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Galactolipids rule in seed plants
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Galactolipids rule in seed plants Peter Dörmann and Christoph Benning Chloroplast membranes contain high levels of the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). The isolation of the genes involved in the biosynthesis of MGDG and DGDG, and the identification of galactolipid-deficient Arabidopsis mutants has greatly facilitated the analysis of galactolipid biosynthesis and function. Galactolipids are found in X-ray structures of photosynthetic complexes, suggesting a direct role in photosynthesis. Furthermore, galactolipids can substitute for phospholipids, as suggested by increases in the galactolipid:phospholipid ratio after phosphate deprivation. The ratio of MGDG to DGDG is also crucial for the physical phase of thylakoid membranes and might be regulated.
Peter Dörmann* Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany. *e-mail: doermann@ mpimp-golm.mpg.de Christoph Benning Dept of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Life in all organisms relies on the presence of a functional plasma membrane enveloping each cell, thereby forming a boundary to the environment. Eukaryotic cells are characterized by their compartmentation, having several organelles. Different polar lipids have evolved that serve as ‘building blocks’ for membranes. In animals and yeast, phospholipids [e.g. phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol (PG)] are the most abundant membrane lipid class. In plants, however, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) (Fig. 1) constitute ~75% of total membrane lipids in leaves and therefore greatly outnumber phospholipids [1,2]. Phosphate is required for plant survival but is also one of the plant nutrients with limited availability in many soil types. Therefore, inorganic phosphate, typically taken up by the roots, can easily become limiting for plant growth [3]. In an Arabidopsis leaf, about a third of the organic phosphate is found in phospholipids [4], even though the bulk of the membrane lipid is nonphosphorous. Therefore, the large amount of galactolipids in green tissues makes plants less dependent on the precious nutrient phosphate than other eukaryotes with predominantly phospholipid-containing membranes. Because of the high abundance of galactolipids in plants, in chloroplasts in particular, and because of their absence from most non-photosynthetic organisms, it has been suggested that galactolipids have specific functions beyond their role as primary membrane building blocks. Strong corroborating evidence became available with the isolation of the relevant plant genes and mutants [5,6], as well as with the recent crystallization of the photosystem I (PSI) complex [7]. No lipids have been identified in the electron density maps of the crystal structures of photosystem II (PSII) from cyanobacteria [8] or plants [9], or in the major light-harvesting complex II (LHCII) from pea [10]. http://plants.trends.com
Enzymes of galactolipid synthesis
Two predominant galactolipids are found in higher plants. The first, MGDG, constitutes up to 50% of chloroplast lipids and contains one galactose residue bound in a β-glycosidic bond to diacylglycerol (Fig. 1). The second, DGDG, accounts for ~20% of chloroplast lipids and contains a second galactose moiety bound in a (1→6) α-glycosidic linkage between the two galactoses. Galactolipids are not charged at physiological pH and thus represent the only neutral membrane lipid class in thylakoids. The other two thylakoid lipids, sulfoquinovosyldiacylglycerol (SQDG) and PG, each carry one negative charge at physiological pH. Our current understanding of the biosynthesis and function of galactolipids was greatly advanced with the isolation of the cDNAs and genes for the MGDG synthase [11] and DGDG synthase [12]. Because of the high similarity between MGD1 [the MGDG synthase of higher plants (EC 2.4.1.46)] and the bacterial MurG protein (a glycosyltransferase involved in cell wall synthesis), it was proposed that MGD1 evolved from a protein of cell wall metabolism of the endosymbiont [2,11]. In Arabidopsis, three genes are known to encode MGDG synthases [13–16] (MGD1, MGD2, MGD3). The galactosyltransferase MGD1 catalyses the synthesis of MGDG from diacylglycerol and UDP–galactose [13]. Based on sequence similarities, the plant MGDG synthase genes belong to the glycosyltransferase family GT28 [17] (http://afmb.cnrs-mrs.fr/CAZY/). The protein localizes to the inner envelope membrane, corroborating previous findings for the localization of MGDG synthase activity [18]. The isolation of an Arabidopsis mutant (mgd1) carrying an insertion in the promoter region of the MGD1 gene showed that the corresponding enzyme synthesizes the bulk of MGDG in chloroplasts, because a 75% reduction in mRNA abundance led to a 75% reduction in MGDG synthase activity [15]. The sequences of the other two MGDG synthases in Arabidopsis, MGD2 and MGD3, are closely related to each other but are less similar to MGD1 [16]. It has been suggested that MGD2 and MGD3 localize to the outer envelope membrane [16]. In Arabidopsis, the diacylglycerol moiety incorporated into galactolipids can be derived from the chloroplast (prokaryote-type lipid) or from the ER (eukaryote-type lipid). Whereas MGDG is predominantly synthesized from prokaryotic diacylglycerol and is thus characterized by a high content of hexadecatrienoic acid, DGDG is mostly of eukaryotic origin and therefore devoid of hexadecatrienoic acid. Analysis of substrate specificity indicates that MGD1 has no particular
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Review
Fig. 1. Galactolipids of higher plants. The structures of the two most abundant galactolipids, monogalactosyldiacylglycerol (MGDG) and β,αdigalactosyldiacylglycerol (DGDG), as well as the structures of oligogalactolipids and modified galactolipids detectable in minor amounts in plants. The conformation of the anomeric carbon atom is indicated in red (α) or yellow (β). Structurally important modifications are highlighted in green. Abbreviations: acylMGDG, sn3-(6-O-acylmonogalactosyl)-diacylglycerol; MGDG-O, sn3monogalactosyl-sn1-(12oxophytodienoyl)-sn2hexadecatrienoyl-glycerol; MGPDG, sn3-(6-phosphomonogalactosyl)diacylglycerol; TetraGDG, α,α,α,β-tetragalactosyldiacylglycerol; TriGDG, α,α,β-trigalactosyldiacylglycerol. R1, R2 and R3 represent different alkyl chains.
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preferences for prokaryotic or eukaryotic molecular species of diacylglycerol, consistent with its role as the main MGDG synthase [13]. Previous experiments with spinach envelopes investigating DGDG synthesis suggested that a second MGDG molecule (but not UDP–galactose) might be the galactose donor for the galactosylation of MGDG [19]. The corresponding activity, the galactolipid:galactolipid galactosyltransferase (EC 2.4.1.184), was localized to the outer envelope of chloroplasts [20,21]. In addition to the DGDG synthase activity, a processive galactosylation activity resulting in the production of oligogalactolipids with three (TriGDG), four (TetraGDG) or even more galactose moieties in the head group has been reported (Fig. 1). Typically, these oligogalactolipids are only found in low concentrations in leaves but accumulate to high concentrations in chloroplast preparations. It is assumed that the additional galactose moieties incorporated into oligogalactolipids by the galactolipid:galactolipid galactosyltransferase form α-glycosidic bonds [22] (Fig. 1). Furthermore, in some species {e.g. Adzuki beans (Vigna angularis) [23]}, the corresponding all-β anomeric forms of DGDG, TriGDG and TetraGDG have been described. This points to the existence of http://plants.trends.com
two different processive galactosyltransferases involved in galactolipid synthesis. The DGDG synthase gene DGD1 was isolated by map-based cloning and complementation of the DGDGdeficient Arabidopsis mutant dgd1 [12]. The amount of DGDG in dgd1 is reduced by 90%, demonstrating that DGD1 is responsible for synthesizing the bulk of DGDG in Arabidopsis [24]. The DGD1 protein is composed of two parts, an N-terminal half (DGD1-N) with no sequence similarities to genes in the databases and a C-terminal half (DGD1-C) that is similar to plant and bacterial glycosyltransferases of primary metabolism (glycosyltransferase family GT4) [12,17]. Therefore, DGD1-C, which supposedly carries the enzymatic domain required for galactosylation, might have evolved from a protein of primary carbohydrate metabolism of the endosymbiont or the plant host. Corroborating previous biochemical studies, the DGD1 protein was found to be associated with the outer envelope of chloroplasts [25]. The protein does not contain a cleavable chloroplast-targeting sequence and is inserted into the outer envelope following a route independent of the general import pathway [26]. The information crucial for import resides in the sequence of the N-terminal half [25]. The fact that chloroplasts
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Fig. 2. Galactolipid trafficking in Arabidopsis. Monogalactosyldiacylglycerol (MGDG) is formed in the inner and outer chloroplast envelope by the action of different MGDG synthases. Digalactosyldiacylglycerol (DGDG) is synthesized in the outer chloroplast envelope and presumably transported to the inner one by an unknown mechanism. The transport of galactolipids from the inner chloroplast envelope to the thylakoids is facilitated by the VIPP1 protein. Under certain conditions, DGDG is also found outside the chloroplasts, but the exact site of synthesis and the mechanism of transport are unknown. Abbreviation: DAG, diacylglycerol.
isolated from dgd1 can still synthesize DGDG and the oligogalactolipids TriGDG and TetraGDG shows that the processive galactosylation activity is not affected in this mutant [24,27]. However, an apparent paralogue of DGD1 (tentatively designated DGD2) is present in the Arabidopsis genome. The DGD2 cDNA encodes an active DGDG synthase, as shown by heterologous expression in E. coli [28]. However, it is not known whether the residual DGDG biosynthetic activity and the biosynthesis of oligogalactolipids are due to the DGD2 protein. Galactolipid trafficking
The localization of the enzymes of galactolipid biosynthesis to the outer and inner chloroplast envelopes raises the question of how galactolipids are transported between the two membranes, to the thylakoids and to other organelles. In Arabidopsis, MGD1 (which localizes to the inner envelope) synthesizes the bulk of MGDG [13] (Fig. 2). Presumably, the outer envelope (MGD2, MGD3) is a second site for MGDG synthesis [16]. MGDG produced in the outer envelope might serve as the precursor for DGDG synthesis by DGD1. It is not known how DGDG produced in the outer envelope is transported to the inner envelope but it has been suggested that the DGDG synthase DGD1 or its N-terminal part might be involved in this process [12]. The E. coli ABC transporter MsaA was recently implicated in the transfer of lipids from the plasma membrane to the outer membrane of E. coli [29,30]. It seems possible that similar proteins, which are abundant in Arabidopsis [31], mediate the transport of galactolipids from the outer to the inner envelope of http://plants.trends.com
chloroplasts in plants, or ‘flip’ galactolipids between membrane leaflets. A vesicular mechanism for the transfer of galactolipids from the inner envelope to the thylakoid membrane has been proposed. In plants adapted to low temperatures, vesicles can be identified in the stroma of chloroplasts that are formed at the inner envelope [32]. Galactolipids synthesized in envelope membranes of isolated chloroplasts are transported to the thylakoids in a temperature-dependent manner [33,34]. An Arabidopsis mutant deficient in vesicleinducing plastid protein 1 (vipp1) is incapable of forming stroma vesicles and is severely lacking in thylakoid membranes [35]. The corresponding pea protein localizes to the inner envelope and the thylakoids [36]. Thus, galactolipids are probably transported in the form of vesicles from their site of synthesis to the thylakoid membranes (Fig. 2). In agreement with the endosymbiont hypothesis, there is an orthologue in cyanobacteria, the mutants of which are deficient in thylakoid formation [37]. Recently, it has been shown that DGDG accumulates in extraplastidic membranes after phosphate deprivation [38] as well as in the chloroplasts. Because the galactolipids produced during phosphate deprivation are accessible to desaturases in the plastid envelopes, they are probably synthesized in the plastid [27]. Thus, galactolipids would have to move from the plastid to other organelles, adding yet another level of complexity. Role of galactolipids in photosynthesis
The high abundance of galactolipids in thylakoids and in cyanobacterial membranes suggests that they play an important role in photosynthesis. However, it was unclear for a long time whether galactolipids are just localized at the periphery of the complexes of the photosynthetic apparatus or whether they are integral constituents of photosynthetic pigment–protein complexes. The recent crystallization of the PSI complex of cyanobacteria revealed that each PSI monomer contains four lipid molecules, three molecules of PG and one of MGDG [7] (Fig. 3). In the electron density map, the lipids were found to form hydrogen bonds with the PsaA or PsaB core proteins at the stromal side of the membrane [7,39]. The crystallographic data might contribute to the elucidation of a long-lasting question: why photosynthetically active organisms contain galactolipids and not glucolipids, particularly given that cyanobacteria (from which PSI was crystallized) first make monoglucosyldiacylglycerol (MGlcDG), which is subsequently epimerized to MGDG. The expanded view of the protein surroundings of PSI in contact with the MGDG head group shows two remarkable interactions (E. Heinz, pers. commun.). The axial hydroxyl group at C4 of galactose is hydrogen-bonded to the amide carbonyl oxygen of Ala562 (part of an often functionally sensitive loop, not of a stable transmembrane helix), whereas the hydrophobic side chain of Val709 supports
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Fig. 3. Localization of galactolipids in the thylakoid membrane. Four lipids are found in thylakoids of higher plants: monogalactosyldiacylglycerol (MGDG), red; digalactosyldiacylglycerol (DGDG), purple; phosphatidylglycerol (PG), blue; sulfoquinovosyldiacylglycerol (SQDG), green. MGDG and PG are integral components of the photosystem I (PSI) complex. DGDG and PG are also found to be associated with the major light-harvesting complex II (LHCII) of photosystem II (PSII). Abbreviations: CP43 and CP47, inner antenna proteins 43 and 47 of PSII; Cyt. b6f, cytochrome b6f complex; D1 and D2, core proteins 1 and 2 of PSII; PsaA and PsaB, protein subunits A and B of PSI; WOC, wateroxidizing complex.
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the galactopyranosyl residue from the bottom (the hydrophobic C3–C4–C5 triangle) and, by this method, keeps the C4 hydroxyl group within hydrogenbonding distance of the peptide loop. None of these interactions would be possible with a corresponding MGlcDG lipid (which has no axial hydroxyl group and no hydrophobic–hydrophilic sidedness). To date, no lipids have been identified in the electron density maps of the crystal structures of PSII from cyanobacteria [8] or plants [9], or in the major LHCII from pea [10]. However, the crystal structure of the light-harvesting complex of the dinoflagellate Amphidinium carterae was found to contain two molecules of DGDG [40]. Two lipids, PG and DGDG, were also found in LHCII preparations isolated from pea chloroplasts [41]. These two lipids were crucial for crystallization and for the stability of the LHCII complex in vitro [42]. Supporting evidence for a role of galactolipids in photosynthesis came from the analysis of Arabidopsis mutants affected in the expression of MGD1 [15] or DGD1 [24]. The mgd1 mutant contains ~50% of the wild-type amount of MGDG. This mutant shows a reduction in chlorophyll content and changes in chloroplast ultrastructure [15]. Similarly, the dgd1 mutant contains less chlorophyll and its chloroplast ultrastructure is changed [24]. Different components of the photosynthetic electron transport chain are affected in the dgd1 mutant, including the intactness of the water-oxidizing complex [43], the amounts of photosynthetic complexes (PSI, PSII and cytochromes b559 and D1) [44] and the xanthophyll-cycle activity [45]. The oxygen evolution rates for wild type and dgd1 were unchanged [44,45], and therefore the observed molecular changes of the components of the photosynthetic electron transport chain cannot be limiting the production of photosynthetic metabolites [45]. Therefore, it is difficult to decide whether the growth reduction of dgd1 mutants is caused by a dysfunctional photosynthetic apparatus or by a reduced capacity to synthesize chloroplast lipids [12,27]. Because the mgd1 and dgd1 mutants still http://plants.trends.com
contain residual amounts of MGDG or DGDG, it would be desirable to obtain lines that are completely devoid of galactolipids by combining several mutations in a single plant. Such mutant plants would be important tools for further unravelling the role of galactolipids in photosynthesis. Regulation of lipid biosynthesis during phosphate deprivation
Many bacteria can adjust their membrane composition according to different growth conditions [46–48]. When grown under phosphate-limiting conditions, neutral and acidic glycolipids accumulate in Pseudomonas diminuta, whereas the amounts of phospholipids decrease [46]. In Rhodobacter sphaeroides, SQDG, a glucosylgalactosyl lipid and the betaine lipid diacylglyceroltrimethylhomoserine (DGTS) replace phospholipids in the membranes during growth under phosphate-limiting conditions [47,49]. Similarly, phosphate deprivation results in the accumulation of different phosphate-free lipids (SQDG, DGTS, ornithine lipid) in Sinorhizobium meliloti [48]. Apparently, these neutral and acidic non-phosphorous lipids can substitute for phospholipids in the membranes. During phosphate deprivation, the amount of phosphate bound to phospholipids in the membranes is reduced to sustain other cellular processes (e.g. DNA and RNA synthesis). Interestingly, higher plants are also capable of this adaptation, increasing the ratio of glycolipids (SQDG and DGDG) to phospholipids when grown on lowphosphate medium [38,50]. The pho1 mutant of Arabidopsis [4], which is deficient in phosphate transport from the root to the shoot, represents an ideal model system to study the effect of permanent phosphate deprivation on membrane lipid biosynthesis (Fig. 4). The accumulation of DGDG under phosphatelimiting conditions is also apparent in the dgd1 mutant of Arabidopsis. There must therefore be a second, DGD1-independent DGDG synthase in Arabidopsis that is induced during phosphate deprivation. Furthermore, this process of low-phosphate adaptation
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Fig. 4. Changes of lipid composition during phosphate deprivation. Under phosphate-limiting conditions, the amounts of phospholipids decrease, whereas the amounts of digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) increase. The amount of monogalactosyldiacylglycerol (MGDG) remains constant. This adaptive process is not limited to the chloroplasts but also involves extraplastidic membranes (compare with Fig. 2). The amounts of lipids are given in mol% for leaves of wild-type Arabidopsis and for the pho1 mutant. Owing to a block in phosphate transport from the root to the shoot, the leaves of the pho1 mutant are constantly deprived of phosphate. Data are from Ref. [36]. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol.
is not limited to chloroplast membranes because, during phosphate starvation, DGDG also accumulates in extraplastidic membranes [37]. The accumulation of DGDG outside the chloroplast raises questions about the site of synthesis and the mechanism of transport of this extraplastidic form of DGDG [27,38] (Fig. 2). Membrane architecture: non-bilayer-forming and bilayer-forming lipids
Owing to its small head group, MGDG has a cone-like geometry, with galactose at the tip and the two fatty acyl chains oriented towards the base of the cone [51]. Therefore, the most stable phase that MGDG adopts in aqueous in vitro mixtures is the hexagonal-II (HII) phase, with the polar head groups facing towards the centre of micellar or tube-like structures [52]. For this reason, MGDG does not form bilayers in mixtures with water at room temperature. In contrast to MGDG, DGDG, which contains two galactose moieties in its head group, has a more cylindrical shape and forms lamellar Lα phases in mixtures with water. Like the other two thylakoid lipids (PG and SQDG), DGDG is a bilayer-forming lipid. As in all organisms, the ratio of non-bilayer-forming to bilayer-forming lipids is crucial for protein folding and insertion [51,53] as well as for intracellular protein trafficking [54]. The ratio of MGDG to DGDG in chloroplasts must therefore be tightly regulated. Indeed, the import of precursor proteins into chloroplasts is affected in the Arabidopsis dgd1 mutant, indicating that DGDG is important for the integrity of the chloroplast-import apparatus [55]. The regulation of the biosynthesis of non-bilayerforming and bilayer-forming lipids has been studied in greater detail in Acholeplasma laidlawii, a prokaryote http://plants.trends.com
that lacks a cell wall and accumulates the glucolipids, MGlcDG and diglucosyldiacylglycerol (DGlcDG) in its membranes [56]. The gene encoding the MGlcDG synthase has been isolated from A. laidlawii [57]. Interestingly, the corresponding sequence is different from the MurG-like MGDG synthase sequences from spinach or Arabidopsis and therefore falls into a different class of glycosyltransferases involved in lipid synthesis. The recombinant MGlcDG synthase is activated by PG and it has been suggested that the specific binding to this negatively charged lipid within the membrane is crucial for enzyme activity [57]. The DGlcDG synthase isolated from A. laidlawii is regulated by different phosphate-containing lipids and metabolites, such as PG, phosphatidic acid, inorganic phosphate, fructose-1,6-bisphosphate and ATP [56]. It has been suggested that the membrane composition and metabolic status of the cells are involved in regulating the balance of MGlcDG and DGlcDG. It is currently not known whether the equivalent plant enzymes are subject to similar regulation. Role for structural modifications of galactolipids?
In addition to the prevalent forms of MGDG and DGDG (Fig. 1), structural derivatives can be found in small amounts in lipid preparations from plants. Galactolipids, especially MGDG, are partially acylated at the C-6 position [58] (acyl-MGDG; Fig. 1). However, it appears that MGDG acylation is induced after cell rupture. Therefore, the physiological relevance of this modification, its function and the enzymes involved, remain unclear [59]. Recently, a phosphorylated form of MGDG was discovered in spinach chloroplasts [60] that has a phosphate ester group at the C-6 position of the galactose ring (MGPDG; Fig. 1). The corresponding phosphorylation activity is CTP dependent and localizes to chloroplast membranes. Phosphorylation of MGDG could drastically affect its biophysical properties because MGPDG is an anionic, probably bilayer-forming lipid. Because the amount of MGPDG in leaves is low, it is difficult to decide whether the synthesis of this lipid might have an effect on the ratio of non-bilayer-forming to bilayerforming lipids in the membranes. Long-chain polyunsaturated fatty acids are the most abundant class of fatty acids attached to galactolipids. Furthermore, fatty acids with three double bonds (e.g. α-linolenic acid, an 18-carbon compound) are the substrate for the lipoxygenase reaction that introduces a hydroperoxy group into the acyl chain [61]. The lipoxygenase pathway leads to the synthesis of several important compounds involved in plant defence, including jasmonic acid. For a long time, it was assumed that free fatty acid, especially free α-linolenic acid, is the most relevant substrate for the lipoxygenase pathway. However, it has recently been shown that most 12-oxophytodienoic acid, an intermediate of the jasmonic acid pathway, is
Review
Acknowledgements Work on lipids in our laboratories is supported by grants from the Deutsche Forschungsgemeinschaft to P.D. and from the US National Science Foundation, the US Dept of Energy and the US Dept of Agriculture to C.B. We would like to thank Ernst Heinz for pointing out how MGDG fits into the PSI crystal structure.
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bound to the sn1 position of MGDG [62] (Fig. 1). Therefore, in Arabidopsis chloroplasts, a large proportion of 12-oxophytodienoic acid synthesized through the lipoxygenase pathway is derived from lipid-bound rather than free α-linolenic acid. Perspectives
Although much is known about the biosynthesis and function of galactolipids in plants, the full complexity of the underlying pathways has not been fully revealed. Paralogues of MGD1 and DGD1 are present in Arabidopsis but their function will remain unclear until mutants in these genes can be isolated and studied. Environmental factors such as phosphate deprivation add yet another level of complexity. Whether this phenomenon is ubiquitous to all plants
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and whether it can be explored to reduce agricultural phosphate fertilizer input remain to be seen. The identification of the enzymes responsible for the biosynthesis of galactolipids under phosphate-limited growth conditions represents a new challenge. Similarly, the regulatory mechanism for galactolipid biosynthesis following phosphate deprivation is unexplored. The most notorious problem is that of lipid transfer between the plastid and extraplastidic membranes. Although there is irrefutable evidence for lipid trafficking between these compartments, no transport protein has been directly implicated in this process. Control of the ratio of non-bilayer-forming to bilayer-forming lipids at the level of galactolipid biosynthetic enzymes is an exciting possibility that needs further exploration.
15 Jarvis, P. et al. (2000) Galactolipid deficiency and abnormal chloroplast development in the Arabidopsis MGD synthase 1 mutant. Proc. Natl. Acad. Sci. U. S. A. 97, 8175–8179 16 Awai, K. et al. (2001) Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 98, 10960–10965 17 Henrissat, B. and Davies, G.J. (2000) Glycoside hydrolases and glycosyltransferases. Families, modules and implications for genomics. Plant Physiol. 124, 1515–1519 18 Block, M.A. et al. (1983) Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. II. Biochemical characterization. J. Biol. Chem. 258, 13281–13286 19 van Besouw, A. and Wintermans, J.F.G.M. (1978) Galactolipid formation in chloroplast envelopes. I. Evidence for two mechanisms in galactosylation. Biochim. Biophys. Acta 529, 44–53 20 Dorne, A-J. et al. (1982) The galactolipid:galactolipid galactosyltransferase is located on the outer membrane of the chloroplast envelope. FEBS Lett. 145, 30–34 21 Cline, K. and Keegstra, K. (1983) Galactosyltransferases involved in galactolipid biosynthesis are located in the outer membrane of pea chloroplast envelopes. Plant Physiol. 71, 366–372 22 Douce, R. and Joyard, J. (1980) Plant galactolipids. In Lipids: Structure and Function (The Biochemistry of Plants) (Vol. 4) (Stumpf, P.K., ed.), pp. 321–362, Academic Press 23 Kojima, M. et al. (1990) Structure of novel glyceroglycolipids in Adzuki bean (Vigna angularis) seeds. Biochem. Cell Biol. 68, 59–64 24 Dörmann, P. et al. (1995) Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacylglycerol. Plant Cell 7, 1801–1810 25 Froehlich, J. et al. (2001) The digalactosyldiacylglycerol synthase DGD1 is inserted into the outer envelope membrane of chloroplasts in a manner independent of the general import pathway and does not depend on direct interaction with MGDG synthase for DGDG biosynthesis. J. Biol. Chem. 276, 31806–31812 26 Keegstra, K. and Cline, K. (1999) Protein import and routing systems of chloroplasts. Plant Cell 11, 557–570
27 Klaus, D. et al. Digalactosyldiacylglycerol synthesis in chloroplasts of the Arabidopsis thaliana dgd1 mutant. Plant Physiol. (in press) 28 Kelly, A.A. and Dörmann, P. (2002) DGD2, an Arabidopsis gene encoding a UDP–galactose dependent digalactosyl–diacylglycerol synthase is expressed during growth under phosphate limiting conditions. J. Biol. Chem. 277, 1166–1173 29 Doerrler, W.T. et al. (2001) An Escherichia coli mutant defective in lipid export. J. Biol. Chem. 276, 11461–11464 30 Chang, G. and Roth, C.B. (2001) Structure of MsbA from E. coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters. Science 293, 1793–1800 31 Sánchez-Fernández, R. et al. (2001) The Arabidopsis thaliana ABC protein superfamily, a complete inventory. J. Biol. Chem. 276, 30231–30244 32 Morre, D.J. et al. (1991) Stromal low temperature compartment derived from the inner membrane of the chloroplast envelope. Plant Physiol. 97, 1558–1564 33 Räntfors, M. et al. (2000) Intraplastidial lipid trafficking: regulation of galactolipid release from isolated chloroplast envelope. Physiol. Plant. 110, 262–270 34 Andersson, M.X. et al. (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 35 Kroll, D. et al. (2001) VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation. Proc. Natl. Acad. Sci. U. S. A. 98, 4238–4242 36 Li, H-M. et al. (1994) Molecular cloning of a chloroplastic protein associated with both the envelope and thylakoid membranes. Plant Mol. Biol. 25, 619–632 37 Westphal, S. et al. (2001) vipp1 deletion mutant of Synechocystis: a connection between bacterial phage shock and thylakoid biogenesis? Proc. Natl. Acad. Sci. U. S. A. 98, 4243–4248 38 Härtel, H. et al. (2000) DGD1-independent biosynthesis of extraplastidic galactolipids following phosphate deprivation in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 97, 10649–10654 39 Klukas, O. et al. (1999) Localization of two phylloquinones, QK and QK′, in an improved electron density map of photosystem I at 4-Å resolution. J. Biol. Chem. 274, 7361–7367
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40 Hofmann, E. et al. (1996) Structural basis of light harvesting by carotenoids: peridinin-chlorophyllprotein from Amphidinium carterae. Science 272, 1788–1791 41 Nußberger, S. et al. (1993) Lipid–protein interactions in crystals of plant light-harvesting complex. J. Mol. Biol. 234, 347–356 42 Reinsberg, D. et al. (2000) Folding, assembly and stability of the major light-harvesting complex of higher plants, LHCII, in the presence of native lipids. Biochemistry 39, 14305–14313 43 Reifarth, F. et al. (1997) Modification of the water oxidizing complex in leaves of the dgd1 mutant of Arabidopsis thaliana deficient in the galactolipid digalactosyldiacylglycerol. Biochemistry 36, 11769–11776 44 Härtel, H. et al. (1997) Changes in the composition of the photosynthetic apparatus in the galactolipid deficient dgd1 mutant of Arabidopsis thaliana. Plant Physiol. 115, 1175–1184 45 Härtel, H. et al. (1998) Photosynthetic light utilization and xanthophyll cycle activity in the galactolipid deficient dgd1 mutant of Arabidopsis thaliana. Plant Physiol. Biochem. 36, 407–417 46 Minnikin, D.E. et al. (1974) Replacement of acidic phospholipids by acidic glycolipids in Pseudomonas diminuta. Nature 249, 268–269 47 Benning, C. et al. (1993) The sulfolipid sulfoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances growth under
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phosphate limitation. Proc. Natl. Acad. Sci. U. S. A. 90, 1516–1565 Geiger, O. et al. (1999) The regulator gene phoB mediates phosphate stress-controlled synthesis of the membrane lipid diacylglyceryl-N,N,Ntrimethylhomoserine in Rhizobium (Sinorhizobium) meliloti. Mol. Microbiol. 32, 63–73 Benning, C. et al. (1995) Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation. Arch. Biochem. Biophys. 327, 103–111 Essigmann, B. et al. (1998) Phosphate availability affects the thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 95, 1950–1955 Gounaris, K. and Barber, J. (1983) Monogalactosyldiacylglycerol: the most abundant polar lipid in nature. Trends Biochem. Sci. 8, 378–381 Webb, M.S. and Green, B.R. (1991) Biochemical and biophysical properties of thylakoid acyl lipids. Biochim. Biophys. Acta 1060, 133–158 Bogdanov, M. and Dowhan, W. (1999) Lipid-assisted protein folding. J. Biol. Chem. 274, 36827–36830 Kusters, R. et al. (1994) A dual role for phosphatidylglycerol in protein translocation across the Escherichia coli inner membrane. J. Biol. Chem. 269, 1560–1563 Chen, L-J. and Li, H-M. (1998) A mutant deficient in the plastid lipid DGD is defective in protein import into chloroplasts. Plant J. 16, 33–39
56 Vikström, S. et al. (2000) The nonbilayer/bilayer lipid balance in membranes. Regulatory enzyme in Acholeplasma laidlawii is stimulated by metabolic phosphates, activator phospholipids and doublestranded DNA. J. Biol. Chem. 13, 9296–9302 57 Berg, S. et al. (2001) Sequence properties of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes. Recognition of a large group of lipid glycosyltransferases in Eubacteria and Archaea. J. Biol. Chem. 276, 22056–22063 58 Myhre, D.V. (1968) Glycolipids of soft wheat flour. I. Isolation and characterization of 1-O-(6-O-acylβ-D-galactosyl)-2,3-di-O-acyl-D-glyceritols and phytosteryl 6-O-acyl-β-D-glucopyranosides. Can. J. Chem. 46, 3071–3077 59 Heinz, E. (1967) Acylgalactosyl diglyceride from leaf homogenates. Biochim. Biophys. Acta 144, 321–332 60 Müller, M-O. et al. (2000) Lipid phosphorylation in chloroplast envelopes. Evidence for galactolipid CTP-dependent kinase activities. J. Biol. Chem. 275, 19475–19481 61 Farmer, E.E. and Ryan, C.A. (1992) Octadecanoid-derived signals in plants. Trends Cell Biol. 2, 236–241 62 Stelmach, B.A. et al. (2001) A novel class of oxylipins, sn1-O-(12-oxophytodienoyl)-sn2-O(hexadecatrienoyl)-monogalactosyl diglyceride, from Arabidopsis thaliana. J. Biol. Chem. 276, 12832–12838
Engineering crop plants: getting a handle on phosphate Henrik Brinch-Pedersen, Lisbeth Dahl Sørensen and Preben Bach Holm In plant seeds, most of the phosphate is in the form of phytic acid. Phytic acid is largely indigestible by monogastric animals and is the single most important factor hindering the uptake of a range of minerals. Engineering crop plants to produce a heterologous phytase improves phosphate bioavailability and reduces phytic acid excretion. This reduces the phosphate load on agricultural ecosystems and thereby alleviates eutrophication of the aquatic environment. Improved phosphate availability also reduces the need to add inorganic phosphate, a non-renewable resource. Iron and zinc uptake might be improved, which is significant for human nutrition in developing countries.
Henrik Brinch-Pedersen* Lisbeth Dahl Sørensen Preben Bach Holm Danish Institute of Agricultural Sciences, Dept of Plant Biology, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark. *e-mail: henrik.brinchpedersen@ agrsci.dk
Phosphate is an essential macronutrient for all living organisms. In natural ecosystems, phosphorus is returned to the soil and converted to inorganic phosphate (Pi) via biological and chemical processes, after which it is available for a new cycle of plant growth. In agricultural ecosystems, two additional factors are introduced: a tapping of the phosphate cycle by the harvest of plant materials for animal and human consumption, and the reintroduction of phosphate in the form of manure or industrially produced fertilizers (Fig. 1). http://plants.trends.com
Because of the importance of Pi as a macronutrient for plant growth and livestock production, and the ability of phosphate compounds to bind important minerals, there is increasing interest in understanding the factors that underlie phosphate uptake and bioavailability in plants used for animal feed and human consumption. Moreover, there is a strong demand for optimization of feeding regimes because of the effect of excess Pi on the aquatic environment (eutrophication). A central component in these contexts is the enzyme phytase, which can release bioavailable phosphate from one of the most important compounds in the phosphate cycle, phytic acid [phytate, or myo-inositol-(1,2,3,4,5,6)hexakisphosphate] (Fig. 2). Phytic acid is largely indigestible by monogastric animals because they have no or limited phytase activity in their digestive tract. Moreover, it is considered to be an important antinutritional factor, preventing the uptake of a range of important minerals. Phytic acid and phytases are
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Galactolipids rule in seed plants Peter Dörmann and Christoph Benning Chloroplast membranes contain high levels of the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). The isolation of the genes involved in the biosynthesis of MGDG and DGDG, and the identification of galactolipid-deficient Arabidopsis mutants has greatly facilitated the analysis of galactolipid biosynthesis and function. Galactolipids are found in X-ray structures of photosynthetic complexes, suggesting a direct role in photosynthesis. Furthermore, galactolipids can substitute for phospholipids, as suggested by increases in the galactolipid:phospholipid ratio after phosphate deprivation. The ratio of MGDG to DGDG is also crucial for the physical phase of thylakoid membranes and might be regulated.
Peter Dörmann* Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany. *e-mail: doermann@ mpimp-golm.mpg.de Christoph Benning Dept of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Life in all organisms relies on the presence of a functional plasma membrane enveloping each cell, thereby forming a boundary to the environment. Eukaryotic cells are characterized by their compartmentation, having several organelles. Different polar lipids have evolved that serve as ‘building blocks’ for membranes. In animals and yeast, phospholipids [e.g. phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol (PG)] are the most abundant membrane lipid class. In plants, however, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) (Fig. 1) constitute ~75% of total membrane lipids in leaves and therefore greatly outnumber phospholipids [1,2]. Phosphate is required for plant survival but is also one of the plant nutrients with limited availability in many soil types. Therefore, inorganic phosphate, typically taken up by the roots, can easily become limiting for plant growth [3]. In an Arabidopsis leaf, about a third of the organic phosphate is found in phospholipids [4], even though the bulk of the membrane lipid is nonphosphorous. Therefore, the large amount of galactolipids in green tissues makes plants less dependent on the precious nutrient phosphate than other eukaryotes with predominantly phospholipid-containing membranes. Because of the high abundance of galactolipids in plants, in chloroplasts in particular, and because of their absence from most non-photosynthetic organisms, it has been suggested that galactolipids have specific functions beyond their role as primary membrane building blocks. Strong corroborating evidence became available with the isolation of the relevant plant genes and mutants [5,6], as well as with the recent crystallization of the photosystem I (PSI) complex [7]. No lipids have been identified in the electron density maps of the crystal structures of photosystem II (PSII) from cyanobacteria [8] or plants [9], or in the major light-harvesting complex II (LHCII) from pea [10]. http://plants.trends.com
Enzymes of galactolipid synthesis
Two predominant galactolipids are found in higher plants. The first, MGDG, constitutes up to 50% of chloroplast lipids and contains one galactose residue bound in a β-glycosidic bond to diacylglycerol (Fig. 1). The second, DGDG, accounts for ~20% of chloroplast lipids and contains a second galactose moiety bound in a (1→6) α-glycosidic linkage between the two galactoses. Galactolipids are not charged at physiological pH and thus represent the only neutral membrane lipid class in thylakoids. The other two thylakoid lipids, sulfoquinovosyldiacylglycerol (SQDG) and PG, each carry one negative charge at physiological pH. Our current understanding of the biosynthesis and function of galactolipids was greatly advanced with the isolation of the cDNAs and genes for the MGDG synthase [11] and DGDG synthase [12]. Because of the high similarity between MGD1 [the MGDG synthase of higher plants (EC 2.4.1.46)] and the bacterial MurG protein (a glycosyltransferase involved in cell wall synthesis), it was proposed that MGD1 evolved from a protein of cell wall metabolism of the endosymbiont [2,11]. In Arabidopsis, three genes are known to encode MGDG synthases [13–16] (MGD1, MGD2, MGD3). The galactosyltransferase MGD1 catalyses the synthesis of MGDG from diacylglycerol and UDP–galactose [13]. Based on sequence similarities, the plant MGDG synthase genes belong to the glycosyltransferase family GT28 [17] (http://afmb.cnrs-mrs.fr/CAZY/). The protein localizes to the inner envelope membrane, corroborating previous findings for the localization of MGDG synthase activity [18]. The isolation of an Arabidopsis mutant (mgd1) carrying an insertion in the promoter region of the MGD1 gene showed that the corresponding enzyme synthesizes the bulk of MGDG in chloroplasts, because a 75% reduction in mRNA abundance led to a 75% reduction in MGDG synthase activity [15]. The sequences of the other two MGDG synthases in Arabidopsis, MGD2 and MGD3, are closely related to each other but are less similar to MGD1 [16]. It has been suggested that MGD2 and MGD3 localize to the outer envelope membrane [16]. In Arabidopsis, the diacylglycerol moiety incorporated into galactolipids can be derived from the chloroplast (prokaryote-type lipid) or from the ER (eukaryote-type lipid). Whereas MGDG is predominantly synthesized from prokaryotic diacylglycerol and is thus characterized by a high content of hexadecatrienoic acid, DGDG is mostly of eukaryotic origin and therefore devoid of hexadecatrienoic acid. Analysis of substrate specificity indicates that MGD1 has no particular
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Review
Fig. 1. Galactolipids of higher plants. The structures of the two most abundant galactolipids, monogalactosyldiacylglycerol (MGDG) and β,αdigalactosyldiacylglycerol (DGDG), as well as the structures of oligogalactolipids and modified galactolipids detectable in minor amounts in plants. The conformation of the anomeric carbon atom is indicated in red (α) or yellow (β). Structurally important modifications are highlighted in green. Abbreviations: acylMGDG, sn3-(6-O-acylmonogalactosyl)-diacylglycerol; MGDG-O, sn3monogalactosyl-sn1-(12oxophytodienoyl)-sn2hexadecatrienoyl-glycerol; MGPDG, sn3-(6-phosphomonogalactosyl)diacylglycerol; TetraGDG, α,α,α,β-tetragalactosyldiacylglycerol; TriGDG, α,α,β-trigalactosyldiacylglycerol. R1, R2 and R3 represent different alkyl chains.
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preferences for prokaryotic or eukaryotic molecular species of diacylglycerol, consistent with its role as the main MGDG synthase [13]. Previous experiments with spinach envelopes investigating DGDG synthesis suggested that a second MGDG molecule (but not UDP–galactose) might be the galactose donor for the galactosylation of MGDG [19]. The corresponding activity, the galactolipid:galactolipid galactosyltransferase (EC 2.4.1.184), was localized to the outer envelope of chloroplasts [20,21]. In addition to the DGDG synthase activity, a processive galactosylation activity resulting in the production of oligogalactolipids with three (TriGDG), four (TetraGDG) or even more galactose moieties in the head group has been reported (Fig. 1). Typically, these oligogalactolipids are only found in low concentrations in leaves but accumulate to high concentrations in chloroplast preparations. It is assumed that the additional galactose moieties incorporated into oligogalactolipids by the galactolipid:galactolipid galactosyltransferase form α-glycosidic bonds [22] (Fig. 1). Furthermore, in some species {e.g. Adzuki beans (Vigna angularis) [23]}, the corresponding all-β anomeric forms of DGDG, TriGDG and TetraGDG have been described. This points to the existence of http://plants.trends.com
two different processive galactosyltransferases involved in galactolipid synthesis. The DGDG synthase gene DGD1 was isolated by map-based cloning and complementation of the DGDGdeficient Arabidopsis mutant dgd1 [12]. The amount of DGDG in dgd1 is reduced by 90%, demonstrating that DGD1 is responsible for synthesizing the bulk of DGDG in Arabidopsis [24]. The DGD1 protein is composed of two parts, an N-terminal half (DGD1-N) with no sequence similarities to genes in the databases and a C-terminal half (DGD1-C) that is similar to plant and bacterial glycosyltransferases of primary metabolism (glycosyltransferase family GT4) [12,17]. Therefore, DGD1-C, which supposedly carries the enzymatic domain required for galactosylation, might have evolved from a protein of primary carbohydrate metabolism of the endosymbiont or the plant host. Corroborating previous biochemical studies, the DGD1 protein was found to be associated with the outer envelope of chloroplasts [25]. The protein does not contain a cleavable chloroplast-targeting sequence and is inserted into the outer envelope following a route independent of the general import pathway [26]. The information crucial for import resides in the sequence of the N-terminal half [25]. The fact that chloroplasts
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Fig. 2. Galactolipid trafficking in Arabidopsis. Monogalactosyldiacylglycerol (MGDG) is formed in the inner and outer chloroplast envelope by the action of different MGDG synthases. Digalactosyldiacylglycerol (DGDG) is synthesized in the outer chloroplast envelope and presumably transported to the inner one by an unknown mechanism. The transport of galactolipids from the inner chloroplast envelope to the thylakoids is facilitated by the VIPP1 protein. Under certain conditions, DGDG is also found outside the chloroplasts, but the exact site of synthesis and the mechanism of transport are unknown. Abbreviation: DAG, diacylglycerol.
isolated from dgd1 can still synthesize DGDG and the oligogalactolipids TriGDG and TetraGDG shows that the processive galactosylation activity is not affected in this mutant [24,27]. However, an apparent paralogue of DGD1 (tentatively designated DGD2) is present in the Arabidopsis genome. The DGD2 cDNA encodes an active DGDG synthase, as shown by heterologous expression in E. coli [28]. However, it is not known whether the residual DGDG biosynthetic activity and the biosynthesis of oligogalactolipids are due to the DGD2 protein. Galactolipid trafficking
The localization of the enzymes of galactolipid biosynthesis to the outer and inner chloroplast envelopes raises the question of how galactolipids are transported between the two membranes, to the thylakoids and to other organelles. In Arabidopsis, MGD1 (which localizes to the inner envelope) synthesizes the bulk of MGDG [13] (Fig. 2). Presumably, the outer envelope (MGD2, MGD3) is a second site for MGDG synthesis [16]. MGDG produced in the outer envelope might serve as the precursor for DGDG synthesis by DGD1. It is not known how DGDG produced in the outer envelope is transported to the inner envelope but it has been suggested that the DGDG synthase DGD1 or its N-terminal part might be involved in this process [12]. The E. coli ABC transporter MsaA was recently implicated in the transfer of lipids from the plasma membrane to the outer membrane of E. coli [29,30]. It seems possible that similar proteins, which are abundant in Arabidopsis [31], mediate the transport of galactolipids from the outer to the inner envelope of http://plants.trends.com
chloroplasts in plants, or ‘flip’ galactolipids between membrane leaflets. A vesicular mechanism for the transfer of galactolipids from the inner envelope to the thylakoid membrane has been proposed. In plants adapted to low temperatures, vesicles can be identified in the stroma of chloroplasts that are formed at the inner envelope [32]. Galactolipids synthesized in envelope membranes of isolated chloroplasts are transported to the thylakoids in a temperature-dependent manner [33,34]. An Arabidopsis mutant deficient in vesicleinducing plastid protein 1 (vipp1) is incapable of forming stroma vesicles and is severely lacking in thylakoid membranes [35]. The corresponding pea protein localizes to the inner envelope and the thylakoids [36]. Thus, galactolipids are probably transported in the form of vesicles from their site of synthesis to the thylakoid membranes (Fig. 2). In agreement with the endosymbiont hypothesis, there is an orthologue in cyanobacteria, the mutants of which are deficient in thylakoid formation [37]. Recently, it has been shown that DGDG accumulates in extraplastidic membranes after phosphate deprivation [38] as well as in the chloroplasts. Because the galactolipids produced during phosphate deprivation are accessible to desaturases in the plastid envelopes, they are probably synthesized in the plastid [27]. Thus, galactolipids would have to move from the plastid to other organelles, adding yet another level of complexity. Role of galactolipids in photosynthesis
The high abundance of galactolipids in thylakoids and in cyanobacterial membranes suggests that they play an important role in photosynthesis. However, it was unclear for a long time whether galactolipids are just localized at the periphery of the complexes of the photosynthetic apparatus or whether they are integral constituents of photosynthetic pigment–protein complexes. The recent crystallization of the PSI complex of cyanobacteria revealed that each PSI monomer contains four lipid molecules, three molecules of PG and one of MGDG [7] (Fig. 3). In the electron density map, the lipids were found to form hydrogen bonds with the PsaA or PsaB core proteins at the stromal side of the membrane [7,39]. The crystallographic data might contribute to the elucidation of a long-lasting question: why photosynthetically active organisms contain galactolipids and not glucolipids, particularly given that cyanobacteria (from which PSI was crystallized) first make monoglucosyldiacylglycerol (MGlcDG), which is subsequently epimerized to MGDG. The expanded view of the protein surroundings of PSI in contact with the MGDG head group shows two remarkable interactions (E. Heinz, pers. commun.). The axial hydroxyl group at C4 of galactose is hydrogen-bonded to the amide carbonyl oxygen of Ala562 (part of an often functionally sensitive loop, not of a stable transmembrane helix), whereas the hydrophobic side chain of Val709 supports
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Fig. 3. Localization of galactolipids in the thylakoid membrane. Four lipids are found in thylakoids of higher plants: monogalactosyldiacylglycerol (MGDG), red; digalactosyldiacylglycerol (DGDG), purple; phosphatidylglycerol (PG), blue; sulfoquinovosyldiacylglycerol (SQDG), green. MGDG and PG are integral components of the photosystem I (PSI) complex. DGDG and PG are also found to be associated with the major light-harvesting complex II (LHCII) of photosystem II (PSII). Abbreviations: CP43 and CP47, inner antenna proteins 43 and 47 of PSII; Cyt. b6f, cytochrome b6f complex; D1 and D2, core proteins 1 and 2 of PSII; PsaA and PsaB, protein subunits A and B of PSI; WOC, wateroxidizing complex.
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the galactopyranosyl residue from the bottom (the hydrophobic C3–C4–C5 triangle) and, by this method, keeps the C4 hydroxyl group within hydrogenbonding distance of the peptide loop. None of these interactions would be possible with a corresponding MGlcDG lipid (which has no axial hydroxyl group and no hydrophobic–hydrophilic sidedness). To date, no lipids have been identified in the electron density maps of the crystal structures of PSII from cyanobacteria [8] or plants [9], or in the major LHCII from pea [10]. However, the crystal structure of the light-harvesting complex of the dinoflagellate Amphidinium carterae was found to contain two molecules of DGDG [40]. Two lipids, PG and DGDG, were also found in LHCII preparations isolated from pea chloroplasts [41]. These two lipids were crucial for crystallization and for the stability of the LHCII complex in vitro [42]. Supporting evidence for a role of galactolipids in photosynthesis came from the analysis of Arabidopsis mutants affected in the expression of MGD1 [15] or DGD1 [24]. The mgd1 mutant contains ~50% of the wild-type amount of MGDG. This mutant shows a reduction in chlorophyll content and changes in chloroplast ultrastructure [15]. Similarly, the dgd1 mutant contains less chlorophyll and its chloroplast ultrastructure is changed [24]. Different components of the photosynthetic electron transport chain are affected in the dgd1 mutant, including the intactness of the water-oxidizing complex [43], the amounts of photosynthetic complexes (PSI, PSII and cytochromes b559 and D1) [44] and the xanthophyll-cycle activity [45]. The oxygen evolution rates for wild type and dgd1 were unchanged [44,45], and therefore the observed molecular changes of the components of the photosynthetic electron transport chain cannot be limiting the production of photosynthetic metabolites [45]. Therefore, it is difficult to decide whether the growth reduction of dgd1 mutants is caused by a dysfunctional photosynthetic apparatus or by a reduced capacity to synthesize chloroplast lipids [12,27]. Because the mgd1 and dgd1 mutants still http://plants.trends.com
contain residual amounts of MGDG or DGDG, it would be desirable to obtain lines that are completely devoid of galactolipids by combining several mutations in a single plant. Such mutant plants would be important tools for further unravelling the role of galactolipids in photosynthesis. Regulation of lipid biosynthesis during phosphate deprivation
Many bacteria can adjust their membrane composition according to different growth conditions [46–48]. When grown under phosphate-limiting conditions, neutral and acidic glycolipids accumulate in Pseudomonas diminuta, whereas the amounts of phospholipids decrease [46]. In Rhodobacter sphaeroides, SQDG, a glucosylgalactosyl lipid and the betaine lipid diacylglyceroltrimethylhomoserine (DGTS) replace phospholipids in the membranes during growth under phosphate-limiting conditions [47,49]. Similarly, phosphate deprivation results in the accumulation of different phosphate-free lipids (SQDG, DGTS, ornithine lipid) in Sinorhizobium meliloti [48]. Apparently, these neutral and acidic non-phosphorous lipids can substitute for phospholipids in the membranes. During phosphate deprivation, the amount of phosphate bound to phospholipids in the membranes is reduced to sustain other cellular processes (e.g. DNA and RNA synthesis). Interestingly, higher plants are also capable of this adaptation, increasing the ratio of glycolipids (SQDG and DGDG) to phospholipids when grown on lowphosphate medium [38,50]. The pho1 mutant of Arabidopsis [4], which is deficient in phosphate transport from the root to the shoot, represents an ideal model system to study the effect of permanent phosphate deprivation on membrane lipid biosynthesis (Fig. 4). The accumulation of DGDG under phosphatelimiting conditions is also apparent in the dgd1 mutant of Arabidopsis. There must therefore be a second, DGD1-independent DGDG synthase in Arabidopsis that is induced during phosphate deprivation. Furthermore, this process of low-phosphate adaptation
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Phospholipids: 19%
Phospholipids: 36%
PC PE PI PG
PC PE
SQDG
PI PG SQDG DGDG
MGDG
Glycolipids: 64% Wild type
DGDG
MGDG
Glycolipids: 81% pho1 mutant TRENDS in Plant Science
Fig. 4. Changes of lipid composition during phosphate deprivation. Under phosphate-limiting conditions, the amounts of phospholipids decrease, whereas the amounts of digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) increase. The amount of monogalactosyldiacylglycerol (MGDG) remains constant. This adaptive process is not limited to the chloroplasts but also involves extraplastidic membranes (compare with Fig. 2). The amounts of lipids are given in mol% for leaves of wild-type Arabidopsis and for the pho1 mutant. Owing to a block in phosphate transport from the root to the shoot, the leaves of the pho1 mutant are constantly deprived of phosphate. Data are from Ref. [36]. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol.
is not limited to chloroplast membranes because, during phosphate starvation, DGDG also accumulates in extraplastidic membranes [37]. The accumulation of DGDG outside the chloroplast raises questions about the site of synthesis and the mechanism of transport of this extraplastidic form of DGDG [27,38] (Fig. 2). Membrane architecture: non-bilayer-forming and bilayer-forming lipids
Owing to its small head group, MGDG has a cone-like geometry, with galactose at the tip and the two fatty acyl chains oriented towards the base of the cone [51]. Therefore, the most stable phase that MGDG adopts in aqueous in vitro mixtures is the hexagonal-II (HII) phase, with the polar head groups facing towards the centre of micellar or tube-like structures [52]. For this reason, MGDG does not form bilayers in mixtures with water at room temperature. In contrast to MGDG, DGDG, which contains two galactose moieties in its head group, has a more cylindrical shape and forms lamellar Lα phases in mixtures with water. Like the other two thylakoid lipids (PG and SQDG), DGDG is a bilayer-forming lipid. As in all organisms, the ratio of non-bilayer-forming to bilayer-forming lipids is crucial for protein folding and insertion [51,53] as well as for intracellular protein trafficking [54]. The ratio of MGDG to DGDG in chloroplasts must therefore be tightly regulated. Indeed, the import of precursor proteins into chloroplasts is affected in the Arabidopsis dgd1 mutant, indicating that DGDG is important for the integrity of the chloroplast-import apparatus [55]. The regulation of the biosynthesis of non-bilayerforming and bilayer-forming lipids has been studied in greater detail in Acholeplasma laidlawii, a prokaryote http://plants.trends.com
that lacks a cell wall and accumulates the glucolipids, MGlcDG and diglucosyldiacylglycerol (DGlcDG) in its membranes [56]. The gene encoding the MGlcDG synthase has been isolated from A. laidlawii [57]. Interestingly, the corresponding sequence is different from the MurG-like MGDG synthase sequences from spinach or Arabidopsis and therefore falls into a different class of glycosyltransferases involved in lipid synthesis. The recombinant MGlcDG synthase is activated by PG and it has been suggested that the specific binding to this negatively charged lipid within the membrane is crucial for enzyme activity [57]. The DGlcDG synthase isolated from A. laidlawii is regulated by different phosphate-containing lipids and metabolites, such as PG, phosphatidic acid, inorganic phosphate, fructose-1,6-bisphosphate and ATP [56]. It has been suggested that the membrane composition and metabolic status of the cells are involved in regulating the balance of MGlcDG and DGlcDG. It is currently not known whether the equivalent plant enzymes are subject to similar regulation. Role for structural modifications of galactolipids?
In addition to the prevalent forms of MGDG and DGDG (Fig. 1), structural derivatives can be found in small amounts in lipid preparations from plants. Galactolipids, especially MGDG, are partially acylated at the C-6 position [58] (acyl-MGDG; Fig. 1). However, it appears that MGDG acylation is induced after cell rupture. Therefore, the physiological relevance of this modification, its function and the enzymes involved, remain unclear [59]. Recently, a phosphorylated form of MGDG was discovered in spinach chloroplasts [60] that has a phosphate ester group at the C-6 position of the galactose ring (MGPDG; Fig. 1). The corresponding phosphorylation activity is CTP dependent and localizes to chloroplast membranes. Phosphorylation of MGDG could drastically affect its biophysical properties because MGPDG is an anionic, probably bilayer-forming lipid. Because the amount of MGPDG in leaves is low, it is difficult to decide whether the synthesis of this lipid might have an effect on the ratio of non-bilayer-forming to bilayerforming lipids in the membranes. Long-chain polyunsaturated fatty acids are the most abundant class of fatty acids attached to galactolipids. Furthermore, fatty acids with three double bonds (e.g. α-linolenic acid, an 18-carbon compound) are the substrate for the lipoxygenase reaction that introduces a hydroperoxy group into the acyl chain [61]. The lipoxygenase pathway leads to the synthesis of several important compounds involved in plant defence, including jasmonic acid. For a long time, it was assumed that free fatty acid, especially free α-linolenic acid, is the most relevant substrate for the lipoxygenase pathway. However, it has recently been shown that most 12-oxophytodienoic acid, an intermediate of the jasmonic acid pathway, is
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Acknowledgements Work on lipids in our laboratories is supported by grants from the Deutsche Forschungsgemeinschaft to P.D. and from the US National Science Foundation, the US Dept of Energy and the US Dept of Agriculture to C.B. We would like to thank Ernst Heinz for pointing out how MGDG fits into the PSI crystal structure.
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bound to the sn1 position of MGDG [62] (Fig. 1). Therefore, in Arabidopsis chloroplasts, a large proportion of 12-oxophytodienoic acid synthesized through the lipoxygenase pathway is derived from lipid-bound rather than free α-linolenic acid. Perspectives
Although much is known about the biosynthesis and function of galactolipids in plants, the full complexity of the underlying pathways has not been fully revealed. Paralogues of MGD1 and DGD1 are present in Arabidopsis but their function will remain unclear until mutants in these genes can be isolated and studied. Environmental factors such as phosphate deprivation add yet another level of complexity. Whether this phenomenon is ubiquitous to all plants
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and whether it can be explored to reduce agricultural phosphate fertilizer input remain to be seen. The identification of the enzymes responsible for the biosynthesis of galactolipids under phosphate-limited growth conditions represents a new challenge. Similarly, the regulatory mechanism for galactolipid biosynthesis following phosphate deprivation is unexplored. The most notorious problem is that of lipid transfer between the plastid and extraplastidic membranes. Although there is irrefutable evidence for lipid trafficking between these compartments, no transport protein has been directly implicated in this process. Control of the ratio of non-bilayer-forming to bilayer-forming lipids at the level of galactolipid biosynthetic enzymes is an exciting possibility that needs further exploration.
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Engineering crop plants: getting a handle on phosphate Henrik Brinch-Pedersen, Lisbeth Dahl Sørensen and Preben Bach Holm In plant seeds, most of the phosphate is in the form of phytic acid. Phytic acid is largely indigestible by monogastric animals and is the single most important factor hindering the uptake of a range of minerals. Engineering crop plants to produce a heterologous phytase improves phosphate bioavailability and reduces phytic acid excretion. This reduces the phosphate load on agricultural ecosystems and thereby alleviates eutrophication of the aquatic environment. Improved phosphate availability also reduces the need to add inorganic phosphate, a non-renewable resource. Iron and zinc uptake might be improved, which is significant for human nutrition in developing countries.
Henrik Brinch-Pedersen* Lisbeth Dahl Sørensen Preben Bach Holm Danish Institute of Agricultural Sciences, Dept of Plant Biology, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark. *e-mail: henrik.brinchpedersen@ agrsci.dk
Phosphate is an essential macronutrient for all living organisms. In natural ecosystems, phosphorus is returned to the soil and converted to inorganic phosphate (Pi) via biological and chemical processes, after which it is available for a new cycle of plant growth. In agricultural ecosystems, two additional factors are introduced: a tapping of the phosphate cycle by the harvest of plant materials for animal and human consumption, and the reintroduction of phosphate in the form of manure or industrially produced fertilizers (Fig. 1). http://plants.trends.com
Because of the importance of Pi as a macronutrient for plant growth and livestock production, and the ability of phosphate compounds to bind important minerals, there is increasing interest in understanding the factors that underlie phosphate uptake and bioavailability in plants used for animal feed and human consumption. Moreover, there is a strong demand for optimization of feeding regimes because of the effect of excess Pi on the aquatic environment (eutrophication). A central component in these contexts is the enzyme phytase, which can release bioavailable phosphate from one of the most important compounds in the phosphate cycle, phytic acid [phytate, or myo-inositol-(1,2,3,4,5,6)hexakisphosphate] (Fig. 2). Phytic acid is largely indigestible by monogastric animals because they have no or limited phytase activity in their digestive tract. Moreover, it is considered to be an important antinutritional factor, preventing the uptake of a range of important minerals. Phytic acid and phytases are
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