to the wild-type value. 0n the other hand, the total length of non-appressed membranes is increased in the mutants by 30%. Discussion W In spite of substantial changes in leaf lipid metabolism and membrane lipid composition the mutant line J825 exhibits comparable or higher growth rates (at temperatures above 30°C) relative to the wild 99 Figure 4-11. Transmission electron micrograph of chloroplasts from rosette leaves of (A) wild type and (8) mutant Ar99i999919. Bar - Ium 100 Table 4-IV. Morphometric analysis of chloroplasts from mutant lines and wild type Arabidopsis. Wild type J825 LK8 Grana/plastid 54.4 1 6.6 90.0 1 7.2 87.6 1 7.6 Thylakoids/granum 6.2 1 3.7 3.7 1 1.6 3.9 1 1.7 Granal width (um) 0.4 1 0.04 0.4 1 0.03 0.4 1 0.04 Stroma thylakoids/plastid 0.2 1 0.01 0.2 1 0.03 0.2 1 0.02 Stroma thylakoid length (um) 103.91 12 99.3 1 10 95.1 1 13 Appressed membrane/plastid (um) 114.1 97.0 104.2 Non-appressed membrane/plastid (um) 40.7 52.8 52.9 Total membrane (um) 154.8 149.8 157.1 Appressed/non-appressed membrane 2.8 1.8 2.0 Surface area (umz/plastid) 9.9 1 2 10.8 1 1 10.7 1 l *Measurements were made on 20 chloroplasts from each line. lOl type. This observation is consistent with the results obtained for two other 99991999919 mutants deficient in fatty acid unsaturation of chloroplast membranes (McCourt et al., 1987, Kunst et al., manuscript in preparation). In each of these cases, a reduction in the degree of lipid unsaturation is correlated with an enhanced growth rate at high temperatures. The acclimation to growth at high temperatures is usually accompanied by an increase in the threshold temperature at which Chl fluorescence enhancement occurs (Schreiber and Berry, 1977; Berry and Bjorkman, 1980). The mechanisms associated with this adaptive response are not known, but include substantial changes in membrane lipid unsaturation. A comparison of the 9911 mutant and wild type by this criterion also indicated increased thermal tolerance of the mutant. On the basis of these results, together with slower inactivation of photosynthetic electron transport in the mutant at elevated temperatures (Figures 4-3,4-4), we conclude that the fatty acid composition of chloroplast membranes may be an important component of the thermal adaptation response characterized in species such as 999199 91999999 (Raison et al., 1982). The fluorescence polariZation measurements performed on J825 indicate that the changes in lipid composition do not affect the fluidity of membranes from the mutant (Figure 4-5). This observation suggests that the superior acclimation to growth at elevated temperatures of J825 is not due to changes in membrane fluidity. Thus, it is not apparent, at this time, why the 9911 mutant exhibits an enhanced thermal tolerance. 102 Wen Hhen expressed on 3 Chi basis, the mutant had a higher rate of electron transport than the wild type. After correction for an 8.5% reduction in Chl content, the rates of whole chain electron transport were identical in the two genotypes. He could also not detect any changes in room temperature fluorescence between mutant and wild type thylakoid membranes (Table 4-III). These results suggest that the photosynthetic capacity of the mutant and the wild type is essentially equivalent. One of the distinguishing characteristics of the mutant is an 8.5% reduction in Chl content (Table 4-1). This phenotype cosegregated with the altered lipid composition, indicating that the 9911 mutation causes both effects. The magnitude of the Chl deficiency parallels a 10% decrease in the protein/lipid ratio of chloroplast membranes. The mutant exhibits preferential loss of Chl a, but Chl b is also decreased by 4.5%. Since LHCP does not accumulate in the absence of chl b, a 4.5% decrease of Chl b in J825 thylakoids suggests a corresponding decrease in LHCP content. However, a 4.5% reduction in the amount of LHCP cannot account for the 10% reduction in the protein/lipid ratio of mutant membranes. Thus, it is apparent that changes must have occurred in the amounts of other thylakoid polypeptides. A comparison of the polypeptide pattern of chloroplast membranes from the mutant and wild type indicated that several polypeptides are obviously reduced in amount in the mutant. However, the role of these polypeptides is not known, and it was not possible to determine by this criterion if the change was adequate to explain the reduced protein/lipid ratio. 103 Another change in 0825 with respect to the wild type concerns 77K fluorescence properties of chloroplast membranes. Measurements on isolated membranes from the mutant lines J825 and LK8, show a decreased fluorescence emission at 734 nm, originating from PSI, and a concomitant increase in fluorescence yield at 685 nm, emitted by LHCP of PSII. This different distribution pattern of excitation energy in the mutant is reflected in a higher F685/F734 ratio in both presence or absence of MgClz (Table 4-III). Fluorescence emission at 734 nm arises from peripheral antennae of PSI (Mullet et al., 1980). Therefore, a reduced fluorescence yield at long wavelengths may be caused by changes within the PSI antennae pigment bed, or it may be attributed to a decrease in energy spillover from P511 to PSI. He favor the latter concept because we observe no significant changes in electron transport rates (after correction for the reduction in Chl content) or PS1 light response. A reduction in the amount of light energy transferred from PSII to PSI can, in principle, be explained in one of several ways. Since mobile LHCP is considered to play a major role in the regulation of excitation energy distribution between the photosystems, reduced LHCP content in mutant membranes would reduce the amount of energy reaching PSI. However, the relatively slight (4.5%) reduction in LHCP (expected on the basis of the reduced Chl b content) is not adequate to account for the fluorescence phenomena. It seems more likely that the structural organization of the chloroplast membranes (Figure 4-11) brought about by compositional changes of thylakoid membranes might impose limitations upon lateral migration of LHCP. 104 The alternate possibility, that a decrease in PSI fluorescence yield is affected by changes in the organization of PS1, cannot be completely ruled out, either. Chlorophyll fluorescence emission is considered a more sensitive monitor of changes in Chl-protein complexes than rates of P51 and PSII photochemistry (Burke et al., 1978). Therefore, our evidence (similar PSI electron transport rates and PSI light response of wild type and mutant thylakoids) might not be sufficient to support the conclusion that PSI antenna of the mutant is not changed. W The most pronounced effects of the 9911 mutation are differences in the amount and arrangement of thylakoids within the chloroplasts. The number of apressed regions per chloroplast in mutant lines J825 and LK8 is increased in comparison with the wild type, but the stacks contain fewer thylakoid membranes. The total length of non-appressed membrane is increased in both mutants. Similar changes in chloroplast ultrastructure in two independently isolated allelic mutants establish a causal relationship between the altered lipid composition and structural differentiation of thylakoid membranes. It has been suggested that membrane stacking in higher plant chloroplasts is mediated by LHCP present in the membrane (Staehelin and Arntzen, 1983). This model is based on observations that greening or mutant plastids which lack or are deficient in LHCP are correspondingly deficient in grana formation (Goodchild et al., 1966, Thornber and Highkin, 1974, Armond et al., 1976), and reconstitution experiments using liposomes and purified LHCP (Ryrie et al., 1980, McDonnel and Staehelin, 1980). 105 Results presented here indicate that the model for membrane appression based on LHCP is incorrect, or incomplete in some important way. Although a careful morphometric analysis does not appear to have been done on the barley 99199199 mutant, or any of the other Chl b deficient mutants, it seems that the changes in ultrastructure in the 9911 mutant are at least as pronounced as in any of the mutant lines deficient in LHCP. Since no major difference in LHCP content between the wild type and mutant was observed, it is not possible to attribute‘ the decrease in the degree of stacking to a reduction in the amount of LHCP. However, there are several quantitative differences between the mutant and wild type in the polypeptide pattern on 2-dimensional gels suggesting that an alteration in the amount of some other protein component of thylakoid membranes might be responsible for the changes in structural features of 9911 plastids. A thorough examination of 2-dimensional polypeptide pattern of chloroplast membranes from the barley 99199199 mutant might, therefore, be informative in relating changes in specific polypeptides with the similar alterations in the degree of membrane appression observed in 9911 and 99199199 mutants. Ultrastructural changes in chloroplasts of the 9911 mutant may also be caused by changes in the organization of PS1 antenna. Optimal efficiency of noncyclic photosynthetic electron flow depends on similar rates of charge separation at the two reaction centers. To ensure the balanced energy distribution between PSI and PSII, light harvesting pigments undergo changes referred to as ”state I-state II” transitions. Preferential light absorption by PSII leads to a reversible phosphorylation of a population of the LHCP particles, and their subsequent migration to non-appressed membrane regions, so that more 106 excitation energy is distributed to PSI reaction centers (Staehelin and Arntzen, 1983). This adaptive response, known as state 11, also results in a decrease of membrane stacking, due to incorporation of negatively charged phosphate groups into LHCP. Therefore, if there are changes in the structural organization of the PSI antenna of 9911 mutant, as suggested by a lower fluorescence emission at 734 nm and a decrease in the amount of Chl a, a reduced absorption by PSI would lead to state 11, and a corresponding decrease in the extent of chloroplast membrane appression. Finally, we cannot rule out the possibility that the fatty acid composition of chloroplast lipids 999 99 plays a role in the organelle biogenesis. WW All described differences between mutant and wild-type plants are relatively minor, and are not deleterious to the mutants. The mutants are also not impaired in growth or development under a variety of environmental conditions. Therefore, we must conclude that under the conditions we have examined there are no major physiological advantages associated with the presence of the prokaryotic pathway. Literature cited Armond PA, CJ Arntzen, J-M Briantais, C Vernotte 1976 Differentiation of chloroplast lamellae. 1. Light harvesting efficiency and grana development. Arch Biochem Biophys 175: 54-63 Armond PA, U Schreiber, O Bjorkmann I978 Photosynthetic acclimation to temperature in a desert shrub 199999 9199919919. Plant Physiol 61: 411-415 107 Barber J, RC Ford, RAC Mitchell, PA Nillner 1984 Chloroplast thylakoid membrane fluidity and its sensitivity to temperature. Planta 161: 375-380 Berry J, 0 Bjorkman 1980 Photosynthetic response and adaptation to temperature in higher plants. Annu Rev Plant Physiol 31:491-543 Browse J, P. McCourt, CR Somerville 1985 A mutant of bi lacking a chloroplast-specific lipid. Science 227: 763-765 Browse J, P. McCourt, CR Somerville 1985 Overall fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152: 141-146 Burke JJ, CL Ditto, CJ Arntzen 1978 Involvement of the light harvesting complex in cation regulation of excitation energy distribution in chloroplasts. Arch Biochem Biophys 187: 252-263 Goodchild DJ, HR Highkin, NK Boardman 1966 The fine structure of ghloroplasts in a barley mutant lacking chlorophyll b. Exp Cell Res 43: 84-688 Haughn GN, CR Somerville 1986 Sulfonylurea-resistant mutants of 9999199991; 19911999. Nol Gen Genet 204: 430-434 Krause GH, J-H Briantais, C Vernotte 1983 Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77K. 1. pH dependent quenching. Biochim Biophys Acta 723: 169-175 Kunst L, JA Browse, CR Somerville 1988 Altered regulation of lipid biosynthesis in a mutant of 5999199991; deficient in chloroplast glycerol phosphate acyltransferase activity. Proc Natl Acad Sci USA, In press Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 MacKinney G 1941 Absorption of light by chlorophyll solutions. J Biol Chem 140: 315-322 Markwell JP, JP Thornber, RT 80995 1979 Higher plant chloroplasts: evidence that all the chlorophyll exists as chlorophyll-protein complexes. Proc Natl Acad Sci USA 76: 1233-1235 Markwell NAK, SM Haas, LL Bieber, NE Tolbert 1978 A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem 87: 206-210 McCourt P, L Kunst, JA Browse, CR Somerville I987 The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of A9991999§1§. Plant Physiol 84: 353-360 108 McDonnel A, LA Staehelin 1980 Adhesion between liposomes mediated by the chlorophyll a/b light harvesting complex isolated from chloroplast membranes. J Cell Biol 84: 40-56 Melis A, GH Harvey 1981 Regulation of photosystem stoichiometry, chlorophyll a and chlorophyll b content and relation to chloroplast ultrastructure. Biochim Biophys Acta 637: 138-145 Paterson DR, CJ Arntzen 1982 Detection of altered inhibition of photosystem 11 reactions in herbicide-resistant plants. In M Edelman, R Hallick, NH Chua, eds, Methods in Chloroplast Molecular Biology. Elsevier, Amsterdam, pp 109-119 Mullet JE, JJ Burke, CJ Arntzen 1980 Chlorophyll proteins of photosystem 1. Plant Physiol 65: 814-822 Quinn PJ, HP Nilliams 1983 The structural role of lipids in photosynthetic membranes. Biochim Biophys Acta 737: 223-266 Quinn PJ, HP Williams 1985 Environmentally induced changes in chloroplast membranes and their effects on photosynthetic function. In J Barber, NR Baker, eds, Photosynthetic Mechanisms and the Environment. Elsevier, Amsterdam, pp 1-47 Raison JK, LKM Roberts, JA Berry 1982 Correlations between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant £9919m891999999 to growth temperature. Biochim Biophys Acta 688: 218-22 Roughan PG, CR Slack 1982 Cellular organization of glycerolipid metabolism. Annu Rev Plant Physiol 33: 97-132 Ryrie IJ, JM Anderson, DJ Goodchild 1980 The role of the light-harvesting chlorophyll a/b-protein complex in chloroplast membrane stacking. Eur J Biochem 107: 345-354 Schreiber U, JA Berry 1977 Heat induced changes in chlorophyll fluorescence in intact leaves correlated with damage in the photosynthetic apparatus. Planta 136: 233-238 Somerville CR, HL Ogren 1982 Isolation of photorespiration mutants in A9991999§1§_Lb9|i9n9. In M Edelman, R Hallick, NH Chua, eds, Methods in Chloroplast Molecular Biology. Elsevier, Amsterdam, pp 129-138 Spurr AR 1969 A low viscosity epoxy embedding medium for electron microscopy. J Ultrastruct Res 26: 31-43 Staehelin LA, CJ Arntzen 1983 Regulation of chloroplast membrane function: protein phosphorylation changes the spatial organization of membrane components. J Cell Biol 97: 1327-1337 109 Steinback KE, JJ Burke, CJ Arntzen 1979 Evidence for the role of surface exposed segments of the light harvesting complex in cation-mediated control of chloroplast structure and function. Arch Biochem Biophys 195: 546-557 Thornber JP, HR Highkin 1974 Composition of the photosynthetic apparatus of normal barley leaves and a mutant lacking chlorophyll b. Eur J Biochem 41: 109-116 CHAPTER 5 A MUTANT 0F ABABLQQEfilfi THAT ACCUMULATES PALMITIC ACID IN LEAF LIPIDS Abstract Leaf membrane lipids of a mutant of A_r9hj_d_9_p_s_i_s, th9|j9n9 accumulate high amounts of palmitic acid (16:0) and show a corresponding decrease in unsaturated 16-carbon fatty acid levels as a consequence of a single nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids suggests that the mutant is deficient in the activity of the chloroplast n-9 fatty acid desaturase which normally introduces a double bond in 16-carbon acyl chains esterified to the 59-2 position of monogalactosyldiacylglycerol (MGD). Both chloroplast and extrachloroplast lipids are affected by the mutation. Thus, either there is substantial transfer of 16:0 acyl groups from MGD to all the leaf polar lipids, or 16:0 which would normally be used for MGD synthesis in the chloroplast is exported to the cytoplasm rather than being elongated to 18:0. Synthesis of MGD by the prokaryotic pathway is reduced 25-30%, but this deficiency is compensated for by the increased production of MGD through the eukaryotic pathway. This change in relative contribution of the two pathways of lipid biosynthesis in the mutant may be a regulated llO lll response to the loss of chloroplast n-9 desaturase which reflects a requirement for polyunsaturated fatty acids for the assembly of chloroplast membranes. Introduction Trienoic fatty acids (18:3 and 16:3) are the predominant fatty acids of chloroplast membranes of higher plants. Typically they account for approximately two thirds of all the thylakoid fatty acids, and over 90% of the fatty acids of M00, the most abundant chloroplast lipid (Gounaris and Barber, 1983). The reason for the high degree of fatty acid unsaturation is not known, but it is thought to be involved in providing an extremely fluid matrix for photosynthetic electron transport (Raison, 1980; Quinn and Hilliams, 1983). Since a relatively large decrease in trienoic fatty acid content in an A9991999§1§ mutant had no effect on chloroplast function, but caused ultrastructural changes, it has also been suggested that lipid unsaturation may be primarily required for the formation of the characteristic ultrastructural features of chloroplasts (McCourt et al., 1987). It is not known with certainty how many desaturase enzymes participate in the synthesis of trienoic acids in plant cells, and their compartmentation has not been precisely established. Isolated chloroplasts of ’16:3’ plants readily synthesize M00 in which 18:1 fatty acids at position 99-1 are converted to 18:2 and 18:3. Similarly, sequential desaturation of 16:0 to 16:3 takes place at position 99-2 of M60 (Roughan et al., 1979). However, desaturation of 18:1 and 18:2 112 fatty acids is not confined to MGD, because PG and SL have also been shown to convert these fatty acids to 18:3 (Roughan, 1985; Joyard et al., 1986). On the other hand, MGD seems to be the sole substrate for 16:0 desaturation, and 16-carbon fatty acids are not desaturated to any extent when esterified to other lipids. The main substrate for the desaturation of 18:1 to 18:2 outside the chloroplast is microsomal PC, on which some 18:3 synthesis also occurs (Slack et al. 1976; Roughan and Slack, 1982). These observations suggest the existence of a family of fatty acid desaturases located in the chloroplast or the endoplasmic reticulum, which use different glycerolipids for the desaturation reactions. The only exception is the soluble chloroplast 18:0-ACP desaturase, which inserts a double bond at the n-9 position of stearic acid while it is still bound to acyl carrier protein. This is also the only desaturase enzyme that has been partially purified (McKeon and Stumpf, 1982). All the other desaturases appear to be membrane bound enzymes that lose activity during membrane solubilization. This is particularly true for chloroplast desaturases which are inactivated by chloroplast rupture or exposure of intact chloroplasts to mild hypotonic conditions (Andrews and Heinz, 1987). Difficulties associated with solubilization and reconstitution of desaturase acitivity in vitro have hindered traditional biochemical investigations of these enzymes. Therefore, we have initiated a genetic approach to study the desaturation process in plant membrane lipids by the isolation of a number of mutants with specific changes in unsaturation of their leaf fatty acids. Ne have previously characterized a mutant deficient in t9999-16:1 synthesis (Browse et al., 1985a), and mutants that lack specific desaturases responsible 113 for the synthesis of n-3 (Browse et al. 1986a) and n-6 fatty acids (Browse et al., manuscript in preparation). Here we describe the biochemical characterization of a mutant deficient in conversion of 16:0 to gig-16:1. Materials and methods W31 The mutant line J867 was isolated from the Columbia wild type of 9999199951; 39911999 (L.) Heynh. following mutagenesis with ethyl methane sulfonate, as previously described (Browse et al., 1985a). It was backcrossed to the wild type four times before being used for the experiments reported here. Plants were grown at 22°C with continuous fluorescent illumination (100-150 uE m'2 s'l) on a perlite:vermiculite:sphagnum (1:1:1) mixture irrigated with a mineral nutrient solution (Haughn and Somerville, 1986). Beagsnn Sodium [14C]-acetate (54 mCi mmol'l) was obtained from Research Products International Corporation, Mt. Prospect, IL, and [14C]-16:0-CoA (58 mCi mmol'l) from DuPont, Wilmington, DE. Rhizopus arrhizus lipase suspension (50000 U ml'l) was purchased from Boehringer Mannheim GmbH. Methanolic-HCl reagent was prepared by diluting a 3M solution (Supelco) to IM with methanol. 114 i i i Leaf material was frozen in liquid N2 and lipids extracted with chloroform:methanol:formic acid (10:10:1 by vol.) as previously described (Browse et al, 1986b). Individual lipids were isolated by thin layer chromatography on silica gel 6 coated plates (Kunst et al., 1988), or (NH4)2S04-impregnated silica gel 6 plates (Khan and Hilliams, 1977), and transmethylated with methanolic-HCl after the addition of 14:0 methyl ester as internal standard. The resulting methyl esters were then quantified by gas chromatography (Browse et al, 1985b). Fatty acid positional distribution of M60 was established following degradation with 39119995, 999111199 lipase (Boehringer Mannheim, triacylglycerol acylhydrolase EC 3.1.1.3) according to Fischer et. al. (1973). M00 (5 umol) was dissolved in chloroform:methanol (2:1 by vol.), and after the addition of Triton X-100 (4 mg in the same solvent), taken to dryness. The mixture was then dissolved in 1 ml 0.04M Tris-HCl buffer (pH 7.2), sonicated fbr 10 nflnutes, and the reaction was started by adding 2000 U of 3131:9995, enzyme. The incubations were carried out at room temperature for 30 minutes with vigorous shaking and stopped by adjusting the pH to 4 with acetic acid. The reaction products were extracted and separated by thin layer chromatography on silica gel 8 plates using two consecutive solvent. systems (Fischer et al., 1973). Law The labeling of intact Arabidopsis plants with [14C1-acetate and “the determinations of distribution of radioactivity in the various lipids were done essentially as described (Browse et al., 1986b). Under 'll5 the conditions used, incorporation of [14C]-acetate did not continue beyond 90 minutes after the label application. MW Chloroplast isolation and fractionation procedures have been described (Kunst et al., 1988). The enzyme activity was assayed at room temperature according to Frentzen et al. (1983). The 80 ul reaction mixture contained 250 It“ Mops-NaOH (pH 7.4), 625 ug ml'1 BSA, 5uM [14CJ-16:0-CoA, 2 mM 1-oleoyl-G3P and 50 ug of chloroplast envelope protein. Results W The isolation and genetic characterization of the mutant line 0867 (£995) was described in Chapter 2. WW As shown in Table 2-IV, the increased levels of palmitic acid in the £993 mutant are accompanied by a decrease in 16:2 and 16:3 fatty acids. This phenotype could, in principle, be caused in one of several ways. First, a deficiency in the chloroplast enzyme monoacyl-G3P acyltransferase, that specifically esterifies the 99-2 position of G3P with a 16:0 acyl group, could lead to accumulation of palmitic acid within the chloroplast. Since fatty acids, with the exception of 18:0, are only desaturated after incorporation into glycerolipids (Roughan 116 and Slack, 1982), the inability of the mutant to synthesize lipids in the chloroplast would result in the absence of 16:2 and 16:3 acyl groups in its leaf lipids. To test this hypothesis we performed the monoacyl-G3P acyltransferase assay by measuring the incorporation of [14C]-16:0-CoA by the envelopes (Figure 5-1). Envelopes from both lines synthesized similar levels of PA, PG and DAG indicating that the mutant has wild type levels of monoacyl-G3P acyltransferase activity. The most likely explanation for the altered lipid composition of the mutant is that the £993 locus controls the activity of a fatty acid desaturase which is responsible for introducing the double bond at position n-9 of 16-carbon acyl groups in wild type plants. Since this desaturase activity has not yet been demonstrated by an 19 91199 assay, the precise enzymatic lesion in the mutant cannot be determined directly. However, it is well established that chloroplast MGD is the substrate for 16:3 synthesis. Therefore we analyzed the fatty acid composition of M00 in detail (Tables 5-1 and 5-II). The data obtained show that 16:2 and 16:3 fatty acids are virtually absent from M00 in the mutant, while 16:0 fatty acid accumulates to more than 12-fold the levels of wild type. The degradation of M60 using 3911999; lipase revealed that more than 94% of palmitic acid occurs at the 99-2 position of M00 in both mutant and wild type (Table 5-11). From these results we infer that the £993 mutation affects the activity of an n-9 desaturase which specifically desaturates 16-carbon acyl chains esterified to the 99-2 position of M60. This headgroup specificity also implies that the desaturase is located in the chloroplast. It is worth noting that the deficiency in n-9 desaturase results in the accumulation of palmitic acid at the 99-2 position of M60, ll7 Figure 5-1.11he distribution of radioactivity among the polar lipids following [ C]-16:0-CoA labeling of isolated chloroplast envelopes of the mutant J867 and wild type Ar991dopgis. The same amount of radioactivity was applied to each lane. H8 m.m ~.¢ w.- ”.mn n.mn m.on m.~_ o.o~ ~.n m.m ~.- ¢.m~ o.~v u.o¢ a ~.¢~ m.w~ c.m~ o.~n o.on m.mm m.nn o.~e m.¢¢ «.mv ~.oo c.o~ ¢.~m o.mm mum" «.mu m.~w m.~m ~.mm n.o~ H.vm m.c ~.s m.m «.mu m.~ o.¢ H.m ~.~ mum“ m.~ m.~ ~.N m.~ ¢.v n.m ~.~ v.m n.m a.“ m.o ~.~ ~.H ~.o mum~ v.~ n.~ a." °.~ m.~ Q.“ m.~ m.o Q.— m." m.~ 5.9 ¢.o I cum“ u n u u a u u u u a ~.o ¢.n ~.~ m.¢m mum" a u u u u a a a u n ~.o ~.o ~.c m.~ Nun" u u u n u . ~.m~ v.e~ n u n u u a uuuog v.~m m.mv o.mm c.om a.om m.- o.on —.m~ ~.e¢ ~.ov m.m~ n.- o.- ~.~ one" nwma h: Nona h: Nana h: Nona #2 Know h: send #3 Roma #3 um um um am am am: am: .uouuouou an: naocu —»Ua «cu «as» ououyuc. mogmao .u poi ago voucomucn mus—o» .uo- an grana m*mnou.a-g< wanna. we. oaxu-v—.3 soc; mu.n.— heap mo co.u’moa-ou u-ua spasm ._um opauh 119 Table 5-11. Fatty acid distribution in M00 from wild type and mutant 99991999919 established by degradation with 39119999 99991199 lipase. The lyso-compounds contain fatty acids only on the 99-2 position. The values are given as weight %. Untreated MGD Lyso-compound Fatty acid HT J867 HT J867 16:0 1.1 13.1 2.0 24.2 other 16C 35.7 0.4 69.0 0.8 18:0 tr. 1.2 - - 18:1 0.6 0.6 0.2 0.2 18:2 2.4 3.5 1.2 3.2 18:3 60.1 81.2 27.6 71.6 120 rather than n-6 and n-3 isomers of 16:1, or n-6,n-3 isomers of 16:2. This observation suggests that the presence of a double bond at the n-9 position is required for the insertion of double bonds in n-6 and n-3 positions by other chloroplast desaturases. Wings In order to investigate if the leaf polar lipids other than MGD are affected by the mutation at £993 locus, we determined the fatty acid composition of individual lipids from both mutant and wild type plants. The analysis showed that the proportions of various lipids are essentially the same in the mutant and wild type leaves (Table 5-1). 0n the other hand, there is a striking increase in the levels of palmitic acid in all the polar lipids of mutant, except for PG, which accumulates mum, and SL which seems relatively unaffected because the amount of 16:0 is already very high in the wild type. Thus, either there is a substantial transfer of acyl groups from MGD other chloroplast and extrachloroplast lipids (PE is entirely an extrachloroplast lipid), or 16:0 which would normally be utilized for MGD synthesis and desaturated to 16:3, becomes available for the synthesis of all the leaf polar lipids. Our results from a different lipid mutant of 59991999919 deficient in GBP-acyltransferase (Kunst et al., 1988), indicate that 16:0 levels in leaf cells are highly regulated, and that greater than normal amounts of 16:0 are not utilized within the chloroplast, but get elongated to 18:0 and desaturated to 18:1 before being exported to the cytoplasm. Thus, the increased amount of 16:0 in the lipids of the £993 mutant are not readily explained. 121 LAW The relatively high levels of palmitic acid present in M60 in the mutant do not fully compensate for the reduction in unsaturated 16-carbon fatty acids. There is still an overall 63% decrease in the total amount of 16-carbon fatty acids in M60 in mutant plants, and therefore, a corresponding decrease in the amount of prokaryotic MGD. This may reflect either an alteration in fluxes through the two pathways of lipid synthesis, or an increased turnover of prokaryotic MGD containing high levels of saturated fatty acids. In an attempt to resolve this question and to determine the consequences of the enzyme deficiency on lipid biosynthesis in the mutant, we labeled the leaves of mutant and wild-type plants with [14C]-acetate, and followed the distribution of radioactivity in polar lipids during the subsequent 142 hours (Figure 5-2). As we have shown previously (Browse et al., 1986b, Kunst et al., 1988), the labeling kinetics of wild-type plants demonstrate the parallel operation of the prokaryotic and eukaryotic pathways. Flux through the prokaryotic pathway leads to substantial ‘ labeling of M60 at the beginning of the experiment, while the increase in MGD label at longer times reflects the transfer of 14C from PC made through the eukaryotic pathway. The distribution of label among various polar lipids in the £993 mutant is extremely similar to that of the wild type (Figure 5-2). However, the reduced rate of incorporation of label into M60 and, conversely, the relatively increased labeling of PC is consistent with a reduced synthesis of M00 by the prokaryotic pathway. The results also indicate that the eukaryotic pathway compensates for this reduction in prokaryotic MGD synthesis, since by the end of the experiment the amount of M60 in leaf tissue of the 122 O) O l l 6()-' '- b O 9 l0 0 Radioactivity incorporated (96) M o o 1 l 1 1 o 1 1 1 1 0.5 2 10 ‘100 0.5 2 10 100 Time(h) Figure 5-2. The distribution of radioactivity in leaf lipids of (A) wild type and (B) J867 mutant of A999id99§is after labeling with [ C]-acetate. Symbols:I, PC;D, MGD;Q, DGD;A, SL;O, PG;O, PE; A, PI. 123 mutant reaches wild-type levels. The decline in total radioactivity per gram fresh weight during the course of the experiment was almost identical in the mutant and wild-type plants, suggesting that there is no major difference in their relative rates of lipid breakdown (data not shown). However, the pathway by which the components of MGD might be recycled are not known. Thus, it does not seem possible at this time to critically evaluate the concept that certain molecular species of MGD are turned over more quickly in the mutant. Discussion Because of the problems related to solubilization, purification and stability of the membrane-bound fatty acid desaturases of higher plants, very little information is available about these enzymes, and desaturation reactions 999 99. The subunit composition of the desaturases has not been elucidated, and the cofactors and electron transport components thought to be involved in the desaturation process have not been identified. Therefore, we could not determine the enzymatic lesion in £993 mutant by direct enzyme assay. Nevertheless, normal monoacyl-G3P acyltransferase activity of the mutant (Figure 5-1), together with the analysis of fatty acid composition of M60 (Tables 5-I and 5-11) support the conclusion that the mutant line J867 is able to synthesize MGD, but is deficient in a chloroplast n-9 desaturase due to a single nuclear mutation at the £993 locus. This enzyme introduces the double bond only in 16-carbon acyl chains esterified to the 99-2 position of MGD synthesized in the chloroplast. 124 .These features make the 16:0 desaturase unique in comparison with the n-6 (Browse et al.,manuscript in preparation) and n-3 enzymes (Browse et al., 1986a), which do not exhibit specificity with respect to acyl Agroup chain length, its point of attachment to the glycerol backbone (99-1 or 99-2), or the lipid head group. The observation that the mutant accumulates MGD containing 16:0 at position 99-2 indicates that introduction of the n-9 double bond is a prerequisite for further desaturation of 16-carbon acyl chains by the other chloroplast desaturases. Similar conclusions were reached for the desaturases of Chlorella presented with various monoenoic fatty acids as substrates (Howling et al., 1972). The accumulation of 16:0 at position 99-2 of M60 is consistent with the expectation for a mutant unable to desaturate 16:0 to 16:1. However, the £993 mutation causes two other changes in lipid composition which are less readily explained. First, all the chloroplast and extrachloroplast polar lipids, except SL, show increased levels of palmitic acid or £9999-hexadecenoic acid. In the case of 060, this may be attributed to a greater proportion of this lipid being synthesized by the prokaryotic pathway. However, for PC, PE and PI, which are thought to be synthesized in the endoplasmic reticulum, the implication is that a greater amount of 16:0 must be available for lipid synthesis by microsomal membranes in the mutant. It is not obvious why this might be the case. On the contrary, we have recently shown that the amount of 16:0 exported from the chloroplast is not regulated simply by availability (Kunst et al., 1988). Thus, it seems necessary to propose that, in some way, the accumulation of M60 125 with 16:0 at the 99-2 position stimulates transport of 16:0 from the chloroplast. The other unusual effect of the £993 mutation is that the amount of prokaryotic M60 is decreased by more than 60%. The implication is that MGD containing 16:0 at 99-2 is either turned over rapidly and does not accumulate, or that this species inhibits the synthesis of M00 from DAG in the chloroplast. The latter explanation seems least likely since it is thought that DAG is normally converted to MGD before the desaturation at the 99-2 position occurs (Siebertz and Heinz, 1977, Heinz and Roughan, 1983). The reduced rate of accumulation of label in M60 at early times (Figure 5-2) is consistent with either possibility. Hhatever the case, the decreased accumulation of prokaryotic M60 in the mutant does not lead to a decrease in the absolute amount of this lipid but, rather, is compensated by increased synthesis of eukaryotic MGD. Thus, by analogy with similar results obtained with a mutant deficient in plastid G3P-acyltransferase (Kunst et al., 1988) it is apparent that that the relative flux through the two pathways of lipid synthesis is tightly regulated in such a way that the physical properties of the lipid component of chloroplast membranes can be adjusted to offset the potentially deleterious effects of a major change in lipid unsaturation in the mutant. He anticipate that the mutant line described here will provide a useful tool for the elucidation of the mechanisms underlying the complex regulation of leaf lipid metabolism. 126 Literature cited Andrews J, E Heinz 1987 Desaturation of newly synthesized monogalactosyldiacylglycerol in spinach chloroplasts. J. Plant Physiol. 131: 75-90 Browse J, P McCourt, CR Somerville 1985a A mutant of lacking a chloroplast specific lipid. Science 227: 763-765 Browse J, P McCourt, CR Somerville 1985b Overall fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152:141-146 Browse J, P McCourt, CR Somerville 1986a A mutant of 3999199951; deficient in C18,3 and C15,3 leaf lipids. Plant Physiol 81: 859-864 Browse J, N Narwick, CR Somerville, CR Slack 1986b Fluxes through the prokaryotic and eukaryotic pathways of lipid synthesis in the 16:3 plant 9999199991; 19911999. Biochem J 235: 25-31 Fischer N, E Heinz, M Zeus 1973 The suitability of lipase from thz99g§ for the analysis of fatty acid distribution in dihexosyl diglycerides, phospholipids and plant sulfolipids. Hoppe-Seyler’s Z Physiol Chem 354: 1115-1123 Frentzen M, E Heinz, TA McKeon, PK Stumpf 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 Gounaris K, J Barber 1983 Monogalactosyldiacylglycerol: the most abundant polar lipid in nature. Trends Biochem Sci 8: 378-381 Haughn GU, CR. Somerville 1986 Sulfonurea-resistant. mutants of 9999199991; 19911999. Mol Gen Genet 204: 430-434 Heinz E, PG Roughan 1983 Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants. Plant Physiol 72: 273-279 Howling 0, LJ Morris, MI Gurr, AT James 1972 The specificity of fatty acid desaturases and hydrolases. The dehydrogenation and hydroxylation of monoenoic acids. Biochim Biophys Acta 260: 10-19 Joyasd J, E Blee, R Douce 1986 Sulfolipid synthesis from 35SO 2' and C]- -acetate in isolated intact spinach chloroplasts. Bischim Biophys Acta 879: 78- 87 127 Khan M, JP Hilliams 1977 Improved thin layer chromatographic method for the separation of the major phospholipids and glycolipids from plant extracts and phosphatidylglycerol and bis(monoacylglycerol) phosphate from animal lipid extracts. J Chromatography 140: 179-185 Kunst L, .1 Browse, CR Somerville 1988 Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol phosphate acyltransferase activity. Proc Natl Acad Sci USA (in press) McCourt P, L Kunst, J Browse, CR Somerville 1987 The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of 99991999919. Plant Physiol 84: 353- 360 McKeon TA, PK Stumpf 1982 Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J Biol Chem 257: 12141-12147 Quinn PJ, HP Williams 1983 The structural role of lipids in photosynthetic membranes. Biochim Biophys Acta 737: 223-266 Raison J 1980 Membrane lipids:structure and function. In PK Stumpf, EE Conn, eds, The Biochemistry of Plants, Vol 4, Academic Press, New York, pp 57-83 Roughan PG, JB Mudd, TT McManus, CR Slack 1979 Linoleate and linolenate synthesis by isolated spinach (Spinacea oleracea) chloroplasts. Biochem J 184: 571-574 Roughan PG, CR Slack 1982 Cellular organization of glycerolipid metabolism. Annu Rev Plant Physiol 33: 97-132 Siebertz HP, E Heinz I977 Labelling experiments on the origin of hexa- and octadecatrienoic acids in galactolipids from leaves. Z Naturforsch 32c: 193-205 Slack CR, PG Roughan, J Terpstra 1976 Some properties of a microsomal oleate desaturase from leaves. Biochem J 155: 71-80 CHAPTER 6 ENHANCED THERMAL TOLERANCE IN A MUTANT 0F ABAELDQE§1§ DEFICIENT IN PALMITIC ACID UNSATURATION Abstract A mutant of 99991999919 19911999, deficient in the activity of a chloroplast n-9 desaturase, accumulates high amounts of palmitic acid (16:0)4, and exhibits an overall reduction in the levels of unsaturation of its leaf lipids. Under standard conditions (22°C, 100-150 uE m'2 s'1 constant illumination) the altered membrane lipid composition has no effect on growth rate of the mutant, net photosynthetic C02 fixation, photosynthetic electron transport, or chloroplast ultrastructure. Similarly, fluorescence polarization measurements indicated that the fluidity of the membranes was not significantly different in the mutant and the wild type. However, at temperatures above 28°C, the mutant grows more rapidly than the wild type. This observation suggests that the altered fatty acid composition results in increased thermal tolerance of the mutant. A comparison of the chloroplast membranes of the mutant and wild type by two additional criteria, temperature-induced fluorescence yield enhancement and whole chain electron transport of membranes preincubated at high 128 129 temperatures, confirmed the superior thermal properties of the mutant. Thus, it is apparent that the lipid composition plays a role in temperature adaptation of chloroplast membranes. Electrophoretic analysis of chlorophyll-protein complexes and Chl fluorescence measurements have shown that the oligomeric form of LHCP is slightly more labile in the mutant. 0n the other hand, the observed change in the efficiency of excitation energy distribution from LHCP to the reaction centers does not seem to affect the normal photosynthetic performance of mutant plants. Introduction The chloroplast membranes of higher plants have a distinct fatty acid composition characterized by an unusually high proportion of polyunsaturated acyl groups. Depending on the plant species, trienoic fatty acids (18:3 and 16:3) comprise up to 80% of total fatty acids in the membrane lipids of this organelle (Murphy, 1986). Furthermore, the atypical fatty acid A3, 19999-16zl is found esterified to the 99-2 position of the major chloroplast phospholipid, phosphatidylglycerol (PG). These features of chloroplast lipids are common and remarkably constant in a wide variety of species, suggesting that the fatty acid composition is important for maintaining photosynthetic function. In an attempt to elucidate the significance of fatty acid composition and unsaturation in photosynthesis, many different approaches have been used. They include reconstitution of photosynthetic components with lipid mixtures (Gounaris et al., 1983), alterations of lipids 19 9jtu 130 by heat (Gounaris et al., 1984), chemical inhibitors (Leech et al., 1985), lipase treatment (Rawyler and Siegenthaler, 1981), or hydrogenation of unsaturated fatty acids (Vigh et al., 1985, Quinn and Williams, 1983), as well as correlation of events during chloroplast development (Leech et al., 1973, Galey et al., 1980). However, these approaches have not proven successful in unequivocally establishing the specific role of acyl group composition in thylakoid membrane function. As an alternative approach to this problem we have isolated a number of mutants with reduced levels of unsaturation of their leaf lipids (Browse et al., 1985a, Browse et al., 1986, Browse et al., manuscript in preparation, Chapter 5). Despite a detailed analysis of these mutants, we have not been able to detect any significant changes in photosynthetic properties of the mutants investigated. On the other hand, we have provided evidence that large decreases in the proportion of trienoic acids lead to changes in chloroplast ultrastructure (McCourt et al., 1987). Here I describe physiological studies of a mutant, designated £993, deficient in the activity of the chloroplast n-9 desaturase, which specifically converts palmitic acid (16:0) at position 99-2 of MGD to 919-16:1 (Chapter 5). As a consequence, the mutant accumulates high levels of palmitic acid, and lacks polyunsaturated 16-carbon fatty acids. Therefore, it should be a useful tool in fUrther evaluating the relationship between trienoic acid content and chloroplast structure and function. 13] Material and methods HAW The mutant line J867 was isolated from the Columbia wild type of 99991999919 99911999 (L.) Heynh. as previously described (Browse et al., 1985a). The mutant carries a defective allele of a locus, designated £993, required for the desaturation of palmitic acid at position 99-2 of MGD (Chapter 5). It was backcrossed to the wild type four times before being used for the physiological experiments described here. Unless otherwise indicated, plants were grown at 22°C 2 5'1) on a under continuous fluorescent illumination (100-150 uE m' perlite:vermiculite:sphagnum mixture (1:1:1) irrigated with mineral nutrients (Haughn and Somerville, 1986). MW Plants were germinated at 22°C and grown under conditions described above. After seven days the temperature was adjusted as noted in the text. Samples of four plants were harvested at two day intervals, and the fresh weights of the aerial portions were measured. The relative growth rate (w'l) was determined as the slope of the natural logarithm of the average fresh weight in mg plotted against time since the temperature adjustment. The increase in fresh weight was linear only during the first six days at all temperatures. The growth slowly ceased afterwards, especially at temperatures above 30°C. Therefore, only the values obtained during the initial 6 days after temperature adjustment were used for relative growth rate determinations. 132 WWW Extracts were prepared by grinding leaves harvested at rosette stage (3 weeks) in cold 20 mM Tricine-KDH (pH 8.4), 5 mM MgCl2 and 2.5 mM EDTA. Insoluble matter was removed by centrifugation at 100 x g for 10 min and Chl, proteins and lipids were assayed essentially as described (Chapter 4). Fatty acid composition of total leaf lipids was determined according to Browse et al. (1985b). WWW Chloroplast membranes were prepared by grinding washed leaves in cold 450 mM sorbitol, 20 mM Tricine-KOH (pH 8.4), 10 mM NaCl, 10 mM EDTA and 0.1% (w/v) BSA. The homogenate was filtered through Miracloth (Calbiochem, La Jolla, CA) and centrifuged at 3000 x g for 5 min. The pellet was washed with cold 10 mM Hepes (pH 7.9), 10 mM NaCl, 5 mM EDTA, and resuspended in 20 mM Hepes (pH 7.9), 10 mM NaCl, 2 mM MgCl2, 2.5 mM EDTA and 0.1% (w/v) BSA. Thylakoid preparations for SDS-PAGE lacked BSA, while MgCl2 was omitted in some fluorescence measurements, as described in the text. Chl was assayed by the method of MacKinney (1941). m n - r h Chloroplast membranes were isolated as described above. Pigment-protein electrophoresis was performed according to the method of Andersson et al. (1982), except that the sodium dodecyl sulfate (SDS, Sequanal Grade, Pierce, Rockford, IL) to Chl weight ratio was adjusted to 3.75 : l. 133 L-[3551-Methionine labeling of thylakoid proteins, protein extraction .1. ,.-. u-. .l. i -.. . .9 .- .- -l- .... - Labeling of chloroplast membrane proteins with [35$]Methionine, protein extraction, and two-dimensional SDS-polyacrylamide gel electrophoresis were performed as described in Chapter 4. Wm An estimation of the microviscosity of the thylakoid membranes was obtained by determining the steady state fluorescence polarization of the hydrophobic fluorophore DPH (Aldrich, Milwaukee, HI)(Barber et al., 1984). DPH (3 mM stock in tetrahydrofuran) was added directly to the thylakoid extract (50 ug Chl ml'l) to a final concentration of 5 uM, and incubated for 40 min in the dark at room temperature. The membranes were then centrifuged at 3000 x g for 5 min and diluted with 100 mM sorbitol, 20 mM Hepes (ph 7.9), 10 mM NaCl to a final concentration of 10 ug ml'l Chl and 1 uM DPH. Fluorescence polarization measurements were carried out as described by McCourt et al. (1987). Wm Hhole chain electron transport and PSI activity were measured in a Rank oxygen electrode with 1200 uE m'2 s'1 PAR, by adding an aliquot of thylakoid membrane extract to 1 ml of resuspension buffer to a final concentration of 20 ug Chl ml'l, as previously described (Kunst et al., manuscript in preparation). PSII activity was assayed as DPIP reductidn at 580 nm using a Hitachi 100-60 spectrophotometer according to Steinback et al., 1979). PSII light response was determined as described in Chapter 4. All assays were performed at 25°C. I34 nc e Room temperature fluorescence induction transients of isolated thylakoids were measured by the method of Paterson and Arntzen (1982). For low temperature (77K) fluorescence determinations, aliquots of thylakoid suspension were diluted to 10 ug ml'1 in 60% glycerol (v/v), and sodium fluorescein was added as an internal standard to a final concentration of 2 uM (Krause et al., 1983). Samples were then frozen in capillary tubes (0.5 mm inner diameter) in liquid N2. Fluorescence was scanned from 470-800 nm at 77K on an SLM spectrofluorometer (McCourt et al., 1985). WWW Temperature induced fluorescence yield enhancement was measured on dark adapted whole detached leaves by minor modifications of the method of Schreiber and Berry (1977). Heak (0.3 uE m'2 s'l) monochromatic light at 480 nm with 4 nm half-bandwidth was directed at a 450 angle to a leaf placed between two sheets of 0.1 mm thick mylar in a water filled cuvette in the SLM spectrofluorometer. Fluorescence emission from the leaf surface was monitored at 700 nm with 2 nm half-bandwidth. The temperature of the sample was increased at a rate of 1°C min'1 and the fluorescence intensity was recorded simultaneously. We: Methods for short term gas exchange measurements on single intact 99991999919 plants have been described (Somerville and Ogren, 1982). For each plant, measurements of dark respiration and photosynthesis at 100 and 300 uE m.2 s'1 were carried out, followed by fresh weight I35 determination and Chl assay (MacKinney, 1941) of the aerial portion of the plant. W Leaves from 3-week old plants were fixed in 2% glutaraldehyde (v/v) in 100 mM sodium cacodylate buffer (pH 7.2) for 2 h at 4°C, washed in the same buffer, and stained with 1% 0504 (w/v) for 1 h at 4°C. The samples were then rinsed with double distilled water and dehydrated in a graded ethanol series (70%, 80%, 95%, 100%). The infiltration with Spurr’s epoxy resin was done in two steps at room temperature. The samples were first incubated with a mixture of 100% ethanol and Spurr’s resin (1:1, v/v) for 1 h, followed by 100% Spurr’s resin for 8 h. Thin sections were poststained with 5% (v/v) uranyl acetate and lead citrate (Reynolds, 1963), and examined in a JEOL 100CX electron microscope. 99W Chloroplast number per cell was counted in isolated protoplasts prepared as described (Kunst et al., 1988). A20 ul sample of protoplast suspension was pipetted onto a microscope slide and the protoplasts were flattened by application of a coverslip. The chloroplasts formed a monolayer within cells and could be easily counted (McCourt et al., 1987). 136 Results f r w Several lines of evidence support the proposal that membrane lipid composition might play a role in thermal adaptation of plants. It was, therefore, of interest to examine the effects of £993 mutation on growth of the £993 mutant at different temperatures ranging from 10-34°C (Figure 6-1). The rate of increase in fresh weight of mutant and wild type was very similar up to 28°C. At temperatures greater than 28°C the mutant plants grew more rapidly than the wild type (Figure 6-1). This observation suggests that the altered fatty acid composition results in increased thermal tolerance of the £993 mutant. However, this was apparent only during the first 6 days at temperatures above 30°C, since the growth rate is greatly reduced after prolonged exposure to high temperatures, and both mutant and wild type plants eventually turn chlorotic. WW Since the £993 mutation primarily affects chloroplast lipids, the apparent effect of the mutation on thermal tolerance is most likely due to an effect on chloroplast membranes. Therefore, we examined the thermal stability of chloroplast membranes as measured by Chl fluorescence in intact leaves of mutant and wild type (Figure 6-2). A heat-induced increase in room temperature fluorescence has been attributed to the physical separation of the LHCP from the PSII core, which blocks the excitation energy transfer and leads to reemission of the energy as fluorescence. Therefore, Chl fluorescence is considered 137 1395 -' - o.4 - 1 T 3 0 1.; 0.3 - _ h 5 fl 3 C) h 9’ 0.2 - _ O .2 H 2 0 “3 o.1 - _ l l 1 l J l J I l 1 1O 13 16 19 22 25 28 31 34 37 Temperature (° C) Figure 6-1. Effect of temperature on the relative growth rate of wild type and mutant 99991999919. 138 0 J36? 1. VVT Relative fluorescence H I 4 I 25 35 45 55 Temperature (°C) Figure 6-2. Temperature induced fluorescence enBancement yield of wild type and mutant leaves. Plants were grown at 22 C. The arrows indicate estimates of threshold temperatures at which fluorescence is enhanced. Each point represents the mean 1 SD (n=-3). 139 an intrinsic probe of lipid-protein interaction (Lynch and Thompson, 1984) and an indicator of photosynthetic membrane stability (Raison et al., 1982, Schreiber and Berry, 1977). The experiment was performed by slowly heating detached leaves at a rate of 1°C min'1 up to 57°C and continuously monitoring fluorescence levels. At approximately 43°C the fluorescence started to rise rapidly in wild type leaves, while the transition in the level of fluorescence did not occur until 45°C in the mutant. Thus, by this criterion, the £993 mutation appears to confer increased thermal stability upon chloroplast membranes. . - .- . - - ., . . . , ,- - - .l ,. .. The effect of temperature on the stability of chloroplast membranes of the mutant line J867 and wild type was also measured by incubating isolated membranes in darkness for 10 min at various temperatures from 25-45°C, and then measuring whole-chain electron transport at 25°C. As shown in Figure 6-3, electron transport rates of both mutant and wild type declined steadily as the preincubation temperature was increased. However, there was a significant difference in the apparent stability of the mutant and wild-type membranes. In the wild type, electron transport activity drops to 60% after 10 min at 35°C, while the mutant exhibits a decrease of only 15% under the same conditions. The kinetics of thermal inactivation of photosynthetic electron transport was examined by incubating isolated chloroplast membranes at 40°C for various times, and then assaying the whole chain activity at 25°C. The results of this experiment (Figure 6-4) are consistant with a 140 l l l l l t 8 m 100— c N :- 80" _ 8 b ‘6; so— 4 .2: :3 O _ ._ 994° 5 I: 20- ‘ >. m 2 o 0- ‘ .C n. l 1 I l l 25 30 35 4O 45 Temperature (°C) Figure 6-3. Effect of temperature on photosynthetic electron transport in chloroplast membranes from wild type and mutant 999bid099i9. Activity is expressed relatige to that obtained with membranes preincubated in darkness at 4 C for 10 min. Th9l ma_xlimal rates for the mutant_ind_wild type were 161.2 umol 0 mg Chl h and 146.9 umol 0 mg Chl h , respectively. Each poing represents the mean 3; SD (n-4 . 141 l I T l l l t . ° 0J867 g 100— : .WT fl 3 so- .. S =9 so 0 > '_ — :2:= ° 3 o - _ 994° 5 c 20- _ > m .9 O O- .. a‘. 1 L l 1 l l O 2 4 6 8' 10 ' Time(min) - Figure 6-4. Photosynthetic electron transport activity in chloroplaft membranes from wild type and mutant Arabidopsis preincubated at 40 C for various times indicated. Activity is expressed regative to that obtained with membranes preincubated in darkness at 4 C for 10 min. Maximal_fates for the mutant and the wild type were 161.2 umol 0 mg Chl h and 146.9 umol 0 mg Chl h , respectively. Each poinz represents the mean i SD (6-4). 142 delayed thermal inactivation of whole chain electron transport in chloroplast membranes of mutant plants. Therefore, it seems apparent that the composition of the lipid matrix plays a role in thermal tolerance of the photosynthetic membranes. W To examine the effect of lipid composition on the fluidity of thylakoid membranes, we carried out fluorescence polarization measurements on isolated membranes from wild type and mutant 99991999919 (Figure 6-5). Polarization (P) values of wild type membranes are similar to those reported for other plant species (Barber et al., 1984), and indicate a highly fluid lipid bilayer. The thylakoid membranes of the £993 mutant had slightly higher P values, suggesting a small decrease in membrane fluidity. A similar result was obtained for another 99991999919 mutant with reduced amounts of polyunsturated fatty acids (McCourt et al., 1987). However, the difference in both mutants was below the limit of statistical significance. o “‘9. ._ ‘ iOQ 01100 ' 01 on .‘o o o ' I 01 ‘I 2 5’1) and various Under standard light conditions (100-150 uE m' temperatures ranging from 10-34°C, the mutant exhibits a slight chlorotic phenotype, due to a 15-20% reduction in Chl per unit fresh weight (Table 6-I). To determine if the reduction in Chl is related to the £993 mutation, 60 F2 plants from a HT x J867 cross were tested for cosegregation of the two phenotypes. 0f 16 plants which showed increased levels of palmitic acid (16:0), all had reduced Chl levels on a fresh weight basis. All other plants had normal levels of Chl and 143 I I l l I 1 0.1867 CDVVT' 0.4— 4 E C .9. f')_ , ~ “03" ' l_ m *0 E 1' .‘2 O c: 0.9.. _ C) 0 C 8 :3 (L1 - _ E- O 2 “- o I I l J I I O 10 20 3O 40 50 Temperature (°C) Figure 6-5. Effect of temperature on DPH fluorescence polarization of chloroplast membranes from wild type and mutant 99a9i9099j9. Each point represents the mean 1 SD (n810). 144 Table 6-1. Relative amounts of lipid, chl and protein in mutant and wild type 99991999919 leaves and chloroplast membranes. Values are means i SD (n-3). Wild type J867 LEM Chl/fwt (mg/g) 1.67 i 0.01 1.39 i 0.07 Chl a/b ratio 2.94 i 0.10 3.21 i 0.05 Lipid/Chl (g/g) 2.48 i 0.07 2.58 1 0.15 Protein/Chl (g/g) 38.46 i 0.15 38.56 i 1.60 Protein/Lipid (g/g) 15.56 9 0.90 14.95 1 0.30 WW Lipid/Chl (g/g) 2.12 i 0.08 2.53 i 0.09 Protein/Chl (g/g) 12.27 9 0.50 16.66 i 0.92 Protein/Lipid (g/g) 5.79 + 0.24 6.79 1 0.29 145 wild type levels of 16:0. Cosegregation of the two traits suggests that both phenotypes are caused by the same mutation. Since all the Chl in higher plants is believed to be associated with proteins (Markwell et al., 1979), a decrease in Chl content suggests changes in one or more of the chl-protein complexes of thylakoid membranes in J867 mutant. The higher a/b ratio of mutant leaves also suggests an alteration in the stoichiometry of LHCP to PSII and PSI. Therefore, we separated the chl-protein complexes of mutant and wild-type membranes by SDS-polyacrylamide gel electrophoresis in order to examine whether certain Chl-protien complexes are preferentially affected (Figure 6-6). The identity of the bands resolved by this method (CP1a, CPI, LHCPl, LHCPZ, CPa, LHCP3 and free pigments) was established by comparing the absorption spectra of individual bands (results not presented) with published values (Anderson et al., 1978). A comparison of the separation patterns of the chl-protein complexes from the wild type and mutant revealed that the mutant contains a slightly lower amount of LHCPl, the presumed LHCP oligomer. A similar observation reported for another lipid mutant of 99991999919, which was deficient in the 19999-16:1 acyl group (McCourt et al., 1985), was interpreted as indicating that an unsaturated fatty acid at sn-2 position of chloroplast PG might be important in stabilizing LHCPl against 505 mediated dissociation. h i ara i To estimate the effects of reduced levels of unsaturation on photosynthetic properties of the £993 mutant, we measured the rates of C0 fixation in mutant and wild-type plants (Table 6-11), as well as 2 146 A AAA AA 3. A 3 Figure 6-6. Chl-protein complexes of chloroplast membranes from wild type and mutant 9r9bid995i9. The nomenclature is from Anderson et al. (1978). ' 147 Table 6-II. Photosynthetic activities in mutant and wild type Arapiggneis- Hild type J867 £9,391.19! -1 -1 - ug C02 mg Chl h Darkness -a487.8 i 13 -498.1 1 34 100 uE m'2 s‘1 2432.5 1 100 2761.5 2 23 300 uE m'2 s'1 4766.9 1 205 5452.2 1 122 ug COZg fwt'1 h'1 Darkness -565.1 i 23 -563.8 1 43 100 uE m'2 s‘1 2802.2 1 140 3127.0 1 56 300 uE m'2 s'1 5535.4 1 235 5015.0 2 197 mm ,1 _1 umol 02 mg Chl h Hhole chain 118.0 1 2 119.0 1 2 PSI 348.0 1 8 351.1 1 7 PSII 347.9 9 35 360.4 1 31 a Net C02 evolution is indicted here as a negative value. 148 electron transport activities in isolated chloroplast membranes. C02 fixation rates of both mutant and wild type were extremely similar when expressed on the basis of fresh weight. However, the mutant did exhibit 15% higher rates per unit Chl relative to the wild type (Table 6-11), probably due to a reduction of similar magnitude in leaf Chl content. There were also no major differences in the whole chain, or P511 and PSII partial electron transport activities between the mutant and wild type (Table 6-11, Figure 6-7). These data indicate that the changes in the fatty acid composition of chloroplast membranes from the mutant do not affect rates of photosynthetic electron transport. Wells; Assuming that the LHCP oligomer is the native form 19 9119 (Kuhlbrandt, 1984), it is possible that the reduced stability of LHCPl in the £993 mutant might be reflected in less efficient transfer of excitons from LHCP to the reaction centers. Low temperature fluorescence spectra are sensitive indicators of the efficiency of energy distribution between Chl-containing components of chloroplast membranes. Therefore, we examined 77K fluorescence spectra of the isolated chloroplast membranes of mutant and wild type. (Table 6-111, Figure 6-8). Reduced excitation energy transfer from LHCP to the photosystems would be expected to result in an increase of LHCP fluorescence (685nm) relative to PSII (695nm) and PS1 (735nm) emission maxima. Indeed, the F685/F734 ratio obtained for the mutant was higher than the wild type (Table 6-111) in both presence or absence of MgClZ. By normalizing emission values of individual peaks at 685 nm and 734 nm to that of the internal standard, fluorescein, it became clear that the 149 (a) g 200 b I I I I I J E O o géisop - 2 o E 3 100 " c: c .2 a 2 so a o .1357 ‘ E 0 WI' 0 o . N o I 1 1 l I c: o 250 500 750 1000 1250 irradiance (uE/ mz/s) E _ I I I I I > 400 (b) .c 0 I: .§ 300- - 2 2 3 200 - 6 - C o z 8 E? 100 a 1' o 3357 g 0 WT o o o I l l l l c? o 250 500 750 -uxx> 1250 irradiance (ILE/mzls) 2m .— l I I I I d (c) 150 - 4 02 evolution (“moles/mg chl/h) 8 i so - O .1367 C WT o l l l l I 0 250 500 750 1000 1 250 lrradiance (118/male) Figure 6-7. Light response curves for (A) whole chain, (8) PSI and (C) PSII electron transport by isolated chloroplast membranes from qud type and mutant Ar99i9999j9. Each point represents the mean i SD (n-3). 150 Table 6-111. Room temperature fluorescence induction and low temperature (77K) fluorescence of isolated thylakoids. Hild type J867 ROOM TEMPERATURE FLUORESCENCEé F0 1213 2 19 1231 i 25 Fm 3530 1 90 3220 2 57 Fv/Fo 1.919 0.05 1.52 1 0.07 77K FLUORESCENCEb 2+ +M9 F585 3122 1 66 3235 2 68 F734 4019 i 84 3505 i 98 Fags/F734 0.78 i 0.001 0.90 3 0.01 _M92+ F585 ‘ 3885 2 96 4105 i 93 F735 5921 1 85 4921 2 117 Fees/F735 0.55 1 0.005 0.84 1 0.005 a Room temperature fluorescence was measured in the presence of 10 uM DCMU, n-10; Values are expressed in arbitrary units. b MgCl 77K fluorescence was measured in the presence and absence of 5 mM , n-6. Values are normalized to the emission peak of 2 uM fluogescein, which was assigned an arbitrary value of 9000. 151 .—’ Relative Fluorescence intensity llllLLlllllll 550 700 - 750 am Wavelength (nm) Figure 6-8. Chl fluorescence spectra of chloroplast membranes from wild type (-—-) and mutant (----) Arabidopsis in the absence of MgClz. The curves were normalized to the same value at 500 nm. 152 change in F685/F734 ratio of the mutant is due to both a lower absolute fluorescence emission at 734 nm and a concomitant absolute increase in Chl fluorescence at 685 nm. This result is consistent with a reduction in the amount of light energy transferred from LHCP to the two photosystems. In order to extend this observation the variable fluorescence of the mutant and wild type was measured at room temperature (Table 6-111). Room temperature Chl fluorescence is emitted from PSII and, in the presence of DCMU, depends only on the exciting light intensity, the number of Chl molecules active in transferring excitation energy to PSII reaction centers and the efficiency of transfer. Fo, the initial fluorescence level, is similar in the wild type and mutant membranes. 0n the other hand, maximal f1uorescence (Fm), and the proportion of Chl active in photochemistry (Fv/Fo), are 15% lower in the £993 mutant (Table 6-111). WWW He have recently provided evidence that fatty acid composition of chloroplast lipids may be an important factor regulating organelle biogenesis (McCourt et al., 1987). Thus, we examined the effect of the £993 mutation on chloroplast ultrastructure. The electron micrographs (Figure 6-9) of the mutant and wild type showed no obvious differences in chloroplast size, the arrangement of chloroplast membranes, the extent of stacking, or any other structural feature of the chloroplast. In addition, the number of chloroplasts per cell in the mutant (44.7 i 15) and wild type (45.0 i 13) were almost identical. 153 Figure 6-9. Transmission electron micrographs of chloroplasts from rosette leaves of (A) wild type and (8) mutant 9rabj9999i9. Bar - 1 um. 154 To examine the effect of the altered fatty acid composition on protein content of chloroplast membranes more thoroughly, we labeled the proteins with 35S-Methionine and analyzed them by SDS-polyacrylamide gel electrophoresis. The polypeptide pattern of mutant and wild type plants (Figure 6-10) was almost identical, providing additional explanation as to why there were no apparent changes in chloroplast organization of the £993 mutant. However, there are subtle quantitative changes in several polypeptides associated with the £993 mutation (Figure 6-10). The amounts of several polypeptides are slightly reduced in comparison with the wild type, but there are also a few proteins that are more abundant in chloroplasts of the mutant. It would be interesting to know if any of these proteins play a role in the increased thermal stability of £993 membranes. Discussion A detailed comparison of the leaf lipid composition of the mutant line J867 and wild type has indicated that the mutant is deficient in the activity of a chloroplast n-9 desaturase, due to a single nuclear mutation at £993 locus (Chapter 2). This lesion results in the accumulation of high amounts of 16:0 acyl group, and an overall reduction in the levels of membrane lipid unsaturation. Despite substantial changes in the membrane lipid composition of the £993 mutant, it exhibits normal growth, chloroplast ultrastructure and photosynthetic activity under standard conditions. 155 . 7.7 5.1 l— 1 WT 116- 84- STAB - 58- - w‘ 485' _ - -030? ’ -. e . a 1 © ' ~13 [36.5- ' 1.67 ' 116- 84_ 3-5 . . 58- - - - g” 485‘ 7"“? ° . 0 .° 2° 5 e '- 0E! 38.5- ’ Figure 6-10. Autoradiographs of [35$]-Methionine labeled proteins of chloroplast membranes from wild type and mutant Arabid9psi9 separated by two—dimensional SDS-polyacrylamide gel electrophoresis. The numbers at the top represent apparent pH, and those at the left apparent molecular mass. El , proteins which are more abundant in the mutant than in the wild type; 0 , proteins which are more abundant in the wild type than in the mutant. 156 The growth rate of the mutant was higher than the wild type at elevated temperatures above 28°C (Figure 6-1). This effect was also observed in our analyses of the two other 99991999919 mutants (McCourt et al., 1987, Chapter 4) with reduced amounts of polyunsaturated fatty acids. The factors involved in thermal stability of chloroplast membranes have not been elucidated. However, the results of Pearcy (1978), who reported the absence of 16:3 acyl group from MGD and a decrease in linolenic acid (18:3) with concomitant increases in more saturated fatty acids in membrane lipids of 91919199 19911£99919 at high temperatures, suggest that changes in physical properites of the membranes might be the basis for thermal acclimation. Additional evidence was provided by Raison et al. (1982), whose work on 999199 91999999 demonstrated that the decline in lipid unsaturation at high temperatures results in a less fluid lipid bilayer. The changes in fatty acid composition of leaf polar lipids of £993 mutant did not have a significant effect on chloroplast membrane fluidity. 0n the other hand, the mutant exhibited superior thermal stability by two criteria: a higher threshold temperature at which fluorescence yield was enhanced, and a slower rate of inactivation of whole chain electron transport at high temperatures. Therefore, it is concievable that the altered composition of the lipid matrix affects the stability of chloroplast membranes in the £993 mutant. It may well be that the changed physical properties of the lipid bilayer 999 99 render the £993 membranes more stable. Alternatively, the changes in lipid-protein interactions, or changes in the amounts of specific proteins, due to alterations in leaf lipid composition, might confer increased thermal stability upon chloroplast membranes of the mutant. 0n the basis of the 157 results presented here, we cannot distinguish among these possibilities. However, there is evidence that the phase separation of non-bilayer lipids and the dissociation of light harvesting Chl-protein complexes of chloroplast membranes occur within the same temperature range (Gounaris et al., 1984). This correlation suggests that the stability of the protein-lipid association might be the most important factor determining the overall thermal stability of photosynthetic membranes. An interesting feature of the £993 mutant is a 15% decrease in the amount of Chl per fresh leaf weight. Cosegregation analysis of altered fatty acid composition with reduced Chl content in the leaves of the mutant indicated that both these phenotypes are caused by the £993 mutation. An increase in Chl a/b ratio (Table 6-1) revealed that Chl b is lost from £993 chloroplasts. Since Chl b is associated with LHCP, a reduction in the amount of Chl b is indicative of a decrease in LHCP Chl-protein complex in chloroplast membranes. However, the loss of Chl b accounts for less than 25% of the total Chl absent from the leaves of the mutant, suggesting that other Chl-protein complexes are affected as well. Chl fluorescence measurements support this conclusion. The fluorescence emission at 695 nm and 734 nm, attributed to PSII snd PSI, respectively, is lower in £993 membranes, and there is a corresponding increase in LHCP fluorescence yield at 685 nm. Therefore, the excitation energy transfer from LHCP to PSII and PS1 reaction centers is either less efficient, or there are structural changes within the PSII and PS1 antennae of the mutant. Lower room temperature fluorescence emission of the mutant relative to the wild type indicates that the amount of Chl active in PSII photochemistry is 158 reduced. Therefore, it seems that £993 related changes in lipid composition result in a reduction of Chl associated with PSII and PS1 reaction center complexes. However, it is apparent that this difference between the mutant and the wild type does not affect the overall photosynthetic performance of £993 mutant under the growth conditions examined. Literature cited Anderson JM, JC Haldron, SH Thorne 1978 Chlorophyll-protein complexes of spinach and barley thylakoids. FEBS Lett 92: 227-233 Andersson 8, JM Anderson, IJ Ryrie 1982 Transbilayer organization of the chlorophyll-proteins of spinach thylakoids. Eur J Biochem 123: 465-472 Barber J, RC Ford, RAC Mitchell, PA Millner 1984 Chloroplast thylakoid membrane fluidity and its sensitivity to temperature. Planta 161: 375-380 Berry J, Bjorkman 1980 Photosynthetic response and adaptation to temperature in higher plants. Annu Rev Plant Physiol 31: 491-543 Browse J, P. McCourt, CR Somerville 1985a A mutant of 99991999919 lacking a chloroplast-specific lipid. Science 227: 763-765 Browse J, P. McCourt, CR Somerville 1985b Overall fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152: 141-146 Browse J, P. McCourt, CR Somerville 1986 A mutant of 99991999919 deficient in C18,3 and C16,3 leaf lipids. Plant Physiol 81: 859-864 Browse J, L Kunst, S Anderson, CR Somerville 1988 A Mutant of Ara9i9999i9 deficient in the chloroplast 16:1/18:1 desaturase. In preparation Galey J, B Francke, J Bahl 1980 Ultrastructure and lipid composition of etioplasts in developing dark-grown wheat leaves. Planta 149: 433-439 159 Gounaris K, DA Mannock, A Sen, APR Brain, NP Nilliams, PJ Quinn 1983 Polyunsaturated fatty acyl residues of galactolipids are involved in the control of bilayer/non-bilayer lipid transitions in higher plant chloroplasts. Biochim Biophys Acta 732: 229-242 Gounaris K, APR Brain, PJ Quinn, NP Williams 1984 Structural reorganization of chloroplast thylakoid membranes in response to heat stress. Biochim Biophys Acta 766: 198-208 Haughn Gil, CR Somerville 1986 Sulfonylurea-resistant mutants of Arabidopsis thaliana. Mol Gen Genet 204: 430-434 Krause GH, J-M Briantais, C Vernotte 1983 Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77K. 1. pH dependent quenching. Biochim Biophys Acta 723: 169-175 Kuhlbrandt N 1984 Three dimensional structure of the light-harvesting chlorophyll a/b protein complex. Nature 307: 478-480 Kunst l” .1 Browse, CR Somerville 1988 Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol phosphate acyltransferase activity. Proc Natl Acad Sci USA, In press Leech RM, MG Rumsby, NH Thomson 1973 Plastid differentiation, acyl lipid, and fatty acid changes in developing green maize leaves. Plant Physiol 52: 240-245 Leech RM, CA Nalton, NR Baker 1985 Some effects of 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (SAN 9785) on development of thylakoid membranes in flgrdgum yulgane L. Planta 165: 277-283 Lynch D, GA Thompson 1984 Chloroplast phospholipid molecular species alterations during low temperature acclimation in W. Plant Physiol 74: 198-203 MacKinney G 1941 Absorption of light by chlorophyll solutions. J Biol Chem 140: 315-322 Markwell JP, JP Thornber, RT Boggs 1979 Higher plant chloroplasts: evidence that all the chlorophyll exists as chlorophyll-protein complexes. Proc Natl Acad Sci USA 76: 1233-1235 McCourt P, J Browse, J Watson, CJ Arntzen, CR Somerville 1985 Analysis of photosynthetic antenna function in a mutant of Arabidopsis thaliana (L.) lacking trans-hexadecenoic acid. Plant Physiol 78: 853-858 McCourt P, L Kunst, J Browse, CR Somerville 1987 The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis. Plant Physiol 84: 353-360 160 Murphy DJ 1986 The molecular organization of the photosynthetic membranes of higher plants. Biochim Biophys Acta 864: 33-94 Paterson DR and CJ Arntzen 1982 Detection of altered inhibition of photosystem 11 reactions in herbicide-resistant plants. In M Edelman, R Hallick, NH Chua, eds, Methods in Chloroplast Molecular Biology. Elsevier, Amsterdam, pp 109-119 Quinn PH, HP Hilliams 1983 The structural role of lipids in photosynthetic membranes. Biochim Biophys Acta 737: 223-266 Pearcy RH 1978 Effect of growth temperature on the fatty acid composition of the leaf lipids in Atniplgx lentifprmis (Torr.) Hats. Plant Physiol 61: 484-486 Raison JK, JKM Roberts, JA Berry 1982 Correlations between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant Ngrium gleam to growth temperature. Biochim Biophys Acta 688: 218-228 Rawyler' A, PA. Siegenthaler 1981 Transmembrane distribution of phospholipids and their involvement in electron transport as revealed in phospholipase A2 treatment of spinach thylakoids. Biochim Biophys Acta 635: 348-368 Reynolds ES 1963 The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17: 208-212 Schreiber U, JA Berry 1977 Heat induced changes in chlorophyll fluorescence in intact leaves correlated *with damage in the photosynthetic apparatus. Planta 136: 233-238 Somerville CR, HL Ogren 1982 Isolation of photorespiration mutants in A_ab1ggp§1§ . In M Edelman, R Hallick, NH Chua, eds, Methods in Chloroplast Molecular Biology. Elsevier, Amsterdam, pp 129-138 Steinback KE, JJ Burke, CJ Arntzen 1979 Evidence for the role of surface exposed segments of the light harvesting complex in cation-mediated control of chloroplast structure and function. Arch Biochem Biophys 195: 546-557 Vigh L, F Joo, M Droppa, L1 Horvath, G Horvath 1985 Modulation of chloroplast membrane lipids by homogeneous catalytic hydrogenation. Eur J Biochem 147: 477-481 161 CHAPTER 7 CONCLUDING REMARKS Summary Each membrane in the cell has a distinct lipid composition with respect to both the head group and the acyl groups. However, the role of lipids and fatty acids in proper functioning of cell membranes remains uncertain. In order to directly address the question of how the lipid composition of thylakoid membranes affects chloroplast structure and function, we isolated a series of mutants of the crucifer Arabidopsis Lhallgna (L.) Heynh., with specific alterations in leaf lipid metabolism. Since relatively large changes in membrane lipids, like changes in the amount of various lipid headgroups could be lethal, we have concentrated on screening for mutants with altered leaf fatty acid composition by direct analysis using gas chromatography. In this dissertation I have described the isolation of two classes of mutants, actl and fagfi, deficient in the activity of chloroplast enzymes G3P acyltransferase and n-9 desaturase, respectively. I have characterized these mutants genetically and biochemically (Chapters 3 and 5), and examined the physiological consequences of the changes in their leaf membrane lipids (Chapters 4 and 6). 162 In the first mutant analyzed, a single recessive nuclear mutation at the actl locus causes a deficiency in the activity of the G3P acyltransferase, the first enzyme of the prokaryotic (plastid) pathway of glycerolipid synthesis. The principal results and conclusions of the work on actl mutant are: (1) The lesion in the plastid pathway of lipid biosynthesis does not cause the accumulation of precursors within the organelle, but results in a redirection of fatty acids towards the cytoplasmic sites of lipid synthesis in the endoplasmic reticulum. (2) The increased synthesis of glycerolipids by the eukaryotic pathway in the endoplasmic reticulum compensates for the loss of the prokaryotic pathway and provides, with the exception of the PG, almost normal amounts of all the lipids required for chloroplast membrane biogenesis. (3) Differences in the distribution of radioactivity in leaf polar lipids between the wild type and the mutant after long term labeling provide definitive evidence for the parallel operation of prokaryotic and eukaryotic pathways in wild-type plants. (4) The fatty acid composition of leaf lipids of the mutant is altered, since the acyltransferases of the two biosynthetic pathways exhibit different substrate specificities. (5) 3911 mutation has a pronounced effect on structural features of chloroplasts. The number of appressed regions is increased in the mutant, but the number of membranes per stack is significantly reduced relative to the wild type. These changes were not associated with a major change in the amount of Chl a/b binding proteins, suggesting that 163 the model for membrane appression based on the properties of LHCP is incorrect or incomplete. (6) Extensive changes in leaf lipid composition do not affect growth or development of the mutant under standard conditions. However, at temperatures above 28°C the mutant grows slightly more rapidly. A comparison of the wild type and the mutant by two additional criteria: temperature-induced fluorescence yield enhancement and the rate of inactivation of whole chain electron transport activity in isolated chloroplast membranes preincubated at various increasing temperatures suggests an increased thermal stability of the photosynthetic membranes of the mutant. The second mutant that 1 studied lacks polyunsaturated l6-carbon fatty acids and shows a corresponding increase in the amount of the 16:0 acyl group. These changes indicate that the mutant is deficient in the activity of a chloroplast n-9 fatty acid desaturase, due to a single nuclear mutation at a locus designated £6513- The major conclusions based on the analysis of the fadfi mutant are: (1) The mutation at £3513 locus affects both chloroplast and extrachloroplast lipids. Thus, there is a substantial transfer of 16:0 acyl groups from MGD to all the leaf polar lipids, or 16:0 which would normally be used for MGD synthesis in the chloroplast is exported to the cytoplasm and utilized for the synthesis of lipids in this compartment. (2) Synthesis of MGD by the prokaryotic pathway is reduced 25-30%, but this deficiency is compensated for by the increased production of this lipid in the endoplasmic reticulum. I64 (3) fagfl related changes in leaf fatty acid composition result in increased thermal tolerance and enhanced stability of chloroplast membranes of the mutant at high temperatures. (4) Under standard conditions the altered membrane lipid composition does not affect the vigor of the mutant, net photosynthetic C02 fixation, photosynthetic electron transport, chloroplast ultrastructure, or fluidity of chloroplast membranes. Future directions W The collection of acyl group mutants analyzed to date contributed considerably to our understanding of the desaturation process in the plant membranes and the control of cellular lipid metabolism. Furthermore, the mutants provided evidence for the possible funCtions of lipids in temperature responses of plants, and their involvement in the formation of chloroplast structure. However, because of the existence of the two pathways.of lipid synthesis, the mutations in the prokaryotic pathway result in the increased compensatory synthesis of lipids through the eukaryotic pathway, so that the phenotypic effects produced by the mutations were not severe under standard growth conditions. Only one mutant has been isolated in the eukaryotic pathway of glycerolipid synthesis so far. It is not clear yet whether this lesion has any consequences on chloroplast lipid metabolism, or perhaps only affects the extrachloroplast lipids. In any case, isolation and characterization of more mutants in the eukaryotic pathway should be 165 useful in investigating the relevance of membrane fatty acid composition to a. wide range of cellular processes including mitochondrial respiration, transport through the plasmalemma and tonoplast, or intracellular transport and secretion mediated by the Golgi apparatus. In addition, double mutants may be constructed with defects in both the prokaryotic and eukaryotic pathways. These mutants would probably have much more severe phenotypes and might be extremely valuable in testing how extensive the changes in lipid composition have to be before photosynthesis is affected, as well as which aspects of photosynthesis are most susceptible to alteration in fatty acid composition. One of the major conclusions of our mutant analyses to date is that plants can withstand relatively large changes in membrane lipid composition without serious effects. Therefore, even the isolation of head group mutants is not inconceivable. It seems likely that the absence of a headgroup would be deleterious to the organism, but a leaky mutant, with a quantitative rather than qualitative variation in a certain headgroup, might provide answers to a lot of interesting questions concerning specific functions of lipids in different membranes. W The fatty acid desaturase enzymes, with the exception of the soluble chloroplast desaturase (McKeon and Stumpf, 1982), have not been purified and characterized. The lack of information about plant lipid desaturases prevents the application of molecular genetic techniques in modifying the composition of storage lipids to suit industrial needs, 166 as well as the alteration of leaf lipids to possibly improve the acclimation of plants to different environmental conditions. The analysis of the fadfl mutant, together with other mutants defective in the activities of chloroplast desaturases, provided important information about the desaturation of chloroplast lipids. The same mutants may be useful tools for cloning the desaturase genes. For example, if the defective gene product is missing or altered in the mutant, it may be possible to identify the wild-type polypeptide by comparing the polypeptide patterns of the mutant and wild type after separating them by 2-dimensional SDS-polyacrylamide gel electrophoresis. Another feasible approach in Arabidopsis is chromosome walking. Once the mutants are mapped, every desaturase gene within 250 kb of the restriction fragment length polymorphism (RFLP) marker on the Arabidopsis map can, in principle, be isolated. Alternatively, the fad mutants may be used for gene isolation by transposon tagging. MW Characteristic changes in chloroplast size and ultrastructure associated with changes in membrane fatty acid composition raise the possibility that high levels of acyl group unsaturation, characteristic of chloroplast membranes, might be an important factor involved in chloroplast structural differentiation and/or division. The polypeptide pattern of the 3911 mutant resolved by 2-dimensional SDS-polyacrylamide gel electrophoresis revealed several quantitative differences wdth respect to the wild type. These differences might reflect an altered capacity of the lipid bilayer for incorporation of proteins. By comparing the protein pattern of another independent actl mutant to the 167 wild type it might be possible to identify (a) specific protein(s) that show the same variation in both mutants and perhaps affect the. chloroplast ultrastructure. The isolation of a gene that codes for this protein and its overexpression in transgenic plants may provide valuable information about the mechanisms involved in the assembly of chloroplast membranes, and/or the formation of appressed regions within chloroplasts. W Chilling sensitivity in plants has been correlated with a reversible phase separation of the lipids within cellular membranes, which effectively prevents the membranes and their constituent enzymes from functioning normally. Although plant membranes contain highly unsaturated lipids, which would not be expected to undergo phase transitions at physiological temperatures, the presence of as little as 1% of a disaturated phospholipid, such as PG, induces a thermal phase transition (Raison and Hright, 1983). In agreement with this, Murata et al. (1982) found a correlation between the high proportion of disaturated molecular species of PG and chilling sensitivity. Further surveys, involving a large number of species, generally support this correlation (Roughan et al., 1985, Bishop et al., 1986), although there were a few exceptions. Since the sn-2 position of PG is always esterified with 16:0 and trans-16:1 (which was also considered as a saturated fatty acid, by virtue of the position and isomerism of the double bond) acyl groups, the formation of disaturated PG depends on the specificity of the chloroplast G3P acyltransferase. Therefore, the ultimate test for the validity of Murata’s hypothesis would be the 168 introduction of the G3P acyltransferase gene from a chilling sensitive species into a chilling resistant plant, to make it chilling sensitive, and subsequent analysis of PG acyl group composition of the transgenic plant. This approach depends upon first being able to eliminate the endogenous gene by the isolation of a null mutation. However, the actl mutant of Arabidopsis (a chilling resistant plant) described in this dissertation, with less than 4% of residual G3P activity, is also a good candidate for such an experiment. W A detailed physiological characterization of actl and fadfl mutants has shown that both mutants exhibit enhanced thermal tolerance relative to wild type plants. A similar result obtained for another mutant of Arabidopsis with altered leaf lipid composition (McCourt et al., 1987) suggests that membrane polar lipids might play a role in acclimation of plants to high temperatures. Thermal tolerance of plants is thought to be determined by the stability of their photosynthetic membranes. Gounaris et al. (1984) have shown that broad bean (M151; faba) is adversely affected by temperatures above 35°C. The ultrastructural changes involve: (I) a decrease in the amount of stacked membrane regions, (2) a decrease in size of EFs particles on freeze-fractured membranes, which was interpreted to indicate a dissociation of light harvesting units from the P511 core complex, and (3) the formation of phase separated aggregates of non-bilayer forming lipids. Ne have no information regarding the structural changes associated with heat-induced damage of chloroplast membranes in the lipid mutants of Arabidopsis. Freeze-fracture electron microscopic studies on actl and l69 fadfl mutants might, therefore, be useful for obtaining additional evidence concerning the function of lipids, or lipid-protein association, in membrane stability at elevated temperatures. An alternate approach to addressing the question of increased thermal stability of chloroplast membranes from 5511 and fadfl mutants would be to measure the physical properties of membrane lipids. In this context, it would be especially interesting to determine the threshold temperature for phase separation of membrane lipids using fluorescence polarization from the probe trans-parinaric acid, since differences in the transition temperature of polar lipids seem to correlate with different physiological performances at high temperatures (Raison et al., 1982). Literature cited Gounaris K, ARR Brain, PJ Quinn, HP Williams 1984 Structural reorganization of chloroplast thylakoid membranes in response to heat stress. Biochim Biophys Acta 766 198-208 McCourt P, L Kunst, JA Browse, CR Somerville 1987 The effects of the reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis. Plant Physiol 84: 353-360 McKeon TA, PK Stumpf 1982 Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J Biol Chem 257: 12141-12147 Murata N, N Sato, N Takahashi, Y Hamazaki 1982 Compositions and positional distributions of fatty acids in phospholipids from leaves from chilling-sensitive and chilling-resistant plants. Plant Cell Physiol 23: 1071-1079 Murata N, J Yamaya 1984 Temperature-dependent phase behavior of phosphatidylglycerols from chilling-sensitive and chilling-resistant plants. Plant Physiol 74:1016-1024 170 Raison (JK, CS Pike, JA Berry 1982 Growth temperature-induced alterations in the thermotropic properties of ugrium gleandgr membrane lipids. Plant Physiol 70: 215-218 Raison JK, LC Wright 1983 Thermal transitions in the polar lipids of plant membranes. Their induction by disaturated phospholipids and their possible relations to chilling injury. Biochim Biophys Acta 731: 69-78 Roughan PG 1985 Phosphatidylglycerol and chilling sensitivity in plants. Plant Physiol 77: 740-746