Three Enzyme Systems for Galactoglycerolipid Biosynthesis Are Coordinately Regulated in Plants*

Galactoglycerolipids, in which galactose is bound at the glycerol sn -3 position in O -glycosidic linkage to dia-cylglycerol, are abundant in plants and photosynthetic bacteria, where they constitute the bulk of the polar lipids of the photosynthetic membranes. Galactoglycerolipid biosynthesis in plants is highly compartmental-ized involving enzymes at the endoplasmic reticulum and the two chloroplast envelopes. This peculiar organization requires extensive trafficking of lipid precursors. It is now increasingly apparent that there are three different sets of lipid galactosyltransferases capable of galactoglycerolipid biosynthesis in the model plant Arabidopsis . Two enzymes, MGD1 and DGD1, provide the bulk of galactoglycerolipids in the chloroplast and in photosynthetic tissues in general. Under phosphate-lim-ited growth conditions and in non-photosynthetic tissues MGD2/3 and DGD2 are highly active. Moreover, galactoglycerolipids produced by this second pathway are often found in extraplastidic membranes. Although these galactosyltransferases use UDP-Gal as the galactose donor, a third pathway involves a processive enzyme, which transfers galactose from one galactolipid to another. mind

In thinking about membrane lipids, phosphoglycerolipids often come to mind first as these are the primary building blocks of many eukaryotic and prokaryotic cell membranes. However, in plant cells the fraction of phosphoglycerolipids is relatively small. Instead, non-phosphorous galactoglycerolipids are predominant, representing up to 50% of polar lipids in extracts of photosynthetic tissues (1). Of the different galactolipids reported for plants (2), most abundant are the monogalactosyldiacylglycerol (MGDG) 1 and digalactosyldiacylglycerol (DGDG) lipids (cf. Fig. 1 for structures). In addition, under certain circumstances, e.g. in the Arabidopsis tgd1 mutant (3), plants can synthesize higher galactosylated forms of galactoglycerolipids. Although most of these lipids are restricted to the chloroplast membranes in plants, it has become increasingly apparent that galactoglycerolipids are present in extraplastidic membranes in non-photosynthetic tissues or under phosphate-limited growth conditions (4). Current evidence suggests that galactoglycerolipids are exclusively synthesized by enzymes associated with the two chloroplast envelopes in plants, a fact that requires the transfer of lipid precursors to, between, and through the envelopes as well as that of galactoglycerolipids from the envelopes to the photosynthetic membranes inside the chloroplast (thylakoids) and to extraplastidic membranes (5). The three classes of glycosyltransferases ( Fig. 1) involved in galactoglycerolipid biosynthesis in plants and their respective functions and interactions in vivo will be discussed.

Monogalactosyldiacylglycerol Synthases
The most abundant plant galactoglycerolipid, 1␤-MGDG, is synthesized in plants by the action of a galactosyltransferase (MGDG synthase, EC 2.4.1.46) catalyzing the transfer of a galactosyl residue from UDP-1␣-Gal to the sn-3-position of sn-1,2-diacylglycerol (DAG) as shown in Fig. 1A. It is important to note that the anomeric configuration of the galactose is inverted from ␣ in the substrate to ␤ in the product and that only one galactosyl moiety is transferred. These properties identify the MGDG synthase of plants as an inverting, non-processive galactosyltransferase of the GT28 class of glycosyltransferases (afmb.cnrs-mrs.fr/CAZY/) according to the classification by Coutinho et al. (6). It has been long known that the activity of MGDG synthase in plants is associated with the chloroplast envelopes (7), but early efforts in purifying the activity were hampered by its apparent very low abundance (8,9). Nevertheless, substantial information on this enzyme, such as its apparent K m for UDP-Gal and DAG, was gathered indirectly on chloroplast envelopes or partially purified and detergent-solubilized enzyme preparations (10, 11) using a "surface dilution" kinetic model (12). In 1997, Shimojima and co-workers (13) succeeded in purifying MGDG synthase from cucumber cotyledons, isolated a corresponding cDNA, and expressed it to produce a functional protein in Escherichia coli, thereby enabling the molecular analysis of plant MGDG synthases. The fully sequenced genome of Arabidopsis (14) contains three genes encoding functional MGDG synthases, designated MGD1, MGD2, and MGD3 (15,16). These three isoforms can be phylogenetically grouped into the A-type (MGD1) and the B-type (MGD2 and MGD3) MGDG synthases, which have different substrate specificities with regard to the DAG precursor and different functions. Mutant analysis has shown that MGD1 is the isoform responsible for the bulk of MGDG synthesis in Arabidopsis because even partial inactivation of the gene led to a drastic reduction in activity and corresponding loss of MGDG (17). All MGDG synthase isoforms of Arabidopsis lack membrane-spanning domains. Nevertheless, MGD1 was found to be associated with the inner envelope ( Fig. 2A), whereas MGD2 and MGD3 were proposed to be associated with the outer chloroplast envelope (15,16). It is widely assumed (but not unambiguously proven) that MGD1 is on the outside of the inner envelope ( Fig. 2A) because the concentration of its substrate, UDP-Gal, is very low inside plastids and high in the cytosol (18).

Digalactosyldiacylglycerol Synthases
Addition of a second galactose from UDP-Gal to the 6-hydroxyl of MGDG is catalyzed by DGDG synthase (Fig. 1B) and gives rise to the digalactoglycerolipid DGDG. The 136 glycosidic linkage between the two galactose moieties results in an ␣-anomeric configuration for the second galactose, the same as for the anomeric configuration of the UDP-Gal substrate. Therefore, the DGDG synthase is a retaining galactosyltransferase. Formation of DGDG has been described for isolated chloroplasts using UDP-Gal as a substrate (19 -23), but purification of the DGDG synthase proved very difficult. The problem was aggravated by the fact that in chloroplast membrane preparations a processive galactolipid:galactolipid galactosyltransferase (GGGT) is observed ( Fig. 1, C and D; see also below), obscuring the activity of the true UDP-Gal:MGDG galactosyltransferase (DGDG synthase). The isolation of the dgd1 mutant of Arabidopsis, which shows a 90% reduction in DGDG content (24), led to the identification of the gene encoding the enzyme responsible for the bulk of DGDG synthesis (25). The DGD1 gene encodes a galactosyltransferase that has been classified along with other retaining glycosyltransferases (GT4, afmb.cnrs-mrs.fr/ CAZY/). A paralog of DGD1 is present in the Arabidopsis genome, which has been designated DGD2 (26). Interestingly, the DGD1 protein consists of an N-terminal domain required for the insertion of the protein into the outer envelope (27) and a C-terminal glycosyltransferase domain (25) although DGD2 lacks the N-terminal domain (26). The recombinant DGD2 protein uses UDP-Gal as the galactose donor and MGDG as the acceptor (26). MGDG in the absence of UDP-Gal did not lead to the formation of DGDG ruling out a GGGT activity originally proposed to be the major DGDG synthase activity in plants (21)(22)(23). By deleting the N-terminal domain it was recently demonstrated that the glycosyltransferase domain of DGD1 also utilizes UDP-Gal as the galactose donor (28). Moreover, fusing the DGD1 N-terminal domain to DGD2 did not alter the substrate specificity of the fusion protein suggesting that the N-terminal domain of DGD1 does not affect the UDP-Gal preference of the DGD1 glycosyltransferase domain. Therefore, both Arabidopsis DGDG synthases catalyze the formation of ␤␣-DGDG and use UDP-Gal and MGDG as shown in Fig. 1B (26,28). In addition, both DGDG synthases of Arabidopsis are associated with the outside of the outer envelope of chloroplasts (Fig. 2, A and B) (27,28). However, although DGD1 does not require ATP for insertion and is not processed (27), DGD2 is processed and is dependent on ATP for insertion into the outer envelope (28). Both enzymes may be located in different domains of the chloroplast envelope and, therefore, may have access to different pools of MGDG. This notion is supported by the comparison of the lipid composition of dgd1 and dgd2 null mutants of Arabidopsis. Although DGD1 seems to act preferentially on MGDG molecular species with 18-carbon fatty acids in the two positions of MGDG in vivo, DGD2 seems to act on MGDG with a 16-carbon fatty acid in the sn-1 position and an 18-carbon fatty acid in the sn-2 position of the DAG moiety (28), a lipid species preferentially formed under phosphate starvation conditions in Arabidopsis (29).

Galactolipid:Galactolipid Galactosyltransferases
A third class of enzymes capable of galactoglycerolipid biosynthesis is the GGGT. This enzyme is processive giving rise to oligogalactolipids such as tri-and tetragalactosyldiacylglycerols. Its activity has been observed in chloroplast and chloroplast envelope preparations, leading some to suggest that it is the major DGDG synthase of plants (21)(22)(23). However, the analysis of dgd1 and dgd2 double mutants of Arabidopsis clearly indicates that GGGT is different from the genuine DGDG synthases DGD1 and DGD2 (28), because chloroplasts of the double mutant still are capable of oligogalactolipid biosynthesis. Independent evidence is derived from the newly isolated tgd mutants of Arabidopsis (3). These mutants accumulate oligogalactolipids in vivo. The detailed structural analysis of the oligogalactolipids accumulating in these mutants (3) revealed that the galactosyl residues are all ␤136-linked (Fig. 1, C  and D). The oligogalactolipid biosynthetic activity in these tgd mutants was processive as opposed to that of the MGDG and DGDG synthases. It seems likely that this activity is due to GGGT previously observed in chloroplast preparations. Indeed, we have analyzed the structures of oligogalactolipids produced by chloroplast preparations of Arabidopsis and found them to be identical to those produced in the tgd mutants in vivo. 2 Sensitivity to thermolysin suggests that this enzyme is located on the outside of the outer envelope (3,20,28) as shown in Fig. 2C.

Three Sets of Galactosyltransferases with
Different Functions MGD1 and DGD1-Mutant analysis in Arabidopsis has shown that the bulk of galactoglycerolipids found in chloroplasts is synthesized by MGD1 and DGD1 (17,24), and their experimentally determined membrane association is shown in Fig. 2A. Chloroplast lipids in many plants are derived from two pathways (eukaryotic (ER) and prokaryotic (plastid) pathways) giving rise to distinct molecular species (30,31). In Arabidopsis the contributions of the two pathways to thylakoid lipid biosynthesis are nearly equal (32), although the relative fluxes are dependent to some extent on the type of tissue and tissue age (15). It is important to note that in the mgd1 and dgd1 mutants of Arabidopsis the biosynthesis of molecular species from both pathways is affected, suggesting that both enzymes are involved in the prokaryotic and the eukaryotic pathways as shown in Fig. 2A. In fact, MGD1 has broad substrate specificity for various DAG molecular species (15). The expression of the MGD1 gene is generally high under conditions or in tissues promoting or requiring rapid chloroplast development, such as in young expanding leaves, following light exposure of etiolated seedlings, or following the application of the greening-stimulating plant growth factor cytokinin (15, 33, 34). The redox state in chloroplasts 2 C. Xu and C. Benning, unpublished data.

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appears to affect the catalytic activity of MGD1. Prolonged enzymatic activity of MGD1 in vitro requires reducing agents (8). Furthermore, thioredoxin, which is often involved in redox reactions in the chloroplast, modulates the activity of MGD1 in vitro. Thus, redox regulation could provide for coordination between galactolipid biosynthesis and chloroplast development (34,35). However, for this regulatory mechanism to occur, MGD1 should be able to respond to redox changes in the chloroplast stroma. Therefore, determining the exact topology of MGD1 at the inner envelope will be critical to test this hypothesis.
It should also be noted that the dgd1 mutant of Arabidopsis is strongly impaired in photosynthesis (24,36,37) and protein import into chloroplasts (38), as is the mgd1 mutant (17). Moreover, the MGD1 gene appears to be essential in Arabidopsis because attempts to isolate an mgd1 null-mutant have failed (15). Taken together, all data support the notion that MGD1 and DGD1 are the major enzymes of galactoglycerolipid biosynthesis (Fig. 3).
MGD2/3 and DGD2-Until recently, it was the general view in the field that the occurrence of galactoglycerolipids is restricted to the photosynthetic membranes and that their biosynthesis was mainly controlled by the demands for the assembly of photosynthetic membranes. As discussed above, MGD1 and DGD1 meet these demands and, therefore, the question for the function of MGD2/3 and DGD2 arises. One possible answer is provided by results of experiments on the phosphate-deprived dgd1 mutant of Arabidopsis, which led to the discovery of a DGD1-independent, phosphate-controlled pathway of galactoglycerolipid biosynthesis (29). Under these conditions, particularly high amounts of DGDG were found in roots that lack chloroplasts, and fractionation experiments were consistent with the accumulation of DGDG in extraplastidic membranes following phosphate deprivation. The presence of small amounts of galactoglycerolipids in the plasma membrane or the tonoplast (vacuolar membrane) has been described previously (39 -41), and recent reexamination of this issue has now firmly established that phosphate deprivation induces the accumulation of DGDG in plasma membranes (42)(43)(44), possibly to substitute for bilayer-forming phospholipids (4,45). Moreover, DGDG has been found in the peribacteroid membrane of legume root nodules (46), and it is also a major glycolipid of non-photosynthetic floral organs in petunia (47). Analysis of MGD2/3 and DGD2 mRNA abundance showed that the expression of these genes is strongly induced by phosphate deprivation and is generally higher in non-photosynthetic tissues with high levels of DGDG in extraplastidic membranes (15,26,33,46). Unlike the phosphate-deprived dgd1 mutant (29), the dgd2 mutant is unable to produce a phosphate stress-induced DGDG molecular species, suggesting that DGD2 is responsible for the biosynthesis of this phosphate stress-specific DGDG pool (28). It should be noted that MGD2 and MGD3 prefer eukaryotic DAG molecular species as substrates (15), which are derived from the ER. Taken together, the data suggest that MGD2/3 and DGD2 are of conditional importance when the plants experience phosphate stress leading to the biosynthesis of a new DGDG pool, which is distributed from the plastid outer envelope to extraplastidic membranes (Fig. 2B). This MGD2/3-DGD2 pathway may also be affected by other environmental factors de-  Fig. 1; OGL, oligogalactolipids. The thickness of the reaction scheme arrows is proportional to the relative flux through the respective reaction. The predominant galactoglycerolipid in leaves is MGDG and in non-photosynthetic tissues (roots) is DGDG.

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pending on the plant species. For example, the expression of a rice gene encoding a MGDG synthase of the B-type (similar to Arabidopsis MGD2/3) is induced following submergence (48). In addition, the plant growth regulator auxin is involved in mediating the induction of B-type MGDG synthase-encoding genes following phosphate starvation. 3 Interestingly, the phosphate stress-induced expression is reversed by application of cytokinin, contrary to the induction of genes encoding A-type MGDG synthases by this plant growth regulator. 3 Thus, contrapuntal regulation of MGDG synthase type A and type B gene expression by auxin and cytokinins might be the key to the regulation of galactoglycerolipid biosynthesis during development and in photosynthetic and non-photosynthetic tissues. The emerging picture for the regulation and distribution of the MGD1-DGD1 and the MGD2/3-DGD2 pathways in the model plant Arabidopsis is summarized in Fig. 3. Galactolipid:Galactolipid Galactosyltransferase-A gene encoding the GGGT has not yet been identified. Under normal growth conditions, the in vivo activity of this enzyme is not detectable in the wild type of Arabidopsis or most other plants but becomes apparent in the tgd mutants of Arabidopsis (3). The primary defect in the tgd mutants is a disruption of a membrane transport complex in the chloroplast envelopes involved in the transfer of lipid precursors from the ER to the plastid (3). It seems possible that the disruption of this transport system leads to the abnormal accumulation or the absence of lipid intermediates at the outer chloroplast envelope, thereby activating the enzyme. A similar situation could arise in chloroplast preparations in which the interaction between the ER and the chloroplast is disrupted. The result with isolated chloroplasts also suggests that the regulation of this activity most likely happens at the level of the enzyme. Processive retaining galactosyltransferase activity has been reported as a side reaction of ␤-galactosidases in vitro (2). The GGGT activity generates a DAG molecule from MGDG (Fig. 1, C and D) and could be involved in the remodeling of galactolipids at the outer envelope as part of the normal process of providing DAG precursors for MGDG synthases (Fig. 2C). One intriguing possibility is that GGGT is involved in turnover of chloroplast lipids under stress conditions. Sakaki et al. (49,50) have shown that ozone fumigation of spinach leaves resulted in the apparent conversion of MGDG into triacylglycerols and oligogalactolipids in vivo. These authors postulated that GGGT provides the DAG precursor for triacylglycerol biosynthesis following ozone fumigation and produces oligogalactolipids in the process. Moreover, it was shown that GGGT was activated by free fatty acids in the presence of divalent cations such as Mg 2ϩ following ozone fumigation, whereas UDP-Gal-dependent galactosyltransferases were inhibited by free fatty acids under these conditions (51). Therefore one can speculate that GGGT plays a role during ozone-induced injury (Fig. 3) or possibly during senescence of leaves when chloroplast membranes are turned over. Because of its possible role in lipid homeostasis of chloroplast membranes and its intricate regulation, finding the gene(s) encoding GGGT has become a high priority in the quest to more fully understand galactoglycerolipid biosynthesis and its regulation in plants.