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.
Göteborg, maj 2004
ISBN 91-88896-48-X
Lipid trafficking: into, within and out of the chloroplast
Mats Andersson
Göteborg 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 ERderived 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
Göteborg, maj 2004
ISBN 91-88896-48-X
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
2.1 Membrane lipids
2.2 Plant membranes
2.3 The plastids
2.4 The secretory pathway
2.5 The plasma membrane
2
2
3
3
5
6
3. Galactolipid biosynthesis
3.1 Fatty acid synthesis
3.2 Lipid synthesis in the ER
3.3 Lipid synthesis in the plastid
3.4 ER to chloroplast lipid transport
3.5 The PLAM
3.6 PC metabolism in the envelope membrane
3.7 Lipid galactosyl transferases in the plastid envelope
7
7
8
9
9
11
13
15
4. Intraplastidial lipid trafficking
4.1 Morphological evidence of intraplastidial vesicles
4.2 Characterisation of in organello lipid transport
4.3 Intraplastidial lipid transport reconstituted in vitro
4.4 Components of an intraplastidial vesicle transport system
4.5 Just one pathway?
16
16
17
18
18
21
5. Galactolipids everywhere
5.1 Phosphate house-holding and galactolipid synthesis
5.2 Phospholipid replacement in the plasma membrane
5.3 Phospholipid replacement in other non-plastid membranes
5.4 Galactolipid export from the plastid
22
22
23
25
26
6. A model for galactolipid synthesis and trafficking
6.1 Lipid transport to, whithin and from the chloroplast
6.2 Where do we go now?
27
27
28
7. Acknowledgements
29
8. References
32
9. Svensk populärsammanfattning
45
Abbreviations
16:0
16:3
18:1
18:2
18:3
ACP
ASG
BLAST
CoA
DAG
DGDG
ER
FS
GlcCer
LPCAT
MAM
MGDG
MS
PA
PAM
PAP
PC
PE
PG
PI
PLAM
PLC
PLD
PS
SG
SQDG
Hexadecanoic acid
all-cis-9,12,15-hexadecatrienoic
cis-9-octadecenoic acid
all-cis-9,12-octadecadienoic acid
all-cis-9,12,15-octadecatrienoic acid
Acyl carrier protein
Acylated sterolglucoside
Basic local alignment search tool
Co-enzyme A
Diacylglycerol
Digalactosyldiacylglycerol
Endoplasmic reticulum
Free sterol
Glucosylcerebroside
Lyso-PC acyl transferase
Mitochondria associated membranes
Monogalactosyldiacylglycerol
Microsomes
Phosphatidic acid
Plasma membrane associated membranes
Phosphatidic acid phosphatase
Phosphatidylcholine
Phosphatidylethanolamine
Phosphatidylglycerol
Phosphatidylinositol
Plastid associated membranes
Phospholipase C
Phospholipase D
Phosphatidylserine
Sterolglucosides
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).
1
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.
O
H3C
O
HO
+
N CH2 CH2 O P
H3C
O
O
CH3
O
O
HO
O O
H
H
H
H
CH3
O
Phosphatidylcholine (PC)
OH
HO
2.1 Membrane lipids
O
O
CH3
O
CH3
Monogalactosyldiacylglycerol (MGDG)
OH
H
OH
HO
OH
O
O
O
H
OH
H
H
OHHO
H
HO
O O
H
O
CH3
O
CH3
H
H
H
H
OH
Digalactosyldiacylglycerol (DGDG)
O
HO
NH
CH2CH
O
H
O H HC
H
OH
H
H
OH
HO
OH
OH
CH
CH
Glucosylcerebroside (GlcCer)
HO
Stigmasterol
Figure 1. The structure of some common plant membrane lipids
The key feature of membrane lipids is
their amphiphilic properties; hydrophilic 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
2
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
Cell Wall
tend to decrease mobility in
the
hydrophobic
region
Plasma membrane
making the membrane thicker
and less permeable to small
molecules
(Jones
and
Central vacuole
Chapman 1995). Sterols also
tend to make the hydrocarbon
chains of the hydrophobic
Thylakoid
part of the membrane less
Chloroplast stroma
likely to crystallise at low
Peroxisome
temperatures (Jones and
Chapman 1995).
Mitochondria
2.2 Plant membranes
All living plant cells contain
basically the same set of
membrane-delimited organFigure 2. Transmission electron micrograph of a pea mesophyll cell. Courtesy
of A. S. Carlsson and A.S. Sandelius.
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
3
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;
4
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
Spinach (Block et al.
1983c)
Total envelope
Outer envelope
Inner envelope
Thylakoid
36
17
49
52
29
29
30
26
6
6
5
6.5
9
10
8
9.5
18
32
6
4.5
2
5
1
1.5
0
0
0
0
Pea (Paper III)
Intact chloroplasts
Thylakoid
46
51
32
33
7
8
6
5
7
2
1
0
0
0
Pea (Cline et al. 1981)
Outer envelope
Inner envelope
6
45
33
31
3
2
6
7
44
10
5
2
2
1
Wheat (Bahl et al. 1976)
Total envelope
Lamellar thylakoid
Grana thylakoid
22
42
47
44
37
36
10
9
7
9
10
9
14
2
1
0
0
0
0
0
0
Broad bean (Mackender
et al. 1974)
Total envelope
Thylakoid
29
65
32
26
n.d.
n.d.
9
6
30
3
n.d.
n.d.
0
0
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
5
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
6
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).
7
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
18:1
insert a C16 or C18 fatty acid on
(16:0)
ER
the sn-1 position and always a
PI, PG
18:1
C18 fatty acid on the sn-2
CDP
position. After synthesis of PA,
CDP-DAG
the pathway branches. The two
18:1
OH
(16:0)
anionic phospholipids phosphOH
18:1
atidylinositol (PI), phosphatidylP
P
glycerol (PG) and PS are formed
Glycerol 3PA
18:1
from free head groups and CDPphospate
(16:0)
PC, PE,
diacylglycerol, whereas the
18:1
PS
18:1-CoA
zwitterionic phospholipids PC
16:0-CoA
OH
and PE are formed from
DAG
diacylglycerol (DAG) and CDP
activated bases. PE can also be
Plastid
formed by decarboxylation of
OH
PG
18:1
PS by an enzyme present in both
OH
18:1
(16:0)
mitochondria and ER (Rontein
P
P
et al. 2001; Rontein et al. 2003).
Glycerol 3PA
The ER-localised desaturases
phospate
are specific for C18 fatty acids
18:1-ACP
16:0-ACP
18:1
18:1
esterified
to
phospholipids
18:1
18:1
(Ohlrogge and Browse 1995).
(16:0)
(16:0)
Phospholipids synthesised in the
Galactose
OH
ER are also modified by head
DAG
MGDG
group exchange (Moore 1982)
or exchange of the acyl groups
Figure 3. Glycerolipid synthesis in the ER and the plastid. The
(Williams et al. 2000). The
circle denotes synthesis pathway active in 16:3 plants.
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.
8
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
9
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; SturboisBalcerzak 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 reisolation 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
10
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.
11
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
12
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 4,5bisphosphate 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.
13
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
14
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.
15
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
16
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).
17
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 coincubation 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
18
recognition and fusion in the secretory pathway. However, target-specific vesicle fusion in
vitro only requires the correct SNARE pairing (Parlati et al. 2002). Every single step in
vesicle trafficking in the secretory pathway seems to be somehow regulated by small GTPases
(Bonifacino and Glick 2004).
Both COPI and COPII coated vesicles can be formed in vitro using purified coat subunits and
chemically defined liposomes (Matsuoka et al. 1998; Spang et al. 1998). The formation of
coated vesicles in both cases, however, required anionic lipids in the liposomes.
Generally, anionic lipids appear to be important in the regulation of cytosolic vesicular
trafficking (Matsuoka et al. 1998; Bankaitis and Morris 2003; Pathre et al. 2003).
Phosphoinositides are involved in many different steps of cytosolic vesicular trafficking
(Simonsen et al. 2001). PI kinase activity has been detected in isolated chloroplasts envelope,
most probably in the outer leaflet of the inner envelope membrane (Bovet et al. 1999; Bovet
et al. 2001). In addition to PI kinase, galactolipid kinase activities have also been detected in
the chloroplast envelope (Bovet et al. 1999; Müller et al. 2000; Bovet et al. 2001).
Scission of clathrin coated vesicles from the plasma membrane or Golgi apparatus require the
small GTPase dynamin (Kirchhausen 2001). The binding of dynamin to the membrane is
probably mediated by phosphatidylinositolphosphate or other anionic lipids. Several dynamin
homologues have been identified in plants (Praefcke and McMahon 2004). At least on is
clearly involved in cytosolic vesicular trafficking (Jin et al. 2001). Two A. thaliana dynamin
homologues, however, have been reported to be plastid localised (Park et al. 1997; Kang et al.
1998; Park et al. 1998; Kim et al. 2001). Thylakoid biogenesis is impaired in antisense plants
for one of the plastid-localised dynamin homologues, implicating its involvement in the
supply of membrane material to the thylakoid membrane (Park et al. 1998).
In summary, a minimum machinery for vesicle trafficking would probably consist of coat
subunits, small GTPases that regulate the recruitment of coat subunit, NSF and a
complementary pair of SNAREs to drive fusion. Some of the putative components needed for
a vesicle transport system inside the plastid have been identified by biochemical means,
however many and in particular putative coat proteins remain uncharacterised.
The availability of the entire A. thaliana hypothetical proteome and subcellular localisation
tools makes it possible to perform a large-scale screen for putative components of a vesicular
trafficking system inside the plastid. To do this, a set of A. thaliana protein sequences was
assembled that were predicted to be chloroplast localised by two subcellular localisation tools.
This set was searched for homologues of proteins involved in cytosolic vesicular trafficking
(Paper IV). The sequences of yeast cytoplasmic vesicular trafficking components were used
for BLAST (Altschul et al. 1990) searches in the plastid protein dataset. This approach
generated putative chloroplast localised COPII coat subunits with a very strong homology to
their cytosolic counterparts (Paper IV). COPI and clathrin coat subunits were however not
found in the putative chloroplast protein sequence dataset (Paper IV). A few putative
GTPases related to cytosolic GTPases involved in vesicle trafficking were also found, but
with much weaker homology than the COPII subunits. However, no putative chloroplast
localised SNAREs were found. This approach generates an interesting starting point for
biochemical experiments which are really essential to proof whether the putative plastid
localised COPII coat subunits really are involved in intraplastidial lipid trafficking.
19
Table 2. Evidence for a vesicular transport system inside the plastid
Stage of
vesicular
trafficking
Vesicle
budding
Component
Type of evidence
Source publication
COPII coatomers
Bioinformatics - sequence
similarity and subcellular
localisation prediction
Paper IV
Lipid modification
In vitro lipid phosphorylation
(Siegenthaler et al.
1997; Müller et al.
2000; Bovet et al.
2001)
GTP-binding proteins
Bioinformatics - sequence
similarity and subcellular
localisation prediction
Paper IV
In vitro GTP binding and GTP
dependent phosphorylation of
envelope proteins
(Räntfors et al. 2000;
Kjellberg and
Sandelius 2002, 2004)
Morphology of envelope
vesicles incubated with ATP
and stroma
(Morré et al. 1991a)
Formation of small
vesicles from envelope
Vesicle accumulation in stroma (Morré et al. 1991c;
in situ or in isolated
Westphal et al. 2001)
chloroplasts
Vesicle
scission
Vesicle
fusion
General
vesicular
trafficking
Dynamin
Sequence similarity and in vitro (Park et al. 1997;
characterisation
Kang et al. 1998; Park
et al. 1998; Kim et al.
2001)
Generation of antisense
mutants impaired in thylakoid
biogenesis
(Park et al. 1998)
NSF
In vitro vesicle fusion assays
and sequence similarity
(Hugueney et al.
1995)
SNAREs
“missing”
-
Lipid release from
envelope
Requirement of ATP, GTP and
stroma for lipid release from
isolated envelope
(Räntfors et al. 2000)
In organello lipid
transfer
Temperature dependence of
Paper III
lipid transport from envelope to
thylakoid
VIPP1
Inactivation causes loss of
thylakoids and no formation of
stromal vesicles in situ
20
(Kroll et al. 2001)
The different evidence for vesicular transport inside the plastid discussed above is
summarised in table 2. The only components of known vesicular transport systems that are
missing are SNAREs that are involved in vesicle targeting and fusion in the secretory
pathway. Vesicular fusion inside the plastid may be mediated by some other mechanism than
the SNAREs in the secretory pathway. This is supported by the fact that fusion of biological
membranes by non-SNARE systems is known to occur in eukaryot cells (Mayer 2002). For
example, the membrane spanning subunit of the v-type ATPase in yeast is probably involved
in homotypic vacuolar fusion in yeast. However, a NSF homologue has been shown to be
required for fusion of inner membrane vesicles isolated from red pepper chromoplast
(Hugueney et al. 1995), which would indicate the presence of something like the SNARE
system inside the plastid. On the other hand, the predictors used in the bioinformatics
approach in Paper IV are far from perfect and give both false positives and false negatives.
Combination of the output from more than one predictor is likely to reduce the number of
false positives but is also likely to yield a higher number of false negatives. Thus, putative
plastidial SNAREs might have been missed in the sequence selection procedure.
Alternatively, plastid SNAREs really exist but the degree of sequence conservation is too low
to allow identification with a BLAST search.
Another crucial question in relation to whether lipids really are transported from the envelope
to the thylakoid as vesicles, is whether these vesicles also contain membrane proteins.
Cytosolic transport vesicles carry membrane and soluble proteins as well as lipids and there is
an active sorting of all vesicle constituents (Kirchhausen 2001; Bonifacino and Glick 2004).
In the chloroplast, protein transport to the thylakoid is thought to occur via soluble
intermediates in the stroma that are inserted into the thylakoid membrane by a machinery
directly related to the bacterial protein export systems (Keegstra and Kline 1999). However,
transport of integral thylakoid membrane proteins from the envelope to the thylakoid has been
suggested to occur by a vesicular mode of transfer in the unicellular algae Chlamydomonas
reinhardtii (Hoober et al. 1994; Hoober and Eggink 1999). Protein-free lipid transport
vesicles also seem unlikely in light of the lipid composition of the inner envelope and the
thylakoid. Lipid mixtures of the same composition as the thylakoid membrane are unable to
form stable bilayers (Sen et al. 1981). It has also been reported that liposomes containing >20
mol% of MGDG are unstable (Pinnaduwage and Bruce 1996). The inherent instability of the
lipid composition of the inner envelope and the thylakoid is likely beneficial for fusion and
fission processes, but also indicate that transport vesicles formed from the inner envelope
probably would contain membrane-spanning proteins.
4.5 Just one pathway?
Several lines of evidence, as outlined above and summarised in Table 2, suggest a vesicular
mode of transfer for galactolipids from the chloroplast envelope to the thylakoid membrane.
However, the existence of one pathway does not necessarily exclude the existence of other
transport pathways. All lipid classes are maybe not at all times be transported by the same
mechanism. Different lipid classes are clearly transported by different mechanisms in the
secretory pathway (Moreau et al. 1998). Transport at contact sites cannot be ruled out,
especially at early stages of thylakoid development. A machinery that can produce vesicles
from a donor membrane could share many features with a machinery that deforms a
membrane into invagination or tubules. Likewise, components that catalyse vesicle fusion
may also catalyse fusions between invaginations of the inner envelope and the thylakoid.
However, the lipid sorting observed to occur during the in organello lipid transport (Paper
21
III) is difficult to account for in a model based on complete or partial fusion of the inner
envelope membrane and the thylakoid.
5. Galactolipids everywhere
Phosphate is involved in numerous different processes in the cell and is in many soil types a
limiting nutrient for plant growth (Vance et al. 2003). Even though the lipid bilayer in the
most prominent membrane in green tissue, the thylakoid, is made from non-phosphorous
galactolipids, as much as one third of all organically bound phosphate in leaf tissues resides in
phospholipids (Poirier et al. 1991). The leaf cell apparently limits its need for phosphate
substantially by using non-phosphorous lipids for the thylakoid membranes. The logical
question following this line of reasoning would be, can plants use even more galactolipids
instead of phospholipids to save phosphate?
That certain bacteria can replace phospholipids with non-phosphorous when subjected to
phosphate starvation has been known for decades (Minnikin et al. 1974; Benning et al. 1993;
Geiger et al. 1999; Klug and Benning 2001). Experiments with the A. thaliana dgd1 mutant
provided the first evidence that plants as well as microrganisms can replace phospholipids
with galactolipids (Härtel et al. 2000).
5.1 Phosphate house-holding and galactolipid synthesis
The A. thaliana dgd1 mutant contains very little DGDG compared to wild type, is severely
stunted in growth (Dörmann et al. 1995), is impaired in photosynthesis (Härtel et al. 1997a;
Härtel et al. 1997b) and protein import to the chloroplast (Chen and Li 1998). However, when
the dgd1 mutant was crossed with the pho1 mutant that is impaired in phosphate translocation
from root to shoot (Poirier et al. 1991). The resulting dgd1/pho1 mutant contained substantial
amounts of DGDG (Härtel et al. 2000). The DGDG content increased in wild type and the
dgd1 mutant when cultivated at low phosphate concentration (Härtel and Benning 2000a;
Härtel et al. 2000). Clearly, phosphate limitation caused up-regulation of DGDG synthesis in
A. thaliana. It was also reported that low phosphate availability induced the DGD2 enzyme
(Kelly and Dörmann 2002) and that this activity probably was responsible for the
accumulation of DGDG during phosphate limited growth (Kelly et al. 2003). Replacement of
phospholipids by DGDG was also observed in Acer pseudoplatanus suspension cells (Jouhet
et al. 2003). When the suspension cells were shifted to low phosphate medium, a transient
increase in PC preceded DGDG accumulation suggesting that PC is the immediate precursor
for galactolipids synthesised as a response to low phosphate availability. Phosphate starvation
also induced expression of the B type MGDG-synthases (Awai et al. 2001; Kobayashi et al.
2004) and sulfolipid biosynthesis enzymes (Essigmann et al. 1998; Yu et al. 2002) in A.
thaliana. Since no increase in MGDG content was observed during phosphate limited growth,
the primary function of the B-type MGDG synthases probably was to provide substrate for
DGDG synthesis (Awai et al. 2001; Kobayashi et al. 2004). The phosphate starvation-induced
DGDG in A. thaliana also had a quite different fatty acid composition compared to “normal”
DGDG (Härtel et al. 2000). Analysis of the position of the fatty acids revealed that the DGDG
produced during phosphate limited growth was derived predominantly from the eukaryotic
lipid synthesis pathway. This is also supported by that the B-type MGDG synthases were
preferred eukaryotic over prokaryotic DAG species as substrate (Awai et al. 2001).
22
An increase in tissue DGDG as a response to phosphate starvation has also been observed in
oat roots (Paper V), Calendula officinalis, Tropaeolum majus, Raphanus sativus (K. E.
Larsson, M. X. Andersson, unpublished) and the unicellular algae C. reinhardtii (M. Lind, M.
X. Andersson, K. Andreasson, A. S. Sandelius, unpublished).
The low phosphate induced DGDG in A. thaliana leaves was shown to be associated not only
with chloroplasts, but was also found in the post-chloroplast supernatant (Härtel et al. 2000a;
Härtel et al. 2000). Other more indirect measurements demonstrated that the low phosphate
induced DGDG was probably not associated with photosynthetic membranes (Härtel et al.
2001). Thus, the DGDG synthesised during phosphate limited growth was suggested to be
localised in membranes outside the plastid (Härtel and Benning 2000; Härtel et al. 2000;
Härtel et al. 2001). This obviously makes very good sense since most of the membrane
phospholipids reside in extraplastidial membranes. That DGDG content increased strongly
also in root tissue at low phosphate availability (Paper V; Härtel and Benning 2000; Härtel et
al. 2000), argues very strongly in favour of that phospholipid replacement occurs outside the
plastid. The alternative explanation would be that phosphate starvation induces a large
expansion of plastid membranes. Why this would occur is hard to imagine. This explanation
can nevertheless not be ruled out when considering data on whole tissue lipid composition.
5.2 Phospholipid replacement in the plasma membrane
Plasma membranes can be isolated from both green and non-green plant tissues at very high
fraction purity. Isolated plant plasma membranes often contain a small proportion of
galactolipids (1-5 mol%), which generally has been ascribed to plastid membrane contamination.
In order to determine whether galactolipids could replace phospholipids in a specific nonplastid membrane, plasma membrane fractions were isolated from shoots and roots of oat
cultivated for up to a month with or without inorganic phosphate (Papers V and VI).
Phosphate starvation clearly induced a very large increase in the proportion of DGDG in
plasma membranes isolated from both shoots and roots (Paper V). The increase in DGDG
was balanced by a decrease in phospholipids. The very different fatty acid composition of
plasma membrane DGDG from that of thylakoid DGDG provide further evidence that the
plasma membrane DGDG is unrelated to thylakoid DGDG.
It is well established that the anionic phospholipid PG is replaced with the anionic
galactolipid SQDG in chloroplast and cyanobacterial thylakoids during phosphate limited
growth (Benning et al. 1993; Benning 1998; Essigmann et al. 1998; Sanda et al. 2001; Yu et
al. 2002). MGDG functions as the major non-bilayer lipid in the thylakoid membrane.
Monoglucosyldiacylglycerol has been shown to be able to replace the major non-bilayer
phospholipid PE in E. coli (Wikstrom et al. 2004). Therefore, one may expect that MGDG
and SQDG should replace non-bilayer and anionic phospholipids, respectively in the plasma
membrane. However, while phosphate starvation caused a very large increase in DGDG
proportion in the plasma membrane, SQDG and MGDG remained present only at very low
and unaltered levels (Papers V and VI). However, after four weeks of phosphate limited
cultivation the DGDG of oat root plasma membranes contained significantly more 18:3 than it
did after only two weeks (Paper VI). As DGDG replaces more phospholipids more
unsaturated C18 DGDG accumulates. The decreased saturation causes a change in the lipid
geometry towards a more inverted conical shape, probably more resembling that of nonbilayer prone phospholipids such as PE. Apparently, the physical properties of the bilayer are
23
maintained by modulating the fatty acid composition of DGDG rather than by modulating the
head group composition. Such a mechanism has been observed to occur in the gram negative
bacteria Eschericha coli, which regulates the bilayer/non-bilayer lipid balance by changing
the fatty acid composition of its membrane lipids rather than changing head group
composition (Rilfors and Linblom 2002).
In addition to glycerolipids, oat root plasma membranes also contain sterols, sterolglucosides
(SG), acylated sterolglucosides (ASG) and glucosylcerebroside (GlcCer) (Norberg and
Liljenberg 1991; Uemura and Steponkus 1994; Paper VI). The molar distribution of the main
lipid classes in plasma membranes isolated from roots of oat grown for 4 weeks with or
without phosphate is shown in figure
4. The shifts in lipid composition
A
PL 45%
were quite dramatic. The DGDG
proportion increased to 26 mol% of
the total lipids and no less than 66
mol% (sic) of the total glycerolipids.
Prolonged phosphate starvation also
DGDG 5%
FS 20%
caused increased proportions of
GlcCer and SG (Paper VI; figure 4).
GlcCer 8%
In effect, the plasma membranes
isolated from phosphate-limited roots
SG 12%
ASG 10%
were no longer built from
phospholipids, sterols and sphingoB
FS 20%
lipids, but from galactolipids, sterols
and sphingolipids. The composition
ASG 11%
of the free sterol pool was also
PL 13%
slightly changed by phosphate
starvation. The proportion of
SG 17%
stigmasterol decreased, balanced
mainly by an increase in β-sitosterol
(Paper VI) A decreased ratio of
DGDG 26%
stigmasterol to other sterols has been
GlcCer 14%
observed as a response to many
different stresses (Grunwald and
Endress
1985;
Guye
1989;
Figure 4. The lipid composition of plasma membranes isolated
Navariizzo et al. 1989; Mansour et
from oat roots grown with (A) or without (B) phosphate for 4
weeks. Redrawn from Paper VI, excluding other (2% in A and
al. 1998; Hellgren et al. 2001). The
4% in B) ASG, acylated sterol glycosides; DGDG,
different sterol classes are known to
digalactosyldiacylglycerol; FS, free sterols; GlcCer, glucosylhave different effects on the physical
cerebroside; PL, phospholipids; SG, sterolglycosides.
properties
of
the
membrane
(Hellgren and Sandelius 2001b). The key question here is; is the change in sterols a direct
effect of phosphate starvation or does it represent a mechanism for the membrane to maintain
its physicochemical properties after extensive phospholipid replacement?
The exact effects of such a large change in plasma membrane lipid composition, as observed
after phosphate starvation, has on membrane function can at this point only be speculated in.
Clearly plasma membrane function was maintained to a degree that allowed the plants to
actually complete there life cycle and eventually set seeds (Paper V). Adverse effects of the
lipid replacement are probably to a large degree masked behind the strong impact of the
general phosphate limitation. In addition, adverse effects of lipid replacement in the plasma
24
membrane may not be apparent until the plant is subjected to other stresses. A system in
which phosphate availability could be maintained at a defined level where the phospholipid
replacement is induced but other effects of phosphate limitation are kept at a minimum would
be useful to settle this issue. A starting point for this kind of experiment is provided by the
report that the DGDG increase in A. thaliana tissue was initiated at phosphate concentrations
below 1 mM in the growth medium (Härtel et al. 2000).
The data presented in paper VI provide a few clues to plasma membrane function after lipid
replacement. The first clue is the stability of plasma membrane DGDG when phosphate is resupplied to the plant. One week after phosphate was re-supplied, the proportion of DGDG in
the root plasma membranes had decreased by approximately 50 %. However, during the week
after phosphate re-supply the root mass almost doubled. The decrease in the proportion of
plasma membrane DGDG could thus be attributed almost entirely to de novo membrane
biogenesis. In other words DGDG is probably not to a very large extent replaced once it has
been introduced into the plasma membranes of root cells. This would speak in favour of that
essential membrane functions are fairly well preserved after lipid replacement.
In contrast to the very large change in lipid composition of the plasma membrane caused by
phosphate limitation, the polypeptide pattern of isolated plasma membranes changed only
marginally (Paper VI). The overall limited change in polypeptide composition again
indicates that the membrane retains its physicochemical properties fairly well. Interestingly,
two of the plasma membrane polypeptides induced by phosphate limitation were identified as
a bacterial-type PLC and a heat shock protein. The induction of heat shock protein in root
plasma membranes of phosphate deficient oat, indicate that membrane proteins are not be
fully stable in the “new” lipid environment. A large change in lipid composition of E. coli
membranes causes induction of heat shock proteins (Mileykovskaya and Dowhan 1997).
At this stage, the function of the putative plasma membrane PLC induced by phosphate
starvation can only be speculated on. The protein is similar in sequence to small bacterial
PLCs but completely unrelated to the plasma membrane polyphosphoinositol-specific PLCs
(Paper VI). Whether the putative PLC exhibits PLC activity remains to be demonstrated as
well as its substrate specificity. A speculative model for the function of a plasma membrane
localised PLC would be that it functions in recycling phospholipids delivered via the
secretory pathway. As discussed below, the plasma membrane appears to be a major target for
phospholipid replacement. It is reasonable to assume that the secretory apparatus (ER, Golgi
and transitory vesicles) is dependent on specific lipid composition, including phospholipids,
for proper function and thus retains a high proportion of phospholipids. Thereby,
phospholipids are constantly delivered to the plasma membrane in the form of Golgi derived
vesicles. It has been shown that exogenously added PA is metabolised to DAG in the plasma
membrane and quickly internalised in mammalian cells (Roberts and Morris 2000). If this
also occurs in plant cells, the plasma membrane PLC would metabolise phospholipids
delivered from the Golgi and thereby enabling their recycling to the ER. Another explanation
would be that the plasma membrane PLC provides DAG for galactolipid synthesis directly in
the plasma membrane. This, however, seems less likely, since all three different MGDG
synthases identified in A. thaliana have been shown to be plastid-localised (Awai et al. 2001).
5.3 Phospholipid replacement in other non-plastid membranes
Based on the degree of phospholipid replacement with DGDG in whole root tissue and
isolated plasma membrane and an approximation of how much of the total membrane area the
25
plasma membrane represents in root cells, it was proposed that the plasma membrane is a
major target for phospholipid replacement (Paper V). To investigate whether this really was
the case, oat root microsomes were fractionated by a ten-step aqueous polymer two-phase
counter current system (Larsson 1983; Sandelius and Hellgren 1990) and the distribution of
different marker enzymes and lipids determined (Paper VI). The majority of the low
phosphate induced DGDG clearly followed the plasma membrane marker, but DGDG was
also enriched in another or other membrane fractions. The second DGDG peak, however, did
not coincide with markers for mitochondria, ER, plasma membrane or Golgi membranes. In
none of the fractions was the ATPase activity inhibited by known inhibitors of the tonoplast
ATPase (Paper VI).
What membrane system the non plasma membrane peak of low phosphate induced DGDG
represents remains to be demonstrated. However, an educated guess would be that it
represents the tonoplast. A few previous reports on tonoplast lipid composition in fact report
rather high proportions of DGDG and GlcCer (Verhoek et al. 1983; Yamaguchi and Kasamo
2001). In most root cells the tonoplast and plasma membrane together probably represent a
large proportion of the total cellular membranes, rendering lipid replacement in these
membranes very lucrative from a phosphate economy point of view. Vital mitochondrial
(Dowhan and Bogdanov 2001; Ostrander et al. 2001; Dowhan and Su 2003; Zhang et al.
2003a) and ER (Bankaitis and Morris 2003) functions are critically dependent on
phospholipids and especially anionic ones. With this in mind it seems likely that the plant cell
will try to concentrate phospholipids to less metabolically active membranes. And again, as
stated above, the benefits of replacing phospholipids in the ER and Golgi are probably limited
since these membranes usually represent only a small proportion of the total cellular
membranes.
5.4 Galactolipid export from the plastid
In conclusion it seems that the phospholipid replacement affects primarily the plasma
membrane and another membrane with as yet uncertain identity. An important question in
relation to phospholipid replacement in non-plastid membranes is where the low phosphate
induced DGDG is synthesised and how it is transported from the site of synthesis to the
plasma membrane. The low phosphate induced DGDG-synthase is probably localised in the
outer plastid envelope (section 3.7). Thus, phosphate deprivation induces a quite substantial
export of lipids from the chloroplast envelope. The identity of the membrane on the receiving
end of galactolipid export from the chloroplast envelope is at this point pure speculation.
However, in light of the close connections between the ER (or the PLAM) and the chloroplast
discussed in section 3, it seems likely that DGDG synthesised in the outer envelope
membrane is exported to the ER. The major plasma membrane phospholipids PC and PE with
16C and 18C fatty acids are delivered to the plasma membrane outside the secretory pathway
(Moreau et al. 1994; Sturbois-Balcerzak et al. 1995; Moreau et al. 1998), probably directly
from the ER. Thus, it seems reasonable to suggest that the DGDG synthesised in the outer
plastid envelope is exported to the ER and subsequently transported directly from the ER to
the plasma membrane. However, since root plastids are known to an form extensive network
throughout the cytosol (Köhler and Hanson 2000; Hans et al. 2004), lipid delivery at sites of
contact between the plastid envelope and the plasma membrane cannot be ruled out.
26
6. A model for galactolipid synthesis and trafficking
In the final section of the thesis a model for galactolipid trafficking to, within and from the
chloroplast will be presented. The model is based on the features discussed in the previous
chapters and a generous dose of speculation. Finally, I will present some thoughts about what
I think are the major blanks in the model.
ER/PLAM
lysoPC
PC
1a
1b
PC
OEM
IEM
DAG
2
1c
PA
3
DAG
5
DGDG
11
DAG 4
PC
9
Induced by phosphate starvation
8
6b
MGDG 6a
11
11
lysoPC
DGDG
11
MGDG
DGDG
7
10
Figure 5. A model for galactolipid synthesis and trafficking.
6.1 Lipid transport to, within and from the chloroplast
The model for galactolipid synthesis and trafficking presented herein is outlined graphically
in figure 5 and I will refer to the numbers in the figure in the following discussion.
Phospholipids are synthesised in the ER and a glycerolipid derived from those is transported
to the outer chloroplast membrane (1). The exact identity of the transported lipid remains a
matter of debate. Most authors however agree on that PC (1a) or a PC-derivative such as
lysoPC (1b) or DAG (1c) is the transported lipid. There appears to be a metabolic interchange
between these lipids in the ER. Perhaps more than one single ER lipid class could function as
galactolipid precursors and there could be differences in precursor preference between
species, tissues and growth conditions. If lysoPC is the transported molecule, a monomeric
diffusion mode of transfer could be proposed. Transport of lysoPC also requires the presence
of the envelope LPCAT (9). Nevertheless, transport of lipids from the ER to the outer
envelope likely occurs at sites of physical contacts between the outer envelope and a
specialised subdomain of the ER, the PLAM. The TGD1 protein in the outer chloroplast
envelope somehow facilitates transfer of the ER-derived galactolipid precursors.
Envelope-localised PC is metabolised in the outer envelope by cytosolic enzymes, first to PA
by a PLD (2) and subsequently to DAG by PAP (3). If DAG is directly transferred from the
ER to chloroplast envelope, these steps are, needless to say, not required. The lipid
environment in the outer envelope membrane probably facilitates the recruitment of the
cytosolic lipases and/or the substrate presentation. The DAG is used for MGDG synthesis by
the A-type MGDG-synthases in the inner chloroplast envelope (4). MGDG is converted to
DGDG by UDP-galactose dependent DGDG synthase(s) in the outer envelope membrane
(6a).
27
Galactolipids that are to be transported to the thylakoid membrane are packaged into vesicles
in the inner envelope membrane (7). The vesicles are released from the inner envelope and
eventually fuse with the thylakoid membrane (10), releasing their lipids into the thylakoid
membrane. Apparently these vesicles share many traits with ER derived transitory vesicles
and bio-informatics evidence suggest a direct evolutionary relationship. However, whether
these vesicles also contain and/or transport proteins to the thylakoid remains an open
question.
Under phosphate-limited conditions (grey areas) a new set of galactolipid synthases are
induced, the outer envelope localised type B MGDG-synthase(s) (5) and additional UDPgalactose dependent DGDG-synthase(s) (6b). The DGDG synthesised during phosphatelimited conditions in the outer envelope is exported to other cellular membranes. It appears
likely that it is exported at the contact sites between the envelope and the PLAM to the ER
(8). ER-localised DGDG would be transported to the plasma membrane and the tonoplast
membrane. In these membranes, phospholipids are replaced and either returned to other
internal membranes and/or degraded in the plasma membrane/tonoplast. The mode of transfer
of DGDG from the ER to the plasma membrane and the tonoplast is pure speculation. But, in
light of the branched nature of the ER and presence of contact sites between the ER and other
compartments DGDG delivery at contact sites would be good guess.
It appears, based on the previous discussion, that during the endosymbiont relationship
between the plastid and the host plant cell, both sides have adopted important features of
membrane biogenesis from each other. The chloroplast adopted the host cell mechanism to
transport membrane material between membrane-delimited compartments by means of
transitory vesicles. On the other hand, the host cell adopted from the chloroplast the practice
of saving phosphate for other uses by substituting phospholipids with non-phosphorous
galactolipids.
6.2 Where do we go now?
There are of course significant blanks in the model outlined above. One outstanding issue that
has been totally disregarded in this thesis and, to my defence usually by other authors as well,
is lipid transport between the two chloroplast envelope membranes (11). Even though the
membranes are closely situated, they are nevertheless quite clearly separated. However, the
two envelope membranes do in fact make contact at sites of protein import (De Boer and
Weisbeek 1993). Could lipid exchange occur at these sites too?
Overall membrane lipid metabolism in the ER is quite poorly understood at the level of
regulation and the different pool sizes of products and intermediates. Many enzymes and
pathways are known, but relatively little is known regarding how they interact and how pools
of intermediates are selectively channelled into different pathways. These are clearly subjects
of high interest for future research.
The actual involvement of the PLAM in lipid transfer is at this stage completely hypothetical.
The molecular details of how the PLAM/chloroplast connection is stabilised are completely
unknown. Identification of specific PLAM-proteins is likely to shed more light on both issues.
Overall the functional compartmentalisation of the ER is a poorly investigated area that
clearly deserves much more research.
28
The molecular details of how lipids are transported between the plastid envelope and the ER
remains to be elucidated. The identification of TGD1 marks an important step forward but the
molecular details remain far from clear. Further characterisation of TGD1 and identification
of binding partners is likely to advance our understanding of ER to plastid lipid trafficking
significantly. However, the advances in the understanding of ER/mitochondria lipid transport
in yeast and the export of lipids to the outer membrane in gram negative bacteria is also likely
to contribute significantly to our understanding of lipid transport between the ER and the
plastid.
Intraplastidial lipid trafficking is in regard to its importance in nature a very under-explored
field of research. Whether lipid transport from the thylakoid really is vesicle-mediated is still
rather speculative. The evidence is more circumstantial than direct. Nevertheless, vesicle
trafficking stands out as the best hypothesis at the moment. The involvement of the COPII
coat protein homologues in intraplastidial lipid trafficking remains, until supported by “real”
biochemical data, also quite speculative.
The phospholipid replacement during phosphate-limited growth has opened a whole new field
of research. Questions concerning everything from basic lipid metabolism, membrane
trafficking and the influence of different lipids on the physicochemical properties of
membranes are to be explored. It is interesting to reflect on that this whole system was
overlooked for so long time because most research tend to be performed on plants cultivated
under optimal or almost optimal conditions. What more interesting pathways and systems are
“missed” when we consider only “pampered” plants?
Finally, future research needs to deal with the issue of regulation. How is lipid metabolism as
a whole regulated? How is the need for certain lipids signalled and how are the individual
enzymes regulated? The phospholipid replacement with DGDG may well turn out to provide
an excellent model system for addressing such questions. Again, knowledge generated from
other biological systems such as yeast and bacteria will most probably help to advance our
understanding of the regulatory mechanisms also in plant cells.
29
7. Acknowledgements
Congratulations! You’ve just made it to the end of the thesis. Alternatively, you just skipped
ahead to the end to see if your name appears in the acknowledgements. In that case, shame on
you! Go back and start over! At least you could read the abstract.
There are a lot of people I should thank for a lot of things. If your name does not appear
below it does not necessarily mean that I don’t like you or that you smell of old goat.
Thank you!!
The first one who should receive credit for this thesis is my supervisor Anna Stina Sandelius,
whose kindness and unbridled enthusiasm for lipids and membranes never stops to amaze me.
I’m also deeply thankful for all the invaluable help with preparing this manuscript.
Christer Sundqvist, Hans Ryberg and Mats Räntfors are gratefully acknowledged for taking
time to critically read the manuscript of my thesis in various stages of completion.
Karin (Larsson) for being such a nice roommate, conference companion and co-worker.
Conny for all the lunches, tea breaks, discussions and good advice.
Magnus, my membrane group substitute big brother, thank you for all your patience and good
advice. I know that I will always admire your apparent calm in the face of urgency and
general stress.
Ralf and Henrik (Aronsson) I guess you count as substitute big brothers too.
Mats Ellerström, for showing honest interest in membrane lipids and believing in my ideas.
The “lunch gang”, Lars, Fredrik, Adrian, Tara and the rest (you know who you are). In
particular Anders Tryggvesson for all interesting discussions on very diverse subjects. Along
the same line of “fun at Botan” I would also like to thank the girls in the room next door,
Jossan, Marianne and Maria.
All other present and former colleagues at the Botanical Institute.
Fakultetsnämnden för att det varit lärorikt och faktiskt (på ett kanske aningens perverst sätt)
roat mig ganska mycket.
Doktorandrådet, Andreas, Johan, Rainer, Therese och Marcus, för att det faktiskt är kul att i
grupp bli upprörd över all orätt på GU.
Tack Louise, mitt lilla murmel, för att du tycker om mig så mycket och visat prov på så stort
tålamod med mitt avhandlingsarbete och allmänt insnöade attityd till världen i stort. Du är
faktiskt bäst!
30
Tack mor, far och bror med äkta eller oäkta hälfter för en uppväxt i helt genomsnittlig lycka,
mer än så kan, i min mening, ingen son eller bror begära.
Tack Anders (Johnsson) för att du är så juste kompis och alltid ställer upp, Terje för att du är
så kul att prata med, Martin för att du är så smart och trevlig och Andreas för att du är en sån
där rolig jävel.
Tack till den utökade familjen, Roland, Ulla, Rune, Engelbert, Birgitta, Gugge, Moa, John
med flera (ni vet vilka ni är). Ni bidrar alla starkt till ökad lycka och välbefinnande hos mig
och, är jag övertygad om, andra.
Tack mormor för de omsorger jag förutan varit svulten, ensam och allmänt olycklig.
Tack familjen Franzen för att ni är så snälla och rara.
Sist ett extra tack till Anders Tryggvesson för att kritiskt granskat ”Acknowledgementet” och
den svenska populärsammanfattningen.
The financial support from the following organisations is gratefully acknowledged: The
Swedish Natural Research Council, C.F. Tryggers Stiftelse, The Swedish Research Council
for Agriculture, Environmental Sciences and Spatial Planning, Helge Ax:son Johnssons
Stiftelse, Wilhelm och Martina Lundgrens Vetenskapsfond, Hvitfeldtska Stiftelsen, C.F.
Lundströms Stiftelse and Adlerbertska forskningsfonden.
31
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9. Svensk populärsammanfattning
Du läser titeln på avhandlingen, skakar på huvudet och tänker ”trafficking” är inte det
olagligt? Nej, trafficking är inte olagligt, i alla fall inte om det är membranlipider som
transporteras omkring i växtcellen, det är intressant. Faktum är att det är intressant nog att låta
sig uppslukas av ämnet i inte mindre än fem år. Ok, nu kanske du knoppat att det handlar om
trafik eller transport av membranlipider. Vad är då en membranlipid, vad gör den och varför
är den intressant? Låt oss börja med den första delen av ”membranlipid”, membran alltså.
Alla levande celler, oavsett om de är bakterier, svampar, djurceller eller växtceller omges av
ett membran, plasmamembranet eller cellmembranet. Poängen med att ha ett membran är lätt
insedd, man måste ha en barriär mot omvärlden. Cellen måste kunna behålla vissa saker på
insidan och samtidigt kunna hålla andra saker utanför cellen. Men, samtidigt kan inte
membranet vara helt tätt, vissa substanser måste kunna tas in i cellen och andra substanser
måste avges. Membranet består av membranlipider och proteiner. Det är dock
membranlipiderna som är den ”magiska” ingrediensen. Blandar man membranlipider i vatten
bildar de alldeles spontant själva grundstrukturen för ett membran, proteinerna finns i
membranet för att utföra alla möjliga uppgifter, men lipiderna är själva grunden i membranet.
Det som gör membranlipiden ”magisk” är det att både kemiska grupper som är lösliga i vatten
och kemiska grupper som inte är lösliga i vatten finns i samma molekyl. En typisk
membranlipid består av ett vattenälskande ”huvud” och vattenavskyende ”svansar”.
Svansarna skiljer sig inte nämnvärt från t.ex. paraffin eller smörjolja som ju alla vet inte löser
sig i vatten. När membranlipid hamnar i vatten klibbar de ihop på ett sådant sätt att kontakten
mellan svansarna och vattnet minimeras, fenomenet skiljer sig inte nämnvärt från när oljan i
salladsdressingen bildar större och större droppar för att till slut flyta upp på ytan. Det bästa
sättet för membranlipider är dock inte att bilda droppar i vattnet som oljan gör, det bästa sättet
för membranlipiderna är istället att aggregera ”svans mot svans” i stora sjok. Om sjoket sluts
till en sfär eller ett ”löklager” om man så vill så har man grunden till ett cellmembran. En
droppe vatten sluts in av ett lipiddubbelskikt och skiljs från det omgivande vattnet.
Membraner är dock långt ifrån bara passiva barriärer. Massor av biokemiska reaktioner har
också sin hemvist i membraner, t.ex. fotosyntesens ljusreaktion och andningskedjan i
mitokondrien. Eukaryota (allt som inte är bakterier) celler har förutom det yttre membranet
även en hel rad olika interna membransystem. Den eukaryota cellen är full med
underavdelningar som avdelas av membraner. En del av de interna membransystemen i den
eukaryota cellen har troligen sitt evolutionära ursprung i att regioner av plasmamembranet
invaginerats och så småningom helt förlorat kontakten med cellens utsida. Två
membranavgränsade delar av växtcellen har dock ett helt annat ursprung. Mitokondrien och
kloroplasten har båda troligen sitt ursprung som fritt levande bakterier som en gång i tiden
ingått ett samarbete med den eukaryota cellen. Numera är båda organellerna helt integrerade i
den eukaryota cellen, men många drag hos de båda minner dock fortfarande om att de en gång
i tiden bara var gäster i cellen. Kloroplaster finns förstås, som du kanske minns från
högstadiet, bara hos gröna växter. I kloroplasten bor fotosyntesen. Den här avhandlingen
handlar om ett par aspekter av de lipider som bygger upp kloroplastens membraner.
Kloroplastens membraner är till största delen uppbyggda av lipider som skiljer sig i ett
fundamentalt avseende från de lipider som bygger upp cellens övriga membraner.
Kloroplastens lipider har i sitt polära ”huvud”, till skillnad från lipider i andra membraner,
ingen fosfatgrupp utan består av enkla sockerarter. Kloroplastens membranlipider kallas för
galaktolipider och de vanliga lipiderna i andra membraner för fosfolipider.
45
Den här avhandlingens första del handlar om hur kloroplastens galaktolipider kan bildas ur
fosfolipider som syntetiserats i det endoplasmatiska nätverket. I detta ingår egentligen två
problem. Det första är att transportera en membranlipid mellan två olika membraner. I teorin
skulle en sådan transport kunna medieras på tre olika sätt: monomerer skulle kunna passivt
diffundera mellan de två membranerna, hela bitar av det ena membranet skulle kunna snöras
av och bilda vesiklar som sedan fuserar med det andra membranet eller så skulle lipider kunna
förflyttas mellan membranen vid punkter där membranen bildar kontaktpunkter med
varandra. Diffusion av fria monomerer är troligen inte av någon större betydelse för
membranbildning i cellen. Detta för att monomerer av membranlipider helt enkelt inte löser
sig i vatten. De två andra varianterna förekommer utan tvekan i olika delar av cellen. De data
som jag presenterar och diskuterar i avhandlingen talar starkt för att lipidtransport mellan
endoplasmatiska nätverket och kloroplasten sker i speciella kontaktpunkter mellan de två
membranerna. När lipiden väl transporterats till kloroplasten återstår dess omvandling från
fosfo- till galaktolipid. Jag presenterar data som talar för att den här omvandlingen delvis
drivs av enzymer (så kallade lipaser) som s.a.s. simmar fritt i lösningen utanför kloroplasten.
Det förstadie som bildas efter inverkan av lipaserna omvandlas av speciella enzymer i
kloroplastens yttermembran, envelopet, till galaktolipider.
Kloroplastens galaktolipider bildas alltså i kloroplastens yttermembran, envelopet och här
uppstår genast nästa problem. Återigen handlar det om transport. Lipiderna skall ju i
slutändan bygga upp kloroplastens inre membransystem, tylakoiden. Här vill jag argumentera
för att transporten medieras av vesiklar. D.v.s. hela bitar av envelopemembranet snörs av och
transporteras till tylakoiden där de sammansmälter med det sistnämnda membranet. Detta sätt
att transportera membranmaterial är ett typiskt drag för den eukaryota cellen. Om vi nu drar
oss till minnes att kloroplasten är en relativ nykomling i växtcellen så tyder det på att en
mekanism för att transportera membranmaterial som är typisk för den eukaryota cellen under
evolutionens gång ”flyttat” in i kloroplasten.
Den allra sista delen av avhandlingen behandlar hur galaktolipider kan ersätta fosfolipider då
växten behöver spara fosfat. Galaktolipider innehåller som sagt inget fosfat medan varje
fosfolipid innehåller en fosfatgrupp. Den rådande dogmen har dock varit att galaktolipid bara
finns i kloroplastens och aldrig i cellens övriga membraner t.ex. cellens yttermembran
(plasmamembranet) som istället byggs upp huvudsakligen av fosfolipider. Fosfor är ett för
växten mycket viktigt näringsämne och i många naturliga miljöer ett för tillväxt begränsande
ämne. Växten har med andra ord allt att vinna på att vara effektiv i sin fosfatanvändning.
Omkring 30 % av allt fosfat som finns organiskt bundet i en växtcell är antagligen bundet till
fosfolipider i cellens icke-kloroplastmembraner. Det vore alltså en ganska stor fördel ur
fosfathushållningssynpunkt om växten kunde ersätta fosfolipider i t.ex. plasmamembranet
med galaktolipider. Att så faktiskt är fallet visar jag och mina medarbetare i två av arbetena
som ingår i avhandlingen. När havre odlas utan fosfat visar det sig att upp till 70 (sic) % av
plasmamembranets fosfolipider kan bytas mot galaktolipider. Alltså samma typ av lipider som
nästan alla seriösa forskare i fältet envist hävdat aldrig finns i membraner utanför kloroplasten
i några nämnvärda mängder. Denna enorma förändring i lipidsammansättning i
plasmamembranet sker utan, tycks det, väsentligt negativa konsekvenser för membranets
funktion. Detta antyder att det inte så mycket är lipidsammansättningen i sig som är viktig för
membranets funktion, utan snarare membranets fysiska egenskaper. Egenskaper som tydligen
kan hållas relativt konstanta med flera olika lipidsammansättningar.
Som slutsats kan man alltså säga att under växtcellens evolution så har kloroplasten adopterat
en mekanism för att transportera membranlipider av värdcellen. Värdcellen har å andra sidan
46
”lärt” sig av kloroplasten att använda galaktolipider i stället för fosfolipider för att bilda
membraner och därmed kunna använda begränsade fosfattillgångar mera effektivt.
47