Plant chloroplasts contain an intricate photosynthetic membrane system, the thylakoids, and are surrounded by two envelope membranes at which thylakoid lipids are assembled. The glycoglycerolipids mono- and digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol as well as phosphatidylglycerol, are present in thylakoid membranes, giving them a unique composition. Fatty acids are synthesized in the chloroplast and are either directly assembled into thylakoid lipids at the envelope membranes or exported to the ER (endoplasmic reticulum) for extraplastidic lipid assembly. A fraction of lipid precursors is reimported into the chloroplast for the synthesis of thylakoid lipids. Thus polar lipid assembly in plants requires tight co-ordination between the chloroplast and the ER and necessitates inter-organelle lipid trafficking. In the present paper, we discuss the current knowledge of the export of fatty acids from the chloroplast and the import of chloroplast lipid precursors assembled at the ER. Direct membrane contact sites between the ER and the chloroplast outer envelopes are discussed as possible conduits for lipid transfer.
- ATP-binding-cassette transporter (ABC transporter)
- fatty acid transport
- lipid transfer
- membrane contact site
Plant photosynthesis is a ubiquitous biochemical process of plants and other photosynthetic organisms converting light into metabolic energy. It is essential for life on earth. The organelle in plant cells responsible for photosynthesis, the chloroplast, is enclosed by two envelopes and contains the inner thylakoid membranes harbouring the photosynthesis apparatus. These intricate membrane systems constitute approximately 70% of total plant cell membranes of which ~80% are galactoglycerolipids . In fact, the chloroplast glycoglycerolipids MGDG (monogalactosyldiacylglycerol), DGDG (digalactosyldiacylglycerol) and the sulfur-containing lipid SQDG (sulfoquinovosyldiacylglycerol) predominate over the monophosphoglycerolipid, PtdGro (phosphatidylglycerol), present in thylakoids . Chloroplast lipids interact directly with photosynthetic complexes. For example, galactoglycerolipids have been found in the crystal structure of Photosystems I and II [3,4]. Indeed, Arabidopsis mutants deficient in MGDG, DGDG, SQDG or PtdGro synthesis, mgd1, dgd1, sqd2 or pgp1 respectively, are impaired in photosynthesis, which is apparent as a decrease in chlorophyll content, defects in chloroplast ultrastructure and reduced photosynthetic activity [5–8]. The assembly of chloroplast lipids primarily takes place at the chloroplast envelope membranes, but is complicated by the fact that lipid precursors originate at different locations/membranes within the cell. Apart from the chloroplast envelope membranes, the ER (endoplasmic reticulum) is also involved in the biosynthesis of chloroplast lipids. Therefore extensive lipid trafficking takes place between the ER and chloroplast envelope membranes.
Structural and functional properties of chloroplast lipids
In leaves of Arabidopsis grown under normal conditions, the ratios of MGDG, DGDG, SQDG and PtdGro relative to total glycerolipids are approximately 50, 20, 2 and 10% respectively . In an MGDG molecule a galactose is linked by a β-glycosidic bond in the sn-3 position of the glyceryl residue of the DAG (diacylglycerol) moiety (Figure 1). The predominant fatty acids in MGDG are α-linolenic acid (C18:3Δ9,12,15; an 18-carbon acyl chain with cis-double bonds at carbons 9, 12 and 15 counting from the carboxy group) and hexadecatrienoic acid (C16:3Δ7,10,13) esterified at the sn-1 and sn-2 position of the glyceryl backbone respectively. Because of the relatively small galactosyl headgroup of MGDG compared with DGDG and the high degree of unsaturation of the respective fatty acids, the MGDG molecule adapts a conical shape (with the headgroup at its tip), which is thought to accommodate the curvature of envelope and thylakoid membranes . In fact, MGDG is a non-bilayer-forming lipid when mixed with water in the absence of other lipids. MGDG is synthesized by MGDG synthase 1, MGD1 in Arabidopsis, which is a UDP-Gal:diacylglycerol galactosyltransferase localized at the inner envelope membrane of the chloroplast facing the intermembrane space [10,11]. In spinach leaves, MGDG constitutes 17% of total lipids on the outer envelope, 55% on the inner envelope and 58% on the thylakoid membranes .
The headgroup of DGDG consists of MGDG with a second galactosyl residue connected in an α1-6 linkage (Figure 1). The major fatty acids in DGDG are α-linolenic acid (C18:3Δ9,12,15) and palmitic acid (C16:0). Since DGDG has two galactosyl moieties in its headgroup, it is more cylindrically shaped than MGDG and, hence, a bilayer-forming lipid . In Arabidopsis, the major DGDG synthase, DGD1, is localized at the outer envelope membrane facing the cytosol  and catalyses the transfer of a galactosyl residue from UDP-Gal to MGDG . In spinach, DGDG makes up 30% of total lipids in both envelope membranes and 27% in the thylakoid membrane .
The sulfolipid SQDG and the phospholipid PtdGro are anionic lipids essential for photosynthesis . In SQDG, the sulfoquinovosyl headgroup is linked to DAG in an α-glycosidic bond (Figure 1). Similar to DGDG, the most abundant fatty acids in SQDG are α-linolenic acid (C18:3Δ9,12,15) and palmitic acid (C16:0). SQDG synthase, SQD2, associated with the inner chloroplast envelope membrane, transfers a sulfoquinovose from UDP-sulfoquinovose to DAG forming SQDG , which accounts for approximately 6% of the lipids in both envelope and thylakoids membranes in spinach . In Arabidopsis, PtdGro is synthesized from CDP-DAG and glycerol-3-phosphate by PtdGro-phosphate synthases PGP1 and PGP2 and subsequently dephosphorylated by PtdGro-phosphate phosphatase associated with the inner envelope membrane of chloroplasts [7,15]. Apart from α-linolenic acid, PtdGro also possesses a unique C16:1Δ3 fatty acid with its double bond in a trans-configuration (Figure 1) . PtdGro represents 10% of polar lipids in both envelope membranes and 7% in thylakoids of spinach .
Two pathways for chloroplast lipid synthesis
During the biogenesis of complex lipids, fatty acids synthesized de novo in the chloroplast can have two fates: either they remain in the plastid where they are assembled into complex lipids by the prokaryotic pathway or they are exported to the ER entering the eukaryotic pathway of lipid assembly (Figure 2) . The two-pathway hypothesis was first proposed by Roughan et al. . In vivo pulse–chase experiment with intact spinach leaves incubated with 14C-labelled acetate showed a biphasic kinetic: an initial labelling of MGDG followed by a decrease and a subsequent increase. The initial rapid incorporation of fatty acids into MGDG is interpreted as de novo biosynthesis by the prokaryotic pathway. The subsequent decrease is due to lipid turnover, e.g. the conversion of MGDG into DGDG. The second increase reflects the import of DAG moieties assembled at the ER into the chloroplast where they serve as the substrate for MGDG synthesis . Glycerolipids originating from the prokaryotic pathway carry a 16-carbon acyl chain at the sn-2 position of the glycerol backbone, while glycerolipids assembled by the eukaryotic pathway contain an 18-carbon acyl chain at the same position (Figure 1) . In Arabidopsis MGDG, DGDG and SQDG can be synthesized from either pathway, while plastid PtdGro is synthesized exclusively by the prokaryotic pathway . Many seed plants, such as pea, only use the eukaryotic pathway for chloroplast glycoglycerolipid assembly. These plants have a high proportion of α-linolenic acid (C18:3) in chloroplast lipids, giving rise to their designation ‘18:3 plants’. In other plants, such as Arabidopsis and spinach, both pathways are involved in the biosynthesis of chloroplast lipids and they are named ‘16:3 plants’ for the abundance of hexadecatrienoic acid (C16:3) in their chloroplast lipids . In Arabidopsis, both pathways contribute approximately equally to galactolipid synthesis . The two-pathway hypothesis was confirmed by the discovery of Arabidopsis mutants impaired in either of the pathways. In the ats1 mutant, the prokaryotic pathway was disrupted converting Arabidopsis essentially into an 18:3 plant without any significant growth phenotype . However, mutants impaired in the eukaryotic pathway such as tgd1 are severely affected in growth and strong alleles cause embryo lethality [11,25]. Pulse–chase labelling experiments have shown that ats1 plants lack the rapid phase of MGDG synthesis, whereas the tgd1 mutant lacks the second phase increase . The eukaryotic pathway involving both the ER and the chloroplast necessitates extensive lipid trafficking between the three membranes, especially during the export of fatty acids from the chloroplast to the ER and the import of DAG moieties from the ER to the chloroplast. The mechanisms of these processes will be discussed below in detail.
Fatty acid export from chloroplast
In seed plants, essentially all fatty acids are synthesized inside the chloroplast attached to acyl-ACP (acyl carrier protein) and become available for complex lipid assembly primarily in the form of oleoyl- and palmitoyl-ACP . Fatty acids must be exported from the chloroplast to the ER where they are assembled into various glycerolipids. The molecular mechanism of fatty acid export is still under debate. Rapid label experiments suggested that phosphatidylcholine is the first acyl-incorporating product following fatty acid export from the plastid and not acyl-CoA as previously assumed . At least two enzymes are expected to be involved in this process: acyl-ACP thioesterases and LACSs (long-chain fatty acyl-CoA synthetases). Acyl-ACP thioesterases catalyse the hydrolysation of acyl-ACP to non-esterified (‘free’) fatty acids that are later activated to acyl-CoA in the presence of ATP by LACSs. Chloroplast fractionation experiments have shown that acyl-ACP thioesterases are localized at the inner envelope membrane, whereas LACSs are associated with the outer envelope, consistent with a mechanism of hydrolysis acyl-ACPs and re-activation of the acyl groups to acyl-CoAs on the respective sides of the membrane . Indeed, experiments following 18O-labelled acetate incorporation into plastid versus extraplastidic lipids were consistent with the export of a free carboxylate anion intermediate produced by intermittent hydrolysis of acyl-ACP . Furthermore, 14C labelling revealed fast turnover of a non-esterified fatty acid pool with a half-life time of less than 1 s . Although long-chain fatty acids can cross the membrane bilayer by simple diffusion, as their membrane permeability is several magnitudes higher than hydrophilic metabolites such as glucose or amino acids [31,32], this process may be facilitated and regulated by the involvement of proteins. It has been shown that in the presence of BSA, which removes non-esterified fatty acids outside of the chloroplast, LACS activity was not affected, suggesting that fatty acid transport is facilitated by a vectorial mechanism coupling transport and re-activation of the acyl group . This mechanism has been postulated also for fatty acid import systems in Escherichia coli and yeast . However, little is known about how non-esterified fatty acids cross the inter-membrane space.
In Arabidopsis two acyl-ACP thioesterases, FatA and FatB, have been described. Although FatA is specific to oleoyl-ACP, FatB has a broader substrate spectrum, but prefers palmitoyl-ACP . There are nine genes encoding LACSs in Arabidopsis, but LACS9 is responsible for 90% of the fatty acyl-CoA synthetase activity . However, neither altered growth nor impaired fatty acid export from chloroplasts was observed in the LACS9 T-DNA (transfer DNA) knockout mutant consistent with functional overlap between different LACSs in Arabidopsis [36,37].
The ER–chloroplast contact site
Scattered throughout the literature are reports of observations of the ER in close vicinity to plastid envelope membranes. In different groups of algae the chloroplast is surrounded by periplastidic ER [38,39]. In specific cells of Acer pseudoplatanus and the resin canal cells from Pinus pinea, immature plastids were completely sheathed by an ER membrane . In resin canal cells, presumed precursors for resin synthesis were observed between the outer envelope membrane of plastids and the sheathing ER. ER–chloroplast contact sites were also reported in embryonic pea leaves where lipid droplets were associated with the developing chloroplast . In the mature chloroplast, the complete sheathing ER disappears and, instead, localized ER–chloroplast attachment is observed with GFP (green fluorescent protein) targeted to the ER or YFP (yellow fluorescent protein) fused to reticulon-like proteins in Arabidopsis [42,43]. The chloroplast-associated ER adapts a reticulum structure rather than cisterna. Chloroplasts isolated from pea or Arabidopsis leaves have ER membrane fragments attached . This attachment is sufficiently strong to withstand a force of 400 pN applied with optical tweezers, suggesting strong protein–protein interactions. Using rapid freeze–fracture, continuity of the ER and the outer envelope of chloroplasts in the green alga Chara globularis var. capillacea and the non-seed vascular plant Equisetum telmateia were observed , but has not been reported for seed plants. In addition, osmiophilic particles, probably lipid droplets, were enriched at the ER-to-chloroplast contact sites, suggesting a possible role of these contact sites in lipid metabolism.
Labelling of cellular compartments with specific fluorescent markers enabled the dynamic imaging of the ER–chloroplast interaction. Following laser stimulation at ER-to-chloroplast contact sites, GFP-labelled ER grew on the surface of the chloroplast forming a denser network . Myosin may be involved in this movement as it is localized in chloroplast-associated ER tubules . In addition, chloroplast stromules, which are extensions of chloroplasts suggested to be involved in inter-plastidic communication and exchange of substances, have been shown to be surrounded by ER tubules and the movement of stromules coincided with the movement of the surrounding ER [48,49].
Although there is compelling evidence for the existence of ER–chloroplast membrane contact sites, the functional significance of this structure remains to be determined. The ribosomes associated with the ER surrounding the plastids observed in early studies with algae led to an early hypothesis that the ER–chloroplast contact sites may serve as a conduit for plastid protein import . However, for seed plants it is well established that proteins are imported directly through the Toc–Tic complex of the envelope membranes of the chloroplasts. That is, with notable exceptions, some, if not all, N-glycosylated proteins, such as NPP (nucleotide pyrophosphatase/phosphodiesterase) in rice and CAH1 (α-carbonic anhydrase) in Arabidopsis, depend on the secretory pathway to reach the chloroplast [51,52]. One can speculate that during early stages of plastid symbiosis as represented by algae, plastid proteins encoded by the nucleus genome may not have had chloroplast-targeting sequences and thus reached the chloroplast via the secretory pathway. In seed plants, although targeting of nuclear encoded plastid proteins is mostly independent of the secretory pathway, those plastid proteins requiring post-translational modification in the ER may still utilize this mechanism. It is unclear how N-glycosylated proteins transfer from the ER to the chloroplast, but ER–chloroplast contact sites may represent a possible structure involved in this process.
Membrane contact sites as lipid conduits have been reported in yeast MAMs (microsomal fractions associated with mitochondria) [53,54]. Phosphatidylserine synthesized at the ER is imported through MAMs into mitochondria where it is utilized for the synthesis of phosphatidylethanolamine. It is possible that similar lipid transfer also takes place at the ER–chloroplast contact sites, as the eukaryotic pathway for chloroplast lipid synthesis requires the participation of the ER. The TGD proteins involved in this process were discovered during a forward genetic screen in Arabidopsis . The tgd mutants accumulate abnormal oligogalactolipids, most prominently trigalactosyldiacylglycerol, after which they are named. Chloroplast lipids derived from the eukaryotic pathway are decreased in these mutants and the above-mentioned pulse–chase labelling experiment with [14C]acetate showed that the tgd1-1 mutant is impaired in the eukaryotic pathway for chloroplast lipid biosynthesis. The accumulation of trigalactosyldiacylglycerol results from the activation of a processive galactosyltransferase named SFR2 (SENSITIVE TO FREEZING 2) . Genes affected in the tgd mutants have been identified and the proteins have been designated TGD1–TGD4 (Figure 2). The TGD1, TGD2 and TGD3 proteins form a bacterial-type ABC transporter (ATP-binding-cassette transporter) in the inner envelope of chloroplasts touching the outer envelope . TGD1 is a permease and TGD3 is an ATPase providing the energy for lipid transport [11,25,56], probably making the lipid transfer from the ER to the chloroplast unidirectional . TGD2 has an N-terminal transmembrane domain and a C-terminal soluble domain facing the intermembrane space . The C-terminal domain specifically binds phosphatidic acid and disturbs adjacent membranes [59,60]. The TGD4 protein lacks known functional domains but its C-terminus may fold into a β-barrel . A GFP–TGD4 fusion protein transiently overproduced in tobacco was localized to the ER membrane . In addition, the TGD4 protein was found in chloroplast envelope fractions during proteomics studies . These conflicting results may indicate that TGD4 is localized at ER–chloroplast contact sites, through which lipid precursors might transfer from the ER to the chloroplast. Besides TGD4, a cytochrome P450 mono-oxygenase, CYP86B1, which is required for suberin biosynthesis in roots and seeds, has also been localized to both ER and the chloroplast using two different approaches. CYP86B1 has an N-terminal sequence rich in serine/threonine residues that is interpreted to be a chloroplast-targeting peptide, and the protein was localized to the outer envelope of the chloroplast facing the cytosol using a chloroplast protein import assay . However, transiently expressing CYP86B1–YFP in tobacco localized the fusion protein to the ER membrane. With prolonged inspection, some of the fluorescence started to associate with chloroplasts . Either overproduction of fluorescent fusion proteins generally leads to mistargeting to the ER or this protein is also localized at the ER chloroplast contact sites, explaining the ambiguous results.
The biosynthesis of chloroplast membrane lipids in seed plants involves at least two organelles and three membrane bilayers: the ER, the chloroplast inner and outer envelope membranes. Using Arabidopsis as a model system, the development of forward genetic screening and the completion of the genome sequence enabled the identification of nearly all of the enzymes involved in chloroplast lipid synthesis during the past two decades. Fluorescent protein fusions, cell fractionation and proteomics revealed the fine localization of these enzymes. However, our understanding of how the lipid intermediates are handed from one enzyme to the next and transferred from one organelle to another has just begun to emerge. Direct membrane contacts between the ER and the chloroplast are suggested as the sites for the transfer of lipids.
Work in the Benning laboratory on membrane lipid biosynthesis and lipid trafficking is supported by the U.S. Department of Energy, the U.S. National Science Foundation and the Michigan Agricultural Experiment Station.
Cellular Traffic of Lipids and Calcium at Membrane Contact Sites: A Biochemical Society held at the Snowbird Ski and Summer Resort, Snowbird, UT, U.S.A., 6–9 October 2011. Organized and Edited by Tim Levine (Institute of Ophthalmology, London, U.K.) and William Prinz (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, U.S.A.).
Abbreviations: ACP, acyl carrier protein; DAG, diacylglycerol; DGDG, digalactosyldiacylglycerol; ER, endoplasmic reticulum; GFP, green fluorescent protein; LACS, long-chain fatty acyl-CoA synthetase; MAM, microsomal fraction associated with mitochondria; MGDG, monogalactosyldiacylglycerol; PtdGro, phosphatidylglycerol; SQDG, sulfoquinovosyldiacylglycerol; YFP, yellow fluorescent protein
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