Lipid trafficking:
into, within and out of the
chloroplast
Mats Andersson
Göteborg University
Department of Botany
Göteborg, Sweden
Göteborg University
Faculty of Science
2004
Lipid trafficking: into, within and out of the
chloroplast
Mats Andersson
Akademisk avhandling för filosofie doktorsexamen i fysiologisk botanik (examinator Christer Sundqvist), som enligt beslut i lärarförslagsnämnden i biologi kommer att offentligen
försvaras fredagen den 28 maj 2004, kl. 10.15 i föreläsningssalen, Botaniska Institutionen, Carl Skottsbergs gata 22B, 413 19 Göteborg.
Fakultetsopponent: Professor Christoph Benning, Michigan State University, East Lansing, USA.
Lipid trafficking: into, within and out of the chloroplast
Mats AnderssonGöteborg University, Department of Botany Box 461, SE-405 30 Göteborg, Sweden
___________________________________________________________________________
Abstract: Plant cellular membranes consist of two different kinds of glycerolipids, phospholipids and galactolipids. The galactolipids make up the bulk of the chloroplast membranes, whereas other membranes such as the plasma membrane, the tonoplast and the endoplasmic reticulum (ER) largely consist of phospholipids. In all plants, the diacylglycerol (DAG) backbones of the chloroplast galactolipids are partially or completely derived from phospholipids synthesised in the ER. Thus, there is a need for transport of phospholipids from the ER to the chloroplast. Evidence is presented and discussed for that the transport of ER-derived galactolipid precursors occurs at sites of physical contact between the chloroplast and a specialised plastid-associated domain of the ER, the PLAM. As galactolipids are synthesised from DAG, there is a need for the enzymatic degradation of ER-derived phospholipids to DAG in the chloroplast envelope. In an in vitro system, the degradation of PC to DAG in the chloroplast envelope was found to be mediated by soluble cytosolic phospholipase D and phosphatidic acid phosphatase acting in sequence. Evidence for that the lipid environment of the outer envelope membrane are important for this process is presented. Galactolipids synthesised in the chloroplast envelope are transported across the aqueous stroma to the thylakoid membrane. The evidence at hand suggest that lipid transport from the envelope to the thylakoid is at least partially mediated by vesicles that are formed at the inner envelope and fuses with the thylakoid. This putative vesicular transport system appears to be evolutionary related to vesicle trafficking in the cytosolic secretory pathway. Finally, the effects of phosphate limitation on the lipid composition of oat plasma membranes were studied. Phosphate limitation caused a very large increase in the proportion of the galactolipid digalactosyldiacylglycerol (DGDG) in the plasma membranes; this increase was balanced by a decreased proportion of phospholipids. After four weeks of cultivation in a phosphate-free medium DGDG, a lipid previously assumed to be strictly plastid localised, made up as much as 66 mol% (sic) of the root plasma membrane glycerolipids. This finding implies that phosphate limitation causes a massive efflux of DGDG from the plastid to other cellular membranes, such as the plasma membrane. In addition the results also demonstrate a much larger degree of plasticity of plant membrane lipid composition than previously recognised. ___________________________________________________________________________
Keywords: chloroplast, endoplasmic reticulum, galactolipid, lipid trafficking, phospholipid, plasma membrane, phosphate, thylakoid
Murmel
|
djur
s.
gulligt djur som lätt
faller i sömn
”Mmmmmm, fattening!”
-H. Simpson
Lipid trafficking: into, within and out of the
chloroplast
Mats Andersson
Göteborg University, Department of Botany
Box 461, SE-405 30 Göteborg, Sweden
This thesis is based on data presented and discussed in the following papers, throughout the thesis referred to by roman numerals.
Paper I Andersson MX,Kjellberg JM, Sandelius AS (2004) The involvement of cytosolic lipases in converting phosphatidylcholine to substrate for galactolipid synthesis in the chloroplast envelope. Submitted
Paper II Andersson MX, Sandelius AS.Isolation and characterisation of PLAM, a subfraction of the endoplasmic reticulum closely associated with the chloroplast. Manuscript
Paper III Andersson MX, Kjellberg JM, Sandelius AS (2001) Chloroplast biogenesis. Regulation of lipid transport to the thylakoid in chloroplasts isolated from expanding and fully expanded leaves of pea. Plant Physiol. 127: 184-193
Paper IV Andersson MX,Sandelius AS (2003)Identification of molecular components of a plastid localised vesicular transport system: a bio-informatics approach. Submitted
Paper V Andersson MX, Stridh MH, Larsson KE, Liljenberg C, Sandelius AS (2003) Phosphate-deficient oat replaces a major portion of the plasma membrane phospholipids with the galactolipid digalactosyldiacylglycerol. Febs lett. 537: 128-132
Paper VI Andersson MX, Larsson KE, Liljenberg C, Sandelius AS. Effects of phosphate limitation on the root cellular membranes of oat (Avena sativa). Manuscript
Contents
Abbreviations 1. Introduction 1 2. Background 2 2.1 Membrane lipids 2 2.2 Plant membranes 3 2.3 The plastids 32.4 The secretory pathway 5
2.5 The plasma membrane 6
3. Galactolipid biosynthesis 7
3.1 Fatty acid synthesis 7
3.2 Lipid synthesis in the ER 8
3.3 Lipid synthesis in the plastid 9 3.4 ER to chloroplast lipid transport 9
3.5 The PLAM 11
3.6 PC metabolism in the envelope membrane 13 3.7 Lipid galactosyl transferases in the plastid envelope 15
4. Intraplastidial lipid trafficking 16
4.1 Morphological evidence of intraplastidial vesicles 16 4.2 Characterisation of in organello lipid transport 17
4.3 Intraplastidial lipid transport reconstituted in vitro 18 4.4 Components of an intraplastidial vesicle transport system 18
4.5 Just one pathway? 21
5. Galactolipids everywhere 22
5.1 Phosphate house-holding and galactolipid synthesis 22 5.2 Phospholipid replacement in the plasma membrane 23 5.3 Phospholipid replacement in other non-plastid membranes 25 5.4 Galactolipid export from the plastid 26 6. A model for galactolipid synthesis and trafficking 27 6.1 Lipid transport to, whithin and from the chloroplast 27
6.2 Where do we go now? 28
7. Acknowledgements 29
8. References 32
Abbreviations 16:0 Hexadecanoic acid 16:3 all-cis-9,12,15-hexadecatrienoic 18:1 cis-9-octadecenoic acid 18:2 all-cis-9,12-octadecadienoic acid 18:3 all-cis-9,12,15-octadecatrienoic acid ACP Acyl carrier protein
ASG Acylated sterolglucoside BLAST Basic local alignment search tool
CoA Co-enzyme A DAG Diacylglycerol DGDG Digalactosyldiacylglycerol ER Endoplasmic reticulum FS Free sterol GlcCer Glucosylcerebroside LPCAT Lyso-PC acyl transferase
MAM Mitochondria associated membranes MGDG Monogalactosyldiacylglycerol
MS Microsomes PA Phosphatidic acid
PAM Plasma membrane associated membranes PAP Phosphatidic acid phosphatase
PC Phosphatidylcholine PE Phosphatidylethanolamine PG Phosphatidylglycerol PI Phosphatidylinositol PLAM Plastid associated membranes PLC Phospholipase C
PLD Phospholipase D PS Phosphatidylserine SG Sterolglucosides SQDG Sulfoquinovosyldiacylglycerol
1. Introduction
To simply state that plants are “important” is an understatement of no less than monumental proportions. Would it not be for photosynthetic plants and algae the world as we know it would not be, and neither would we. The energy that powers (almost) every living creature on earth has its origin in the sun and has been captured and made available to biological life by the chemical process known as photosynthesis. Oxygenic photosynthesis is the defining feature of plants, algae and cyanobacteria (popularly known as blue green algae).
To briefly state the “importance” of the lipid bilayer to biological life may be an even grander understatement. Would it not be for this fundamental biological structure no life at all would exist on earth, not even the anaerobic chemoautotrophic bacteria that would populate the barren hypothetical world without oxygenic photosynthetic organisms. Among the basic features that all known living organisms share is a barrier against the surrounding environment. This barrier has to be permeable enough to allow the entry of substances needed from the environment and the excretion of waste products, while impermeable enough to prevent the uncontrolled leakage of the cellular constituents. All organisms, no matter how primitive or sophisticated, have membranes surrounding their cells, plasma membranes. The story, however, does not end with the plasma membrane. Over the course of evolution the cellular membranes gained many more functions than the presumably original barrier function, they also became the home of numerous chemical reactions. Regions of the outer membrane of the progenitors of modern day eukaryots probably became specialised in carrying out certain biochemical tasks, these regions invaginated and eventually they lost contact completely with the plasma membrane. The pinched off membrane areas gave rise to the “modern” endomembrane system. At later stages in the evolution, the eukaryotic ancestors also obtained the precursors for mitochondria and, in case of plants and algae, chloroplasts. Both the chloroplast and the mitochondrion probably originate from free-living bacteria that became incorporated into the eucaryotic ancestors through a symbiotic relationship. The chloroplast and mitochondria are completely integrated into modern day eucaryotic cells, but several features, however, still reflect their bacterial origin.
All biological membranes are built from lipids and proteins but the all important “magic” building blocks for constructing membranes are the membrane lipids. Membrane lipids in an aqueous environment spontaneously self assemble into the basic bilayer structure of biological membranes.
This thesis deals with three important aspects of the lipids that chloroplast membranes are built from. How the precursors of chloroplast lipids are transferred from the endoplasmic reticulum to the chloroplast and how they are retailored into chloroplast lipids (Papers I and II), how the chloroplast lipids are transported from their site of synthesis, the chloroplast envelope, to the thylakoid membrane (Papers III and IV) and finally how lipids usually considered to be chloroplast localised function in other cellular membranes to replace phosphate-containing lipids during phosphate-limited conditions. Thereby, the plant cell is enabled to better survive conditions of low phosphate availability (Papers V and VI).
2. Background
The current model for the structure of biological membranes was proposed in 1972 by Singer and Nicolson (Singer and Nicolson 1972). Remarkably little in the general perception of membranes has changed since the original “fluid mosaic model” was first proposed. The model proposes that the membrane lipids form a bilayer that acts like a two dimensional liquid. Immersed in this liquid are integral membrane proteins that behave much like mobile islands in a “sea” of membrane lipids. Lateral diffusion and rotation of lipids and membrane spanning proteins is fast whereas movement across (flip/flop) the bilayer of lipids is very slow. Even though it has its limitations, this model has proved to be of very high explanatory value. Biological membranes are not really just two dimensional liquids, lateral diffusion of lipids is not always completely unrestricted and all components of the membrane are probably not always ideally mixed (Pike 2004). Lipid bilayers are rather impermeable to large, polar or charged molecules (Jones and Chapman 1995). Water permeates easily but not unrestricted through a lipid bilayer (Hill and Zeidel 2000; Krylov et al. 2001). All biological membranes contain transporters that allow the controlled passage of various substances.
2.1 Membrane lipids
The key feature of membrane lipids is their amphiphilic properties; hydro-philic and hydrophobic chemical functions are present within the same molecule. This feature causes the membrane forming lipids to spontaneously self assemble into structures that minimise the contact area between the aqueous environment and the hydrophobic portions of the lipids. Which structure that will be formed depends on the geometrical shape of the lipids, the temperature and the degree of hydration of the system. The lamellar
α-structure is probably the most physiologically relevant membrane structure, but other structures such as the inverted hexagonal and the cubic phases are probably also highly relevant to various physiological processes (Williams 1998). Biological membranes in fact contain a number of lipids that have a strong propensity to form other phases than the lamellar
α-phase (Israelachvili et al. 1980). In theory an almost endless range of substances could function as membrane forming lipids. In nature, however, only a rather limited set function as membrane lipids (Gurr and Harwood 1991). The majority of membrane forming lipids are based on glycerol or the sphingosine base (Fig. 1). Glycerolipids usually
Figure 1. The structure of some common plant membrane lipids Stigmasterol Glucosylcerebroside (GlcCer) Monogalactosyldiacylglycerol (MGDG) Phosphatidylcholine (PC) Digalactosyldiacylglycerol (DGDG) O H O H H H O H H O H H OH O OH O O O O CH3 CH3 C H3 N+CH2CH2 CH3 C H3 O P O H O O O O O O CH3 O O H H O H H OH H OH H OH O H H H O H H O H H OH O O O O O CH3 CH3 O OH H H H O H OH H OH H O CH2 NH O O H CH C H OH CH CH
contain two acyl groups esterified to the sn-1 and sn-2 position of glycerol. Glycerolipids containing only one fatty acid are known as lyso lipids. The sn-3 position of the glycerol backbone is esterified to a polar head group. Plant membranes contain two quite distinct classes of glycerolipids, glycerophospholipids and glycerogalactolipids. The head group of the former class consists of a phosphate group esterified to a small organic molecule, the common ones being choline, ethanolamine, serine, glycerol and inositol. The head group of the galactolipids is composed of one or two galactose units linked by glucosidic bonds and directly esterified to the glycerol backbone. The galactolipids constitute the major portion of the chloroplast membranes (see below). Plant membrane glycerolipids usually contain C16 and/or C18 fatty acids, the latter usually mono- or poly-unsaturated. The sphingolipids carry sugar containing head groups and fatty acid moieties with varying degrees of hydroxylation (Sperling and Heinz 2003; Lynch and Dunn 2004). The third important group of membrane lipids are the sterols (Fig. 1) and sterol derivatives. Mixing of sterols into lipid bilayers tend to decrease mobility in the hydrophobic region making the membrane thicker and less permeable to small molecules (Jones and Chapman 1995). Sterols also tend to make the hydrocarbon chains of the hydrophobic part of the membrane less likely to crystallise at low temperatures (Jones and Chapman 1995).
2.2 Plant membranes
All living plant cells contain basically the same set of membrane-delimited organ-elles. However, depending on tissue type, plant species and developmental stage the proportion of different organelles and membranes differs widely. On the ultrastructural level membranes are visible as 5-10 nm thick, electron dense bands. At the electron microscopy level many different membrane bound compartments can be observed (Fig 2). The plasma membrane just inside the cell wall delimits the cell from the surrounding environment. In green tissue the chloroplast membranes and the central vacuole that is delimited by the tonoplast membrane dominate the picture. Due to the large size of plant cells and the central vacuole, the plasma membrane and the tonoplast membrane constitute a large portion of the non-plastid membranes of the cell. Mitochondria, stacks of Golgi cisternae and ER-tubules are usually visible as are peroxisomes and unidentified vesicles.
2.3 The plastids
The plastid is the one organelle that separates plants and algae from all other eukaryot organisms. The plastid type in green tissue, chloroplasts, are the home of the entire photosynthesis pathway from light absorption to reduced carbon compounds. Modern day
Central vacuole Cell Wall Plasma membrane Thylakoid Chloroplast stroma Mitochondria Peroxisome
Figure 2. Transmission electron micrograph of a pea mesophyll cell. Courtesy of A. S. Carlsson and A.S. Sandelius.
plastids are considered to be the descendents of once free-living cyanobacteria-like organisms (McFadden 1999). Thus, plastids are still in many respects semiautonomous organelles and share many traits with their free living cyanobacterial cousins. Over the course of evolution most of the genes in the endosymbiont genome has been lost to the nucleus and the organelle genome retain only a small number of genes (Martin and Herrmann 1998).
The chloroplast consists of three different membrane systems. The outer chloroplast envelope delimits the organelle. The inner chloroplast envelope is situated with only a narrow spacing from the outer chloroplast envelope. The envelope membranes contain the machinery required to import nucleus encoded proteins into the stroma (Jarvis and Soll 2002), the biosynthetic machinery for many of the chloroplasts hydrophobic substances (Joyard et al. 1998a) and transporters for various solutes (Ferro et al. 2003; Froehlich et al. 2003; Schroeder and Kieselbach 2003). The inner chloroplast envelope delimits the aqueous stroma. Inside the stroma lies the thylakoid membrane system. The thylakoid consists of multiple stacks of interconnected flattened membranes. The thylakoid contains photosynthetic pigments bound to the multi-protein complexes of the photosynthetic machinery (Åkerlund 1993; Andersen and Scheller 1993; Paulsen 1993). The thylakoid membrane contains protein complexes that function in importing proteins into the thylakoid lumen and insert integral proteins into the thylakoid membrane (Keegstra and Cline 1999). The protein import machinery in the thylakoid membrane is directly related to the bacterial plasma membrane protein export machinery (Keegstra et al. 1999). The chloroplast envelope import complexes, in contrast, seem to originate from other bacterial trans membrane transport systems (Reumann and Keegstra 1999). The thylakoid lumen is a continuous aqueous compartment. By electron microscopy, chloroplasts are observed as kidney shaped organelles 4-8 µm long and 1-2 µm across (Ryberg et al. 1993). Recent studies in which GFP was expressed in the chloroplast stroma reveal that chloroplast structure may be more complex and that chloroplasts may be connected through envelope delimited strands of stroma; so called stromules (Kohler and Hanson 2000; Hanson and Kohler 2001).
In addition to chloroplasts, plants contain many types of non-green plastids, such as starch containing amyloplasts, pigment-rich chromoplasts and the proplastids found in meristems, roots and developing leaves. The proplastids of non-green tissues are traditionally regarded as just smaller versions of chloroplasts devoid of thylakoids, but recent studies with GFP localised to the plastid stroma reveals that the proplastids sometimes form extensive networks throughout the cell (Köhler and Hanson 2000; Hanson and Köhler 2001; Hans et al. 2004). In the absence of light, plastids develop into etioplasts. The etioplast is characterised by that instead of a thylakoid system, the plastid contains one or several prolamellar bodies. The prolamellar body is a semi crystalline structure and contains large amounts of the enzyme NADH:protochlorophyllide oxidoreductase (Ryberg et al. 1993). The prolamellar body is built from the same membrane lipids in essentially the same proportions, as are thylakoids (Ryberg et al. 1983; Selstam and Sandelius 1984). These lipids probably, in the absence of enough membrane spanning helices, form a cubic phase yielding the semi crystalline appearance (Selstam and Sandelius 1984; Brentel et al. 1985; Williams et al. 1998). Upon illumination the prolamellar body quickly “dissolves” into lamellar structures (Virgin et al. 1963; Ryberg et al. 1993).
Since the first reports on chloroplast lipid composition in the 1960s (Lichtenthaler and Park 1963), the lipid composition of the different membranes from chloroplasts isolated from various plant species has been elucidated in detail (Table 1). Chloroplast membranes are rich in the galactolipids mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively;
Fig. 1) and contain the negatively charged glucolipid sulfoquinovosyldiacylglycerol (SQDG). The phospholipid content of chloroplast membranes is low. Phosphatidylethanolamine (PE) and phosphatidylserine (PS) are usually not found at all in isolated chloroplast and when found, ascribed to extraplastidial membrane contamination. Sterols and sterol derivatives are likewise not found in isolated chloroplast membranes. The lipid composition of chloroplast membranes is in good agreement with the bacterial background of the plastid, as the photosynthetic membranes of cyanobacteria contain essentially the same lipids as higher plant chloroplasts (Murata and Nishida 1987; Harwood and Jones 1989). The thylakoid galactolipids contain a high proportion of polyunsaturated fatty acids (Mackender and Leech 1974; Bahl et al. 1976; Cline et al. 1981; Block et al. 1983c). The phospholipid content is higher in the envelope membranes than in the thylakoid with the highest content in the outer envelope membrane. The lipid to protein ratio is relatively high in the outer envelope membrane (2.5-3), while the inner envelope and the thylakoid membranes have lower lipid to protein ratios (0.8-1 and 0.4, respectively; (Block et al. 1983b)).
Table 1. Lipid composition (mol%) of chloroplast membranes.
Species Membrane MGDG DGDG SQDG PG PC PI PE Total envelope 36 29 6 9 18 2 0 Outer envelope 17 29 6 10 32 5 0 Inner envelope 49 30 5 8 6 1 0
Thylakoid 52 26 6.5 9.5 4.5 1.5 0 Spinach(Block et al.
1983c)
Intact chloroplasts 46 32 7 6 7 1 0
Thylakoid 51 33 8 5 2 0 0
Pea (Paper III)
Outer envelope 6 33 3 6 44 5 2 Inner envelope 45 31 2 7 10 2 1 Pea (Cline et al. 1981)
Total envelope 22 44 10 9 14 0 0 Lamellar thylakoid 42 37 9 10 2 0 0 Grana thylakoid 47 36 7 9 1 0 0 Wheat(Bahl et al. 1976)
Total envelope 29 32 n.d. 9 30 n.d. 0
Thylakoid 65 26 n.d. 6 3 n.d. 0 Broad bean (Mackender
et al. 1974)
Dark-grown wheat
(Ryberg et al. 1983) Etioplast inner membranes 50 32 9 5 5 - -
Small amounts of “exotic” lipids have been found in isolated chloroplasts. Tri- and tetragalactosyldiacylglycerol are normally not found in lipids extracted from intact leaf tissue, but can be found as minor constituents of isolated chloroplasts (Cline et al. 1981; Wintermans et al. 1981). Acylated MGDG (Heinz et al. 1978), phosphorylated MGDG (Müller et al. 2000) and the mono and diphosphorylated derivatives of PI (Siegenthaler et al. 1997) have all been identified as radiolabelled products after incubation of isolated chloroplast membranes with radiolabelled substrates. MGDG with oxophytodienoic acid esterified to the sn-1 position that probably functions as a precursor for jasmonates is also present in low concentrations in plant tissue (Stelmach et al. 2001).
2.4 The secretory pathway
The ER is the site where the bulk of the material handled by the secretory apparatus is produced. Secreted proteins, and the membrane proteins of the ER, the Golgi apparatus, the plasma membrane and the tonoplast are produced by ribosomes attached to the rough ER and
the bulk of the membrane lipids are assembled in the smooth ER. Proteins destined for the plasma membrane and the tonoplast passes through the Golgi apparatus where they are sorted and modified before reaching their final destination (Morré and Mollenhauer 1976; Neumann et al. 2003). Protein trafficking in the secretory apparatus is strictly dependent on transport of membrane vesicles. Transport vesicles are formed at a donor compartment and fuses with a specific target membrane (Bonifacino and Glick 2004). Thus, soluble proteins, membrane spanning proteins and membrane lipids are transferred from one compartment to the other. The molecular details of the secretory pathway in mammalian and yeast cells have been elucidated in great detail. Three different types of transport vesicles are known to mediate vesicle trafficking in the secretory apparatus; COPI, COPII and clathrin coated vesicles (Kirchhausen 2001; Bonifacino and Glick 2004). Lipid transport from site of synthesis (the ER) to target membrane is however not entirely dependent upon vesicle trafficking (Moreau et al. 1998; Voelker 2000; Voelker 2003). Lipid delivery to the mitochondria (Voelker 2000; Wu and Voelker 2001) and chloroplasts (see section 3) appears to occur completely outside the secretory system. There is evidence that the most common phospholipids of the plant plasma membrane are delivered to the plasma membrane outside the secretory pathway (Moreau et al. 1994; Sturbois-Balcerzak et al. 1995). Lipids are also sorted in the secretory pathway; the lipid composition of the plasma membrane is distinct from that of the ER membrane (Moreau et al. 1998). Sorting occurs with regard to both lipid class and species (Moreau et al. 1998; Schneiter et al. 1999).
The plant ER is a complex system of interconnected membrane tubules and cisternae that extends all through the cytosol (Staehelin 1997). The tubules observed by light microscopy have been correlated to ribosome-free smooth ER as revealed by electron microscopy, whereas the cisternae corresponds to ribosome-clad rough ER. The ER network is also directly connected to the nuclear envelope, which can be viewed as a functional domain of the ER. In addition to the division of the ER into rough and smooth regions as many as 16 different functional and/or structural domains of the ER are recognised (Staehelin 1997). The plant ER has been observed to make close contacts with mitochondria (Lichtscheidl et al. 1990), developing plastids (Whatley 1974; Kaneko and Keegstra 1996) and the plasma membrane (Lichtscheidl et al. 1990). In yeast, specialised subdomains of the ER associated with mitochondria (Gaigg et al. 1995; Achleitner et al. 1999) and plasma membrane (Pichler et al. 2001) have been isolated. These ER fractions were named Mitochondria Associated Membranes (MAM) and Plasma Membrane Associated Membranes (PAM). The MAM and PAM fractions differ from bulk ER in polypeptide composition and lipid synthesis capacity. The MAM fraction has been shown to participate in lipid delivery from the ER to the mitochondrion in yeast (Gaigg et al. 1995; Achleitner et al. 1999). By analogy to the MAM fraction in yeast, it was suggested that plant cells contain a specific ER subdomain that is associated to the chloroplast; Plastid Associated Membranes (PLAM; see section 3 and Paper II).
2.5 The plasma membrane
The plasma membrane is, second to the cell wall, the primary outer surface of the cell and thereby the first structure to be affected by toxins, pathogens, drought, high salinity and many other stress factors that the plant may encounter, but not physically evade. Non-membrane permeable signalling molecules that reach the cell surface will be relayed through the plasma membrane. All water and nutrients has to cross the plasma membrane at some point. Thus, the importance of the plasma membrane and its integrity can hardly be overestimated. Great progress has been made in the understanding of the plant plasma membrane over the past
decades. Plasma membranes can be isolated at very high yield and purity from many different plant tissues using aqueous polymer two phase partitioning (Larsson 1983; Sandelius and Morré 1990).
The plant plasma membrane contains many different small molecule-conducting channels. Active transport across the plasma membrane is almost universally powered by a H+-ATPase (Serrano 1990; Maathuis and Sanders 1999; Arango et al. 2003).
The polypeptide composition of plasma membranes isolated from different plant tissues and species is apparently rather well conserved (Larsson et al. 1990). In contrast to this, the lipid composition appears to vary between species (Larsson et al. 1990; Uemura and Steponkus 1994) and growth conditions (Norberg and Liljenberg 1991a; Uemura and Steponkus 1994; Uemura et al. 1995; Quartacci et al. 2002). Beside phospholipids, plant plasma membranes contain a high proportion of sterols, sterol glucosides (SG), acylated sterol glucosides (ASG) and glucosylcerebroside (GlcCer) (Norberg and Liljenberg 1991; Norberg et al. 1991; Uemura and Steponkus 1994; Norberg et al. 1996; Paper VI). The dogma that plant plasma membranes always consist of phospholipids, sterols and sphingolipids has been challenged in recent studies (se further section 5 and Papers V and VI).
3. Galactolipid biosynthesis
Plant lipid metabolism is complicated and only partly understood; there is a complex interplay between different membranes and organelles. The fluxes through the different pathways are influenced by a large array of both membrane-bound and soluble substrates. There appears to be some degree of functional redundancy between pathways and enzymes. However, the advent of molecular biology and especially the identification of A. thaliana lipid biosynthesis mutants have helped to greatly increase the understanding of plant lipid biosynthesis (Miquel and Browse 1998). The recent attempt to identify and organise all lipid synthesis related genes in the A. thaliana genome in a publicly available database (Beisson et al. 2003) is likely to facilitate the further exploration of plant lipid metabolism.
3.1 Fatty acid synthesis
De novo fatty acid synthesis in plants occurs in the plastid stroma (Ohlrogge and Browse 1995) and in mitochondria (Wada et al. 1997; Gueguen et al. 2000). The significance of the fatty acid synthesis in mitochondria for bulk acyl lipid synthesis is rather hypothetical and the general consensus is that the fatty acids synthesised within the plastid constitute the bulk of the membrane lipid acyl groups in the plant cell (Ohlrogge et al. 1995). The exact identity of the carbon source for fatty acid synthesis inside the plastid is a matter of some debate (Bao et al. 2000). A detailed discussion of fatty acid synthesis is beyond the scope of this thesis and the interested reader is referred to reviews on the subject (Ohlrogge and Jaworski 1997; Rawsthorne 2002). The end products of plastidial fatty acid synthesis are saturated C16 and C18 fatty acids bound to acyl carrier protein (ACP). A specialised soluble stroma localised ∆9 desaturase catalyses the desaturation of 18:0-ACP to 18:1-ACP (Ohlrogge and Browse 1995). The 16:0 and 18:1 fatty acids are exported from the chloroplast and esterified to CoA by an acyl-CoA synthase in the outer plastid envelope (Andrews and Keegstra 1983; Schnurr et al. 2002).
3.2 Lipid synthesis in the ER
The ER is the main site of acyl lipid synthesis in the plant cell (Ohlrogge and Browse 1995). Sphingolipid (Sperling and Heinz 2003; Lynch and Dunn 2004) and sterol (Brown 1998; Piironen et al. 2000) synthesis also take place in the ER, but lies outside the scope of this discussion. Acyl lipid synthesis in the ER is outlined in figure 3. Phosphatidic acid (PA) is formed by the sequential transfer of two acyl groups from acyl-CoA to glycerol-3-phosphate. The acyl transferases in the ER insert a C16 or C18 fatty acid on the sn-1 position and always a C18 fatty acid on the sn-2
position. After synthesis of PA, the pathway branches. The two anionic phospholipids phosph-atidylinositol (PI), phosphatidyl-glycerol (PG) and PS are formed from free head groups and CDP-diacylglycerol, whereas the zwitterionic phospholipids PC and PE are formed from diacylglycerol (DAG) and CDP activated bases. PE can also be formed by decarboxylation of PS by an enzyme present in both mitochondria and ER (Rontein et al. 2001; Rontein et al. 2003). The ER-localised desaturases are specific for C18 fatty acids esterified to phospholipids (Ohlrogge and Browse 1995). Phospholipids synthesised in the ER are also modified by head group exchange (Moore 1982) or exchange of the acyl groups (Williams et al. 2000). The compartmentalisation of the ER membrane discussed in the previous section may complicate matters substantially. So far no comprehensive data exist on compartmentalisation of lipid metabolism in the plant ER. In yeast, however, the ER fractions associated to the mitochondrion and the plasma membrane have quite different capacity for lipid synthesis compared to each other as well as bulk ER (Gaigg et al. 1995; Pichler et al. 2001).
Many in vivo pulse chase studies indicate that PC synthesised in the ER is the immediate precursor for galactolipid synthesis in the chloroplast (Slack et al. 1977; Heinz and Roughan 1983; Roughan et al. 1987; Hellgren et al. 1995; Hellgren and Sandelius 2001a). Since both the outer chloroplast envelope and the ER membrane contain a substantial proportion of PC, it seems reasonable to suggest that PC is transported from the ER to the chloroplast envelope to function as a precursor for galactolipid synthesis. However, both lysoPC (Mongrand et al.
OH OH P Glycerol 3-phospate 18:1 P 18:1 (16:0) PA OH 18:1 18:1 (16:0) DAG CDP 18:1 18:1 (16:0) CDP-DAG 18:1-CoA 16:0-CoA PI, PG PC, PE, PS
ER
OH OH P Glycerol 3-phospate P 18:1 18:1 (16:0) PA OH 18:1 18:1 (16:0) DAG PG 18:1-ACP 16:0-ACP Galactose 18:1 18:1 (16:0) MGDGPlastid
Figure 3. Glycerolipid synthesis in the ER and the plastid. The circle denotes synthesis pathway active in 16:3 plants.
1997; Mongrand et al. 2000) and DAG (Williams et al. 2000) have been proposed to be the ER derived lipid precursor transported to the plastid.
3.3 Lipid synthesis in the plastid
PA is synthesised in the inner envelope membrane by a pathway similar to that in the ER except that acyl-ACP functions as acyl donor (Ohlrogge and Browse 1995). Due to differing specificities of the plastid acyl transferases from those in the ER, the fatty acid configuration of acyl lipids synthesised in the plastid differs from that of those synthesised in the ER. This pathway appears to be more or less conserved from the cyanobacterial origin of the plastid. The glycerolipids synthesised within the plastid always carry a C18 fatty acid at the sn-1 and if they contain a C16 fatty acid it is esterified to the sn-2 position. Thus, it is possible to differentiate between ER- and plastid-synthesised glycerolipids by fatty acid positional analysis. Thus, a high content of C16 fatty acids on the sn-2 position is an indication for an intraplastidial origin. In so-called 16:3 plants, the intraplastidial glycerolipid synthesis pathway contributes significantly to the synthesis of thylakoid galactolipids (Heinz and Roughan 1983). Most plants are so called 18:3 plants (Mongrand et al. 1998) in which this pathways only contribution to the plastid membrane lipid pool is PG (Sparace and Mudd 1982; Andrews and Mudd 1985). In fact, intraplastidial glycerolipid synthesis can be completely inactivated without any apparent consequences for the plant (Kunst et al. 1988; Kunst et al. 1989). The main reason that PA synthesised in the plastid do not contribute to galactolipid synthesis in 18:3 plants is thought to be that the inner envelope PA phosphatase (PAP) that provide DAG for MGDG synthesis is present at only very low levels in 18:3 plants (Heinz and Roughan 1983; Gardiner et al. 1984a). Fatty acids esterified to glycerolipids synthesised in the chloroplast envelope are desaturated by desaturases located in the envelope (Schmidt and Heinz 1993; Ohlrogge and Browse 1995).
3.4 ER to chloroplast lipid transport
Any successful model for the lipid trafficking from the ER to the plastid has to account for lipid sorting and directionality in the transport process. The ER membrane contains PE, PS, sphingolipids and sterols, none of which are considered to be chloroplast constituents. The only phospholipids shared in significant proportions between the chloroplast envelope and the ER are PC, PI and PG.
Lipid transport can, in theory, be mediated by three different mechanisms: monomeric diffusion (unassisted or assisted by soluble proteins), vesicle transport or transport at sites of physical contact between the membranes. Unassisted monomeric diffusion is not very likely to account for the bulk flow of lipids, since the common phospholipids have very low solubility in water. One way to avoid the solubility problem and still get away with a model based on unassisted monomer diffusion is the “lysoPC-hypothesis” (Testet et al. 1996; Mongrand et al. 1997; Moreau et al. 1998; Mongrand et al. 2000). According to this model, lysoPC would be produced in the ER by the action of a phospholipase A2. LysoPC is fairly
water-soluble and could partition freely between the ER and the chloroplast envelope. LysoPC would then be reacylated to PC by an acyl-CoA dependent lysoPC acyltransferase (LPCAT) in the chloroplast envelope. An LPCAT activity is indeed present in the chloroplast envelope in pea (Kjellberg et al. 2000) and leek (Mongrand et al. 1997). The model is supported by in vivo pulse chase studies performed on leek seedlings (Mongrand et al. 1997; Mongrand et al. 2000). However, the model does not explain why lysoPC is not reacylated in
the ER in a futile cycle of hydrolysis and reacylation when the ER apparently also contain LPCAT activity (Kjellberg et al. 2000).
Monomeric diffusion assisted by non specific lipid transfer proteins has been proposed and was demonstrated in vitro between liposomes (Oursel et al. 1987) or microsomes (Dubacq et al. 1984) and chloroplasts. However, the non-specific lipid transfer protein was later shown to contain a signal peptide directing the protein into the secretory pathway (Bernhard et al. 1991; Madrid 1991) and was immunolocalised to the cell wall (Kader 1997). Of the many lipid transport proteins found in A. thaliana (Beisson et al. 2003), the great majority (>95 %) are predicted by TargetP (Emanuelsson et al. 2000) as targeted to the secretory pathway. Thus, the non-specific lipid transport proteins are probably not involved in lipid transport between ER and chloroplast.
Lipids are transported by vesicle trafficking in the secretory pathway. Reconstituted lipid transport (presumably vesicle-mediated) between ER and Golgi in plant (Morré et al. 1991b; Sturbois-Balcerzak et al. 1994) and animal (Moreau and Morré 1991; Moreau et al. 1991; Morré 1998) cells require soluble proteins and ATP. Purified ER derived transport vesicles were found to contain approximately equal proportions of PC and PE, were slightly enriched in PS and their formation required ATP and soluble proteins (leek seedlings; Sturbois-Balcerzak et al. 1999). Since chloroplast membranes contain neither PE nor PS, it seems unlikely that these or similar ER-derived vesicles are directly involved in the supply of lipids to the chloroplast envelope.
Lipid transport at sites of physical contact requires the physical closeness of the ER and the chloroplast envelope. Tubules of ER were observed in close proximity to plastids in Vicia fabia cotyledons (Kaneko and Keegstra 1996) and mature tobacco leaves (Hanson and Köhler 2001). Thus, the physical prerequisites for lipid transport between ER and chloroplast at sites of physical contact exist in higher plants.
Regarding the transport of lipids from ER to chloroplasts in plants, biochemical evidence is rather scarce. An attempt to reconstitute the transport of lipids from ER to chloroplasts used enriched ER fractions containing 14C-labelled lipids or 35S-labelled proteins and unlabelled intact chloroplasts (Paper I). After co-incubation with radiolabelled enriched ER, the chloroplasts were re-purified by Percoll gradient centrifugation. Labelled lipids were retained in the re-isolated chloroplasts approximately twice as efficiently as 35S labelled ER proteins, indicating that some of the donor membrane remained attached to the chloroplasts after re-isolation but also that lipid transfer occurred during the co-incubation. No additions were required for lipid transfer from ER to chloroplasts. Radiolabel in PC and PI was found to increase in the re-isolated chloroplasts with incubation time, while radiolabel in PE did not, indicating active transport of the two former phospholipids.
The lack of dependence of nucleotides or cytosolic proteins for the in vitro lipid delivery from ER to chloroplast strongly argues against lipid delivery by ER-derived transitory vesicles. Generally, the in vitro characteristics are much more consistent with lipid delivery at sites of physical contact between the membranes than with any other model.
Cytosolic proteins did not stimulate the lipid transfer, but had other interesting effects. When lipid transfer was reconstituted from 14C-labelled ER to chloroplasts without additions, almost no radiolabel was found associated with galactolipids in the re-isolated chloroplasts. If the reconstituted transfer is to be of any significance for in planta chloroplast lipid biogenesis, the
transferred lipids should function as precursors for galactolipid synthesis in the chloroplast envelope. If radiolabel transfer from phospholipids to galactolipids could be observed it would also provide further evidence that intermembrane lipid transport really had occurred in the in vitro system. Cytosolic proteins were found to be the key component required for synthesis of radiolabelled MGDG in chloroplasts during co-incubation with ER containing radiolabelled phospholipids in the presence of UDP-galactose (Paper I). Clearly, the cytosol provided enzymatic activity that allowed the DAG backbone of ER-derived phospholipids to function as substrate for the inner envelope-localised MGDG synthase (see further below). Lipid transport from ER to mitochondria in yeast has been extensively studied by various in vitro approaches (Gaigg et al. 1995; Voelker 2000; de Kroon et al. 2003; Voelker 2003). The general conclusion is that no nucleotides or soluble proteins are required for lipid transport from ER to mitochondria and that lipid transfer probably occurs at sites of physical contact between the ER and the mitochondrion. Apparently, there are similarities between lipid transport from ER to mitochondria in yeast and ER to chloroplast lipid transport in higher plants. Recently published data demonstrate that ubiquitination is involved in lipid transport from the yeast ER to the mitochondria (Schumacher et al. 2002). The first molecular clue to how ER to chloroplast lipid transport in plants actually work was recently published (Xu et al. 2003). A permease-like protein localised in the outer chloroplast envelope was shown to be required for ER to chloroplast lipid transport in A. thaliana. Strangely, inactivation of this protein caused the activation of a processive galactolipid synthase and the plant accumulated trigalactosyldiacylglycerol (Xu et al. 2003). Consequently the gene was named TGD1 for trigalactosyldiacylglycerol 1.
Other evidence for lipid transport at contact sites include that the yeast vacuole localised PS decarboxylase contain a domain that is unrelated to catalytic activity but functions in extracting lipids from a physically close donor membrane and deliver them to the active site in the vacuolar membrane (Wu and Voelker 2004). In gram negative bacteria, lipids have to be exported from their site of synthesis in the inner membrane to the outer membrane (Huijbregts et al. 2000). Two components of a putative lipid export system in bacteria have been identified. One is an ATPase located in the inner membrane with similarity to ABC transporters (Doerrler et al. 2001) and the other is an outer membrane protein (Genevrois et al. 2003). The bacterial lipid export system, the components identified as involved in interorganellar lipid transport in yeast and the A. thaliana TGD1 all bear no obvious resemblance to each other. Nevertheless, they all by some mechanism solve the same problem. They all move glycerolipids between closely positioned membranes. The molecular details of the transport process remains to be elucidated but it would not be surprising if, at some level, similarities between the systems were to be found.
3.5 The PLAM
As discussed, the plant ER is comprised of many different functionally and/or structurally distinct domains. Enzymatic activities considered to be associated to the ER were present in measurable amounts in intact chloroplasts isolated from pea seedlings (Kjellberg et al. 2000). The amount of the ER enzyme activity per chloroplast equivalent decreased with the age of the plant material used for chloroplast isolation (Kjellberg et al. 2000). At earlier stages of leaf development there is probably a large expansion of the thylakoid membrane area and since pea is an 18:3 plant (Heinz et al. 1983; Gardiner et al. 1984b; Mongrand et al. 1998) there is a large need for import of galactolipid precursors from the ER to the chloroplast.
As the plant ER forms close connections with the plastid envelope, a functional equivalent of the MAM was proposed (Kjellberg et al. 2000). These Plastid Associated Membranes (PLAM) would represent a specialised domain of the ER that is closely associated to the chloroplast and presumably involved in lipid transport between the ER and the chloroplast. The MAM-fraction can be released from yeast mitochondria by incubating isolated mitochondria at pH 6 and separated from the mitochondria by sucrose gradient centrifugation (Gaigg et al. 1995). The yeast MAM is enriched in lipid synthesis enzymes and is superior to bulk light microsomes as an in vitro lipid donor to mitochondria (Gaigg et al. 1995; Achleitner et al. 1999). A MAM fraction with similar properties has also been isolated from rat liver mitochondria (Vance 1990).
Since MAM fractions could be isolated from intact yeast mitochondria an attempt was made to isolate the PLAM fraction from intact chloroplast isolated from young pea seedlings (Paper II). Young plant material was chosen because the presence of ER associated activities in isolated chloroplasts was highest in the young leaves (Kjellberg et al. 2000). Highly purified fully intact chloroplasts were isolated by Percoll™ gradient centrifugation (Räntfors et al. 2000). The chloroplasts were incubated at pH 6 and loaded onto sucrose gradients. After centrifugation, a light membrane fraction was recovered at the top of the gradient and was collected as the PLAM fraction. To obtain a suitable bulk light membrane fraction for comparison, a microsomal fraction obtained from the post chloroplast supernatant was treated in the same way as the chloroplasts, loaded on an identical gradient and the top band was collected. The resulting fraction was considered as representative of bulk light microsomes (light MS). The light MS and the PLAM fractions were compared with respect to lipid and polypeptide composition and marker enzyme activities.
Chloroplasts are fragile and can easily break during manipulation and at least outer envelope membranes are also of quite low density (Block et al. 1983c). Thus, special attention should be paid to the presence of envelope membranes in the light membrane fractions. Both the PLAM and the light MS fractions were rich in phospholipids and contained only minor proportions of the chloroplast galactolipids MGDG and DGDG. The specific activity of MGDG-synthase was, compared with isolated envelope membranes, very low in both fractions. The specific activity of two different ER marker enzymes was approximately the same in the PLAM as in the light MS fraction. Taken together, this demonstrates that the PLAM fraction is probably more related to the ER than the envelope. In other words a predominantly non-plastid fraction could be washed off and purified from “highly purified” intact chloroplasts.
Interesting differences between the light MS and the PLAM fraction regarding the lipid composition were also observed (Paper II). The PC/PE ratio was about twice as high in the PLAM fraction as in the light microsomes and the polypeptide composition was very different between the two fractions. The higher PC content in the PLAM fraction is very interesting. If the PLAM is involved in exporting lipids from the ER to the chloroplast it seems logical that it should be enriched in the molecule that could function as a galactolipid precursor and slightly depleted in a lipid that ideally should not be exported to the chloroplast at all. Another aspect is that PC is a cylindrical lipid and therefore bilayer prone, whereas the shape of PE is more of an inverted cone and consequently the lipid is prone to form other structures than bilayers (Israelachvili et al. 1980). The light MS fraction is likely to largely represent the tubular structures of the smooth ER and tubules require membranes of high curvature; a structure that would benefit from a high PE to PC ratio. On the other hand, a domain of the
ER that associates to the rather flat surface of the outer envelope would probably resemble a flattened sack; a low curvature structure that conversely would benefit from a higher PC to PE ratio. Support for that the ER actually forms a flattened sack in contact with the plastid comes from electron microscopy where the strands of ER along the plastid envelope most likely represent sections through a flattened sack of ER.
What the in planta function of the PLAM is can at this point only be speculated on. However, it seems reasonable to suggest that it is, like the yeast MAM, involved in lipid transport between the ER and the chloroplast envelope.
3.6 PC metabolism in the envelope membrane
The enzymatic degradation of PC to DAG is required if PC, transported to or synthesised by LPCAT in the envelope, is to function as precursor for MGDG synthesis. DAG is the immediate lipid precursor for galactolipid synthesis in the plastid envelope (see further below). PC (or any other phospholipid) could be metabolised to DAG by two different pathways. Phospholipase C (PLC) could directly hydrolyse the phospholipid to DAG or alternatively, a phospholipase D could hydrolyse the phospholipid to PA that a phosphatidic acid phosphatase (PAP) could metabolise further to DAG.
It was found that soluble proteins were required for ER-derived PC to function as precursor for in vitro MGDG synthesis in pea chloroplasts (Paper I). To further characterise the role of the soluble proteins in the PC to MGDG conversion in the chloroplast envelope, a system was set up which supplied the chloroplast envelope with radiolabelled PC. The LPCAT activity present in the inner envelope of pea chloroplasts was used to introduce radiolabelled PC into isolated chloroplasts or chloroplast envelopes (Paper I). It should be noted that this approach provide no information concerning the involvement of the chloroplast localised LPCAT in lipid transport to the chloroplast. In this particular case it was just a convenient way of introducing radiolabelled PC into the chloroplast envelope. Essentially the same results were obtained using intact pea chloroplasts or isolated envelope membranes. When a cytosolic protein fraction and UDP-galactose were added to chloroplasts or envelopes containing [14C]PC, radiolabel appeared in MGDG. Size fractionation of the cytosolic fraction revealed that a fraction of <100 kD proteins was without effect. Stroma could not substitute for cytosol. When a PLD inhibitor (AEBSF) was included, the amount of radiolabel in MGDG decreased markedly. The results indicate that the pathway from PC to DAG more likely utilises PLD and PAP than direct degradation of PC to DAG by PLC.
To test the phospholipase activity present in cytosolic fractions, [14C]PC-containing lipid mixtures were incubated with cytosolic fractions in a mixed micellar assay (Paper I). Three different lipid mixtures were tested: a mixture of PC, PE and phosphatidylinositol 4,5-bisphosphate known to stimulate PLD activity (Pappan et al. 1997), a mixture made to resemble the outer envelope membrane (Block et al. 1983c) and a mixture containing the same proportion of PC as the outer envelope membrane mixture but only DGDG as the additional component. Total PC hydrolysis was highest in the outer envelope lipid mixture and the main hydrolysis product was DAG. The DAG producing activity was found to be highly enriched in the >100 kD fraction. The DAG producing activity could be inhibited by approximately 50 % by the PLD inhibitor AEBSF. This indicates that the DAG was produced from PA formed by PLD catalysed hydrolysis of PC.
All this taken together suggests that in the in vitro system, [14C]PC was degraded by cytosolic PLD followed by PAP to DAG that could function as substrate for the MGDG synthase. When intact chloroplasts were used, the lipolytic reactions most probably took place on the outer surface of the outer chloroplast envelope, since the outer envelope is impermeable to substances larger than 10 kD (Flügge and Benz 1984). Interestingly, the lipid environment of the outer envelope membrane appeared to stimulate the cytosolic lipases. What properties of the outer envelope lipids stimulate the cytosolic lipases? The outer envelope lipid mixture contains PG and MGDG in addition to PC and DGDG. PC and DGDG are both bilayer prone lipids that carry no net electrical charge, whereas PG is anionic and MGDG (given the usual degree of unsaturation) quite prone to form other structures than bilayers. However, a mixture that contained the essentially the same amount of anionic lipids as the outer envelope lipid mixture provided a much poorer environment for PC-hydrolysis than the outer envelope mixture (Paper I). Thus, the stimulatory properties of the outer envelope lipids on cytosolic PC hydrolysis enzymes could probably be ascribed mainly to the presence of the non-bilayer lipid MGDG. Specific interaction between MGDG and chloroplast protein transit peptides has been reported (Pinnaduwage and Bruce 1996), suggesting that the presence of MGDG could help recruiting specific cytosolic lipases. Lipid biosynthesis in bacterial membranes (Rilfors and Lindblom 2002) and the mammalian CTP:phosphocholine cytidylyltransferase (Attard et al. 2000) are known to be regulated by the bilayer/non-bilayer lipid balance in the membrane. The presence of PLD in plant tissues has been recognised for a very long time (Wang 2001), but only recently the whole family of plant PLDs were identified. The 13 PLDs identified in
A. thaliana are, based on sequence similarity and biochemical characteristics, divided into five different groups, α, β, γ, δ and ς (Qin and Wang 2002). A number of these PLDs have been linked to signalling events (Wang et al. 2000; Sang et al. 2001; Wang 2002; Dhonukshe et al. 2003; Potocky et al. 2003; Zhang et al. 2003b; Zhao and Wang 2004). PLDα, β, γ and δ
all contain a C2 domain that is known to tether proteins to membrane phospholipids in a Ca2+ -dependent manner, linking PLD activation to calcium signalling. These A. thaliana PLDs have predicted sizes of <100 kD. PLDς, in contrast, does not contain the C2 domain, is indeed independent of Ca2+ for catalytic activity, but highly specific for PC. It is slightly larger than the other A. thaliana PLDs with a predicted size of 125 kD (Qin and Wang 2002). PLDς has been found to be expressed also in Oryza sativa, Medicago truncatula and Lycopersicum esculentum (Elias et al. 2002). Taken together, PLDς is the identified PLD isoform that best fits the description for the soluble PLD activity required for metabolising PC in the chloroplast envelope to PA (Paper I). Characterisation of PLDς mutants would settle this issue.
The information regarding plant PAPs is rather scarce and research has focused mainly on the PAP in the chloroplast inner envelope responsible for formation of the DAG used for prokaryotic galactolipid synthesis in 16:3 plants (Block et al. 1983a; Andrews et al. 1985). However, the envelope PAP-activity in pea, an 18:3 plant, is very low or completely absent (Heinz et al. 1983). Furthermore, soluble protein fractions obtained from pea seedlings contained the whole machinery for PC to DAG metabolism (Paper I). Thus, envelope localised PAP was probably not involved in supplying [14C]DAG for MGDG synthesis in the experiments described in Paper I. Vicia fabia leaves (Königs and Heinz 1974) and developing seeds of Brassica napus (Kocsis et al. 1996; Furukawa-Stoffer et al. 1998) contain both soluble and membrane bound phosphatidic acid phosphatases. Unfortunately, soluble plant PAPs have not received much attention and no candidate genes or proteins are known. The membrane bound PAPs identified in A. thaliana appears to be involved in lipid signalling rather than bulk membrane lipid synthesis (Pierrugues et al. 2001). However, the data
presented in Paper I predicts that a soluble NEM-insensitive PAP of native size exceeding 100 kD is responsible for providing DAG for eukaryotic plastid galactolipid synthesis.
3.7 Lipid galactosyl transferases in the plastid envelope
DAG is used for MGDG synthesis in the chloroplast envelope where a galactosyltransferase transfers a galactosyl moiety from UDP-galactose supplied from the cytosol (Bertrams et al. 1981; Maréchal et al. 2000). The MGDG synthase activity has been localised to the inner chloroplast envelope in spinach a 16:3 plant (Block et al. 1983c; Miege et al. 1999) and both the inner and outer envelope membrane in pea chloroplasts (Tietje and Heinz 1998; Kjellberg et al. 2000). The MGDG synthase genes identified to date fall into two distinct categories (Maréchal et al. 2000; Awai et al. 2001). The A type MGDG-synthases are localised in the inner envelope membrane and provide the major portion of thylakoid MGDG in green tissue (Jarvis et al. 2000; Awai et al. 2001). The B type of MGDG-synthases are mainly expressed in non green tissues and also contribute to MGDG synthase under phosphate limited conditions (Awai et al. 2001; Kobayashi et al. 2004). The A type MGDG-synthases are expressed as a precursor with a cleavable N-terminal chloroplast transit peptide, whereas the B type MGDG synthases lack apparent cleavable transit peptides (Maréchal et al. 2000). Nevertheless, both the A and B type MGDG synthases in A. thaliana have been shown to be chloroplast localised (Awai et al. 2001). The A type MGDG synthases are probably localised to the inner chloroplast envelope, whereas the B type MGDG-synthases probably are localised to the outer envelope.
Isolated chloroplasts contain an enzymatic activity that transfers one galactose moiety from one molecule of MGDG to another MGDG molecule yielding DGDG and DAG (Wintermans et al. 1981; Heemskerk et al. 1990; Kelly et al. 2003). It was accepted for a long time that this activity provided the bulk of the chloroplast DGDG. However recent findings indicate that in fact UDP-galactose dependent galactosyl transferases are responsible for the majority of in planta DGDG synthesis (Kelly et al. 2003). The DGDG synthase activities seem to be strictly localised to the outer chloroplast envelope (Kjellberg et al. 2000; Froehlich et al. 2001). Like the MGDG synthases, the DGDG synthases characterised to date also fall into two different classes (Dörmann and Benning 2002; Kelly and Dörmann 2002). DGD1 in A. thaliana
accounts for the bulk of DGDG synthesis in green tissue (Dörmann et al. 1995; Härtel et al. 2000b; Kelly et al. 2003). DGD1 contains an N-terminal extension not found in the DGD2, which is not required for catalytic activity but essential for in planta function of DGD1 (A. A. Kelly, personal communication). A. thaliana DGD1 contains a predicted transit peptide and is targeted to the outer envelope of isolated chloroplasts (Froehlich et al. 2001). DGD2 lack predicted chloroplast transit peptide, but is nevertheless targeted to the outer chloroplast envelope in in vitro assays (Kelly et al. 2003). The processive DGDG synthase activity found in isolated chloroplasts (Wintermans et al. 1981; Heemskerk et al. 1990; Kelly et al. 2003), however, appears unrelated to both DGDG synthases identified in A. thaliana to date (Kelly et al. 2003). ER to chloroplast lipid transport is probably disrupted in the tgd1 mutant and this mutant accumulates the oligogalactosyldiacylglycerol products of the processive galactolipid synthase in planta (Xu et al. 2003). Thus, it is striking that severing the ER-plastid link either by mechanical isolation of the chloroplast or genetic disruption of lipid import activates the processive galactolipid synthase.
4. Intraplastidial lipid trafficking
As discussed in the previous section the galactolipids, that make up the majority of the thylakoid lipids are synthesised in the chloroplast envelope. Membrane lipids are not the only nor the most hydrophobic thylakoid constituents synthesised in the envelope. Carotenoids and quinones are also synthesised in the chloroplast envelope (Joyard et al. 1998b). Thus, there is a need for transport of membrane lipids and other hydrophobic substances from the inner envelope membrane to the thylakoid. In theory, again as discussed in the previous section, lipids could be transported at contact sites between the membranes, by assisted or unassisted monomeric diffusion or by vesicles that are formed from the donor membrane and fuse with the target membrane.
There are, at least in mature chloroplasts, no apparent physical contacts between the thylakoid and the envelope membranes. In contrast, contacts between the thylakoid and the inner envelope are quite frequently observed in developing chloroplasts (Carde et al. 1982). Thus, transport at contact sites is likely to occur during early stages of chloroplast development. There is, however, turnover of thylakoid lipids in mature chloroplasts too (O'Sullivan and Dalling 1989; Hellgren and Sandelius 2001a), and thus a need for lipid transport from the envelope to the thylakoid in mature chloroplasts where the thylakoid and inner envelope are well separated by the aqueous stroma (Ryberg et al. 1993). The lipids synthesised in the envelope have to cross a distance of at least 50-100 nm (Morré et al. 1991c; Ryberg et al. 1993) of aqueous solution to reach the thylakoid. For an acyl lipid this is in terms of thermodynamics no less than an energetic disaster. Thus, monomeric unassisted diffusion across the distance separating the inner envelope and the thylakoid is not likely to account for any significant proportion of bulk lipid flow from envelope to thylakoid. Monomeric diffusion assisted by soluble proteins in the stroma would be more plausible. A 28 kD soluble protein that was able to catalyse the in vitro transfer of MGDG between liposomes has been isolated from spinach chloroplast stroma (Nishida and Yamada 1985). The in vivo
significance of this protein for the bulk flow of galactolipids from envelope to thylakoid, however, remains to be demonstrated.
4.1 Morphological evidence of intraplastidial vesicles
When leaf pieces were incubated at low temperature, an accumulation of vesicular structures in the chloroplast stroma was observed (Morré et al. 1991c). Accumulation of vesicles was also observed in isolated chloroplasts incubated at low temperature (Westphal et al. 2001). These results bear resemblance to the accumulation of transitory ER-derived vesicles in animal cells incubated at low temperature (Moreau et al. 1992). The molecular basis for formation of the ER-Golgi low temperature compartment is that the fusion of transport vesicles with the Golgi membrane is inhibited while transport vesicle formation is unaffected by the low temperature (Moreau et al. 1992). The vesicles that accumulated in the stroma of isolated chloroplasts or the chloroplasts of leaf discs incubated at low temperature quickly disappeared when the temperature was increased again (Morré et al. 1991c; Westphal et al. 2001), The latter observation lends further support for the similarity between the vesicles accumulated in the chloroplast stroma and the ER-Golgi low temperature compartment. Furthermore, the amount of vesicles observed in the stroma of isolated chloroplasts could be modulated by inhibitors of vesicle trafficking in the secretory pathway (Westphal et al. 2001). The in situ low temperature-induced accumulation of vesicles in the stroma was impaired in an A. thaliana mutant that contained drastically reduced amounts of thylakoid membranes (Kroll et al. 2001). The mutated protein was named VIPP1 for Vesicle-Inducing Protein in
Plastids, and had been shown previously to be localised to both the inner envelope and the thylakoid (Li et al. 1994).
4.2 Characterisation of in organello lipid transport
Lipid transfer from the envelope to the thylakoid can be studied in isolated chloroplasts by a method developed by Rawyler and co-workers (Rawyler et al. 1992). Intact spinach chloroplasts were isolated and incubated with radiolabelled UDP-galactose and the radiolabel was transferred to the head groups of MGDG and DGDG. After the incubation the chloroplasts were washed, lysed and the thylakoids isolated. The transport of galactolipids from the envelope to the thylakoid could be determined from the ratio of lipid radiolabel in the thylakoid to that in the whole chloroplasts. The method was utilised to study lipid transport in pea chloroplasts (Paper III). Generally, lipid transfer from envelope to thylakoid in isolated chloroplasts was rapid (Rawyler et al. 1992; Rawyler et al. 1995; Paper III). Exogenous nucleotides or soluble proteins did not affect the export of lipids from the envelope membrane to the thylakoid (Paper III). KF, which generally inhibits phosphatases, increased the transfer of MGDG and decreased the transfer of DGDG from the envelope to the thylakoid (Paper III). This result indicates the involvement of phosphatases in regulating the transport process. Other phosphatase inhibitors were shown to affect the accumulation of vesicles in isolated chloroplasts (Westphal et al. 2001). When the outer envelope-localised DGDG synthesis activity was inactivated by protease treatment, the transport of MGDG remained unaffected, indicating that the lipid transport from the inner envelope to the thylakoid was independent of protease sensitive factors in the outer envelope membrane and ongoing DGDG synthesis.
A sorting of lipids prior to export to the thylakoid from the inner envelope was observed in the in organello system (Paper III). In chloroplasts isolated from mature leaves, MGDG was clearly preferred over DGDG. The preferential export of MGDG may be at least in part explained by the different sites of synthesis. MGDG is synthesised in the inner envelope while DGDG is synthesised in the outer envelope membrane. However, MGDG was strongly preferred over PC synthesised in the inner envelope by LPCAT activity, which clearly indicates that lipid sorting occurred at the level of the inner envelope membrane (Paper III). Whether PC is an actual thylakoid membrane constituent has been a matter of debate. To my knowledge, all studies published to date regarding the lipid composition of thylakoid membranes include 1-5 mol% of PC (Dorne et al. 1985; Dorne et al. 1990) (Paper III). Dorne and co-workers (Dorne et al. 1990) demonstrated that mild phospholipase C treatment of intact chloroplasts removed all PC from the chloroplasts, indicating that all PC was exposed to the outside of the chloroplast. This result may also indicate that thylakoid PC is not stationary to the thylakoid but continuously recycled to the envelope membrane. The PC of highly purified thylakoid membranes appears to have a rather different fatty acid composition than bulk chloroplast PC (Paper III). Furthermore, a portion of radiolabelled PC produced by incorporation of [14C]Acyl-CoA by LPCAT in the inner envelope (Kjellberg et al. 2000) was rapidly transferred to the thylakoid membrane (Paper III). In conclusion, the available evidence suggest that PC is in fact an authentic albeit minor and perhaps transient thylakoid lipid constituent.
To determine whether there is relation between the accumulation of vesicular structures in the stroma in leaf discs or isolated chloroplasts (cf. above) and lipid transport to the thylakoid, the temperature dependence of galactolipid transport in organello was assayed (Paper III).
Interestingly, the transfer of newly synthesised galactolipids from the envelope membrane to the thylakoid was inhibited to ca. 50 % at the same temperature interval as accumulation of vesicles in the stroma was observed. This result indicates that the observed vesicles may in fact be responsible for lipid transport form the envelope to the thylakoid. However, inhibition by low temperature (<12°C) was never complete and at the lowest temperatures tested, 25-30 % of the newly synthesised radiolabelled galactolipids were transported to the thylakoid. The latter finding may indicate the presence of other not as temperature sensitive transport pathways inside the chloroplast.
4.3 Intraplastidial lipid transport reconstituted in vitro
Transport of galactolipids has been reconstituted in vitro using membranes immobilised on nitrocellulose strips as acceptors and membranes containing radiolabelled galactolipids as donors (Morré et al. 1991a). The envelope to thylakoid specific transfer was found to be stimulated by ATP and to some extent by stromal proteins. Furthermore, incubation of isolated chloroplast envelope with stromal proteins and ATP resulted in the formation of vesicles similar in size to ER transitory vesicles (Morré et al. 1991a). Räntfors and co-workers used a slightly different approach to study intraplastidial lipid trafficking (Räntfors et al. 2000). Isolated envelope membranes containing radiolabelled galactolipids were immobilised on nitrocellulose strips and the requirements for release of radiolabel from the strips into solution were analysed. The release of lipid radiolabel from the immobilised envelope was found to be strongly stimulated by ATP, GTP and stromal proteins. Similar characteristics were found for envelope fractions isolated from pea, wheat and spinach. Requirement of nucleotides and soluble proteins is a hallmark of cytosolic vesicular trafficking (Kirchhausen 2001; Bonifacino and Glick 2004). GTP binding proteins and proteins that are phosphorylated by GTP are present in the chloroplast envelope (Kjellberg and Sandelius 2002; Kjellberg and Sandelius 2004), stroma and thylakoid (Kjellberg and Sandelius 2004). Both phosphorylation and the GTP binding pattern to proteins in pea chloroplast envelopes were modified by co-incubation with stromal proteins (Räntfors et al. 2000).
4.4 Components of an intraplastidial vesicle transport system
If indeed vesicular membrane trafficking occurs inside the plastid, there would have to be molecular machineries present inside the chloroplast that drives formation of vesicles at the inner envelope and vesicle fusion with the thylakoid membrane, respectively. The biochemical evidence discussed above suggests that such a vesicular system would share several characteristics with vesicle trafficking in the secretory pathway. The important first question would be, what are the minimum requirements for vesicle formation and targeting in the secretory pathway?
In the cytosol, membranes are deformed into vesicles by the force generated when a protein coat is assembled. Three different protein coats are known from the secretory pathway, clathrin coats, COPI and COPII (Kirchhausen 2001; Bonifacino and Glick 2004). Of these the simplest system seems to be the COPII coat, which requires only two soluble protein complexes and a small GTPase for vesicle coat formation. Once released from the donor membrane, the secretory vesicle shed its coat. Correct targeting and fusion of the vesicle are mediated by so called SNAREs. Once primed by a specialised ATPas called NSF, SNAREs in the vesicle (v-SNAREs) and SNAREs in the acceptor membrane (t-SNAREs) recognise each other and bind very tightly bringing the membranes close enough to allow fusion of the bilayers (Bonifacino and Glick 2004). Additional factors may well be involved in both
